controlled temperature at 23 ± 2 °C in the dark for four weeks, and then transferred to the same medium after 2 weeks. After this period the explants were transferred to basal medium with WPM, 20 g L⁻<fnref n="1" /> sucrose, 0.1 g L⁻<fnref n="1" /> myo-inositol, 5 µM BAP, 0.05 µM NAA and 7 g L⁻<fnref n="1" /> agar and placed under a photoperiod of 16 hours. The pH of all media was adjusted to 5.8 before autoclaving. After two months explants oxidation, callus formation, shoot formation in explants with callus and number of shoots per explants with callus were evaluated. Each treatment consisted of five Petri dishes with 10 explants. Data were analyzed by an analysis of variance. Comparisons between treatments were made by orthogonal contrasts.

**Results and conclusions:** In the first experiment, there was no callus formation or shoot regeneration on media containing zeatin in any tested concentration. These results contradict those found by [5] who observed shoot formation on leaf explants on medium containing the combination of zeatin and NAA. However, these authors have used salts formulations other than MS. In the treatment with TDZ, 10% of explants regenerated shoots. The number of shoots per callus ranged from one to more than 10. In the second experiment, B5 medium was not favorable for shoot induction. All explants oxidized, although they formed small calli. In the comparison between WPM and MS N/2, in both media 6% of explants regenerated shoots. However, the WPM showed less oxidation than MS N/2. In the third experiment, where several concentrations of TDZ were compared, the number of explants with callus induction was lower in treatment with 0.25 µM TDZ (83%). In all other treatments all explants formed callus. The number of explants with shoots ranged from 7.5% in the treatment with 0.75 µM to 14% in treatments with 0.25 and 0.50 µM TDZ. Treatments with 1 and 2 µM TDZ showed 11% and 12% of calli with shoots, respectively. In *E. globulus* [6] observed buds induction on hypocotyls when placed on medium containing 0.05 µM TDZ and 5 µM NAA or 0.2 µM 2,4-D in. In *E. saligna* 40% of the calli induced on cotyledon explants regenerated shoots on medium containing 1 µM TDZ and 0.1 µM NAA [7]. Among the media tested, the WPM medium containing 0.5 µM TDZ and 0.1 µM NAA was the most suitable for *in vitro* organogenesis of the *Eucalyptus* sp. clone tested. However, regeneration rates (14%) are still low and further research is needed.

References

1. Quoirin M, Quisen R: Advances in genetic transformation of *Eucalyptus* species. *Molecular Biology of Tropical Plants* 2006, 41-56.

2. Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue culture. *Plant Physiol* 1962, 15:473-497.

3. Lloyd G, McCown B: Commercially feasible micropropagation of mountains laurel, *Kalmia latifolia* by use of shoot-tip cultures. *Plant Prop Soc Proc* 1981, 30:421-427.

4. Gamborg OL, Miller RA, Ojima K: Nutrient requirements of suspension cultures of soybean root cells. *Exp Cell Res* 1968, 50:151-158.

5. Lainé E, David A: Regeneration of plants from leaf explants of micropropagated clonal *Eucalyptus grandis*. *Plant Cell Reports* 1994, 13:473-47.

6. Nugent G, Chandler SF, Whiteman P, Stevenson TW: Adventitious bud induction in *Eucalyptus globulus* Labill. *Vitro Cell Dev Biol_Plant* 2001, 37:388-391.

7. Dibax R, Quisen RC, Bona C, Quoirin M: Plant regeneration from cotyledonary explants of *Eucalyptus camaldulensis* and histological study of organogenesis in vitro. *Brazilian Archives of Biology and Technology* 2010, 53:311-318.

P135

Characterization of herbicide-resistant *Eucalyptus* plants expressing phosphinothricin acetyltransferase gene

Esteban González<fnref n="1" />*, Carla Gugliermoni<fnref n="1" />, Milton Galvão<fnref n="1" />, Maria Fagundes<fnref n="1" />,
Maria Ferreira<fnref n="1" />, Guilherme Almeida<fnref n="1" />, Henrique Alves<fnref n="1" />, José Gonsalves<fnref n="1" />,
Fernando Silva², Sergio Bentivenha², Shinitiro Oda², Eduardo Mello<fnref n="1" />

E-mail: estebang@suzano.com.br

BMC Proceedings 2011, 5(Suppl 7):P135

Herbicide resistant crops are commercially advantageous for efficient field productivity by enabling use of non-selective herbicides for weed management. Herbicide resistant trees could be used to improve productivity and reduce the costs of forest management through to the first and second years post tree establishment. We describe the

introduction of the *phosphinothricin acetyltransferase (bar)* gene, which confers resistance to the herbicide glufosinate, into *Eucalyptus pellita* (clone EP11) and hybrid *Eucalyptus* (*E. grandis* x *E. dunnii* - clone SP1383) under the control of a constitutive promoter. The transgenic lines produced showed a copy number ranging from 1 to 3 copies by Southern blot analysis. Herbicide resistance of the transgenic clones was assessed in the greenhouse by application of 200g/L phosphinotricin at the level of 6.0 L/ha and 4.0 L/ha (commercial rate). The effect of the two herbicide treatments was tested on 4-week and 6-month old plants derived from several transgenic events from the two different *Eucalyptus* clones. All 4-week old non-transgenic control plants, as well as whole shoots of 6-month old control plants were killed by the herbicide by fifteen days after application. The same herbicide treatment effectively killed all the weeds in field conditions. All the transgenic plants showed strong resistance to the herbicide in all treatments with no observed negative effects. The use of herbicide resistant trees may provide an additional means for improving the economic efficiency of plantation forest management.

P136
Expression pattern of the GRAS gene family during somatic embryogenesis in pine

Inmaculada Hernández<fnref n="1" />*, Elena Carneros<fnref n="1" />, Alberto Pizarro<fnref n="1" />, Dolores Abarca²,
Carmen Díaz-Sala²

E-mail: inmaculada.hernandez@uah.es

BMC Proceedings 2011, 5(Suppl 7):P136

The GRAS protein family of putative transcription factors, which includes SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE (SCL) proteins, is involved in root development in *Arabidopsisthaliana* and other plant species [1]. In forest species, genes with homology to the *A. thaliana* SCR gene have been involved in the induction of somatic embryogenesis in *Picea glauca* (Moench) Voss [2] and *Pinus taeda* L. [3] as well as in the development of radial patterning of roots in *Pinus sylvestris* L. [4]. Schrader et al [5] also reported the expression of genes with homology to the *A. thaliana* SHR gene in cambial region of *Populus tremula* x *tremuloides*. Increased levels of mRNA of *Pinus radiata* SHR (PrSHR), *Pinus radiata* SCARECROW-LIKE1 (PrSCL1) and *Castanea sativa* SCARECROW-LIKE1 (CsSCL1) have been associated with the early stages of adventitious root induction in *Pinus radiata* D. Don and *Castanea sativa* Mill., respectively [6-9].

In addition to PrSHR and PrSCL1, we have identified 13 new GRAS genes belonging to the different GRAS clades in the pine genome. The objective of this work is the analysis of the spatiotemporal expression patterns of the pine GRAS gene family during somatic embryogenesis in *Pinus radiata* D. Don. Somatic embryogenesis has become the first biotechnology showing great potential for mass propagation of conifers for application in forestry, allowing the implementation of multivarietal forestry (MVF) [10,11]. Despite major advances in clonal regeneration by somatic embryogenesis or organogenesis, many forestry species are recalcitrant [12]. More knowledge of the regeneration process regulation is necessary to improve the capacity of vegetative regeneration.

The expression pattern of the genes was analyzed by qRT-PCR following the methodology described by Sánchez *et al* [6] and Solé *et al* [7]. For expression analysis, total RNA was extracted from four stages of the somatic embryogenic process: proliferative tissue after 7 and 14 days from the last transference to proliferation medium, somatic embryos at the beginning of differentiation and cotyledonary somatic embryos.

In general, the transcripts of the pine GRAS genes accumulated at the highest levels in cotyledonary somatic embryos. In addition, the transcript levels of PrSCR, PrSHR, PrSCL1, PrSCL6, PrSCL8, PrSCL11 andPrSCL12 showed an increase in somatic embryos at the beginning of differentiation. No differences in PrSCL10 transcript levels were found between the four stages analyzed. Transcript levels of PrSCL16 were undetectable at all stages. *In situ* hybridization for spatial expression analysis will confirm differential expression domains.

This work has been funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR).

Embryogenic lines were provided by C. Walter (Scion).

<footnote n="1">¹FuturaGene Brazil Ltda, Brazil; ²Suzano Papel e Celulose, Brazil</footnote>

<footnote n="1">¹Department of Plant Biology, University of Alcalá, Alcalá de Henares, Madrid, 28871, Spain; ²Department of Plant Biology, Universtiy of Alcalá, Alcalá de Henares, Madrid, 28871, Spain</footnote>
## References

1. Bolle C: The role of GRAS proteins in plant signal transduction and development. *Planta* 2004, 218:683-692.

2. Stasolla C, van Zyl L, Egertsdotter U, Craig D, Liu W, Sederoff RR: The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos. *Plant Physiol* 2003, 131:49-60.

3. Cairney J, Zheng L, Cowels A, Hsiao J, Zismann V, et al: Expressed sequence tags from loblolly pine embryos reveal similarities with angiosperm embryogenesis. *Plant Mol Biol* 2006, 62:485-501.

4. Laajanan K, Vuorinen I, Salo V, Juuti J, Raudaskoski M: Cloning of *Pinus sylvestris* SCARECROW gene and its expression pattern in the pine root system, mycorrhiza and NPA-treated short roots. *New Phytol* 2007, 175:230-243.

5. Schrader J, Nilsson J, Mellerowicz E, Berglund A, Nilsson P, Hertzberg M, Sandberg G: A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity. *Plant Cell* 2004, 16:2278-2292.

6. Sánchez C, Vielba JM, Ferro E, Covelo G, Solé A, Abarca D, de Mier BS, Diaz-Sala C: Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species. *Tree Physiol* 2007, 27:1459-1470.

7. Solé A, Sánchez C, Vielba JM, Valladares S, Abarca D, Diaz-Sala C: Characterization and expression of a *Pinus radiata* putative ortholog to the Arabidopsis *SHORT-ROOT* gene. *Tree Physiol* 2008, 28:1629-1639.

8. Abarca D, Diaz-Sala C: Adventitious root formation in conifers. *Adventitious Root Formation of Forest Trees and Horticultural Plants- From Genes to Applications* Kerala, India: Research Signpost: Niemi K & Scagel C 2009, 227-257.

9. Abarca D, Diaz-Sala C: Reprogramming adult cells during organ regeneration in forest species. *Plant Signal Behav* 2009, 4:8793-795.

10. Park YS: Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and deployment considerations. *Ann For Sci* 2002, 59:651-656.

11. Klimaszewska K, Trontin J-F, Becwar MR, Devillard C, Park Y-S, Lelu-Walter M-A: Recent progress in somatic embryogenesis of four *Pinus* spp. *Tree For Sci Biotech* 2007, 1:11-25.

12. Bonga JM, Klimaszewska K, von Aderkas P: Recalcitrance in clonal propagation, in particular of conifers. *Plant Cell Tissue Organ Cult* 2010, 100:241-254.

## P137
### Tissue culture of two medicinal trees native to Japan

Katsuaki Ishii¹, Naoki Takata, Manabu Kurita, Toru Taniguchi
Forest Bio-research Center, Forestry and Forest Products Research Institute, Japan

E-mail: katsuaki@ffpri.affrc.go.jp

BMC Proceedings 2011, 5(Suppl 7):P137

Wadatsuminoki (Nothapodytes amamianus) is an endangered tree species observed in only Amami Oshima Island located in southern part of Japan. According to the Red list published by Ministry of Environment, it is classified as 1A (Critically endangered) and naturally remaining number is only 20. It contains camptotesin which is used for anti-cancer drugs. Kagikazura (Uncaria rhynchophylla) is an medicinal tree species observed widely in Japan and China. It contains alkaloids (rhynchophylline, isorhynchophylline, hirstine and so on) which are good for remedy of high blood pressure and dementia. For the purpose of micropropagation and development of basis of useful substance production by cell culture as well as conservation of endangered species, tissue culture procedure was developed for those two species.

Excised shoots of 2 years old seedling of Wadatsuminoki rooted in the 1/2DCR medium containing 3 g/l activated charcoal powder. Newly shoots were induced from in vitro root segments subcultured to 1/2MS medium containing 2uM BAP. This cycle can be used for micropropagation of Wadatsuminoki. We have succeeded in micropropagation by tissue culture of Wadatsuminoki (Figure 1). Callus proliferation from stem or root segments was observed on the 1/2LP medium containing 0.5uM BAP and 1 uM 2,4-D, this subculture cell line may be used for the possible production of secondary metabolites in vitro.

Shoots were induced from stem spine (thorn) of Kagikazura in the 1/2MS medium containing BAP or Zeatin. Regenerated plants were obtained by

rooting of these shoots on 1/2MS medium containing 1 uM IBA. Callus induced around the stem segments were continuously subcultured in the fresh 1/2LP medium containing 0.5 uM BAP and 1 uM 2, 4-D. These cell lines can be used for the possible secondary metabolite production and for breeding by somaclonal variation or genetic engineering.

## P138
### Establishment of molecular markers for early selection of embryogenic cultures with high embryogenic potential in brazilian pine (*Araucaria angustifolia* (BERT) O. KTZE)

Leonardo Jo¹*, Andre Luis Wendt dos Santos¹, Paulo Sérgio Schlögl², Miguel Pedro Guerra², Maria Magdalena Rossi³, Eny Iochevet Segal Floh¹
¹Laboratory of Plant Cell Biology, Department of Botany, University of São Paulo, São Paulo-SP, Brazil; ²Plant Developmental Physiology and Genetics Laboratory, Department of Plant Science, Federal University of Santa Catarina, Florianópolis-SC, Brazil; ³Laboratory of Plant Molecular Genetic, Department of Botany, University of São Paulo, São Paulo-SP, Brazil
E-mail: jo.leonardo85@gmail.com
BMC Proceedings 2011, 5(Suppl 7):P138

**Background:** Brazilian pine (*Araucaria angustifolia* (Bert) O. KTze) is the only native conifer species with economic importance in Brazil. Recently, due to intensive exploitation Brazilian pine was included in the official list of endangered Brazilian plants. Biotechnology tools, like somatic embryogenesis (SE), may become a potentially useful tool for mass clonal propagation and *ex situ* conservation of commercial and endangered plant species, especially conifers. SE involves the coordinated execution of four steps (embryogenic culture (EC) induction, proliferation, maturation, and plant regeneration). As observed for other conifers, the presence of well-developed early somatic embryos (SE) in EC of Brazilian pine can be considered the pre-requisite for embryo maturation in a medium supplemented with abscisic acid (ABA) and osmotic agents. However, in some genotypes even the presence of bipolar SE does not guarantee embryo maturation. Since SE morphology cannot be used as the only factor for EC selection, the development of molecular markers for early detection of embryogenic cultures responsive to maturation promoters (ABA and osmotic agents) is highly desirable. Polyamines (putrescine (Put), spermidine (Spd), and spermine (Spm)) have been classified as plant growth regulators and hormonal second-messengers playing a critical role in various growth and developmental processes in plants, such as the differentiation and development of somatic embryos. The relation Put/Spd has demonstrated the best answers for predicting embryogenic potential in different plant species. Apart from polyamines quantification, the analysis of gene expression has been used to detect the expression of embryogenesis regulating genes like somatic embryogenesis receptor kinase (SERK), wuschel-related WOX (WOX), and ABA insensitive-1 (ABI1) during conifer somatic embryogenesis. In order to develop molecular markers for early detection of EC with high embryogenic potential in Brazilian pine, we measured the polyamine content (free and conjugate) and the expression of three embryogenesis-regulating genes (SERK, WOX and ABI1) during the proliferation phase of ECs with different maturation capabilities.

**Methodology:** For induction of ECs, immature zygotic embryos were inoculated in MSG medium free of growth regulators supplemented with 1.46 g L⁻¹ filter-sterilized L-glutamine, 30 g L⁻¹ sucrose, 3 g L⁻¹ Gelrite® (Sigma) in the dark at 25 ±2°C. After one year of EC proliferation, maturation tests were performed using MSG semi-solid medium supplemented with 120 mM abscisic acid, 9% (w/v) maltose, 7% (w/v) PEG 4000, 3% (w/v) sucrose, and 0.15% (w/v) active charcoal. All ECs were maintained in the dark at 25 ±2°C and subcultured every four weeks by transferring ECs to fresh maturation medium. Total RNA from ECs with different maturation capabilities (0.3 g fresh weight) were extracted with Trizol® (Invitrogen, Carlsbad, CA). cDNA was synthesized using 2 µg of total RNA, digested with DNase I (Fermentas), and reverse transcribed with 500 ng oligo-dT25-ancored primer (5'-T(25)VN-3') using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Primers were designed from nucleotide sequences of Brazilian pine cloned cDNA fragments. The template cDNA were synthesized and the dilutions adjusted with Ubiquitin 1 as an endogenous normalization factor. PCR reactions were carried out in a final volume of 25 µL and the PCR products had an average length of 175 - 200 bp. The RT-PCR products were resolved on 2% (w/v) agarose gels stained with ethidium bromide
and photographed. The methodology for the determination of free PAs and conjugate was based on that developed by [1]. Samples (0.2 g fresh weight) were ground in perchloric acid (PCA) 5%. The conjugated forms of the PAs were obtained by hydrolysis (18 h at 110°C) in 12 N chloridric acid (HCl). The samples were then derivatized using dansyl chloride and partitioned with toluene. PAs levels were obtained by means of HPLC using a C18 reverse phase column.

**Results and discussion:** Despite of the maturation capability and as observed in other conifer species, the levels of free PAs in all Brazilian pine ECs tested were higher than the conjugated form, and the most abundant PA found was Put followed by Spd and Spm. However, ECs responsive to maturation conditions (with development of mature somatic embryos) showed significantly lower Put/Spd ratios, when compared to non-responsive ECs. A similar profile was observed in embryogenic cultures of *Oryza sativa* L. [2]. In somatic embryos of *Vitis vinifera*, an abnormal growth and a disorganized cellular proliferation were associated to an inadequate Put/Spd ratio [3].

Concerning gene analysis, only the expression of ABI1 gene could be detected during proliferation phase of the ECs cultures. Although ABI-1 gene is normally associated to events mediated by ABA [4], both ECs responsive or not to ABA showed the expression of ABI1. No expression of SERK and WOX could be detected during the proliferation phase of ECs tested, although the expression of these genes was already detected in somatic embryos, late stage zygotic embryos and seedlings of Brazilian pine.

**Conclusions:** Based on our results, we can suggest that the Put/Spd ratio can be used as a molecular marker for early selection during proliferation phase of Brazilian pine ECs with high embryogenic potential. However, selected embryogenesis regulating genes (ABI1, SERK-1, and WOX) did not show any association with the embryogenic potential in the ECs tested.

### References

1. Silveira V, Floh EIS, Handro W, Guerra MP: Effect of plant growth regulators on the cellular growth and levels of intracellular protein, starch and polyamines in embryogenic suspension cultures of *Pinus taeda*. *Plant Cell, Tiss. Org. Cult* 2004, 76:53-60.

2. Faure O, Mengoli M, Nougarède A, Bagni N: Polyamine pattern and biosynthesis in zygotic and somatic embryo stages of *Vitis vinifera*. *J. of Plant Physiol* 1991, 138:545-549.

3. Shoeb F, Yadav JS, Bajaj S, Rajam MV: Polyamines as biomarkers for plant regeneration capacity improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice. *Plant Science* 2001, **160**:1229-1235.

4. Leung J, Merlot S, Giraudat J: The *Arabidopsis* ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 Genes Encode Homologous Protein Phosphatases 2C involved in Abscisic Acid Signal Transduction. *The Plant Cell* 1997, **9**:759-77.

## P139
### Chemical screening for promotion of adventitious root formation in *Eucalyptus globulus*

Naoki Negishi¹*, Masatoshi Oishi, Akiyoshi Kawaoka
Nippon Paper Industries Co., LTD., Agri-Biotechnology Research Laboratory
E-mail: negishi.n@np-g.com

*BMC Proceedings* 2011, **5**(Suppl 7):P139

*Eucalyptus globulus* is one of the most profitable trees for pulp and paper industries due to its fast growth and short harvesting cycle. This species is easily pulped with high yield, and its fiber qualities are among the best for paper production. However, it is difficult to vegetatively propagate *E. globulus*. So far, we have developed "photoautotropic culture method" that promotes rooting percentage by feeding approximately three times higher level CO₂ level (1000 µmol mol⁻¹) and suitable culture condition [1]. However, several lines showed poor rooting percentage even in the higher CO₂ conditions. Therefore, it was necessary to develop novel method for promotion of adventitious root (AR) formation in *E. globulus*. First, we measured endogenous levels of 20 kinds of hormones such as abscisic acid, auxins, cytokinines and gibberellins, at basal part of stem of easy-rooting line and poor-rooting line by UPLC-ESI-qMS/MS. As a result, the Indole-3-acetic acid (IAA) level of easy-rooting line was two times higher than that of the poor-rooting line, suggesting that endogenous IAA level may regulate ability of AR formation.

Next, we focused on the cytochrome P450s that are involved in a vast array of reactions of many different metabolic pathways. Several triazole-containing chemical compounds have previously been shown act as efficient inhibitors of cytochrome P450 monooxygenases. A chemical library of triazole derivatives to find chemicals which have the effect of promoting AR formation was screened. Consequently, five compounds effectively promoted AR formation.

Finally, we investigated how these chemicals affected the growth of *Arabidopsis thaliana*. *Arabidopsis* seedlings were grown on agar medium containing 1 µM selected chemicals. One of the selected chemicals, MA65 increased the number of roots in wild-type *Arabidopsis* seedlings and this phenotype was similar to a mutant *superroot2* (sur2) [2]. The *SUR2* gene encodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis. The amounts of endogenous IAA in 14-d-old *Arabidopsis* grown in the presence of 1 mM MA65 were analyzed. The IAA content was increased two-fold in the presence of MA65 as compared with untreated *Arabidopsis*. In addition, 1.0 cm explants of *Arabidopsis* stems were incubated for 7 d on MS medium containing 1 mMMA65. Stimulation of an AR formation was observed as compared to untreated samples. Taken together, MA65 may increase endogenous IAA level in a plant cell and promotes an AR formation.

**Acknowledgements:** We thank Dr Tadao Asami and Dr Hitoshi Sakakibara for technical help.

### References

1. Nagae S, Takamura T, Tanabe T, Murakami A, Murakami K, Tanaka M: In vitro shoot development of *Eucalyptus citriodora* on Rockwool in the film culture vessel under CO₂ enrichment. *J. Forest Research* 1996, 1:227-230.

2. Delarue M, Prinsen E, Van Onckelen H, Caboche M, Ballini C: *Sur2* mutations of *Arabidopsis thaliana* define a new locus involved in the control of auxin homeostasis. *Plant J* 1998, 14:603-611.

## P140
### Gene expression in cultured primordial shoots of adult white spruce (*Picea glauca*) in somatic embryogenesis responsive and non responsive genotypes

Krystyna Klimaszewska¹*, Brian Boyle², Sebastien Caron², Don Stewart¹, Catherine Overton¹, John MacKay², Robert Rutledge¹

¹Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.R.S., Québec, QC G1V 4C7, Canada; ²Arborea Project, Centre d'études de la Forêt, Institut de Biologie Intégrative et des Systèmes, Université Laval, 1030 Rue de la Médecine, Québec, QC, G1V 0A6, Canada
E-mail: Krystyna.klimaszewska@nrcan.gc.ca

BMC Proceedings 2011, **5**(Suppl 7):P140

**Background:** Modern forest management relies on extensive breeding and reforestation programs to sustain forest productivity and conservation of natural forests. Plantation forestry, with its increased productivity and improved wood quality, is likely to become an important source of wood products in the future. Vegetative propagation of superior coniferous forest trees through tissue culture has the potential to deliver a stable supply of superior seedlings for forest plantation. To clone adult (mature) conifer trees by means of tissue culture has been a cherished goal for the last several decades. The benefits derived from improving the genetic make-up of planting stock would be significant if such clonal propagation could be achieved at a high efficiency and without growth abnormalities of regenerated plants. Although, somatic embryogenesis (SE) technology has worked well for many conifer species using zygotic embryos as starting material, attempts to achieve the same in adult conifers have failed. The basis of this failure is not exactly understood.

**Results and discussion:** Recently, we have been successful in inducing SE from primordial shoots (PS) of 10 years-old somatic white spruce, genotype 893-6 [1]. We have also identified a few genes (VP1, WOX2, CHAP3A, SAP2C) that were expressed exclusively or significantly up regulated in embryonal mass (EM). In an attempt to gain some understanding of the underlying general molecular events occurring during SE induction phase, we used microarray technology to examine gene expression in responding PS of 893-6 genotype and in non responding PS of 893-12 genotype. Shoot buds were collected from trees in early spring. Some of them were immediately frozen in liquid nitrogen (day 0) and others were disinfected. PS were excised, subdivided into two or four sections and cultured on a standard medium that is used for SE induction from zygotic embryos of white spruce [1]. After 3, 7, 15 and
21 days of culture the PS explants were collected and frozen in liquid nitrogen. RNA was extracted from shoot bud samples at day 0 and from PS explants after 7 days of culture. cDNA was then subjected to microarray analysis using an oligo-based microarray developed by the Arborea project that contains 32,000 probes.

Microarray analysis lead to identification of a number of genes that was up-regulated in each genotype (1-45 fold) in response to day 7 of culture on SE induction medium. Absolute quantification qPCR of the four most up-regulated genes in each genotype confirmed the microarray results, although the magnitude of up-regulation determined by qPCR tended to be greater than that predicted by microarray analysis. In order to provide a more detailed perspective into the dynamics of gene expression, qPCR analysis of these eight genes was expanded to include 3, 15 and 21 days samples. This revealed that while differences in the magnitude of up-regulation for most of these genes differed, the overall trend in expression dynamics were very similar for both genotypes. The one exception was a gene of unknown function, which was rapidly induced in the responsive genotype, reaching a maximum at day 3 followed by progressive reduction over the remaining 18 days. In contrast, expression of this gene remained very low in the non-responsive genotype throughout the entire culture period. The expression level for three other genes peaked between 7-15 days, followed by a reduction at day 21 in both genotypes, whereas the remaining four genes showed a progressive increase up to the last day in culture in both genotypes. This project also included analysis of nine candidate reference genes, which demonstrated that expression of five were highly stable (≤±20% CV).

Reference

1. Klimaszewska K, Overton C, Stewart D, Rutledge RG: Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process. *Planta* 2011, 233:635-647.

P141

Effect of inorganic nitrogen concentration in co-culture and regeneration media on *Agrobacterium tumefaciens* growth and on the regenerative capacity of transformed *Pinus radiata* embryonal mass

Regis Le-Feuvre¹*, Claudia Triviño², Ana María Sabja², Krystyna Klimaszewska³
¹Genomica Forestal S.A., CBT Universidad de Concepción, Concepción, Chile;
²Genfor S.A., Fundación Chile, sucursal Valdivia, Valdivia, Chile; ³Natural
Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055
du P.E.P.S., Quebec, QC G1V 4C7, Canada
E-mail: rlefeuvre@gfsachile.cl
BMC Proceedings 2011, 5(Suppl 7):P141

The Genetic Engineering program at Genomica Forestal SA, Chile (GFSA) has a goal of generating stably transformed radiata pine for *in planta* evaluation of candidate genes. Regeneration of transgenic plants depends mainly on two factors: regeneration ability of transformed cells and stable transgene integration and expression.

In several conifer species, including radiata pine, transgenics have been
regenerated through cocultivation of *Agrobacterium tumefaciens* with
embryogenic cells [1-3]. However, in our first experiments using MSG [4]
culture medium we found that radiata pine embryonal masses did not
recover easily after co cultivation and that there was an excessive
overgrowth of bacterial cells in spite of using bacteriostatics in the medium.
This impediment prompted our study on testing other culture medium
formulations, routinely used in conifer somatic embryogenesis, on the
growth of *A. tumefaciens* GV3101. The tested media were: MSG, DCR [5], and
modified Litvay [6] MLV. Of the three media MSG supported significantly
higher bacterial growth than the other two media. One of the major
differences in the composition of these media is inorganic nitrogen
concentration (NH₄NO₃ and KNO₃). Compared with MLV and DCR, MSG has
the lowest concentration of inorganic nitrogen (100 mgl⁻¹ compared with
340 in DCR and 950 mgl⁻¹ in MLV) provided in the sole form of KNO₃. Based
on our results and the work of others, we concluded that low nitrate
concentration in MSG medium promoted *A. tumefaciens* growth and this
had a deleterious influence on the viability of radiata pine cells during co
cultivation, and also rendered eradication of bacterial cells difficult.
Comparison of growth of radiata pine embryonal mass on the three media
did not show statistically significant differences. A strategy for producing
transgenic radiata pine for *in planta* transgene expression and stability study
will be presented.

References

1. Levée V, Lelu M, Jouanin L, Cornu D, Pilate G: *Agrobacterium tumefaciens*-mediated transformation of hybrid larch (*Larix kaempferi* x *L. deciduas*) and transgenic plant regeneration. *Plant Cell Reports* 1997, **16**:680-685.

2. Klimaszewska K, Park Y, Overton C, MacEacheron I, Bonga J: Optimized somatic embryogenesis in *Pinus strobes* L. *In vitro Cell Dev. Biol* 2001, **37**:392-399.

3. Grace L, Charity J, Gresham B, Kay N, Walter C: Insect-resistant transgenic Pinus radiata. *Plant Cell Reports* 2005, **24**:103-111.

4. Beckwar M, Nagmani R, Wann S: Initiation of embryogenic cultures and somatic embryo development in loblolly pine (*Pinus taeda*). *Can. J. For. Res* 1990, **20**:810-817.

5. Gupta P, Durzan D: Shoot multiplication for mature trees of Douglas fir (*Pseudotsuga menziesii*) and sugar pine (*Pinus lambertiana*). *Plant Cell. Rep* 1985, **4**:177-179.

6. Litvay J, Verma D, Johnson M: Influence of a loblolly pine (*Pinus taeda* L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (*Daucus carrot* L.). *Plant Cell. Rep* 1985, **4**:325-328.

P142

"Vegetative propagation – knowhow and technology for enhancing bioeconomy " - A new project launched in Finland

Tuija Aronen, Martti Venäläinen, Anni Harju, Teijo Nikkanen*
Finnish Forest Research Institute, Punkaharju, Finland
E-mail: teijo.nikkanen@metla.fi
BMC Proceedings 2011, 5(Suppl 7):P142

A three-year project, financed by EU, European Regional Development Fund, will be carried out in 2011-14 as a collaborative effort with Finnish Forest Research Institute as a main performer, and Eastern Finland University and a commercial company Taimityllilä Oy as partners. Three new researchers are engaged by the project. The aim of the project is to deepen knowhow and develop technology for vegetative propagation of forest trees in Finland, and to enhance collaboration among research institutions and practical plant producers. The project strengthens infrastructure for research and development, and benefits industrial and commercial activities in the field. A target group of the project includes forest owners, forest and ornamental nurseries, professionals in landscaping and home gardeners, wood product industry, and research sector. The objectives of the project are divided into five theme areas:

**New solutions for producing forest regeneration material:** The main aim of the project is to develop propagation methods for Nordic conifers, especially Norway spruce with a goal set for commercial mass-propagation. This will be done in collaboration with practical plant producers. Tissue culture approach using somatic embryogenesis, potentially combined with cutting technology, will be studied. Also issues related to user rights and royalty fees when using selected clones or bred families will be touched.

**Production of special forms of forest trees for ornamental use and landscaping:** Methods of vegetative propagation for special forms of forest trees are developed to meet the increasing demand of hardy conifers for ornamental purposes and landscaping in Northern conditions. Both cutting and tissue culture technology will be tested, and commercial plant production piloted together with a company partner. User right and royalty issues for both natural mutants found or special forms created by breeding are also covered.

**Potentials of vegetative propagation in improving the quality of Scots pine heartwood:** The success of tissue culture propagation of Scots pine families producing abundantly extractives, and thus resistant to wood decay, will be compared with the success of families producing only small amounts of extractives. Furthermore, analyses of secondary compounds induced in tissue cultures will be carried out and their use in selection *in vitro* will be studied.

**In vitro -testing of wood quality using mould and decay fungi:**
Methods of *in vitro* -testing of wood decay resistance will be developed and used to enhance the selection of materials more resistant against fungal degradation. Better decay resistance leads to longer service life of wooden constructions and thus improves the environmental performance of wood.

**Vegetatively propagated materials in enhancing research activities:**
Clonally propagated materials would greatly benefit e.g. pathological and entomological studies in Nordic conifers. In addition, developed technologies and knowhow can be utilised in forest tree breeding programmes by adopting clonal testing options.
P143

Novel BAP degradation pathway during adventitious caulogenesis in *Pinus pinea* L. *cotyledons*

Ondrej Novak¹*, Candela Cuesta², Karel Dolezal¹, Lucie Szucova¹, Lukas Spichal¹, Belen Fernandez², Ana Rodriguez², Miroslav Strnad¹

¹Laboratory of Growth Regulators, Faculty of Science, Palacky University & Institute of Experimental Botany AS CR, Slechtitelu 11, Olomouc, CZ-783 71, Czech Republic; ²Area de Fisiologia Vegetal, Departamento Biologia de Organismos y Sistemas, Instituto de Biotecnologia de Asturias, Universidad de Oviedo, Oviedo, E-33071, Spain

E-mail: ondrej.novak@upol.cz

*BMC Proceedings* 2011, **5**(Suppl 7):P143

**Background:** Cytokinins (CKs) are a group of phytohormones which probably regulate the growth, development, and metabolism of all plants. The aromatic CK benzyladenine (BA) has been widely applied in *in vitro* culture for inducing shoot organogenesis. Our study of endogenous cytokinin profiles during the caulogenic process based on mature cotyledons of stone pine (*Pinus pinea* L.) showed a novel metabolic pathway of aromatic cytokinins based on modification of purine skeleton.

**Methods:** Three-year-old mature seeds from two half-sibling selected families and open-pollinated trees of *P. pinea* were used [1] and the samples were collected following the Alonso et al.[2] procedure. Extraction and purification of cytokinins was based on the method described by Novák et al. [3], including modifications published later [4]. The samples were purified using a combination of a cation (SCX-cartridge) and anion [DEAE-Sephadex/C18-cartridge] exchangers. Combination of high performance liquid chromatography (HPLC) with quadrupole-time of flight mass spectrometry (QqTOF) was used for accurate and sensitive identification and quantification of cytokinins.

**Results and discussion:** Cortizo et al. [5] published the dynamics of BA uptake and metabolism in *P. pinea* cotyledons excised from embryos precultured for 2 and 4 days and cultured *in vitro* in modified Le Poivre media with 4.4 µM BA. In our experiment, we used 44.4 µM concentration of BA and samples were collected at different periods (0; 1; 2; 6; 16; 24 h and 2; 4; 6 d). Using high-resolution MS, the naturally-occurring BA metabolites as well as new BA forms were identified. In comparison with previously published profiles of the BA metabolite pool [5,6], the ribosyl and glycosyl forms were quantified as the most abundant metabolites. Moreover the biological activity of identified BA and its derivatives were compared in various CK bioassays. The results indicate that BA uptake during the caulogenic process may be possible to regulate not only by known cytokinin pathways. Finally, the feeding experiment with stable isotope-labelled standard, ¹⁵N₄-BA, confirmed our identification of the novel metabolic CK pathway.

**Conclusions:** The identification of the novel BA forms demonstrates that the novel cytokinin pathway is used as a control mechanism of BA uptake from the bud induction medium during adventitious caulogenesis in cotyledons of *P. pinea*. Our results help to understand the processes associated with embryo germination in plant tissue culture.

**References**

1. Cuesta C, Ordas RJ, Fernandez B, Rodriguez A: Clonal micropropagation of six selected half-sibling families of *Pinus pinea* and somaclonal variation analysis. *Plant Cell Tiss. Org* 2008, **95**:125-130.

2. Alonso P, Moncalean P, Fernandez B, Rodriguez A, Centeno ML, Ordas RJ: *An improved micropropagation protocol for stone pine (Pinus pinea* L.). *Ann. Forest Sci* 2006, **63**:879-885.

3. Novak O, Tarkowski P, Tarkowska D, Dolezal K, Lenobel R, Strnad M: *Quantitative analysis of cytokinins in plants by liquid chromatography/ single-quadrupole mass spectrometry*. *Anal. Chim. Acta* 2003, **480**:207-218.

4. Novak O, Hauserova E, Amakorova P, Dolezal K, Strnad M: Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. *Phytochem* 2008, **69**:2214-2224.

5. Cortizo M, Cuesta C, Centeno ML, Rodriguez A, Fernandez B, Ordas RJ: *Benzyladenine metabolism and temporal capacity of Pinus pinea L. cotyledons to form buds in vitro*. *J Plant Physiol* 2009, **166**:1069-107.

6. Zhang H, Horgan KJ, Reynolds PHS, Jameson PE: Benzyladenine metabolism during reinvigoration of mature *Pinus radiate* buds in vitro. *Tree Physiol* 2010, **30**:514-526.

P144

Micropropagation of *Pinus taeda* L. via axillary buds

Leandro Francisdo de Oliveira¹*, Luciana Lopes Fortes Ribas²,
Marguerite Quoirin², Henrique Soares Koehler³, Antonio Rioyei Higa⁴

¹Programa de Pós-Graduação em Botânica, Departamento de Botânica,
Universidade Federal do Paraná, Curitiba, Paraná, 81531-980, Brazil;
²Departamento de Botânica, Universidade Federal do Paraná, Curitiba, Paraná,
81531-980, Brazil; ³Departamento de Fitotecnia e Fitossanitarismo, Universidade
Federal do Paraná, Curitiba, Paraná, 80210-170, Brazil; ⁴Departamento de
Ciências Florestais, Universidade Federal do Paraná, Curitiba, 80035-050, Brazil
E-mail: lebio.oliveira@yahoo.com.br

*BMC Proceedings* 2011, **5**(Suppl 7):P144

**Introduction:** *Pinus taeda* stands for productivity and quality of its timber [1]. Researches using biotechnology are of great importance and have been applied to the improvement of its timber and plantation [2]. The main method of *Pinus* propagation is by seeds, once the minicuttings depends on the season of the year or depends of juvenile material [3-5]. Thus, researches on micropropagation of *Pinus* taeda are currently a priority in Brazil [6]. Micropropagation is the best method for mass production of superior genotypes and represents a strategy for tree improvement and capture of genetic gains [7]. Studies on *Pinus taeda* micropropagation by axillary bud proliferation are quite few. The purpose of this study was to develop a protocol for micropropagation of this species from juvenile material.

**Materials and methods:** For *in vitro* establishment two to four month old seedlings were used. Apical shoots and nodal segments of 3 cm length were inoculated in MS [8], DCR [9], WV3 [10] or WV5 [11] medium. For axillary shoots induction, the explants were inoculated in WV3, WV5 or DCR medium, with BAP (0, 0.12, 0.25 and 0.50 µM). For the induction of roots, we tested the effect of double-layer medium, with semi-solid phase consisting of agar and water or GDm/2 [12] medium and the liquid phase containing water or GDm/2 medium. Both phases were supplemented with 2.69 µM NAA and 0.44 µM BAP for 9 days, followed by transfer to growth regulator-free GDm/2 medium. The rooted plants were planted in Plantmax® Forestry substrate and maintained in a greenhouse.

**Results and Discussion:** Nodal segments showed better responses during *in vitro* establishment, with up to 100% of explants forming

<figure><img src="image_63.png" /><figcaption>**Figure 1(abstract P144)** Nodal segments of *Pinus taeda* inoculated in WV5 medium, after nine weeks in the *in vitro* establishment. Bar: 1 cm.</figcaption></figure>
axillary shoots and an average of 4.3 to 5.8 shoots per explant. The WV5
media proved better and presented the highest survival rate (86.0%) and
highest elongation percentage (85.2%) (Figure 1).

The balance of salts in WV5 and WV3 culture media favored an optimal
development of *in vitro* cultures of *Pinus taeda* due to its lower concentration
of N in comparison with MS medium and to higher concentrations of
thiamine and inositol, which are growth promoters. Elongated shoots were
subdivided into segments, increasing the multiplication rate to 3 segments
per shoot. The majority of BAP treatments did not promote better
multiplication when compared to control. However, the alternate use of
0.12 µM BAP added to WV5 culture medium during initial culture and a BAP-
free medium during the 1st subculture can increase the multiplication rate.
The estimated production was 1024 shoots from 100 explants, in seven
months of cultivation. The best rooting percentage (37.5%) was obtained in
shoots treated with 2.69 µM NAA and 0.44 µM BAP for 9 days in culture
medium composed of water and agar without liquid phase, followed by
transfer to growth regulator-free GDM/2 medium. The double-layer medium
did not increase the rooting percentage. This result was higher than that

found in *Pinus virginiana*, when the same combination of plant growth
regulators was used [13]. The roots originated directly and indirectly from the
stem with callus formation. After 90 days of acclimatization, the survival rate
was 90% and an average of 4.6 roots per plant was obtained (Figure 2). This
result was better than that obtained in other study with *Pinus taeda*
that reported 38% of necrosis five weeks after transplantation [14].
Micropropagation of *Pinus taeda* from axillary buds and juvenile material is
feasible, but requires further studies to optimize the rooting stage.

**Acknowledgments:** The autors are grateful to Battistella Florestal
Company for the financial assistance, to FINEP (Financiadora de Estudos e
Projetos), to REUNI (Reestruturação e Expansão das Universidades
Federais) and the Federal University of Paraná, Brazil.

References

1. Schultz RI: Genetics and tree improvement. *Loblolly pine: the ecology and culture of loblolly pine (Pinus taeda) L.* U.S. Department of Agriculture, Forest Service: Schultz RI. New Orleans 1997, 1-50.

2. Gonzalez-Benecke CA, Martin TA, Clark III A, Peter GF: Water availability and genetic effects on wood properties of loblolly pine (*Pinus taeda*). Canadian Journal of Forest Research 2010, **40**:2265-2277.

3. Stojičić D, Budimir S, Čulafić L: Micropropagation of *Pinus heldreichii*. *Plant Cell, Tissue and Organ Culture* 1999, **59**:147-150.

4. Alcantara GB, Ribas LLF, Higa AR, Ribas KCZ, Koehler HS: Efeito da idade da muda e da estação do ano no enraizamento de miniestacas de *Pinus taeda* L. *Revista Árvore* 2007, **31**:399-404.

5. Andrejow GMP, Higa AR: Potencial de enraizamento de miniestacas de *Pinus taeda* L. provenientes de brotação apical de mudas jovens. *Floresta* 2009, **39**:897-903.

6. Faleiros M: Plantação a partir de clones de pínus já é feita no Brazil.
2011, Available in http://www.revistaopapel.org.br/noticia-anexos/
1272649863_aae1535dc2037f6433ca5e0011dcd609_138912915.pdf.

7. Gupta PK, Durzan DJ: Loblolly pine (*Pinus taeda* L.). *Biotechnology in Agriculture and Forestry Trees III* Verlag Berlin Heidelberg New York: Springer: Bajaj YPS 1991, **16**:383-407.

8. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. *Physiologia Plantarum* 1962, **15**:473-497.

9. Gupta PK, Durzan DJ: Shoot multiplication from mature trees of Douglas-fir (*Pseudotsuga menziesii*) and sugar pine (*Pinus lambertiana*). *Plant Cell Reports* 1985, **4**:177-179.

10. Coke JE: Basal nutrient medium for in vitro cultures of loblolly pines. USA Patent 5,534,434 1996, Available in http://www.freepatentsonline.com/5534434.pdf accessed in May 16 2011.

11. Coke JE: Basal nutrient medium for in vitro cultures of loblolly pines. USA Patent 5,534,434 1996, Available in http://www.freepatentsonline.com/5534433.pdf accessed in May 16 2011.

12. Mehra-Palta A, Smeltzer RH, Mott RL: Hormonal control of induced organogenesis experiments with excised plant parts of loblolly pine. *Tappi Journal* 1978, **61**:37-40.

13. Chang S, Sen S, McKinley CR, Aimers-Halliday J, Newton RJ: Clonal propagation of Virginia Pine (*Pinus virginiana* Mill.) by organogenesis. *Plant Cell Reports* 1991, **10**:131-134.

14. Leach GN: Growth in soil of plantlets produced by tissue culture. *Tappi Journal* 1979, **62**:59-61.

<figure><img src="image_64.png" /><figcaption>**Figure 2(abstract P144)** Micropropagated plants of *Pinus taeda*, 60 days after transplanted and acclimated. Bar: 5 cm.</figcaption></figure>

P145

Indirect organogenesis from leaf explants of *Eucalyptus benthamii* x *Eucalyptus dunnii* and shoot multiplication

Yohana de Oliveira<fnref n="1" />*, Laís Gomes Adamuchio², Cassiana de Oliveira³,
Juliana Degenhardt-Goldbach⁴, Isabel Gerhardt⁵, João Carlos Bespalhok Filho⁶,
Roberson Dibax⁵, Marguerite Quoirin⁷

¹Departamento de Fitotecnia e Fitossanitarismo, Pós-Graduação em
Agronomia-Produção Vegetal, Universidade Federal do Paraná (UFPR), 80035-
050, Curitiba, Paraná, Brazil; ²Graduação em Agronomia, UFPR, Curitiba, Paraná,
Brazil; ³Departamento de Fitotecnia e Fitossanitarismo, Pós-graduação em
Agronomia-Produção Vegetal, UFPR, Curitiba, Paraná, Brazil; ⁴Embrapa Florestas,
Estrada da Ribeira, km 111, Caixa Postal 319, Colombo, Paraná, Brazil; ⁵Embrapa
Florestas, Colombo, Paraná, Brazil; ⁶Departamento de Fitotecnia e
Fitossanitarismo, Pós-graduação em Agronomia-Produção Vegetal, Universidade
Federal do Paraná (UFPR), 80035-050, Curitiba, Paraná, Brazil; ⁷Departamento de
Botânica, UFPR, Caixa Postal 19031, 81531-990, Curitiba, Paraná, Brazil

<footnote n="1">¹Departamento de Fitotecnia e Fitossanitarismo, Pós-Graduação em
Agronomia-Produção Vegetal, Universidade Federal do Paraná (UFPR), 80035-
050, Curitiba, Paraná, Brazil; ²Graduação em Agronomia, UFPR, Curitiba, Paraná,
Brazil; ³Departamento de Fitotecnia e Fitossanitarismo, Pós-graduação em
Agronomia-Produção Vegetal, UFPR, Curitiba, Paraná, Brazil; ⁴Embrapa Florestas,
Estrada da Ribeira, km 111, Caixa Postal 319, Colombo, Paraná, Brazil; ⁵Embrapa
Florestas, Colombo, Paraná, Brazil; ⁶Departamento de Fitotecnia e
Fitossanitarismo, Pós-graduação em Agronomia-Produção Vegetal, Universidade
Federal do Paraná (UFPR), 80035-050, Curitiba, Paraná, Brazil; ⁷Departamento de
Botânica, UFPR, Caixa Postal 19031, 81531-990, Curitiba, Paraná, Brazil
E-mail: yohana@ufpr.br
BMC Proceedings 2011, **5**(Suppl 7):P145</footnote>
**Background:** In Brazil, especially in the Southern region, stresses caused by cold and eventual frost are those that exert the most negative effect on the productivity of *Eucalyptus* spp. The genetic transformation techniques may contribute to forestry improvement programs in order to obtain genotypes expressing new interesting characteristics. They allow shortening the long breeding cycles and avoiding manipulation of adult trees. Their efficiency depends on establishment of regeneration procedures that allow the development of shoots from the transformed tissues.

*E. benthamiix* E. dunnii hybrids have shown superiority to their parents concerning growth and frost tolerance [1], but no information about their *in vitro* organogenesis has been reported in the literature.

The objective of this study was to evaluate the effect of some factors of culture medium on indirect organogenesis and shoot multiplication of an*E. benthamii* x *E. dunnii* clone.

**Methods:** *In vitro*established shoots,provided by EMBRAPA-Florestas (Colombo, PR, Brazil), were used as explant source. Cultures were maintained under white fluorescent tubes providing a photon flux density (PFD) of approximately 20 µmol m⁻² s⁻¹, a 16-h photoperiod and a temperature of 25±2 °C.

The cultures were performed in glass flasks containing 25 mL MS [2] medium supplemented with 1.11µM BA and sealed with rigid polypropylene caps. For the indirect organogenesis, leaves were excised from shoots at the petiole base, split into two halves and inoculated in culture media. The cultures were done in Petri dishes kept in a growth chamber in the dark throughout the experiment.

The statistical design was performed in a factorial scheme (2:2:2) and a comparison was done between two culture media (MS-N/2, with half concentration of potassium and ammonium nitrates, and JADS [3] with 0.1 µM NAA, with and without PVP-40 (250 mg L⁻¹) and two TDZ concentrations (0.1 and 0.5 µM).

After 70 days, the percentages of explants forming callus, oxidized explants, explants producing anthocyanin, explants forming buds and shoots, and the number of shoots per explant were evaluated.

For the multiplication test, the statistical design was performed in a factorial scheme (3:2) with three culture media (MS, WPM [4] and JADS, with 1.11µM BA) and two subcultures (28 and 56 days after the initial culture period). The analyzed variables for each subculture were: percentage of oxidation, of explants showing chlorosis, fresh weight and number of shoots.

**Results and discussion:** Regarding the oxidation, the higher rates (100%) were observed on JADS medium in presence or absence of PVP-40 and on MS-N/2 medium (68.3%) in presence of PVP-40. However, the JADS medium showed the highest percentage of callus formation (83.3%). In MS-N/2 medium the highest percentage of callus formation (55%) was found in the presence of PVP-40 and 0.5µM TDZ. Similar results were observed [5] with *E. saligna* leaf explants cultured on MS-N/2 medium supplemented with 0.1µM NAA and 1.0µM TDZ. Anthocyanin was only observed in explants cultured on MS-N/2 medium without PVP and containing 0.5µM TDZ. The lowest percentage of explants presenting anthocyanin was observed on MS-N/2 with PVP-40 that gave the best result for organogenesis (8.3%).

For the multiplication experiment, the variance analysis did not reveal significant interaction between the factors. During the first subculture MS medium with 1.11µM BA promoted the highest number of shoots/explant (9.2). The explants cultured on MS medium showed the highest fresh weight (0.384g) and highest percentage of chlorotic explants (88%). The percentage of oxidized explants was the same on JADS and MS media (40%). In a study on *E. urophylla* x *E. globulus* shoot multiplication [6], an increase of oxidation was also observed on JADS medium in comparison with MS medium.

With regard to the second subculture, the number of shoots per explant and percentage of oxidation did not differ among the treatments. The explants cultured on MS medium showed the same behavior for the chlorosis than during the first subculture and the percentage of chlorotic explants was superior to that observed in others treatments (64%). On WPM medium, the mean number of shoots/explant was the highest (9.08).

**Conclusions:** *E. benthamiix* E. dunnii hybrid has a low organogenic response to the tested combinations of growth regulators and high tendency to oxidation even when an antioxidant is present in the culture medium. However, the concentration of PVP-40 used in this study may

have been low and other types of antioxidants and concentrations need to be compared.

The MS medium with 1.11 µM BA promoted the highest number of shoots per explant after a 28 day culture period, and after 56 days, there were no differences among the treatments.

**Ackoledgements:** The authors thank CNPq (Conselho Nacional de Pesquisa, Brazil) and Fundação Araucaria (Parana State, Brazil) for funding the research.

**References**

1. Paludyszyn Filho E, Santos dos PET, Ferreira CA: Eucaliptos indicados para plantio no Estado do Paraná. *Colombo: Embrapa Florestas* CNPF 2006, 45, (Documentos, 129).

2. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue culture. *Physiologia Plantarum* 1962, 15:473-497.

3. Correia D: Crescimento e desenvolvimento de gemas na multiplicação de *Eucalyptus* spp. em meio de cultura líquido e sólido. (Dissertação - Mestrado). USP/ESALQ 1993, 113.

4. Lloyd G, McCown B: Commercially-feasible micropropagation of mountain laurel, *Kalmia latifolia*, by use of shoot-tip culture. *International Plant Propagation Society Proceedings* 1980, 30:421-427.

5. Dobax R, Deschamps C, Bespalhok Filho JC, Viera LGE, Molinari HBC, Campos MKF, Quorin M: Organogenesis and *Agrobacterium tumefaciens*-mediated transformation of *Eucalyptus saligna* with P5CS gene. *Biologia Plantarum* 2010, 54:6-12.

6. Borges SR: Micropropagação e enraizamento de miniestacas de clones híbridos de *Eucalyptus globulus*. (Dissertação – Mestrado). Universidade Federal de Viçosa, MG, Brazil. Programa de Pós-Graduação em Ciência Florestal 2009, 72.

P146

Characterization of metabolic differences between embryogenic and non-embryogenic cells in forest trees

So-Young Park¹, Wi-Young Lee, Yong-Wook Kim, Heung-Kyu Moon

Korea Forest Research Institute

E-mail: soypark7@korea.kr

BMC Proceedings 2011, 5(Suppl 7):P146

Somatic embryogenesis (SE) in forest trees is considered as one powerful approach in cloning elite genotypes. However, SE is difficult to achieve in tissue or cell beyond the mature embryo phase in trees and its physiological process involved are also poorly understood. Recently metabolic profiling studies have been contributed to understand the mechanism involved in SE process of plant [1-3]. In addition, there are also a number of reports indicating that polyamines (PAs) play a crucial role in SE process [4,5]. As part of an ongoing study of the transition of somatic cells to an embryogenic state in adult trees, embryogenic (EC) and non-embryogenic cells (NEC) in various forest species were investigated for its metabolic compositions including PAs. A comparison of metabolic compositions of NEC and EC using gas chromatography/mass spectrometry (GC/MS) identified around 50 compounds, partly displaying significant changes in metabolite levels, e.g., highly elevated levels of xanthosine and methyloxazole in EC compared to NEC of broadleaves and conifer species (Fig.1). Changes in the polyamine content were also analyzed in both cell types. Free polyamine contents varied according to the species and cell types, the highest levels occurring in the NEC on proliferation medium, when putrescine and spermidine were most abundant. However, the putrescine/spermine+spermidine (put/spm+spd) ratio was higher in EC of yellow poplar and hybrid pine. Analysis of PAs in both cell types indicated that total polyamine concentration was always higher in NEC than in EC, and spermine was present in only minute quantities and showed only a small change. From this analysis, we have identified numerous compounds involved with embryogenic state, and could characterize its differences between broadleaves and conifer trees.

**References**

1. Dowlatbadi R, Weljie AM, Thorpe TA, Yeung EC, Vogel HJ: Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy. *Plant physiol biochem* 2009, 47:343-350.

2. Hosp J, Tashpulatov A, Roessner U, Barsova E, Katholnigg H, Steinborn R, Melikant B, Lukyanov S, Heberle-Bors E, Touraev A: Transcriptional and metabolic profiles of stress-induced, embryogenic tobacco microspores. *Plant Mol Biol* 2007, 63:137-149.
<figure><img src="image_65.png" /></figure>

3. Robinson AR, Dauwe R, Ukrainetz NK, Cullis IF, White R, Mansfield SD: Predicting the regenerative capacity of conifer somatic embryogenic cultures by metabolomics. *Plant Biotech J* 2009, 7:952-963.

4. Minocha SC, Minocha R: Role of polyamines in somatic embryogenesis. *Biotechnology in Agriculture and Forestry* Heidelberg: Springer-Verlag; Bajaj YPS 1995, 30:55-72.

5. Mala J, Cvikrova M, Machova P, Martincova O: Polyamines during somatic embryo development in Norway spruce (*Picea abies* [L.]). *J For Sci* 2009, 55:75-80.

P147

**Use of kanamycin for selection of *Eucalyptus saligna* genetically transformed plants**

Yohana Oliveira¹*, Lais Adamuchio¹, Juliana Degenhardt-Goldbach²,
Isabel Gerhardt², João Bespalhok¹, Roberson Dibax¹, Marguerite Quoirin¹
¹UFPR, Brazil; ²EMBRAPA-Florestas, Brazil

E-mail: yoyohana@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):P147

**Background:** Several factors may affect the genetic transformation efficiency of woody species. One factor is the use of an efficient selective agent that inhibits the development of non-transformed cells and just allows the development of transformed tissues. The most used selection agent is the neomycin phosphotransferase II (NPTII) gene, which confers resistance to aminoglycoside antibiotics kanamycin, neomycin and G-418 [1].

The selective agent concentration in culture medium may have influence on shoot regeneration and high concentrations may promote adverse effects on organogenic potential [2]. The kanamycin effects in *Eucalyptus* are variable and depend on the species and genotypes [3]. The purpose of this study was to evaluate the effect of kanamycin concentration on transformation efficiency for *Eucalyptus saligna* cotyledons after co-culture with *Agrobacterium tumefaciens*.

**Methods:** The bacterial strain was EHA 105, containing a binary vector carrying the GUS gene under control of CaMV35S promoter and NPTII gene under control of the same promoter. Cotyledons from twelve days old *E. saligna* plantlets were co-cultured for 30 min in the bacterial suspension (OD<sub>600nm</sub> = 0.5) followed by a 5 day co-culture on MS culture medium containing 2.7 µM NAA + 4.4 µM BAP in the dark. The explants were then transferred on the same medium supplemented with (1) 12.5 mg L<sup>-1</sup> kanamycin (Km) + 300 mg L<sup>-1</sup> Augmentin (Aug); (2) 25 mg L<sup>-1</sup>Km+ 300 mg L<sup>-1</sup> Aug and (3) 50 mg L<sup>-1</sup>Km+ 300 mg L<sup>-1</sup> Aug. The explants were subcultured in the same culture medium every 15 days and, after 60 days, the percentage of oxidation, callus and shoot formation, and shoot number per explant were evaluated. DNA was extracted from fresh young leaves and processed according to the specific protocol [4]. The presence of GUS gene in the putative transformed plants was confirmed by PCR 10 months after inoculation.Gus expression was studied by histochemical test for β-glucuronidase, three and 120 days after inoculation.

**Results:** With regard to the percentage of oxidation, percentage of callus formation and percentage of shoot formation, there was no significant difference among the three treatments. The percentages of explants
regenerating shoots were 24, 15.9 and 14.6 respectively for the three Km concentrations, after 60 days of co-culture period. The lower concentration of kanamycin (12.5 mg L⁻¹) showed best results for shoot regeneration (24%) and number of shoots per explant (3.9) and these results were statistically different of those obtained with other treatments. Three gus positive events were regenerated from explants cultured on medium containing 12.5 mg L⁻¹Kmand the transformation efficiency was 0.0075%.

**Conclusion:** The concentration of 12.5 mg L⁻¹Kmallowed the shoot induction from genetic transformed tissues and was considered satisfactory for selection of transformed tissues. The information presented here may constitute the basis for optimization of the genetic transformation of other *E. saligna* genotypes.

**Acknowledgements:** The authors thank CNPq (Conselho Nacional de Pesquisa, Brazil) and Fundação Araucaria (Sciences Secretary, Parana State, Brazil) for funding the research.

## References

1. Joersbo M, Okkels FT: A novel principle for selection of transgenic plant cells: positive selection. *Plant Cell Reports* 1996, 16:219-221.

2. Gonzalez ER: Transformação genética de *Eucalyptus grandis* e do híbrido *E. grandis* x *E. urophylla* via *Agrobacterium*. *Tese (Doutorado em Agronomia)* Universidade de São Paulo 2002.

3. Gonzales ER, Andrade A, Bertolo AL, Lacerda GC, Carneiro RT, Prado Defávari VA, Labate CA: The efficiency of aminoglycoside antibiotics in the regeneration and selection of *Eucalyptus* spp. *In: International Conference Eucalyptus in the Mediterranean Basin: Perspectives and New Utilizations* Taormina, 1999. Anais. Taormina: Centro Promozione Publicità Press 2001, 45-48.

4. Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. *Focus* 1990, 12:13-15.

# P148
## Induction and repetitive embryogenesis of *Ocotea porosa*

Marguerite Quoirin¹*, Luciana Pelegrini², Luciana Rivas²

¹Universidade Federal do Paraná, Brazil; ²UFPRM Brazil

E-mail: mquoirin@ufpr.br

BMC Proceedings 2011, 5(Suppl 7):P148

**Introduction:** *Ocotea porosa* (Liberato Barroso) known as "imbuia" belongs to the Lauraceae family and is native to the Mixed Ombrophilous Forest (Araucaria Forest) where it was heavily exploited due to the high quality and worldwide value of his hardwood that is exported in large quantities for luxury furnishing manufactures [1]. The sexual propagation of *O. porosa* at its natural occurrence area is difficult, due to the strong tegumentary dormancy and its irregular germination. Moreover, the seed viability is short by virtue of being a recalcitrant species, showing high level of humidity [1]. Another limiting factor for its vegetative propagation is the low response of cuttings to the induction of adventitious roots (4%) [2]. The aim of this study was to establish repetitive cycles of secondary somatic embryogenesis from *O. porosa* embryonic axes.

**Material and methods:** Zygotic embryonic axes from immature seeds were used as explants. The seed disinfection was performed in a laminar flow chamber, through immersion in ethanol 70% (v/v) for 5 min, followed by 20 min in NaOCI 4% (v/v) supplemented with 0,1% of Tween® 20. After that, the seeds were rinsed five times with sterile water. The WPM [3] culture medium was used in all stages. Embryonic axes were inoculated on culture medium supplemented with sucrose (20 g L⁻¹), activated charcoal (1.5 g L⁻¹), agar Vetec® (4 g L⁻¹) and 2,4-D (200 µM) combined or not with hydrolyzed caseinor glutamine (0.5 or 1 g L⁻¹) during 90 days. The somatic embryos obtained in the induction phase were multiplied on medium with sucrose (20 g L⁻¹), agar Vetec® (3.5 g L⁻¹) and 2,4-D (22.62 µM) combined with 2-iP (2.46 µM) for 90 days followed by transfer to culture medium with 0.5 g L⁻¹ of hydrolyzed casein combined with 0.5 g L⁻¹ glutamine or 1 g L⁻¹ hydrolyzed casein or 1 g L⁻¹ glutamine for 30 days. The maturation of somatic embryos was tested in WPM culture medium containing sucrose (20 g L⁻¹), agar Vetec® (3.5 g L⁻¹) and polyethylene glycol (PEG 6000) (control, 3.5 and 7%) during 30 days. The experiment was repeated twice. All the cultures were maintained in the dark at 27±2°C (day) and 18±2°C (night).

**Results and discussion:** Patterns of direct and indirect induction of somatic embryos were observed with low frequency in culture medium

containing 200 µM 2,4-D alone or combined with 1 g L⁻¹ hydrolyzed casein or glutamine. The mean percentage of calli with somatic embryos varied between 4.2% and 8.3% for primary somatic embryogenesis without differences between treatments (after 90 days). In the present study, the maximum percentage of somatic embryos induction was 8.3%. Similar results were found with *O. odorifera* that presented a mean percentage of 6.5% [4]. On the culture medium containing 200 µM 2,4-D, the percentage of calli with somatic embryos was 6.3% and the formation of one globular embryo was visible. When 200 µM 2,4-D was combined with 1 g L⁻¹ hydrolyzed casein this percentage was 8.3% and two globular somatic embryos were developed per callus. However, in the medium containing 200 µM 2,4-D and 1 g L⁻¹ glutamine, after 90 days the percentage was smaller (4.2%) and two globular and one cordiform shape embryos were observed. After three months, the primary somatic embryos subcultured in culture media without plant growth regulators with or without activated charcoal did not progress to torpedo and cotyledonary stages. In repetitive embryogenesis the combination of 0.5 g L⁻¹ of hydrolyzed casein and glutamine promoted an average of 46.6 new globular embryos per callus and 75% of callus presented embryogenic mass com pro-embryos. The subculture in media containing 1 g L⁻¹ hydrolyzed casein promoted an average formation of 58 globular embryos, 2 cordiform, 2.5 torpedo and 1 cotyledonary per callus at the end of every subculture and 72.5% of explants formed mass containing pro-embryos. When 1 g L⁻¹ glutamine was added into the media, 57.5% of the mass presented pro-embryos and the induction of globular somatic embryos was lower (23.3 globular embryos per explant). During maturation phase, the somatic embryos develop from initial to late stages. However, in the present conditions of maturation, the percentage of embryos developing to cordiform, torpedo and cotyledonary ontogenetic stages was low, revealing an asynchronous development. In both treatments (3.5 and 7% PEG) as well as in the control, the first response was the formation of pro-embryogenic masses in 100% of calluses. In the control and in the medium supplemented with 3.5% PEG, an average of 32.7 and 27 new globular embryos were formed per explant, respectively. When PEG concentration in culture medium was 7%, the formation of new globular embryos decreased (12.7), but the development of cordiform (1.5), torpedo (2) and cotyledonary embryos (2) was observed.

## References

1. Carvalho PER: Espécies arbóreas Brazileiras. Colombo: Embrapa Florestas 2003, 1:1040.

2. Inoue MT, Putton V: Macropropagação de 12 espécies arbóreas da Floresta Ombrófila Mista. Floresta 2007, 37:55-61.

3. Lloyd G, McCown B: Commercially feasible micropropagation of mountain laurel, *Kalmia latifolia*, by use of shoot tip culture. *Proceed Int Plant Propagation Soc* 1980, 30:421-427.

4. Santa-Catarina C, Maciel SC, Pedrotti EL: Germinação *in vitro* e embriogênese somática a partir de embriões imaturos de canela sassafrás (*Ocotea odorifera Mez*). *Revista Brazileira de Botanica* 2001, 24:501-510.

# P149
## Establishment of endomycorrhizal fungi on micropropagated teak (*Tectona grandis* L.f.)

Maria Isabel Ramírez Caro¹*, Ian Bennett², Nicholas Malajczuk³

¹School of natural Sciences, Edith Cowan University, 270 Joondalup Drive, Joondalup, 6027, Western Australia; ²School of natural Sciences, Edith Cowan University, 270 Joondalup Drive, Joondalup, Western Australia;

³Mycorrhizal Applications International (Australia) Pty Ltd. PO Box 1046. Bunbury, 6231, Western Australia

E-mail: mramire@our.ecu.edu.au

BMC Proceedings 2011, 5(Suppl 7):P149

**Background:** Commercial micropropagation of teak (*Tectona grandis* L.f.) has been achieved for a number of years in countries such as Thailand, India and Malaysia. This has led to the availability of elite genotypes for large scale plantation production. Teak has been shown to develop arbuscular mycorrhizal (AM) fungal associations [1], and this has been attributed to increasing productivity [2]. It is likely that the establishment of mycorrhiza, through the introduction of *Glomusspecies*, on the roots of micropropagated plantlets will improve the productivity of selected
clones. Established hyphal networks can speed up plant colonization [3] and in the long term may also increase carbon storage through glomalin production [4]. Our research is pursuing the development of a mycorrhization protocol for micropropagated teak at the acclimatization stage.

**Methods:** Shoots of two clones of teak were multiplied on media containing Murashige and Skoog [5] nutrients and organics, 30 g L⁻¹ sucrose, 0.5 µM benzyl amino purine, 0.5 µM kinetin, 2.5 g L⁻¹ agar, 2.5 g L⁻¹ gelitre and a pH of 5.8. Individual shoots were subsequently exposed to a medium containing ¼ strength M&S macronutrients, full strength M&S micronutrients, 60 mM sucrose, 2.5 g L⁻¹ of agar and 2.5 g L⁻¹ of gelrite [6], and a range of indole butyric acid (IBA) concentrations (0-160 µM) for varying lengths of time (4-28 days) to produce roots *in vitro*. Rooted plantlets were transferred to three pasteurised soil types (1sand:1perlite;1sand:1peat;and 1sand:1perlite:1peat) under the following conditions: they were maintained under mist (covered 75-95% humidity) for 5 weeks followed by hardening through gradual reduction in humidity (50-65%) on greenhouse benches for a further 5 weeks. Plant survival, height and root area were measured.

Two clones of *T. grandis* were exposed to different inoculum sources and various inoculation techniques during the acclimatization period. Unprocessed commercial AM inoculum 100 g kg⁻¹ from a legume/grass pot culture (chopped mycorrhizal roots and soil) or processed commercial

<figure><img src="image_66.png" /><figcaption>**Figure 2(abstract P149)** Glomoid mycorrhiza in a cleared teak root and stained with trypan blue, (X400) scale bar = 0.01 mm.</figcaption></figure>

<figure><img src="image_67.png" /></figure>

AM inoculum 10 g kg⁻¹ were both mixed with pasteurised inert soil (1sand:1perlite) and watered to field capacity with ½ strength sorghum nutrient solution [7]. When the processed inoculum was used, roots were also dipped into the inoculum powder before being planted. All plants were acclimatised under high humidity as above and periodically fertilized with ½ strength sorghum nutrient solution. Plantlet height, root area and mycorrhizal development were assessed at 10 and 20 weeks and means compared using analysis of variance and Tukey's multiple range test.

**Results and discussion:** Optimum root production, (8.6 ± 0.7 roots per shoot) and tallest plants were obtained from shoots exposed to 80 µM IBA for 8 days. However, shoots from higher IBA (160 µM) concentrations had lower survival rates but there was no effect on the growth of the plantlets that survived. Soil type used for acclimatisation did not affect plant height and root area measurements. This allowed the acclimatisation to be conducted in a soil type that is considered most appropriate for mycorrhization development [7].

At 10 weeks, 100% of inoculated plantlets had survived. Mycorrhizal inoculation using unprocessed AM commercial inoculum increased root area (609.9 ± 46.2 mm²; Fig 1 A & B), a similar result to that of [8] for micropropagated grapes. This was achieved, however, for one clone only. Teak root infection was also achieved using the high concentration of the unprocessed AM commercial inoculum 20 weeks after inoculation. Control plants were not infected. A preliminary comparison of the inocula used showed that early evidence of mycorrhization can also be achieved using soil pot culture (Figure 2) which agrees with [3].

**Conclusions:** *T. grandis* can be inoculated and *exvitromycorrhization* achieved during the acclimatization phase of micropropagation. The time required for the mycorrhization appears to be dependent upon inoculum source, and perhaps other factors that are currently being investigated such as inoculum concentration and substrate phosphorus contents. In addition, early results indicate that successful mycorrhization may vary between host genotypes.

The availability of clonal, mycorrhizal teak may lead to greater plantation sustainability through more appropriate fertilizer regimes and greater carbon storage.

**References**

1. Dhar PP, Mridha MAU: Biodiversity of arbuscular mycorrhizal fungi in different trees of madhupur forest, Bangladesh. *J For Res* 2006, **17**(3):201-205.

2. Rajan SK, Reddy BJD, Bagyaraj DJ: Screening of arbuscular mycorrhizal fungi for their symbiotic efficiency with *Tectona grandis*. *Forest Ecol Manag* 2000, **126**:91-95.

3. Smith SE, Read DJ: Mycorrhizal symbiosis. Burlington: Elsevier 2008.
4. Garcia MO, Ovasapyan T, Greas M, Treseder KK: Mycorrhizal dynamics under elevated CO2and nitrogen fertilization in a warm temperate forest. *Plant Soil* 2008, **303**:301-310.

5. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. *Physiol Plantarum* 1962, **15**:473-497.

6. Bennett IJ, McDavid DAJ, McComb JA: The influence of ammonium nitrate, pH and indole butyric acid on root induction and survival in soil of micropropagated *Eucalyptus globulus*. *Biol Plantarum* 2003/4, **47**:355-360.

7. Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N: Working with mycorrhizas in forestry and agriculture. Canberra: Australian Centre for International Agricultural Research 1996.

8. Krishna H, Singh SK, Sharma RR, Khawale RN, Grover M, Patel VB: Biochemical changes in micropropagated grape (*Vitis vinifera* L. plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during *ex vitro* acclimatization. *Sci Hortic* 2005, **106**(4):554-567.

P150

Effect of PGR pulse treatments and ethylene precursor and inhibitors on Scots pine micropropagation

Vadim Lebedev, Konstantin Shestibratov*

Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Pushchino, Russia

E-mail: schestibratov@rambler.ru

BMC Proceedings 2011, **5**(Suppl 7):P150

**Background:** Conifer clonal forestry as a form of plantation forestry has great potential advantages. However, vegetative propagation of conifers, especially that of mature trees, is quite problematic. These species also are most recalcitrant objects for cultivation in vitro. For this reason the development of an effective system for clonal micropropagation of some conifers is still an important aim. Scots pine (*Pinus sylvestris* L.) is one of the most widespread conifers in the world and in Russia. This important species has high economic value. Among *Pinus* species, Scots pine is especially difficult to deal with in culture [1]. We investigated influence of pulse treatment with BA, GA and auxins on clonal micropropagation of Scots pine. In addition effects of ethylene precursor and inhibitors were analyzed.

**Methods:** Seeds were collected in 2008 from open pollinated trees growing in the Republic of Belarus (Gomel Region) and were stored at 4-5 °C until used. Seeds were washed under running tap water for 24 h, surface-sterilized in 15% H₂O₂ for 30 min, rinsed in sterile water and germinated on wet filter paper in plastic containers. The germinating seeds were incubated for 10-11 days at 23±1 °C in the dark. Hypocotyls of the seedlings were trimmed to 5-7 mm upper cotyledons, sterilized in 0.2% Hg(NO₃)₂ for 4 min, rinsed in sterile water and placed vertically on

hormone-free medium PM1 [2], containing 3% sucrose, 0.25% Gelrite gellan gum (Sigma, USA). After one week (BA treatment) or 6-7 weeks (auxin treatment) explants were used for experiments. For shoot induction explants were treated in an aqueous 50 or 100 mg/l BA solution buffered with 0.1 M MES during 0.5, 1.5 or 4 h. For elongation newly formed shoots were treated in 0.2, 1 or 5 mg/l GA3 during 0.5, 1, 2 or 4 h. For rooting pulse treatment by 50 or 100 mg/l NAA and IBA for 6 or 24 h were used. After treatments explants were transplanted on hormone-free medium: MS and PM1 (BA treatment), PM1 (GA treatment), 1/2 DCR (auxin treatment). Effects of various ethylene inhibitors, including AgNO₃ (10 and 40 µM) and CoCl₂ (10 and 100 µM), and the ethylene precursor ethephon (3 and 10 mg/l) also were studied in our experiments. Culture conditions were 23±1 °C and a photoperiod of 16 h. Rooted shoots were transferred to a peat-perlite (3:1) mixture and acclimatized in a greenhouse under gradually decreasing humidity conditions during 3-4 weeks.

**Results and conclusions:** The best results after BA pulse treatment were obtained at 4 h exposure to 100 mg/l solution (Table 1). Using this time, about 74.2% of explants formed an average of 3.5 buds. The proportion of vitrified explants was higher on MS medium (up to 76%) than on PM1 medium (up to 33%). It is interesting that we observed shoot regeneration on tips of cotyledons.

The previous studies showed that optimal treatment duration and the concentration of cytokinin are different for each conifer species. For instance, for explants of *P.canariensis* the best exposure time was 4-8 h in 22.5 mg/l BA solution [3]. High concentrations or prolonged exposure can completely suppress shoot formation. Bud induction in *P. wallichiana* was not observed at concentrations above 135 mg/l BA and time more than 3 h [4].

Pulse treatment with 0.2, 1 or 5 mg/l GA solution did not improve shoot elongation. Addition of AgNO₃ to medium decreased vitrification and death of explants in comparisons with CoCl₂ and ethephon. The treatment with 50 mg/l NAA during 6 h resulted in 44% rooted shoots after 12 weeks of culture, whereas pulse treatments with IBA resulted in 13-19% root formation (Table 2). An average of 1.5-3.2 roots per shoot were produced after NAA treatment and only single root per shoot after IBA treatment.

Similar results were obtained when auxin pulse treatment was applied on other *Pinus* species. When shoots of *P. ayacahuite* were incubated in 18.6 mg/l NAA for 8 h, up to 40% of the shoots were rooted [5]. An exposure of 6 h to 203 mg/l IBA solution resulted in 27.4% rooted shoots of *P. wallichiana* [4]. Rooted plants of Scots pine were successfully acclimatized in the greenhouse with the survival rate 80-90% (Figure 1).

References

1. Hohtola A: Seasonal changes in explant viability and contamination of tissue cultures from mature Scots pine. *Plant Cell Tissue Organ Cult* 1988, **15**:211-222.

<table><caption>Table 1(abstract P150) Effect of BA pulse treatment on shoot formation in Scots pine</caption><thead><tr><th>Treatment, mg/l - hours</th><th>Medium</th><th>Vitrification, %</th><th>Necrosis, %</th><th>Explants with shoots, %</th><th>Shoots/explant</th></tr></thead><tbody><tr><td rowspan="2">0-1.5</td><td>PM1</td><td>33.3</td><td>16.7</td><td>0.0</td><td>-</td></tr><tr><td>MS</td><td>63.6</td><td>50.0</td><td>0.0</td><td>-</td></tr><tr><td rowspan="2">50-0.5</td><td>PM1</td><td>20.5</td><td>12.2</td><td>75.6</td><td>3.1</td></tr><tr><td>MS</td><td>75.5</td><td>15.5</td><td>71.4</td><td>3.1</td></tr><tr><td rowspan="2">50-1.5</td><td>PM1</td><td>12.8</td><td>2.1</td><td>53.2</td><td>2.6</td></tr><tr><td>MS</td><td>73.5</td><td>11.0</td><td>40.8</td><td>2.8</td></tr><tr><td rowspan="2">50-4</td><td>PM1</td><td>5.7</td><td>3.6</td><td>62.3</td><td>3.0</td></tr><tr><td>MS</td><td>50.0</td><td>12.5</td><td>69.0</td><td>2.4</td></tr><tr><td rowspan="2">100-0.5</td><td>PM1</td><td>27.8</td><td>3.6</td><td>64.8</td><td>3.2</td></tr><tr><td>MS</td><td>44.6</td><td>6.7</td><td>60.7</td><td>2.9</td></tr><tr><td rowspan="2">100-1.5</td><td>PM1</td><td>6.3</td><td>11.1</td><td>56.3</td><td>2.8</td></tr><tr><td>MS</td><td>45.2</td><td>13.9</td><td>54.8</td><td>2.6</td></tr><tr><td rowspan="2">100-4</td><td>PM1</td><td>3.2</td><td>8.8</td><td>74.2</td><td>3.5</td></tr><tr><td>MS</td><td>23.8</td><td>12.5</td><td>50.0</td><td>1.7</td></tr></tbody></table>
<table><caption>Table 2(abstract P150) Effect of auxin pulse treatment on root formation in Scots pine</caption><thead><tr><th>Auxin</th><th>Treatment, mg/l-hours</th><th>Rooting, %</th><th>Root/shoot</th><th>Root length, mm</th></tr></thead><tbody><tr><td>control</td><td>water-24</td><td>3.1</td><td>1.0</td><td>2.0</td></tr><tr><td rowspan="4">NAA</td><td>50-6</td><td>43.8</td><td>1.5</td><td>16.5</td></tr><tr><td>50-24</td><td>34.4</td><td>1.8</td><td>17.5</td></tr><tr><td>100-6</td><td>37.5</td><td>3.2</td><td>14.9</td></tr><tr><td>100-24</td><td>21.9</td><td>2.6</td><td>15.8</td></tr><tr><td rowspan="4">IBA</td><td>50-6</td><td>15.6</td><td>1.0</td><td>40.8</td></tr><tr><td>50-24</td><td>12.5</td><td>1.0</td><td>21.5</td></tr><tr><td>100-6</td><td>18.8</td><td>1.0</td><td>14.2</td></tr><tr><td>100-24</td><td>12.5</td><td>1.0</td><td>12.0</td></tr></tbody></table>

<figure><img src="image_68.png" /><figcaption>Figure 1(abstract P150) Acclimatized plants of Scots pine in the greenhouse.</figcaption></figure>

2. Schestibratov K, Mikhailov R, Dolgov S: Plantlet regeneration from subculturable nodular callus of *Pinus radiate*. *Plant Cell Tissue Organ Cult* 2003, **72**:139-146.

3. Pulido C, Harry I, Thorpe T: Optimization of bud induction in cotyledonary explants of *Pinus canariensis*. *Plant Cell Tissue Organ Cult* 1992, **29**:247-255.

4. Mathur G, Nadgauda R: In vitro plantlet regeneration from mature zygotic embryos of *Pinus wallichiana* A.B. Jacks. *Plant Cell Rep* 1999, **19**:74-80.

5. Saborio F, Dvorak W, Donahue J, Thorpe T: In vitro regeneration of plantlets from mature embryos of *Pinus ayacahuite*. *Tree Physiol* 1997, **17**:787-796.

07, Sweden; ²Evolutionary Biology Center, Physiological Botany, Uppsala University, Uppsala, SE-752 36, Sweden; ³Department of Plant Physiology, Instituto de Investigaciones Agrobiologicas de Galicia (CSIC), Santiago de Compostela, Apartado 122, 15780, Spain

E-mail: daniel.uddenberg@slu.se

BMC Proceedings 2011, **5**(Suppl 7):P151

**Background:** Somatic embryogenesis is a useful method to propagate conifers vegetatively. In many coniferous species embryogenic cultures can be established from zygotic embryos, while the embryogenic potential decreases during germination. Embryo formation is thought to require a signal that induces a somatic cell to dedifferentiate and gain embryogenic potential as well as the expression of an appropriate cellular environment for the response of the inductive signal. In *Arabidopsis thaliana* (*Arabidopsis*) *LEAFY COTYLEDON* (LEC) genes are expressed during the embryonic stage but must be repressed to allow germination [1]. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. In addition, ectopic post-embryonic expression of LEC1 is sufficient to induce differentiation of embryo-like structures from vegetative cells [2]. *ABSCISIC ACID3* (*ABI3*) and its maize (*Zea mays*) orthologue *VIVIPAROUS1* (*VP1*) act together with the LEC genes

P151

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor

Daniel Uddenberg¹*, Silvia Valladares¹,³, Malin Abrahamsson¹, Jens F Sundström¹, Annika Sundås-Larsson², Sara von Arnold¹
¹Uppsala Biocenter, Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Uppsala, Box 7080, SE-750
to promote embryo maturation [3]. Knowledge about the molecular mechanisms underlying embryogenic competence in conifers is largely uncharacterized, although this is the foundation for propagation of conifers through somatic embryos. However, we have recently shown that TSA-treatment affects both the embryogenic potential and the expression of embryogenesis-related genes in Norway spruce [4].

**Materials and methods:** Conifer sequences of LEC1-type *HAP3* and *ABI3/VP1* homologues were retrieved from public databases and isolated in Norway spruce (*Picea abies*) and Scots Pine (*Pinus sylvestris*). Phylogenetic analyses were done on nucleotide alignments of conserved domains made using Baysian inference (MrBayes) and maximum parsimony (PAUP*). Embryogenic cell lines 06.28.05 of Norway spruce and 12:12 of Scots pine were used in this study. To analyze the effect of TSA (Sigma-Aldrich) during maturation and germination of somatic embryos of Norway spruce, the growth media were supplemented with 10µM TSA. Expression levels of the conifer *HAP3a* and *VP1* genes were assessed using quantitative real-time PCR.

**Results and discussion:** We isolated two conifer LEC1-type *HAP3* genes, *HAP3A* and *HAP3B*, from Norway spruce and Scots pine. A comparative phylogenetic analysis of plant *HAP3* genes suggests that *HAP3A* and *HAP3B* are paralogous genes originating from a duplication event in the conifer lineage. The angiosperm *ABI3/VP1* genes belong to the plant specific B3 gene family and phylogenetic relationship position the conifer homologs closest to *ABI3* and *VP1*. The expression of *HAP3A* is high during early embryo development but decreases during late embryogeny, while the expression of *VP1* is initially low and increases during late embryogeny, in both Norway spruce and Scots pine. The expression levels for both genes are similar during somatic and zygotic embryogenesis. When embryogenic cultures of Norway spruce were exposed to TSA during embryo maturation, maturation was arrested and the expression levels of *PaHAP3A* and *PaVP1* were maintained. Furthermore, when germinating somatic embryos of Norway spruce were treated with TSA, the germination progression was partially inhibited and the embryogenic potential was maintained at a similar level as embryos before germination.

**Conclusions:** Taken together, our results suggest that important regulators of embryogenesis are conserved between angiosperms and gymnosperms, and, assuming that TSA affects histone acetylation in conifers, our results indicate a connection between between chromatin structure and expression of embryogenesis-related genes in conifers.

References

1. Braybrook SA, Harada JJ: LECs go crazy in embryo development. *Trends Plant Sci* 2008, 13(12):624-630.

2. Tanaka M, Kikuchi A, Kamada H: The Arabidopsis histone deacetylases HDA6 and HDA19 contribute to the repression of embryonic properties after germination. *Plant Physiol* 2008, 146:149-161.

3. To A, Valon C, Savino G, Guilleminot J, Devic M, Giraudat J, Parcy F: A network of local and redundant gene regulation governs *Arabidopsis* seed maturation. *Plant Cell* 2006, 18:1642-1651.

4. Uddenberg D, Valladares S, Abrahamsson M, Sundström JF, Sundås-Larsson A, von Arnold S: Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. *Planta* 2011, DOI10.1007/s00425-011-1418-8.

P152

Transgene copy number estimation and analysis of gene expression levels in *Populus* spp. transgenic lines

Francesca Donnarumma¹, Donatella Paffetti², Matthias Fladung³,

Stefano Biricolti⁴, Ernst Dieter⁵, Illimar Altosaar⁶, Cristina Vettori¹*

¹Plant Genetics Institute, CNR, Italy; ²Department of Agricultural and Forest

Economics, Engineering, Sciences and Technologies, University of Florence,

Italy; ³Johann Heinrich von Thuenen Institute (vTI), Forestry and Fisheries

Institute for Forest Genetics, Germany; ⁴Dipartimento di Ortoflorofrutticoltura,

Università degli Studi di Firenze, Italy; ⁵Helmholtz Zentrum München,

German Research Center for Environmental Health (GmbH), Germany;

⁶Department of Biochemistry, Microbiology and Immunology, University of

Ottawa, Canada

E-mail: cristina.vettori@cnr.it

BMC Proceedings 2011, 5(Suppl 7):P152

**Background:** The genus *Populus* has certain important features, such as a relatively small nuclear genome, it can be easily regenerated easily *in vitro*

<figure><img src="image_69.png" /></figure>

and genetically transformed by *Agrobacterium* vector system, which make it ideal for gene transfer and molecular genetic studies in forest trees [1]. Insect-tolerant poplars have been obtained using several types of insecticidal genes coding for *Bacillus thuringiensis*-toxins. Regenerated plants with insect-resistance were obtained in different studies. *Agrobacterium*-mediated transformation has been the favored method for the introduction of foreign genes into plants. The effectiveness of insect-resistance in transgenic plants is related to the side effects of gene transfer (site of gene insertion, copy number, gene silencing etc.). Moreover intragenic plants, transgene copy number can greatly affect the expression level and genetic stability of the target gene, making estimation of transgene copy numbers an important area of genetically modified plant research [2]. Thus molecular biological analysis of transgenic plants, like real time PCR, widely used to detect and quantify DNA and cDNA [3], could represent an useful tool to investigate the genetic stability of transgenic forest trees having a long life cycle as well as for determining copy number in transformed plants.

**Material and methods:** The present study was undertaken to investigate *Populus alba* and *P. tremula* × *P. tremuloides* transgenic lines, obtained via *Agrobacterium*-mediated transformation, carrying *cry1Ab* and *nptII* genes in the T-DNA region. The plants were vegetatively propagated in growth chambers over 2 years. Ten individuals from each clone were planted in containers with "forest soil", and grown in a climate chamber.

Extraction of genomic DNA and RNA from leaves was performed for PCR and Real Time PCR (RT-PCR) analysis to estimate the transgene copy number [4] as well as expression of the inserted gene [5]in transgenic poplar, respectively.

**Results and discussion:** All lines contained one copy of *cry* gene and two of them showed that the copy number was different for the *cry1Ab* and *nptII* genes, suggesting rearrangements or multiple but incomplete copies of the transferred DNA (Figure 1). The copy number was concordant among the 3 individuals of each lines analysed and with those determined from the same transgenic lines kept in micropropagation for 2 years.

<figure><img src="image_70.png" /><figcaption>**Figure 2(abstract P152)** Relative expression obtained through qRT-PCR for the transgenes *cry1Ab* and *nptII* in transgenic *Populus* lines. The data are shown relative to the endogenous *actin* gene. Boxes: interquartile range, or the middle 50% of observations; dotted line: median gene expression; whiskers: minimum and maximum observations. Data are expressed as mean ± 95% confidence limit after 10000 permutations and have a *p* = 0.000 - 0.002.</figcaption></figure>
The transcript levels from both genes were determined in 3 individuals for each line growing in climatic chambers. High levels of mRNA expression were detected with respect to the stable endogenous *actin* gene for both transgenic lines (Figure 2). Comparing the transcript level of inserted genes among lines, a significant low level of *nptII* gene (*p* = 0.005) in the line carrying 3 copies was observed.

Preliminary results indicate a differential expression of endogenous genes among transgenic lines and towards their isogenic form.

**Conclusions:** The evaluation of the copy number of the inserted genes has indicated their stability after 2 years of micropropagation. The lower expression level of the *nptII* inserted gene in one line could suggest that factors like position effects or DNA rearrangements lead to differential expression.

The screening of the transcriptomic variations in transgenic plants carrying the *cry* gene and the comparison with position effects or DNA rearrangements is in course. The final aim is to unravel possible pleiotropic transcriptomic effects following *cry* gene expression in *P. alba* and *P. tremula* x *P. tremuloides* transgenic lines.

### References

1. Fladung M, Kumar S, Ahuja RA: Genetic transformation of *Populus* genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis. *Transg Res* 1997, 6:111-121.

2. Yang L, Ding J, Zhang C, Junwey J, Weng H, Liu W, Zhang D: Estimating the copy number of transgenes in transformed rice by real time quantitative PCR. *Plant Cell Rep* 2005, 23:759-763.

3. Bubner B, Gase K, Baldwin T: Two fold differences are the detection limit for determining trascgene copy numbers in plants by real time PCR. *BMC Biotechnol* 2004, 4:14.

4. Weng H, Weng H, Pan A, Yang L, Zhang C, Liu Z, Zhang D: Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. *Plant Mol Biol Rep* 2004, 22:289-300.

5. Pfaffl M: A new mathematical model for relative quantification in real-time RT-PCR. *Nucleic Acids Res* 2001, 29:e45.

BAP and thidiazuron - TDZ). We tested different *Agrobacterium* trans- formation protocol enhancements including the use of acetorsyringone and two different co-cultivation techniques (whole explant immersion versus pipetting of *Agrobacterium* suspension onto cut leaf edges). We also tested the suitability of kanamycin and hygromycin as selectable markers during transformation.

**Results and discussion:** Light conditions during shoot regeneration were critical to the rate of organ regeneration, with more than 50% of explants producing shoots in the presence of reduced or indirect light, compared to approximately 30% incubated under normal light conditions. In addition, shoot differentiation occurred earlier (10-14% at 40 days) under reduced or indirect light compared to normal light (1%; Fig. 1).

We have also begun to test the use of muslin screens to identify the best level of light exposure. The optimal level of GA3 in the micropropagation medium for the production of sturdy elongated internodes was found to be 0.05 mg/L. It was also apparent that the recovery of micropropagated plantlets on medium without GA3 for 21 days prior to internode harvesting improved plant health and produced better quality stem sections for transformation. Ascorbic acid, used as a means of reduction of phenolic browning, proved to be ineffective when used alone, even though it produced no adverse effects on callus health. By comparing several shoot induction media (SIM) for shoot regeneration from stem and leaf explants of the *E. grandis* x *urophylla* containing different cytokinin combinations, we found that the highest rates of shoot regeneration (11.8%) occurred when using stem explants incubated on SIM containing 1 mg/L NAA (nicotinamide) and 10 mg/L zeatin. Of the five tested *E. grandis* clones, we found that the highest rates of shoot regeneration (52%) were from leaf explants of clone p207 in the presence of SIM containing 3 µM TDZ and 0.1 µM NAA. Neither the use of two different acetosyringone concentrations (250 and 750 µM) during co-cultivation, nor either of the two co-cultivation techniques (immersion and pipetting) had a significant effect on transient gene expression measured shortly after cocultivation. Testing of different kanamycin and hygromycin concentrations ("kill curve"), revealed that 30 mg/L kanamycin or 5 mg/L hygromycin can be used for the selection of putative transgenics after transformation (Fig. 2).

**Conclusions:** There is significant variability in the performance and response of different clones of *Eucalyptus* during micropropagation, organogenesis and transformation. Testing of multiple *Eucalyptus* clones confirmed that the genus is relatively unwieldy in tissue culture compared, for example, to *Populus*. We found that explant type (stem vs leaf for example), and quality (age, general health, absence callus browning) as well as finely-tuned phytohormone concentrations, will play critical roles in enhancing the probability of the successful generation of stable transgenic lines.

## P153

### Development of *Eucalyptus* tissue culture conditions for improved in vitro plant health and transformability

Cathleen Ma¹, Raj Deepika², Alexander A Myburg², Martin Ranik¹,
Steven H Strauss¹

¹Department of Forest Ecosystems and Society, 321 Richardson Hall, Oregon State University, Corvallis, OR, 97331-5752, USA; ²Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa

E-mail: Steve.Strauss@oregonstate.edu

BMC Proceedings 2011, 5(Suppl 7):P153

**Background:** Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform *in vitro*. Compared to other plant species, few non-commercial laboratories are proficient at *Eucalyptus* tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.

**Methods:** Five different clones of *E. grandis* (including the clone of which the genome was recently sequenced - Brasuz1) as well as a single *E. grandis* x *urophylla* hybrid were established in the laboratory. Light intensity was studied as a means to reduce browning and promote callus growth and shoot regeneration. Gibberelic acid (GA3) concentrations in the micropropagation medium (Murashige and Skoog's (MS) basal medium containing 0.05 mg/L benzylaminopurine - BAP) were studied to help produce long internodes to aid transformation *in vitro*. To mitigate phenolic production we used several different antioxidants including ascorbic acid, PVP and PVPP. Shoot regeneration rates were studied by testing a concentration range of cytokinin (zeatin, benzyl aminopurine -

<figure><img src="image_71.png" /><figcaption>**Figure 1 (abstract P153)** Percentage of shoot regeneration from co-cultivated leaf explants of a *E. grandis* x *urophylla* clone under different light conditions.</figcaption></figure>
<figure><img src="image_72.png" /><figcaption>**Figure 2**(abstract P153) **A.** Percentage of non-transgenic explants forming callus after 30 days on shoot induction medium (SIM) with different concentrations of hygromycin. **B.** Percentage of explants forming roots (black bars) and exhibiting shoot yellowing (white bars). This experiment was conducted on a single kanamycin-resistant transgenic line of a *E. grandis* x *urophylla* clone after 30 days on rooting medium.</figcaption></figure>

# S8. GENOMIC, PROTEOMIC AND METABOLOMIC TECHNOLOGIES

P154

**Functional markers development and genetic diversity analysis in *Eucalyptus globulus* with emphasis in wood quality candidate genes**

Cintia Acuña*, Pamela Villalba, Martín García, Esteban Hopp, Susana Marcucci
Poltri Instituto de Biotecnología, CICVyA, INTA Castelar, CC 25, Castelar
(B1712WAA), Argentina

E-mail: cacunia@cnia.inta.gov.ar

BMC Proceedings 2011, 5(Suppl 7):P154

**Background:** *Eucalyptus globulus* is the most planted hardwood species for pulpwood in temperate regions. Genomic researches in *Eucalyptus* have increased the information available in DNA sequences public databases and several structural and regulatory genes involved in the cellulose and lignin pathways are known.

Functional genetic markers, while frequent in crop, are still scarce in forest species. Hence the detection and validation of SSRs in interesting genes to be used in future projects of marker-assisted breeding are needed.

The present study aimed the development of novel functional markers (SSRs) in ESTs and wood quality candidate genes (CG) from *Eucalyptus globulus*, and analyzes their potential for genetic diversity and individual identification studies.

We report the design of SSR primers flanking simple sequence repeats in ESTs and CG, the validation of a subset of randomly selected EST-SSRs

using eight *E. globulus* genotypes and the screening of a sample of 60 trees with the polymorphic SSRs. Also, SSRs cross-transferability was tested in seven *Eucalyptus* species coming from three sections: *E. grandis*, *E. saligna* (section Latoangulatae); *E. globulus*, *E. dunnii*, *E. viminalis* (section Maidenaria); and *E. camaldulensis*, *E. tereticornis* (section Exsertaria).

**Material and methods: Plant material:** A total of 60 trees, each from a different OP family of *E. globulus*, were analyzed for their variability. These sixty trees represented major geographical races of the species' natural distribution that were grown in a field trial in the Province of Buenos Aires, Argentina, between 1995 and 1997. For validation analyses one individual of each race (except Furneaux) was included.

A total of 47 individuals from seven species (including *E. globulus*) of the genus were sampled for the transferability analyses.

**Methods:** Novel microsatellites were identified mainly by two different methods:

- SSR Mining software (GDR Server, http://www.rosaceae.org/bio/content?title=&url=/cgi-bin/gdr/gdr_ssr) on selected candidate gene sequences identified at the GenBank.

- SSRs detection from non redundant ESTs of *E. globulus* from GenBank: Annotations of these SSR-ESTs were based on the Gene Ontology (GO) (http://www.geneontology.org/) using Blast2GO (http://www.blast2go.org/) [1].

For validation and diversity analyses, amplification products were silver-stained or analyzed through an ABI3100 Genetic Analyzers (Applied Biosystems, USA) with fluorescent dyes respectively.

SSR statistics for determining number and frequency of alleles, effective number of alleles ($N_e$), observed heterozygosity ($H_o$) and unbiased expected heterozygosity estimates ($UH_o$), fixation index (FI) and probability of identity (PI) were determined with the GenAIEx 6.4 program [2]. Tests for Hardy-Weinberg equilibrium were conducted using GENEPOP 4.0.10 [3]. Null allele frequencies were estimated with INEST software (Inbreeding/Null allele estimation) [4], using and Individual Inbreeding Model (IIM) with 10,000 iterations.

Structure analysis was explored through UPGMA and DAS (Shared allele distance) indices as well as Structure [5] software.

**Results:** From 12,690 updated *E. globulus* EST database published in National Center for Biotechnology Information a total of 4,924 non-redundant sequences were identified. From these ones, 952 unigenes (19.3%) contained 1,140 SSRs. A new set of 979 primers was designed. The predicted functions of these EST-SSRs were adjudged, including biological process, molecular function and cellular component Gene Ontology (GO) categories.

Twenty four structural and regulatory candidate genes for wood quality carrying 29 SSR were indentified. Microsatellite sequences were located in UTR, introns and exons from candidate genes (CG) from: phenylpropanoid biosynthesis, cellulose biosynthetic process, hemicellulose metabolism, shikimate pathway, methionine metabolism, tubulin genes and the transcriptor factor LIM1.

Sixty five percent out of atotal of 85 SSR (56 EST-SSRs and 29 SSR contaging GC) detected in this study were validated for actual PCR amplification of tree DNA samples in eight genotypes of *E. globulus*. From this assessment a total of 17 polymorphic EST-SSRs and 12 polymorphic CG-SSRs markers were obtained. These ones were selected for further analyses, so as to accurately estimate genetic information content in a larger sample of 60 non related trees represented major geographical races of the species' natural distribution.

PIC, $H_o$ and $UH_o$ values varied over a wide range from around 0.02 to 0.9, whereas the allele number ranged from 2 to 16, with and average of 7.55. A set of 49 *loci* (37 validated EST-SSRs (polymorphic and monomorphic) and 12 polymorphic CG-SSRs) were also tested for cross-transferability to other six *Eucalyptus* species (*E. grandis*, *E. saligna*, *E. dunnii*, *E. viminalis*, *E. camaldulensis*, *E. tereticornis*). A total of 33 out of the 49 validated markers in *E. globulus* amplified in the six other species and six markers amplified in at least other five.

Finally, the analyses of polymorphism and transferability of functional markers, enabled the selection of a set of 13 (7 EST-SSRs and 6 GC-SSRs) highly informative and transferable to six other species of *Eucalyptus*.

**Conclusions:** The set of highly informative markers developed here will have potential use in studies of genetic diversity, taxonomy, gene mapping and will help the improvement of *Eucalyptus* trough the assisted selection.
Thirty percent of EST-SSRs (17 from 56) are expected to be polymorphic in *E. globulus* and 25% (14 polymorphic from 56) are expected also to be transferable to other six species (*E. grandis*, *E. saligna*, *E. dunnii*, *E. viminalis*, *E. camaldulensis* and *E. tereticornis*). Under these proportions, potentially more than 200 new EST-SSRs described here may contribute to the verification of synteny and collinearity between different *E. globulus* maps, and would allow the validation of gene and QTL positions in multiple pedigrees in the botanical sections *Maidenaria*, *Exsertaria*, and *Latoangulatae*, to which most of the commercially planted eucalypt species belong.

## References

1. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. *Bioinformatics* 2005, 21(18):3674-3676.

2. Peakall R, Smouse PE: Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. *Molecular Ecology Notes* 2006, 6(1):288-295.

3. Raymond M, Rousset F: GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. *J. Hered* 1995, 86:248-249.

4. Chybicki IJ, Burczyk J: Simultaneous estimation of null alleles and inbreeding coefficients. *J. Hered* 2009, 100:106-113.

5. Pritchard JK, Stephens M, Donnelly P: Inference of population structure using multilocus genotype data. *Genetics* 2000, 155:945-959.

## P155

### Proteomics reveals proteins linked to the quality of wood in contrasting xylem of *Eucalyptus* clones

Dahyana Britto¹*, Carlos Pirovani¹, Esteban Gonzalez², João Silva², Abelmon Gesteira³, Júlio Cascardo⁴

¹Center of Biothecnology and Genetics, Dep. of Genetics and Molecular Biology, Universidade Estadual de Santa Cruz - UESC, Ilhéus, BA, 45662-900, Brazil; ²Suzano Paper and Cellulose S.A., Mucuri, BA, 45936-000, Brazil;

³Brazilian Enterprise for Agricultural Research cassava and fruit - EMBRAPA, Cruz das Almas, BA, 44380-000, Brazil; ⁴in memorian

E-mail: dahyanas@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P155

**Background:** Eucalyptus spp. is a genus widely planted in many tropical and subtropical regions of the world as a major source in the production of pulp and paper. Aiming to meet the demand required by the world market papermaker, with the need for greater productivity in forests, as well as higher quality of wood, reducing the amount of lignin and extractive, is that proteomics emerges as an additional tool to help accelerate progress in the selection of elite clones.

**Materials and methods:** In this study, using the combination of the two-dimensional gel electrophoresis (2D-PAGE) and electrospray mass spectrometry on quadrupole-time of flight (ESI-QUAD-TOF) identify differentially expressed proteins between four genotypes of eucalyptus, brothers siblings, contrasting with differences for components of wood quality. The components evaluated in this study for four xylems were: wood density, which was higher in clones X1 and X2, total lignin content, with levels higher in the X3 and X4 clones, extractive content, with higher values for the clones X2 and X4, and gross yield in pulp, which was higher in clones X1 and X3. Whereas the desirable characteristics for application in pulp and paper industry is the clone that best combines the highest density of wood and pulp yield, with lower lignin content and extractive, the X1 is the clone that best meets these criteria, the most suitable for such application, and X4 clones, considered inadequate for the same process.

**Results:** The profile followed by 2D gel analysis software with 3D Image Master Platinum xylem protein extracts of these clones resulted in identification of 30 spots differentially expressed, with 3 proteins in clone X1, 4 proteins in clone X2, 11 proteins in clone X3 and 12 proteins in clone X4. These differential spots were excised, subjected to tryptic digestion and processing, followed by mass spectrometry analysis. The identified proteins are involved in various biological processes, including polyphosphate biosynthetic process, catalytic activity, nucleotide-excision repair, cellular metabolic process, cell redox homeostasis, response to salt stress, response to heat, oxidation-reduction process, potassium ion transport, electron transport chain, response to wounding, superoxide metabolic process, response to water deprivation, protein phosphorylation.

It is noteworthy that the proteins involved in oxidative stress were up regulated in clone X4, followed by clones X2 and X3, and to a lesser extent in clone X1. Among the proteins differentially expressed in clone X4 can highlight catalase, superoxide dismutase, annexins, heat shock protein (chaperone) and ABC transporter. Catalase is an enzyme that converts 2H2O2 in O2 + 2H2O, primarily by preventing the potential damage caused by changes in H2O2 homeostasis [1]. The superoxide dismutase is the first line of plant defense against reactive species of O2, 2H- removing the cellular compartments where this radical is formed [2]. Annexin peroxidase may play a role as the process of oxidative stress in plants [3]. The first reviews of plant ABC transporter showed that they participate in detoxification processes, but evidence already exists of their participation in the flow of ions, and being involved in the process of growth and development of plants [4]. The molecular chaperones known as heat shock proteins (Hsp) are stress proteins. Under stress conditions, Hsp facilitates protein folding and help stabilize polypeptides and membranes [5].

**Conclusions:** The proteins identified in the study involved in redox process in the plant, as well as the extractives, including polyphenols, tannins and resins, components characterized as inadequate in the pulp and paper industry, are induced as a defense response to pathogen attacks as well as to abiotic stress [1,6]. Furthermore, it has been demonstrated that oxidative stress can also induce lignification in plant tissue [7,8]. Given this, we infer that the presence of proteins involved in oxidative stress in the xylem of a eucalyptus is a strong predictor of inappropriate clones in the pulp and paper industry.

Selection of candidate genes and validation study of gene expression through genetic materials in the wood quality will be known the next step in the selection of genetic markers to identify elite clones of eucalyptus for the pulp and paper production.

## References

1. Breusegem FV, Vranová E, Dat JF, Inzé D: The role of active oxygen species in plant signal transduction. *Plant Science* 2001, 161:405-414.

2. Alscher RG, Erturk N, Heath LS: The role of active oxygen species in plant signal transduction. *Journal of Experimental Botany* 2002, 53(372):1331-1341.

3. Delmer DP, Potikha TS: Structures and functions of annexins in plants. *Cellular Molecular Life Sciences* 1997, 53:546-553.

4. Martinioia E, Klein M, Geisler M, Forestier C, Kolukisaoglu U, Müller-Röber B, Schulz B: Multifunctionality of plant ABC transporters-more than just detoxifiers. *Planta* 2002, 214(3):345-55.

5. Scarpeci TE, Zanor MI, Valle EM: Investigating the role of plant heat shock proteins during oxidative stress. *Plant Signaling and Behavior* 2008, 3(10):856-857.

6. Scalbert A: Tannis in woods and their contribution to microbial decay prevention. *In Plant polyphenols: synthesis, properties, significance* New York: Plenum Press: Hemingway RW, Laks PE 1992.

7. Kovacic J, Klejdrus B: Dynamics of phenolic acids and lignin accumulation in metal-treated *Matricaria chamomilla* roots. *Plant Cell Reports* 2008, 27:605-615.

8. Durcekova K, Huttová J, Mistrík I, Ollé M, Tamás L: Cadmium induces premature xylogenesis in barley roots. *Plant Soil* 2007, 290:61-68.

## P156

### Oxidative stress as an indicator of lower quality eucalyptus for pulp and paper industry

Dahyana Britto¹*, Carlos Pirovani¹, Esteban Gonzalez², João Silva², Abelmon Gesteira³, Júlio Cascardo⁴

¹Center of Biothecnology and Genetics, Dep. of Genetics and Molecular Biology, Universidade Estadual de Santa Cruz - UESC, Ilhéus, BA, 45662-900, Brazil; ²Suzano Paper and Cellulose S.A., Mucuri, BA, 45936-000, Brazil;

³Brazilian Enterprise for Agricultural Research cassava and fruit - EMBRAPA, Cruz das Almas, BA, 44380-000, Brazil; ⁴in memorian
E-mail: dahyanas@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P156

**Background:** Wood is the most important natural and renewable source of infinite energy, but this is not their only function, it also has great significance to the buildings and other products [1]. Eucalyptus, the genus that includes more than 500 species, is among the key forest species planted around the world to solid wood, and mainly for the pulp and paper industries. Aiming at the sustainable use of timber, supplying the
demand and meeting the requirements of the paper industry, genetic
breeding programs looking for, among other agronomic traits of interest,
improve the quality of wood, that is, lower lignin and extractive, and
increase the density wood and pulp yield. Therefore, it is interesting to
identify genetic markers that characterize the major events of xylem
differentiation, as well as stress response, because it is key factors to
determine some properties of wood, affecting their performance and
industrial value. Thus, this study aimed to identify genes specifically
involved in oxidative stress response and characterization of genes in
determining the quality of the wood.

**Materials and methods:** To this end, we constructed a Suppression Subtractive Hybridization 3 – SSH3 using two samples of xylem. These samples were extracted from trees of a population of sib eucalyptus from a commercial plantation Suzano with contrasting characteristics to wood quality, among them, lignin content and total amount of extractive, higher for the clone X4, and basic density and cellulose yield, for the largest clone X1, the latter considered as higher for the production of pulp and paper. After identification and selection of genes of interest was the analysis of the expression for Real Time PCR with the same clones of Eucalyptus used in making the library.

**Results:** From the sequencing and bioinformatics analysis of the library has been possible to identify a total of 449 sequences and 90 contigs in SSH3. Among the most representative unigenes, annotated by the program Blast2GO versão1.2.7 in SSH3 were identified proteins involved in oxidative stress response and heat shock protein, ABC transporter and glutamine synthetase. After analyzing for real-time PCR for validation of these sequences more representative, was unable to confirm the decreased expression of all genes in clone X1 compared with the X4 clone.

**Conclusions:** The result was expected for these clones, since the plants when in oxidative stress caused by abiotic and biotic agents, activates the defense mechanism through the expression of genes involved in stress response, as well as accumulated extractives as polyphenols, tannins and resins [2]. So, the ABC transporter, for example, between genes possibly involved in this process, carries lignin monomers to enhance the cell wall making it difficult the entry of pathogens [3,4]. The small heat shock proteins, also known as molecular chaperones, although not expressed constitutively, are abundant in plants, especially when under stress, as observed by Harndahl and collaborators, in transgenic Arabdopsis thaliana [5]. As glutamine synthetase, this protein is involved in improving the assimilation of nitrogen by the plant [6]. Other words, in a soil poor in nitrogen, such as soil Formation Barriers/Spodosol where these clones were grown, these proteins must play important role in plant growth, once the availability of this nutrient is limiting factor growth of eucalypts [7]. In addition, this protein has an important role in maintaining the health of the plant to attack by pathogens [8]. Considering this, it was note worthy that the signaling proteins were oxidative stress in Eucalyptus clone, and that the result reinforces the idea that proteins involved in oxidative stress response is related to the high concentration of extractive and lignin.

Preliminary tests in Real Time PCR with the juvenile offspring of these clones are already underway. But in order to reaffirm the presence of these genes involved in oxidative stress to the condition of poor quality for the production of pulp and paper, the expression analysis performed in the progenies of appropriate age and assessment of components involved in wood quality are needed.

References

1. Plomion C, Leprovost G, Stokes A: Wood Formation in Trees. *Plant Physiology* 2001, 127:1513-1523.

2. Scalbert A: Tannis in woods and their contribution to microbial decay prevention. *Plant polyphenols: synthesis, properties, significance* New York: Plenum Press: Hemingway RW, Laks PE 1992.

3. Liu C-J, Miao Y-C, Zhang K-W: Sequestration and Transport of Lignin Monomeric Precursors. *Molecules* 2011, 16:710-727.

4. Kaneda M, Schuetz M, Lin BSP, Chanis C, Hamberger T, Western TL, Ehling J, Samuels AL: ABC transporters coordinately expressed during lignification of Arabidopsis stems include a set of ABCBs associated with auxin transport. *Journal of Experimental Botany* 2011, 1-15.

5. Harndahl U, Hall RB, Ostyeryoung KW, Vierling E, Bornman JF, Sundby C: The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress. *Cell Stress and Chaperones* 1999, 4(2):129-138.

6. Miflin BJ, Habash DZ: The role of glutamisne synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. *Journal of Experimental Botany* 2002, 53(370):979-987.

7. Araújo RF: in: [http://www.rragroflorestal.com.br/documents/29cbcs/N-BA.pdf]. 2011.

8. Pageau K, Reisdorf-Cren M, Morot-Gaudry J-F, Masclaux-Daubresse C: The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in *Nicotiana tabacum* L. leaves. *Journal of Experimental Botany* 2006, 57(3):547-557.

P157

Development, characterization and use of microsatellite markers for
genetic analysis of Cashew tree (Anacardium occidentale)

Gláucia Buso¹*, Natalia Lamas¹, José Jaime Cavalcanti²,
Marco Antonio Ferreira¹

¹Embrapa Cenargen, Brazilia, DF, Brazil; ²Embrapa Agroindustria Tropical, Fortaleza, CE, Brazil

E-mail: buso@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P157

**Background:** The cashew tree (*Anacardium occidentale*) is found throughout the Brazilian territory, although it is better adapted to the northeastern coast climate. This fruit crop is essential to the agroindustry of the States of Ceará, Piauí and Rio Grande do Norte, where around 95% of the production is harvested and where the whole processing of the chestnut is done. It currently represents 157 million dollars in exports of nuts. Despite its socio-economic importance it still lags behind in the adoption of breeding technologies. There is very little information about the genetics of this species and no molecular tools yet developed. In this work we developed and characterized a set of microsatellite markers for *Anacardium occidentale* from the construction of genomic libraries enriched for repetitive sequences.

**Material and methods:** The markers developed were used in studies of genetic relationships among samples of the germplasm bank, and saturation of existing genetic maps of the species. Genomic libraries enriched for microsatellites were constructed and out of the 5,472 selected clones, 540 were sequenced and analyzed for the presence of microsatellites. A total of 117 sequences containing microsatellites were selected to design PCR primers and screen the resulting markers. One hundred pairs of primers were synthesized and initially tested for amplification in agarose gel. Fourteen markers were selected and characterized by fluorescent detection in an ABI 377 sequencer based on genotyping a representative set of 35 samples derived from the germplasm collection. Genetic parameters such as observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC) and power of exclusion (PE) for paretage testing were estimated.

**Results:** Great variability in analytical performance was seen among the tested microsatellite markers. Marker AOBR48, for example, proved to be the marker with the highest polymorphic information content. Analysis of accessions from the germplasm collection using microsatellite markers indicated that cashew and a commonly used dwarf clone likely have distinct origins, since they were separated into different groups based on similarity coefficients. Additionally, A. microcarpum, used as an outgroup control, clustered with accessions of the giant type. An F1 population derived from a cross between the dwarf cashew variety (CCP1001) and the giant type (CP 96) was used to map these microsatellites. Among the hundred new markers developed in this study, 11 showed segregation of alleles from the female parent (CCP 1001) in the F1 population, while 21 showed segregation of alleles from the male parent (CP 96). Only three of the 100 tested markers were fully informative showing segregation of alleles from both parents in the F1 population. In total 29 markers could be mapped in this population and 11 markers were positioned in both, the male and female maps.

**Conclusions:** An initial set of microsatellites was developed for the Cashew tree. These new markers can be useful for a number of applications in germplasm analysis and breeding. The identification of duplicate accessions in the current germplasm collection, the study
of genetic diversity in natural populations, represent immediate potential applications to better understand the existing diversity available to breed better clones. Genetic mapping of QTLs for some economically important traits such as increased yield and quality of fruits and cashew, and resistance to diseases could be future targets provided a higher marker density is achieved.

P158

Promoting a functional and comparative understanding of the conifer
genome- implementing applied aspects for more productive and
adapted forests (ProCoGen)

Carmen Díaz-Sala¹, Teresa Cervera²*

¹Dpto. Biología Vegetal, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain; ²Dpto. Ecología y Genetica Forestal, CIFOR-INIA, 28040 Madrid, Spain

E-mail: cervera@inia.es

BMC Proceedings 2011, 5(Suppl 7):P158

In the midst of a climatic change scenario, the genetics of adaptive response in conifers becomes essential to ensure a sustainable management of genetic resources and an effective breeding. Conifers are the target of major tree breeding efforts worldwide. Advances in molecular technologies, such as next-generation DNA sequencing technologies, could have an enormous impact on the rate of progress and achievements made by tree breeding programmes. These new technologies might be used not only to improve our understanding of fundamental conifer biology, but also to address practical problems for the forest industry as well as problems related to the adaptation and management of conifer forests. In this context, the FP7-KBBE-2011-5 project "Promoting a functional and comparative understanding of the conifer genome- implementing applied aspects for more productive and adapted forests" (ProCoGen), granted in 2011 by the European Commission, will address genome sequencing of two keystone European conifer species. Genome re-sequencing approaches will be used to obtain two reference pine genomes. Comparative genomics and genetic diversity will be closely integrated and linked to targeted functional genomics investigations to identify genes and gene networks that efficiently help to develop or enhance applications related to forest productivity, forest stewardship in response to environmental change or conservation efforts. The development of high-throughput genotyping tools will produce an array of pre-breeding tools to be implemented in forest tree breeding programmes. ProCoGen will also develop comparative studies based on orthologous sequences, genes and markers, which will allow guiding re-sequencing initiatives and exploiting the research accumulated on each of the species under consideration to accelerate the use of genomic tools in diverse species. ProCoGen will integrate fragmented activities developed by European research groups involved in several ongoing international conifer genome initiatives and contribute to strengthening international collaboration with North American initiatives (US and Canada). Partners involved in this project are:

Carmen Díaz-Sala (financial and administrative coordinator, Universidad de Alcalá, UAH, Spain)

María-Teresa Cervera (scientific coordinator, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA-CIFOR, also including Toni Gabaldón from Centro de Regulación Genómica, CRG; Álvaro Soto from Universidad Politécnica de Madrid, UPM, and Isabel Arrillaga from Universidad de Valencia, UV, Spain)

Francisco Cánovas (Universidad de Málaga, UMA, Spain)

Leopoldo Sanchez, Catherine Bastian and Christophe Plomion (Institut
National de la Recherche Agronomique, INRA, France)

Luc Harvengt (Institut Technologique Forêt Cellulose Boisconstruction Ameublement, FCBA, France)

Pär Ingvarsson (Umea University, UMU, also including Sara Von Arnold from Swedish University of Agricultural Sciences, SLU, Sweden)

Yves Van de Peer (Flanders Institute for Biotechnology, VIB, Belgium)

Berthold Heinze (Federal Research and Training Centre for Forests, Natural Hazards and Landscape, BFW, Austria)

Outi Savolainen (University of Oulu, UOULU, Finland)

Giovanni G. Vendramin (Italian National Research Council, CNR-Firenze, Italy)

Célia Miguel (Instituto de Biología Experimental e Tecnológica, IBET, also including Jorge Paiva from Forest Center of the Tropical Research Institute, Portugal)

John Wooll Williams (University of Edinburgh, UEDIN, United Kingdom)

Marco Bink (Stichting Dienst Landbouwkundig Onderzoek, DLO, The Netherlands)

Carl Gunnar Fossdal (Norwegian Forest and Landscape Institute, NFLI, Norway)

David Torrents (Barcelona Supercomputing Center, BSC, Spain)

Steve Lee (Forest Research, FR, United Kingdom)

John MacKay (Université Laval, ULaval, Canada)

Kermit Ritland (University of British Columbia, UBC, Canada)

Jeffrey Dean (University of Georgia, UGA, US)

Daniel Peterson (Mississippi State University, MS State, US).

P159

Phylogenetic comparative and expression analysis of genes encoding
dof transcription factors from *Eucalyptus grandis*

Gabriel d'Almeida¹*, Michèle Breton¹, Sandro Camargo², Jeverson Frazzon³,
Giancarlo Pasquali¹

¹Biotechnology Center, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil; ²Institute of Informatics, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil; ³Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

E-mail: gsdalmeida@cbiot.ufrgs.br

BMC Proceedings 2011, 5(Suppl 7):P159

Dof proteins are a family of transcription factors specific to the plant
kingdom that contain a particular class of zinc finger DNA binding
domain. Members of this family are involved in the regulation of genes
related to a plethora of metabolic processes including stress or hormone
response, seed and endosperm development, flowering, carbohydrate
metabolism, and cell or tissue specificity. Dof proteins and encoding
genes were characterized in several plant species. Nevertheless poplar is
the only woody species whose *Dof* genes were better characterized. The
recent availability of the *Eucalyptus grandis* genome and transcriptome,
along with transcription factor databases of several plant species allowed
us to identify and run a valuable comparative analysis of the Dof protein
family in this tree. These species included *Arabidopsis thaliana*, *Arabidopsis
lyrata*, *Carica papaya*, *Populus trichocarpa*, *Vitis vinifera*, *Sorghum bicolor*,
*Chlamydomonas reinhardtii*, *Oryza sativa indica* and *Zea mays*. The
phylogenetic relationships among Dof proteins from *E. grandis* and
*Arabidopsis thaliana* - a popular model for studying the genomics of
many plants - is a fundamental step to unravel functionality of new *Dof*
genes not yet characterized. Twenty-three distinct DNA sequences were
predicted to belong to the *Dof* gene family after the analysis of the
complete available genome of *E. grandis*. The deduced protein sequences
of 22 members do contain a conserved Dof domain. One sequence
seemed to have lost the conserved Dof domain, suggesting it to be a
pseudogene or to present an activity not directly linked to the Dof family.
Gene structures, including exon/intron positions, and amino acid
sequences were predicted for each gene based on the available
*E. grandis* transcriptome. In order to determine the relationship and
function of the genes putatively encoding Dof proteins, we carried out a
phylogenetic analysis with 43 Dof protein sequences from *A. thaliana*.
Our analysis allowed us to classify the *E. grandis* *Dof* sequences into five
groups of orthologous genes. Gene expression analysis via real time,
quantitative PCR was also conducted with ten of the *E. grandis* *Dof* genes,
using samples obtained from flowers, leaves and vascular tissue.
Generally, *Dof* steady-state mRNA levels were higher in *E. grandis* vascular
tissues, with more reduced levels in flowers. *Dof* genes showed an
increase in steady-state mRNA levels after hormone signaling, and
reduced levels following abiotic stress. This is the first study that aimed
the identification of *Dof* genes in *E. grandis* which are possibly involved
in numerous plant metabolic processes. The phylogenetic relationship to
*A. thaliana* counterparts and the patterns of mRNA accumulation
in *E. grandis* allowed us to speculate on possible roles for some of the
Dof-encoding genes.

Financial support: Brazilian Ministry of Agriculture, Farming & Supply (MAPA), and The National Council for the Development of Science & Technology (CNPq), Brazilian Ministry of Science & Technology (MCT).
P160

Mitochondrial iron-sulfur cluster genes in *Eucalyptus*

Luisa de Oliveira*, Giancarlo Pasquali, Jeverson Frazzon
Biotechnology Center, Federal University of Rio Grande do Sul, Porto Alegre,
RS, Brazil
E-mail: lu_abruzzi@yahoo.com.br
*BMC Proceedings* 2011, **5**(Suppl 7):P160

Iron-sulfur [Fe-S] clusters are prosthetic groups required to maintain life
processes including respiration, photosynthesis, metabolic reactions,
sensing, signaling, and gene regulation. In plants the biogenesis of Fe-S
proteins is compartmentalized and adapted to specific needs of the
eukaryotic and photosynthetic cell. Although critical to so many
fundamental metabolic pathways and drastically affecting plant
adaptability and productivity, Fe-S proteins were never investigated in
woody species. *Eucalyptus grandis* is an important economical tree widely
cultivated in subtropical regions which suffers under low temperature
stress. Here we describe a transcriptional analysis of the *E. grandis* NFS1,
ISU1 and ISA1, three genes involved in the biogenesis of [Fe-S] clusters.
Microarray analyses were carried out for the comparison of global gene
expression in leaves and vascular tissues (xylem) of E. grandis and vascular
tissues of E. globulus. In general, leaves from *E. grandis* demonstrated
higher expression of these genes than xylem. *EgrISU1* had a constitutive
expression in E. grandis, but its expression pattern was higher in this
species than in *E. globulus* xylem. Differences observed in the relative gene
expression profile between xylem tissues from the two Eucalyptus species
suggest that these genes may be implicated in the contrasting phenotypic
characteristics of their wood. The response of these genes to a series of
hormonal and stress signals over *E. grandis* seedlings was also evaluated
by RT-qPCR. After the chilling treatment of seedlings, *EgrNFS1* and *EgrISU1*
showed 6 to 8-fold and 0.6 to 1.7-fold increase respectively; and *EgrISA1*
exhibited a drastic 69 to 114-fold increase. These data are the same
observed in *Arabidopsis* microarrays available in GeneVestigator database.
These results suggest that (i) NFS1 and ISA1 may be related to the cellular
response to stress caused by chilling, and (ii) the increase in the expression
is probably due to sulfur metabolism. A time-course chilling experiment
was also carried out. The ISU1 gene expression was higher in the first two

hours of treatment and decreased right after that period. The ISA1 gene,
which showed the highest expression in the previous experiment, didn't
show significant differences in the expression pattern during the 16 hours
of chilling, as well as the NFS1 gene. Our data indicated that Fe-S proteins
are possibly involved in the recovery of plants after chilling stress.

P161

Gravitropic response in radiata pine seedlings. Searching molecular keys

Patricio Ramos, Nicolas Cruz, Alvaro González, Raúl Herrera*
Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Chile
E-mail: raherre@utalca.cl
*BMC Proceedings* 2011, **5**(Suppl 7):P161

Coniferous trees develop compression wood in response to gravitropic
stimuli. In nature this response can be generated by growth in slope,
exposure to snow or high winds [1]. However, genes and molecular
mechanism involved in this phenomenon are still unknown. We studied
gene expression in response to gravitropic stimulation induced by 45°
inclination in *Pinus radiata* D. Don one year old seedlings. To characterize
the gravitropic response, whole seedlings were inclined and transversal cuts
were performed in order to identify morphological wood characteristics.
Xylem cells were visualized by optical microscopy in a time course
experiment (fig. 1). On the other hand, a transcriptomic approach was
assayed generating libraries based on the Suppressive Subtractive
Hybridization (SSH) strategy [2]. This technique allows the isolation of genes
differentially expressed between two samples. The libraries were generated
from total RNA extracted at 2.5, 10, 24 hours and 30 days from the inferior
and superior stem half. The sequences obtained were assembled, analyzed
and ontology classified by biological process, molecular function and cell
components. The information give clue about the molecular mechanism
related to this phenomenon. To validate the differential gene expression by
qPCR analyses, housekeeping genes were evaluated in order to have
normalization genes for the gravitropic stress response. We could identify a
large number of genes activation involved in different initial process,
previous to the anatomical hallmarks of compression wood formation.

<figure><img src="image_73.png" /></figure>
One-year old *Pinus radiata* seedlings were inclined during 15 days (d) and transversally cut at 5d, 8d and 15d. Stem slices were stained using a solution of phloroglucinol (2A). From these preparations, wall thickness (2B) and cell diameter (2C) of xylem cells were measured.

**Acknowledgement:** This project was supported by FONDECYT (1071026), DIAT (U. de Talca), PBCT Anillo (ACT-41), Ecos-Conicyt (CB07-01) and postdoctoral PBCT-PSD61 projects. P.R. thanks to Conicyt for doctoral fellowship.

## References

1. Larson PR, Kretschmann DE, Clark A, Isebrands JG: Formation and properties of juvenile wood in southern pines: a synopsis. *Gen. Tech. Rep. FPL-GTR-129* Madison, WI: U.S. Department of Agriculture, Forest Service, Forest Products Laboratory 2001, 42.

2. Diatchenko L, Lau Y-FC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD: Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. *P Natl Acad Sci, USA* 1996, **93**:6025-6030.

## P162

**Proteomics, a systems biology based approach to investigations of *Jatropha curcas* seeds**

Fatemeh Maghuly¹*, Stefan Kogler¹, Gorji Marzban¹, Katharina Nöbauer², Ebrahim Razzazi², Margit Laimer¹

¹Plant Biotechnology Unit (PBU), Dept. Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; ²VetCore Facility of Technology, Proteomics Unit, University of Veterinary Medicine, 1210 Vienna, Austria

E-mail: efatemeh.maghuly@boku.ac.at

BMC Proceedings 2011, 5(Suppl 7):P162

**Background:** *Jatropha curcas* (*Euphorbiaceae*) is cultivated for harvesting the unique oil contained in its seeds, which can be used as raw material for the production of biodiesel [1]. The seed cake remaining after pressing can not be used for animal feed, because it contains toxic proteins and other compounds, e.g. curcin, phorbol esters. The development of genotypes better suited for the production of biodiesel showing lower levels of toxic/allergenic proteins is being hampered by a lack of understanding the a) metabolic pathways and enzymes leading to the production of fatty acids during seed development and b) role of proteins deposited during seed development. In recent years the field of proteomic research has become a fast growing discipline with high relevance to biological sciences.

While genomic and transcript-profiling studies have provided a wealth of information about different plant developmental processes, there is growing awareness that the abundance of mRNA transcripts is not always representative of protein levels and that mechanisms of post-translational regulation must also play an important role.

Proteomics is a systems biology based approach investigating the whole expressed proteins at a given time point and under certain condition [2]. It has been shown to be a valuable tool for studying the biology of living organisms and their interaction with the environment in post genomics area. Proteomics has been shown to have potential values to deliver knowledge about complex biochemical processes and is being used in various fields of modern botany and agriculture like plant biomarker discovery related to resistance as well as drought and water stress. Furthermore proteomics offer major advantages linked to its high throughput capacity and its ability to perform simultaneously the analysis of hundreds of proteins from the same samples.

**Methods:** The method of choice for proteome analysis is the combination of high resolution protein separation like two dimensional gel electrophoresis (2-DE) with tandem mass spectrometric (MS/MS) identification of proteins. The two dimensional gel electrophoresis separates proteins based on their isoelectric points (isoelectric focusing) and in a second dimension based on their molecular weight [3].

In this study a proteomic approach was conducted in order to identify the expression patterns of interesting proteins during seed development, as well as toxic and/or allergenic proteins in *Jatropha*, 2-DE coupled with mass spectrometry and *de novo* sequencing, were employed to analyze whole seed proteins of six developmental stages, covering the essential ontological phases of these important plant organs.

Prominent spots identified in 2-DE analyses were excised, washed, reduced with DTT and alkylated with iodoacetamide. The in-gel digest with trypsin was carried out for 8 hours. The peptides were de-salted using µZip-Tips C18 (Millipore) and 0.5 µl were spotted onto a disposable AnchorChip MALDI target plate pre-spotted with a-cyano-4-hydroxycinnamic acid (PAC target, Bruker Daltonics). Data were acquired on a Matrix Assisted Laser Desorption Ionisation Tandem Time-of-Flight (MALDI-TOF/TOF) mass spectrometer (Ultraflux II, Bruker Daltonics) in MS and MS/MS modes. Spectra processing and peak annotation were carried out using FlexAnalysis and Biotools (Bruker Daltonics).

In order to enhance the quality of tandem mass spectrometry (MS/MS) spectra for de novo sequencing, N-terminal chemical modification using 4-sulfophenyl isothiocyanate (SPITC) was carried out for 1 h at 56°C [4]. The derivatised peptides were de-salted a second time. Again 0.5 µL were spotted onto a PAC target and analyzed as described above.

For standard database searches, processed spectra were searched via Mascot2 in the Swiss-Prot database and in NCBIr. Identifications were considered statistically significant where p < 0.05. Peptide deNovo-sequencing was carried out manually using FlexAnalysis. The sequences were then used for a homology search using MS-homology with the following search parameters: taxonomy Viridiplantae using pre-search parameter 9VIRI (organism code from the UniProt Knowledgebase); Database UniProtKB.2011.01.11; Score Matrix BLOSUM62; 30% amino acid exchanges allowed.

**Results and conclusions:** In 2-DE analyses it was possible to identify 100 non-redundant protein spots. Identified proteins belonged to different functional classes, including proteins involved in metabolism, protein destination and storage, and energy metabolism, which were highly representative.

The significant change in abundance of protein spots during the different developmental stages of seeds indicated that several pathways were involved in the biosynthesis of these interesting compounds, but vary during the growth phases of plant organs, in this case seeds.

**Acknowledgements:** The financial support of FFG project 820132 and of Bioplan R&D is kindly acknowledged.

## References

1. Vollmann J, Laimer M: Novel and traditional oil crops and their biorefinery potential. *Bioprocessing Technologies in Integrated Biorefinery for Production of Biofuels, Biochemicals, and Biopolymers from Biomass John Wiley & Sons, Inc. (USA): Yang S-T, El Enshasy HA, Thongchul N, Martin Lo Y 2010.

2. Wilkins MR, Sanchez J-C, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams KL: Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. *Biotechnology and Genetic Engineering Reviews* 1995, 13:19-50.

3. López JL: Two-dimensional electrophoresis in proteome expression analysis. *Journal of Chromatography B* 2007, 849:190-202.

4. García-Murria MJ, Valero ML, Sánchez del Pino MM: Simple chemical tools to expand the range of proteomics applications. *Journal of Proteomics* 2011, 74:137-150.

## P163

**Cloning and Functional characterization of the promoter of a high affinity potassium transporter gene from *Eucalyptus grandis***

Carolina Costa¹, Flávio Tetsuo Sassaki², Juliana Bravo¹, Ivan Maia¹*

¹IB - UNESP - Botucatu, Brazil; ²IB-UNESP-Botucatu, Brazil

E-mail: igmaia@ibb.unesp.br

BMC Proceedings 2011, 5(Suppl 7):P163

The characterization of organ/tissue-specific promoters is of great interest to transgenic production. The construction of expression cassettes containing tissue-specific promoters is a viable alternative to limited transgene expression to specific organs and cell types. In this context, the purpose of this study was to functionally characterize the promoter of a *Eucalyptus grandis* gene encoding a high affinity potassium transporter (named EgHAK) shown to be specifically expressed in roots. For that, the 5'-flanking region of EgHAK (1,3 kb) was cloned and transcriptionally fused to the b-glucuronidase reporter gene (GUS), and then used to transform tobacco leaf discs. Histochemical analysis of GUS activity in transgenic plants showed that GUS staining was mainly detected in vascular tissues of leaf and root. To investigate the response of the
studied promoter to potassium starvation, a hydroponic system was employed. In this case, enhanced GUS staining was observed in the roots of plants starved for 6 days when compared to control ones. Moreover, a weak induction of the promoter at low potassium conditions was observed using fluorimetric assays. Thus, our results indicate that, in a heterologous system, the studied promoter shows preferential expression in roots in the absence of potassium.

P164

Identification of potential transcriptionally active Copia LTR retrotransposons in *Eucalyptus*

Helena Marcon¹,²*, Douglas Domingues¹*, Celso Marino¹*

¹Departamento de Genética, UNESP, Botucatu, São Paulo, Brazil; ²IAPAR, Londrina, Paraná, Brazil

E-mail: clmarino@ibb.unesp.br

BMC Proceedings 2011, 5(Suppl 7):P164

**Background:** Long Terminal Repeat retrotransposons (LTR-RTs) represent the most abundant genomic component in all plant genomes thus far investigated. They are transposable elements that replicate through a "copy/paste" mechanism that relies on reverse transcription and integration of a RNA intermediate. Plant LTR-RTs can be divided in two major superfamilies: *Copia* and *Gypsy* [1]. LTR-RTs have impact on genome size variation, as well as in the expression of adjacent genes in their host genomes, providing a "genomic plasticity" [2]. Their transcription was believed to be extremely repressed in plants. However, despite their potential mutagenic and deleterious effects, LTR-RTs were proven to be transcriptionally active in several plant species [3].

*Eucalyptus* is one of the most commercially important forest genus in the world, due to their superior growth, broad adaptability and multipurpose wood properties. Most molecular studies in *Eucalyptus* are focused on cellulose production and wood development, and there are few works on genome composition, structure and evolution. *Pinus* and *Populus*, the tree genera with most available genomic resources, have several works analyzing their repertoire of LTR-RTs [i. e 4, 5], but only one study characterized LTR-RTs in *Eucalyptus* [6], with no detailed manual checking or phylogenetic analysis. Here, we used FOREST database as a starting point to identify transcriptionally active *Copia* LTR-RTs in *Eucalyptus*, that were further analyzed regarding their *in silico* expression, evolutionary diversity, and distribution in public genomic databases.

**Methods:** A previous survey with 88 *Copia* LTR-RTs from diverse plants defined six major common evolutionary *Copialineages* [7]. The 22 *Arabidopsis thaliana* families analyzed in that study were used as queries to the identify *Eucalyptus* EST sequences related to *Copia* elements in *FORESTS* database [8], by tBLASTx (e-value >1e-50). Sequences were then analyzed in *RepBase* [9] to confirm their similarity to *Copia* LTR-RTs. *Eucalyptus* ESTs with >200bp of *copia*-like retrotransposon fragments were used to identify complete copies in *Eucalyptus grandis* genome v 1.0 in a BLASTn search (identity >80%; in a region >250bp). We picked up 10000bp surrounding the aligned region, that were analyzed using LTR-

Finder [10] and LTR_STRUC [11]. Full-length LTR-RTs were then used as queries in GenBank to retrieve related *Eucalyptus* EST sequences (>200bp; >80% identity). Phylogenetic analyses using the reverse transcriptase of these elements (alignment in MUSCLE, Maximum Likelihood method, bootstrap 1000 replicates) were done using MEGA 5.01 [12].

<table><caption>**Table 1(abstract P164)** Overall features of LTR-RTs analyzed</caption><thead><tr><th>Family</th><th>Lineage</th><th>Genomic copy number</th><th>FOREST cDNA libraries</th><th>GenBank cDNA libraries</th></tr></thead><tbody><tr><td>RTE_copia_Eu_1</td><td>Ale</td><td>28</td><td>seedlings</td><td>xylem</td></tr><tr><td>RTE_copia_Eu_2</td><td>Ale</td><td>262</td><td>roots, leaves</td><td>xylem</td></tr><tr><td>RTE_copia_Eu_3</td><td>Ale</td><td>24</td><td>root</td><td>xylem, cold-stressed</td></tr><tr><td>RTE_copia_Eu_4</td><td>Angela</td><td>243</td><td>seedlings, calli</td><td>xylem</td></tr><tr><td>RTE_copia_Eu_5</td><td>IVana</td><td>54</td><td>leaves, root, calli, wood</td><td>xylem</td></tr><tr><td>RTE_copia_Eu_6</td><td>GMR</td><td>63</td><td>leaves, seedlings</td><td>xylem</td></tr></tbody></table>

<figure><img src="image_74.png" /><figcaption>**Figure 1(abstract P164)** Phylogenetic tree of Copia LTR-RTs elements. Based on Du and collaborators (2010).</figcaption></figure>
**Results:** Stem, calli and seedlings were the cDNA libraries from FOREST database with most EST sequences, in this *Copia* LTR-RT search. We identified 20 consensus sequences (total: 36 ESTs) from 3 tissues, roots, leaves and flower-buds. We also identified 29 ESTs in GenBank from xylem, root apex and cold-stressed plants (Table 1). Using EST data, we identified six full-length retrotransposons families that had different copy number in the *Eucalyptus* genome, estimated by BLAST searches (cutoff 1e-50). Copy number ranged from 24 to 262 (Table 1). Phylogenetic analyses showed that they are members of the *Ale*, *Angela*, *GMR* and *Ivana* evolutionary lineages (figure 1). *Ale* was the evolutionary lineage encompassing families with highest and lowest copy number (Table 1).

**Conclusion:** In summary, the present data demonstrate the potential impact of future studies about functional and genomic analysis of LTR-RTs in *Eucalyptus*. This is the first characterization of full-length *Copia* LTR-RTs families in *Eucalyptus* genome with potential transcriptional activity, giving insights about phylogenetic diversity and copy number variation of retrotransposons in this tree.

**Acknowledgements:** HSM was supported by a fellowship from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and CNPq.

**References**

1. Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH: A unified classification system for eukaryotic transposable elements. *Nat Rev 2007*, 8:973-982.

2. Du J, Tian Z, Hans CS, Laten HM, Cannon SB, Jackson SA, Shoemaker RC, Ma J: Evolutionary conservation, diversity and specificity of LTR-retrotransposons in flowering plants: insights from genome-wide analysis and multi-specific comparison. *Plant J* 2010, 63:584-598.

3. Vicient CM, Jaaskelainen MJ, Kalendar R, Schulman AH: Active retrotransposons are a common feature of grass genomes. *Plant Phys 2001*, 125:1283-1292.

4. L'Homme Y, Séguin A, Tremblay FM: Different classes of retrotransposons in coniferous spruce species. *Genome* 2000, 43(6):1084-1089.

5. Zhou F, Xu Y: Reppuc: a database for repetitive elements in *Populus trichocarpa*. *BMC Genomics* 2009, 10:14-23.

6. Bacci Júnior M, Soares RBS, Tajara E, Ambar G, Fischer CN, Guilherme IR, Costa EP, Miranda VFO: Identification and frequency of transposable elements in *Eucalyptus*. *Genet Mol Biol* 2005, 28(3):634-639.

7. Wicker T, Keller B: Genome-wide comparative analysis of retrotansposons in Triticeae, rice, and *Arabidopsis* reveals conserved ancient evolutionary lineages and distinct dynamics of individual *copia* families. *Genome Research* 2007, 17:1072-1081.

8. Vicentini R, Sassaki FT, Gimenes MA, Maia IG, Menossi M: *In silico* evaluation of the *Eucalyptus* transcriptome. *Gen Mol Biol* 2005, 28:487-495.

9. Jurka J, Kapitonov VV, Pavlíček A, Klonowski P, Kohany O, Wallichiewicz J: Repbase Update, a database of eukaryotic repetitive elements. *Cytogenetics and Genome Research* 2005, 110:462-467.

10. Xu Z, Wang H: LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. *Nuc Acids Res* 2007, 35:265-268.

11. McCarthy EM, McDonald JF: LTR STRUC: a novel search and identification program for LTR retrotransposons. *Bioinformatics* 2003, 19:362-367.

12. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGAS: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. *Mol Biol Evol* 2011, (submitted).

P165

Bioinformatic prediction of the AP2/ERF family genes in *Eucalyptus grandis*: focus on the CBF family

Sahar Azar, Helène SanClemente, Gilles Marque, Christophe Dunand, Christiane Marque*, Chantal Teulieres
UMR5546 University of Toulouse, France
E-mail: teulieres@lrsv.ups-tlse.fr
BMC Proceedings 2011, 5(Suppl 7):P165

**Background:** Due to their very high economic importance, Eucalyptus tree species are among the most planted hardwoods in the world with over 20 million hectares. However, as long-lived evergreen species, this genus is particularly exposed to cold. Frost tolerance varies among species and is inversely correlated to productivity. The AP2/ERF gene family includes

developmentally and physiologically important transcription factors characterized by the presence of the AP2/ERF DNA-binding domain. AP2 proteins contain two AP2-like domains and RAV family proteins contain one AP2 domain and one B3 domain. ERF family proteins exhibit only one AP2 domain and are further divided into the DREB subfamily and the ERF subfamily [1]. The CBF/DREB1 protein differ from the other DREB proteins by the presence of "signature sequences" (PKK/RPAGRxFKxETRHP and DSAWR) flanking the DNA-binding AP2 domain [2]. The DREB factors recognize the C-repeat or dehydration response element (DRE) in the promoters of low temperature and/or water deficit responsive genes and would play a crucial role in response to abiotic stresses. CBF/DREB1 are the key regulators of the cold-responsive (COR) genes. So far CBF transcription factors have been mainly characterized in model plants such as Arabidopsis, but lately they were identified in several tree species including Eucalyptus [2]. The Eucalyptus cold tolerance was greatly improved in our hands when two genes from the four CBF members isolated from a tolerant species *E. gunnii* were individually constitutively overexpressed in the frost sensitive *E. urophylla* x *E. grandis* hybrid [3]. In the present study *E. grandis* AP2/ERF family genes were identified based on the presence of putative encoding AP2-domain(s) and were studied with regard to the model herbaceous Arabidopsis as well as the main sequenced woody plants. Within this family, a part of the study focused on the CBF/DREB1 subfamily which was compared to the four genes already characterized in *E. gunnii* [2].

**Methods:** We used the available annotated *E. grandis* genome sequence at phytozome (http://www.phytozome.net/eucalyptus.php), the *Arabidopsis thaliana* AP2/ERF family downloaded from the DATF (Database of Arabidopsis Transcription Factors) database website (http://datf.cbi.pku.edu.cn/), and the sequences of AP2/ERF gene family from the grapevine, and the poplar obtained from several publications. In order to get the exhaustive gene AP2 family and the location of the genes of the *E. grandis* genome, we used Scipio [4]. Every identified protein was analyzed for structural motifs by scanning them against PROSITE patterns and profiles (http://www.expasy.org/prosite) and against Pfam (http://pfam.sanger.ac.uk/) to make sure of the presence of AP2 and/or the B3 domains. A fine correction of the predicted AP2/ERF proteins was performed. In order to identify all the members of CBF subfamily, a search within the *E. grandis* annotated AP2/ERF family was performed using consensus sequence including the two highly conserved signatures (PKKPAGR and DSAWR) surrounding the DNA-binding AP2-domain. As a check, we confirmed by blasting the sequences on the NCBI blast page. From a multiple alignment analysis performed with Clustal W, the phylogenetic trees were generated using MEGA version 5. The resulting phylogenetic trees were based on the maximum likelihood (ML) method [5].

**Results and main conclusions:** Based on the sequence alignment, the phylogenetic analyses, and the known criteria described in literature, this study revealed that the annotated AP2 genes from *E. grandis* were divided into the four subgroups already described for Arabidopsis (DREB, ERF subfamilies, AP2 subfamily and RAV subfamily). The two subfamilies DREB and ERF were separated according to the similarity of the sequences of the AP2/ERF domain and to the amino acid at 14 and 19 positions. Distribution of the DREB members in the four dicotyledonous plant genomes (Vitis, Arabidopsis, Populus and Eucalyptus) shows similarities between grapevine and *E. grandis* except for the A1 subgroup which corresponds to the CBF genes. Interestingly this group is much larger in *E. grandis* compared to the three other plant species. The 17 CBF genes (including one pseudogene) identified on *E. grandis* genome exhibit a very good conservation when compared to the *E. gunnii* CBF genes. The *E. grandis* CBF genes could be classified into the four groups (a, b, c and d) described for *E. gunnii* CBF. Most of the *EgrCBF* were localized on the chromosome 1, one member is on the chromosome 4 and the last two on the chromosome 5.

**References**

1. Sakuma Y, Liu Q, Dubouzet J, Abe H, Shinozaki K, Yamaguchi-Shinozaki K: DNA-binding specificity of the ERF/AP2 domain of *Arabidopsis* DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. *Biochem Biophys Res Commun* 2002, 290:998-1009.

2. Navarro M, Marque G, Ayax C, Keller G, Borges JP, Marque C, Teulieres C: Complementary regulation of four Eucalyptus CBF genes under various cold conditions. *J Exp Bot* 2009, 60:2713-2724.

3. Gamboa MC, Rasmussen-Poblete S, Valenzuela PD, Krauskopf E: Isolation and characterization of a cDNA encoding a CBF transcription factor from *E. globules*. *Plant Physiol Biochem* 2007, 45:1-5.
4. Odronitz F, Pillmann H, Keller O, Waack S, Kollmar M: Webscipio: An online tool for the determination of gene structures using protein sequences. *BMC Genomics* 2008, 9(1):422.

5. Felsenstein J: Confidence limits on phylogenies: An approach using the bootstrap. *Evolution* 1985, 39:783-791.

P166

Exploring a new model of cell wall regulation: identification and expression of two putative SHINEs transcription factors in *Eucalyptus*

Wesley L Marques¹*, Marcela Salazar¹*, Eduardo Camargo¹*,
Jorge Lepikson-Neto¹*, Danieli Cristina Gonçalves¹*,
Leandro Costa do Nascimento¹*, Carla Garcia²*, Adriano Almeida²*,
Gonçalo Pereira¹*

¹State University of Campinas – UNICAMP, Brazil; ²International Paper

E-mail: goncalo@unicamp.br

BMC Proceedings 2011, 5(Suppl 7):P166

**Background:** *Eucalyptus* forests are a competitive and efficient alternative to convert carbon from the atmosphere in cellulose, an important source for paper manufacture and bioenergy production.

To obtain transgenic *Eucalyptus* with important traits improved it is necessary to make modifications in genes that affect the final phenotype. One interesting gene that follows this requisite was recently found: this is the *AtSHN2* gene (*Arabidopsis thaliana* SHINE 2).

*AtSHN2* codifies to a Transcription Factor known as "Arabidopsis SHINE/WAX INDUCER". Instead of inducing drought tolerance in transgenic rice (*Oryza sativa*), *AtSHN2* overexpression causes: i) 34% increase in the cellulose content; ii) 45% reduction in lignin content and iii) increase in wood digestibly (elevated S:G ratio) with no compromise in plant strength and performance [1].

The discovery of *AtSHN2* function in plant cell wall formation, led Ambavaram and collaborators [1] to perform other studies and ultimately to propose the following model: *AtSHN2* regulates positively *MYB* transcription factors (TF) related to cellulose synthesis and it down-regulates *MYBTF's* related to lignin formation. At the same time, *SHINE* can repress *NAC TFs* that controls *MYB* expression [1].

As a consequence of the interesting phenotype achieved through *AtSHN2* overexpression in rice, this work focused on the identification and analyses of *AtSHN* orthologues in *Eucalyptus*. Bioinformatics tools were used to search for *AtSHN* similar genes in *Eucalyptus*. Moreover, the expression profile of the corresponding genes in *Eucalyptus* was evaluated to prove their role as *AtSHN*. To carry it on, the expression experiments were done with flower, leaf and xylem. If the *Eucalyptus* putativeSHINE's has the same function of the *AtSHN's*, gene expression in flower tissues will be the highest [2]. This is because it is known that *AtSHN*'s genes are preferentially expressed in abscission and dehiscence zones, a phenomenon that usually occurs in lots of flower tissues.

<figure><img src="image_75.png" /><figcaption>**Figure 2 (abstract P166)** *EgSHN1a* expression ratio in three different tissues: flower (rich in abscission and dehiscence zones), leaf and xylem assessed trough qRT-PCR. Data are expressed as fold change and leaf was chosen as reference condition. Error bars represent SE (n=3).</figcaption></figure>

**Material and methods:** Putative transcription factors of the SHINE family were searched in *Eucalyptus* by comparing the *AtSHN* amino acid sequences with the *Eucalyptus* genome assembly (obtained from *Eucalyptus grandis* – http://eucalyptusdb.bi.up.ac.za).

This analysis revealed the existence of two sequences with high similarity to *AtSHN* proteins. Quantitative RT-PCR assays were carried out to verify the expression profile of these genes in different tissues (leaves, flowers and xylem) of the hybrid variety *E. urograndis* (*E. urophyla* x *E. grandis*). The RNA extraction was carried out following the protocol described by Zeng & Yang (2002) [3]. The qRT-PCR was performed with the *SYBR® Green PCR Master Mix*kit from *Applied Biosystems*. The Results were analyzed according to the mathematical method described by Pfaffl, 2001 [4].

Plant materials were provided by the International Paper Company, Brazil.
**Results and discussion:** The comparison of the *AtSHN* amino acids sequence with *E. grandis* genome revealed the existence of two putative *SHN* genes in *Eucalyptus* (named *EgSHN1a* and *EgSHN1b*). A phylogenetic

<figure><img src="image_76.png" /><figcaption>**Figure 1 (abstract P166)** Phylogenetic analysis of SHN genes from *A. thaliana* (*AtSHN1*, *AtSHN2* and *AtSHN3*) and the new SHN genes identified in *Eucalyptus grandis* (*EgSHN1a* and *EgSHN1b*). The scale bar of 0,1 corresponds to 10% sequence divergence. Bootstrap values are given for nodes and are considered as value of significance of the branches.</figcaption></figure>
analysis showed that these *Eucalyptus* SHN genes are orthologs to the *AtSHN1* of *Arabidopsis thaliana* (Figure 1).

The proteins encoded by *EgSHN1a* and *EgSHN1b* share approximately 58% identity to *AtSHN1* and a conserved gene structure is found between *AtSHN* and both *EgSHN* genes: a single intron is present and located approximately 80 bp from the start codon. In addition, as in *AtSHN1*, *EgSHN* genes contain an AP2 DNA binding domain, which is in accordance to their putative role as transcription factors. More importantly, the domains “mm” and “cm”, which are exclusive of SHN genes, could be identified in the *Eucalyptus* sequences. In the same way that the gene sequence is very correlated between the species, it's probable that their function are the same two.

Gene expression analysis revealed a higher expression of *EgSHN1a* in flowers (Figure 2). This result is similar to the *AtSHN1* expression profile [2], suggesting a similar function for *EgSHN1a*.

The expression pattern of the gene *EgSHN1b* is being carried on.

**Conclusions:** There are two SHN genes in *Eucalyptus* (*EgSHN1a* and *EgSHN1b*), which are orthologs to *AtSHN1*;

The *EgSHN1a* function might be the same of *AtSHN1* as suggested by their similar expression patterns. It is possible that, in *Eucalyptus*, biosynthesis of cuticle and cell wall in abscission and dehiscence zones is regulated by *EgSHN1s*, as already described in *A. thaliana* [1].

The new genes described here are interesting candidate for the development of transgenic *Eucalyptus*. Overexpression of SHN genes in *A. thaliana* increased tolerance to drought and pathogen attacks and greatly improved cell wall quality.

**References**

1. Ambavaram MMR, Krishnan A, Trijatmiko KR, Pereira A: Coordinated Activation of Cellulose and Repression of Lignin Biosynthesis Pathways in Rice. Plant physiology 2010, 155:916-931.

2. Aharoni A, Dixit S, Jetter R, et al: The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance when Overexpressed in Arabidopsis. Society 2004, 16:2463-2480.

3. Zeng Y, Yang T: RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Molecular Biology Reporter 2002, 20:417-417.

4. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic acids research 2001, 29:e45.

P167

**Transgenic *Arabidopsis* thaliana and *Nicotiana tabacum* overexpressing the *Eucalyptus grandis* Cellulose Synthase 3 and its expression pattern in different *Eucalyptus* species and tissues**

Wesley L. Marques¹*, Marcela Salazar¹*, Eduardo Camargo¹*, Jorge Lepikson-Neto¹*, Danieli Cristina Gonçalves¹*, Leandro Costa do Nascimento¹*, Carla Garcia²*, Adriano Almeida²*, Gonçalo Pereira¹*

¹State University of Campinas – UNICAMP, Brazil; ²International Paper

E-mail: goncalo@unicamp.br

BMC Proceedings 2011, 5(Suppl 7):P167

**Background:** In Brazil, the forest industry accounts for 4,5% from the Gross Domestic Product and the country is the biggest *Eucalyptus* cellulose exporter. That's really good news because *Eucalyptus* forests are a competitive and efficient alternative to convert carbon from the atmosphere in cellulose, an important source for paper and bioenergy production.

<figure><img src="image_77.png" /><figcaption>**Figure 1(abstract P167)** *Eucalyptus Cellulose Synthase 3* expression pattern. On the top, expression pattern analyzed through qRT-PCR and, below, the expression analyzed through Northern-Blot. The tissues studied were leaves and xylem from three different *Eucalyptus* species: *E. globulus*, *E. grandis* and *E. urophyla*.</figcaption></figure>
The cellulose biosynthesis happens through the *Cellulose Synthase Complex* activity. This complex is composed by different *Cellulose Synthase* genes (*CesA*) that work together in a non redundant way [1]. It is also known that some of these isoforms act in the primary cell wall synthesis while others, in the secondary cell wall. In this last group there is the gene *Eucalyptus Cellulose Synthase 3* (*EgCesA3*), the most expressed *CesA* gene during xylogenesis [2]. Besides, knockout experiments proved that the *AtCesA7* (*EgCesA3* ortholog in *Arabidopsis thaliana*) activity is essential for the xylem formation and for plant vertical growth [3].

In front of these evidences, the *EgCesA3* gene had it expression pattern evaluated in leaf and xylem tissues among the three most economic important *Eucalyptus* species in Brazil: *E. grandis*, *E. globulus* and *E. urophylla*.

As demonstrated in this work, the expression experiment provided enough information about the *EgCesA3* function, that's why this gene was chosen to be overexpressed in model plants (*Arabidopsis thaliana* and *Nicotiana tabacum*). It's expected increased cellulose content in the transgenic plants xylem.

**Material and methods:** The *EgCesA3* expression pattern was examined trough qRT-PCR and Northern-Blot in xylem and leaf from the tree most commercially important *Eucalyptus* species: *E. grandis*, *E. urophylla* and *E. globulus*. The qRT-PCR was performed using SYBR Green and amplicons with approximated size of 100pb. On the other hand, the Northern-Blot was made with probes containing phosphorous-32 radioactive.

To create transgenic plants with improved cellulose content this work overexpressed the gene *EgCesA3* under the control of the CaMV 35S promoter in *A. thaliana* and *Nicotiana tabacum* trough *Agrobacterium* transformation by floral-spray.

**Results and discussion:** The results show that the *EgCesA3* is strongly more expressed in xylem than in leaves among the three most important economical *Eucalyptus* species for the Brazilian forest industry (Fig. 1). This data corroborate to the theory that this gene is related to the secondary cell wall formation during the xylem development.

However, it's necessary to mention that the gene expression data obtained through the northern-blot related to the *E. globulus* leaf can't be analyzed. This because the amount of RNA used in this sample was different from the others. It can be seen through the rRNA amounts in the RNA electrophoresis (Fig. 1).

Besides, comparing the *EgCesA3* expression pattern between the xylem from the three species studied, it's possible to conclude that should exist a difference: the *EgCesA3* gene is apparently more expressed in *E. grandis*

and *E. urophylla* xylem than in *E. globulus* xylem (Fig. 1). However, this conclusion isn't distant from doubts: the problem is the large error bars that happened in the experiment. In order to solve it, the qRT-PCR repetition is being carried on.

In front of the *EgCesA3* rolein the secondary cell wall synthesis, this gene was overexpressed in model plants (Fig. 2). The genetic modified plants were successfully obtained. Thus, after the generation advance, the homozygous transgenic plants will be evaluated to measure the cell wall chemical composition and morphology.

**Conclusion:** The *Eucalyptus Cellulose Synthase 3* function in the secondary cell wall synthesis was confirmed by this work;

The cellulose content variation trough the species studied should be a consequence of the *Eucalyptus CesA3* expression level;

Transgenic *A. thaliana* and *N. tabacum* overexpressing the *EgCesA3* gene were successfully obtained. The transgenic plants analyses are being performed; however it's already possible to notice, by empirical view, growth improvement in the transgenic plants in comparison to the wild type.

**References**

1. Doblin MS, Kurek I, Jacob-Wilk D, Delmer DP: Cellulose biosynthesis in plants: from genes to rosettes. *Plant & cell physiology* 2002, 43:1407-20.

2. Ranik M, Myburg A: a: Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cell wall biosynthesis. *Tree physiology* 2006, 26:545-56.

3. Brown DM, Zeef LAH, Ellis J, Goodacre R, Turner SR: Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics. *Cellulose* 2005, 17:2281-2295.

P168

In silico comparative analysis of glycoside hydrolase (GH) family 10 endo-(1-4)-beta-xylanase genes from *Eucalyptus grandis* and *Arabidopsis thaliana*

Ritesh Mewalal*, Desre Pinard, Eshchar Mizrachi, Alexander A Myburg
Department of Genetics, Forestry and Agricultural Biotechnology Institute
(FABI), University of Pretoria, Pretoria, 0002, South Africa

E-mail: ritesh.mewalal@tuks.co.za

BMC Proceedings 2011, 5(Suppl 7):P168

**Background:** The hemicellulose xylan constitutes the major non-cellulosic component of plant secondary cell walls. It has been shown

<figure><img src="image_78.png" /></figure>
that xylan adsorbs to cellulose fibres and also covalently binds a carbon moiety of lignin [1,2]. *Eucalyptus* is an important hardwood tree genus used in the pulp and paper industry and has potential as biofuel feedstock. Xylan removal is expensive and uses environmentally harsh chemical treatments [3]. Previous studies have shown that endo-(1-4)-β-

xylanase enzymes belonging to glycoside hydrolase (GH) family 10 internally attacks the xylan backbone resulting in shorter xylo-saccharide chains [4]. The recently sequenced *Eucalyptusgrandis* genome (DOE-JGI, http://www.phytozome.net) provides a unique opportunity to analyze the native endo-(1-4)-beta-xylanase proteins involved in xylan modification in

<figure><img src="image_79.png" /></figure>
<figure><img src="image_80.png" /><figcaption>Figure 2(abstract P168) Mining of EgrXYN1 and AtXYN1 for novel motifs using MEME resulted in three additional motifs [6].</figcaption></figure>

eucalypt fibre cell walls. Detailed knowledge of endogenous xylanolytic
enzymes from *Eucalyptus* could facilitate the development of strategies
to enhance the processing of woody biomass for cellulose and biofuel
production. The aims of this study are to identify xylem secondary cell
wall-related endo-(1-4)-β-xylanase genes in the *E. grandis* genome and to
perform a comparative analysis of the *Eucalyptus* xylanasepeptide
sequences with those of previously studied *Arabidopsis* orthologs to
provide a framework for assigning function to the *Eucalyptus* enzymes.

**Results:** Analysis of the *E. grandis* genome sequence on Phytozome v7.0 (http://www.phytozome.net) for putative endo-(1-4)-β-xylanase genes resulted in the identification of 18 putative GH10 family members. The expression profile of each family member was assessed (via mRNA-Seq analysis, http://eucspresso.bi.up.ac.za/) to identify members with putative roles in xylem secondary cell wall metabolism. Egrandis_v1_0.001952m (designated *EgrXYN1*) showed the highest xylem to phloem and xylem to leaf expression ratios of the expressed *E. grandis* GH10 genes [5]. BLAST analysis (<1e-10) of the *A. thaliana* genome for putative orthologs to *EgrXYN1* and co-phylogenetic analysis of all 18 *E. grandis* enzymes with the putative *A. thaliana* xylanases revealed that AtXYN1 (At1g58370) [4] was one of the closest putative orthologs to *EgrXYN1* (Figure 1). Alignment of the predicted amino acid sequences of *EgrXYN1* and AtXYN1 Jalview 2.6.1 revealed 68.76% identity between the two sequences.

In silico biochemical analysis predicted that EgrXYN1 has a molecular weight of 103 kDa with a pl of 6.08. This is very similar to AtXYN1 which is 102 kDa with a pl of 6.1. The protein domain view in Phytozome (http://www.phytozome.net) revealed that EgrXYN1 contains three successive N-terminal β-sandwich carbohydrate binding modules IV (at amino acid positions 53-185, 216-357 and 387-532) which were also observed in AtXYN1. A protein motif search (http://motif.genome.jp) revealed that EgrXYN1 also contained a conserved and identical C-terminal GH10 active site sequence "GLPIWFTELVD" at amino acid position 802-812. Finally, de novo motif search of both AtXYN1 and EgrXYN1 using MEME revealed the presence of three additional novel C-terminal motifs present within both enzymes (Figure 2).

**Conclusion:** The *E. grandis* genome contains 18 putative GH10 family members (at a BLAST threshold of 1e⁻¹⁰). One of these, *EgrXYN1* is highly preferentially expressed in *Eucalyptus* xylem tissues and shows highest similarity to *AtXYN1*. The similarities between *AtXYN1* and *EgrXYN1* suggest similar biochemical properties and biological functions. Previous studies showed that AtXYN1::eGFP localized to the cell wall providing support for its function in cell wall modification. AtXYN1prom::GUS constructs expressed predominately in the vascular bundles suggesting that AtXYN1 (and therefore putatively EgrXYN1) is involved in secondary cell wall modification [4,7]. Future work will involve experimental validation of the biochemical properties and enzyme kinetics of EgrXYN1.

References

1. Paananen A: Interaction between cellulose and xylan: An atomic force microscope and quartz crystal microbalance study. *In Hemicelluloses: Science and Technology* ACS Pub 2004, 864:269-291.

2. Barakat A, Winter H, Rondeau-Mouro C: Studies of xylan interactions and cross-linking to synthetic lignins formed by bulk and end-wise polymerization: a model study of lignin carbohydrate complex formation. *Planta* 2007, 226:226-267.

3. Battan B, Sharma J, Dhiman S: Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry. *Enz. Mic. Tech* 2007, 41:733-739.

4. Suzuki M, Kato A, Nagata N: A xylanase, AtXYN1, is predominantly expressed in vascular bundles, and four putative xylanase genes were

identified in the *Arabidopsis thaliana* genome. *Plant Cell Phys* 2002, 43:759-767.

5. Mizrachi E, Hefer C, Ranik M: De novo assembled expressed gene catalog of a fast-growing *Eucalyptus* tree produced by Illumina mRNA-Seq. *BMC Gen* 2010, 11:681-693.

6. Bailey T, Elkan C: Fitting a mixture model by expectation maximization to discover motifs in biopolymers. *Proc. Int. Conf. Intell. Syst. Mol. Biol* 1994, 2:28-36.

7. Oikawa A, Joshi H, Rennie E: An integrative approach to the identification of *Arabidopsis* and rice genes involved in xylan and secondary wall development. *PloS One* 2010, 5:263-679.

P169

Expression analysis of DNA methyltransferase and co-repressor genes
in *Quercus suber* phellogen: an attempt to correlate with cork quality

Miguel Ramos¹, Margarida Rocheta¹, Luísa Carvalho¹, José Graça²,
Leonor Morais-Cecílio¹*

¹CBAA, ISA, Technical University Lisbon, Tapada da Ajuda, 1349-017 Lisbon, Portugal; ²CEF, ISA Technical University Lisbon, Tapada da Ajuda, 1349-017 Lisbon, Portugal

E-mail: lmorais@isa.utl.pt

BMC Proceedings 2011, 5(Suppl 7):P169

**Background:** Cork oak (*Quercus suber*) is one of the most important forest species in Portugal. Cork oak produces a thick cork bark which is harvested for industrial uses. Cork quality is the most important factor that affects its uses technical performance and economic value. Cork quality is associated with many features, the most relevant being the porosity resulting from the phellogen's differentiation in filling tissue which will degenerate to lenticels. Good cork has few pores of very thin diameter being the opposite valid for low cork quality. Aiming to understand the mechanisms responsible for controlling of the molecular machinery involved in cork production, namely at the epigenetic level, it is relevant to study the expression profiles of the enzymes involved in DNA methylation in the phellogen.

Methylation of cytosines in the DNA, achieved by DNMT methyltransferases, is one of the most important factors in the regulation of gene expression [1]. In plants three DNMT classes have been identified, each one with its function: the CMT (chromomethylase) class found only in plants, is responsible for maintaining the methylation in CpHpG sequences [2]; the DRM (Domain-Rearranged-Methyltransferase) class is associated with *de novo* methylation in any context [3]; and the MET class, responsible for the maintenance of methylation in CpG zones [1]. Proteins, such as the DNA methyltransferase-associated protein (DMAP1), are known to form stable complexes with DNMTs and act as co-repressors of gene expression.

In this work, we report the transcriptional profile of three putative DNA
methyltransferase genes from the CMT, DRM and MET classes, and one
DMAP in *Q. suber* phellogen of trees producing good or bad quality cork.

**Methods:** Three *Quercus suber* potential producers of good quality cork and three producers of bad quality cork were selected. Samples of cork and phellogen were collected from these trees to further estimate cork quality and perform a transcriptional analysis of selected genes. Previously an *in silico* analysis was performed on a cork oak EST library (COEC, Cork Oak ESTs consortium). Three EST sequences, referred as DNA methyltransferases, and one as DMAP1 were selected. Each one was identified and characterized against available databases. Primers were designed to specifically amplify each sequence in real-time-PCR analysis. The actin gene was chosen as internal reference. RNA was extracted from phellogen
samples, using Spectrum Plant Total RNA kit and cDNA was obtained with
Retroscript kit. Gene expression was analyzed in the six phellogen samples,
using qRT-PCR in triplicate reactions performed for each cDNA template
with each primer pair. The NormFinder algorithm was used to evaluate the
gene expression's stability using the sample 75 as reference.

**Results:** Measurement of cork density allowed forming two groups of trees: (1) three individuals with the lower average density (0.289 g.cm-3 ± 0.037) classified as good cork quality producers, (2) three trees with the highest average density (0.388 g.cm-3 ± 0.036)classified as bad cork quality producers.

Homology search using BLASTP algorithm against available databases showed significant similarities between selected our putatively translated ESTs and known DNMTs (CMT3 - ABW96889, DRM2 - ABW96890 and MET2 - XP_002874265) and DNMT associated proteins (DMAP1-XP_002515237) with e-values of 2e-50, 5e-95, 1e-92, 9e-64, respectively
The expression of the three putative DNMTs and DMAP1 genes was evaluated in the phellogen of both tree groups.

The gene expression stability, evaluated through the NormFinder
algorithm, showed values ranging from 0.4 for QsDRM2 to 1.5 for DMAP1.
Actin also showed an appropriate value (0.7) to be used as internal control.
Bad cork quality tree #75 was used as reference. QsMET2 and QsDMAP1
genes showed the highest expression values, while QsCMT3 and QsDRM2
presented the lowest. Comparison of both group trees revealed that, in
average, the expression level of each gene is higher in the bad cork
quality group than in the good cork quality (Fig1).

**Conclusions:** In this work we identified one functional DNA methyl-transferase of each class (QsCMT3, QsDRM2 and QsMET2) and the MET1 associated protein (QsDMAP1) in cork oak phellogen. This protein has been described in humans [4] as a co-repressor of gene expression, capable of

binding with other regulatory proteins such as histone deacetylases [4]
which are also associated with gene silencing.

The general tendency of DNMTs to express less in trees able to produce good cork quality suggests a globally lower methylation level and therefore a lower phellogen gene repression. We can hypothesize that gene silencing is higher in bad quality producers, leading to a higher weight of genes whose expression originates defects.

Gene stability analyses lead us to consider QsDRM2 as the most stable, as was already reported in other species. Therefore, this gene may be a good candidate to be used as reference gene, in future real-time-PCR analyses. This high stability seems to indicate that de novo methylation although occurring, has no influence in good or bad cork quality production. Conversely, the more variable expression, and therefore the genes that can potentially affect cork quality are QsMET2 and QsDMAP1 genes.

**Acknowledgments:** This work was supported by FCT Projects Sobreiro/
0019/2009, PTDC/AGR-CFL/104197/2008, PTDC/AGR-GPL/101785/2008

**References**

1. Finnegan EJ, Kovac KA: Plant DNA methyltransferase. *Plant Mol Biol* 2000, 43:189-201.

2. Cao X, Jacobsen SE: Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. *PNAS* 2002, 99:16491-16498.

3. Cao X, Spring NM, Muszynski MG, Phillips RL, Kaepler S, Jacobsen SE: Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. *PNAS* 2000, 97:4979-4984.

4. Rountree MR, Bachman KE, Baylin SB: DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. *Nat Genet* 2000, 25:269-277.

<figure><img src="image_81.png" /></figure>
P170

An integrated database of *Eucalyptus* spp. genome project

Leandro Costa Nascimento¹*, Jorge Lepikson Neto¹, Marcela Mendes Salaza¹,
Eduardo Leal Oliveira Camargo¹, Wesley Leoricy Marques¹,
Danieli Cristina Gonçalves¹, Ramon Oliveira Vidal²,

Gonçalo Amarante Guimarães Pereira¹, Marcelo Falsarella Carazzolle³

¹Laboratório de Genômica e Expressão - Instituto de Biologia - Universidade Estadual de Campinas – UNICAMP, Brazil; ²Laboratório de Genômica e Expressão - Instituto de Biologia - Universidade Estadual de Campinas - UNICAMP/LNBio - Laboratório Nacional de Biociências – ABTLuS, Brazil;

³Laboratório de Genômica e Expressão - Instituto de Biologia - Universidade Estadual de Campinas - UNICAMP/Centro Nacional de Processamento de Alto Desempenho em São Paulo, Universidade Estadual de Campinas- UNICAMP, Brazil

E-mail: leandro@lge.ibi.unicamp.br

BMC Proceedings 2011, 5(Suppl 7):P170

**Background:** The species of the genus *Eucalyptus* are the most planted for the fiber crop in the world. They are mainly utilized for timber, pulp and paper production. Brazil, helped by the favorable weather conditions, appears as a big producer and exporter of eucalyptus derivates. In 2002, the Brazilian network research of the *Eucalyptus* Genome (Genolyptus) was established with the goal of integrating several academic and private institutions currently working with eucalyptus genomics in Brazil. This project generated around 200.000 ESTs from several tissues and conditions. Consequently, several individual projects have been implemented generating other transcriptome databases, in special, using RNA-Seq technology. In 2010, a draft genome (http://eucalyptusdb.bi.up.ac.za) of the specie *E. grandis* was produced by researches of the Joint Genome Institute (DOE-JGI) and the *Eucalyptus* Genome Network (EUCAGEN). The main goal of this work is to develop an *Eucalyptus* database (http://www.lge.ibi.unicamp.br/genolyptus) integrating public and private data in a friendly and secure web interface with bioinformatics tools that allowing the users perform complex searches.

**Results and discussion:** First, the public and private ESTs (130,290 from Genolyptus and 36,981 from NCBI) were assembled producing 48,760 unigenes (17,795 contigs and 30,765 singlets). Basically, the bdtrimmer [1] and CAP3 [2] programs were used to perform sequence trimming

(exclude vector, ribosomal, low quality and too short reads) and sequence assembly, respectively.

The autofact pipeline [3] was used to perform an automatic annotation of the assembled unigenes based on BLAST [4] searches, e-value cutoff of 1e-5, against some protein databases, including: non-redundant (NR) database of NCBI, uniref90 and uniref100 – databases containing only curated proteins [5], pfam – database of proteins families [6], kegg – database of metabolic pathways [7] and Gene Ontology (GO) – database of functional annotation [8].

The Genomic and Expression Laboratory at State University of Campinas (http://www.lge.ibi.unicamp.br) sequenced ten RNA-Seq libraries from four species (*E. Urograndis*, *E. globulus*, *E. grandis* and *E. urophylla*) using the Illumina/Solexa technology. Additionally, three RNA-seq libraries [9] were downloaded from NCBI (SRA – sequence read archive). All RNA-seq reads were aligned against the assembled unigenes and genome assembly using the SOAP2 [10] and TopHat [11] aligners, configured to allow up two mismatches, discard sequences with "N"s and return all optimal alignments. In order to perform a differential expression analysis between ESTs or RNA-seq libraries some normalization pipelines and statistical tests have been implemented.From ESTs, the differentially expressed genes between libraries were performed applying AC test [12] in assembled unigenes. The results are available to the users by a web interface (called Electronic Northern) that allows searches by gene or library name. Furthermore, it is possible to compare the gene expression between two or more libraries. From the RNA-seq libraries, the DEG-seq software [13] was used to perform normalization and statistical analysis considering 99% of confidence rate (cut-off of 0.01).

To integrate all data described above, we developed a web site (Fig. 1) hosted in a Fedora Linux machine with MySQL database server. The web interface is based on a combination of CGI scripts using PERL language (including BioPerl module) and the Apache Web Server. The site contains many bioinformatics tools allowing the user perform keyword or local BLAST search in assembled unigenes. Also it is possible to connect these results with gene expression analysis. Moreover, the Gbrowse software (Generic Genome Browser) (Fig. 2) was used to visualization the data in a genomic context, integrating the different information by clickable tracks. The top track is the reference genome assembly and the other tracks correspond to assembled unigenes and RNA-seq data mapped into reference.

<figure><img src="image_82.png" /></figure>
<figure><img src="image_83.png" /></figure>

**Acknowledgments:** The authors would like to acknowledge all researches of the Joint Genome Institute (DOE-JGI) and the *Eucalyptus* Genome Network (EUCAGEN), responsible to produce the draft genome of the *E. grandis*. Moreover, we thank the CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brazil) for the financial support of this work.

**References**

1. Baudet C, Dias Z: New EST Trimming Strategy. *Brazilian Symposium on Bioinformatics* Lecture Notes in Bioinformatics – Berlin – Germany: Springer – Verlag 2005, 3594:206-209.

2. Huang X, Madan A: CAP3: A DNA Sequence Assembly Program. *Genome Res* 1999, 9:868-877.

3. Koski LB, Gray LW, Lang BF, Burger G: AutoFACT: An Automatic Functional Annotation and Classification Tool. *BMC Bioinformatics* 2005, 6:151.

4. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. *Nucl. Acids Res* 1997, 25(17):3389-3402.

5. Suzek BE, Huang H, McGarvey P, Mazumber R, Wu CH: Uniref: comprehensive and non-redundant UniProt reference clusters. *Bioinformatics* 2007, 23(10):1282-1288.

6. Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer ELL: The Pfam Protein Families Database. *Nucl. Acids Res* 2002, 30(1):276-280.

7. Kanehisa M, Goto S: KEGG: Kyoto Encyclopedia of Genes and Genomes. *Nucl. Acids Res* 2000, 28(1):27-30.

8. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene Ontology: tool for the unification of biology. *Nature Genetics* 2000, 25:25-29.

9. Mizrachi E, Hefer CA, Ranik M, Joubert F, Myburg AA: De novoassembled expressed gene catalogue f a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq. *BMC Genomics* 2010, 11(681):1471-2164.

10. Li R, Yu C, Li Y, Lam T-W, Yiu S-M, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short read alignment. *Bioinformatics* 2009, 25(15):1966-1967.

11. Trapnell C, Pachter L, Salzberg SL: TopHat: discovering splice junctions with RNA-Seq. *Bioinformatics* 2009, 25(9):1105-1111.

12. Audic S, Claverie JM: The significance of Digital Gene Expression Profiles. *Genome Res* 1997, 7:986-995.

13. Wang L, Feng Z, Wang X, Wang X, Zhang X: DEGSeq: an R package for identifying differentially expressed genes from RNA-seq data. *Bioinformatics* 2010, 26(1):136-138.

P171

Effects of high levels of CO2 on gene expression in two different genotypes of *Fagus sylvatica*

Donatella Paffetti¹, Olbrich Maren², Matthias Fladung³, Dieter Ernst², Thorsten Markussen³, Manfred Forstreuter⁴, Francesca Donnarumma⁵, Veronika Kučerová⁶, Maik Veste², Cristina Vettori⁵

¹Department of Agricultural and Forest Economics, Engineering, Sciences and Technologies, University of Florence, Via San Bonaventura 13, 50145 Florence, Italy; ²Helmholtz Zentrum München, - Institute of Biochemical Plant Pathology, Neuherberg, Germany; ³Johann Heinrich von Thuenen Institute (vTI), Institute of Forest Genetics, Sieker Landstr. 2, D-22927 Grosshansdorf, Germany; ⁴Freie Universitaet Berlin, Institut fuer Biologie, Altensteinstr. 6, D-14195 Berlin, Germany; ⁵Plant Genetics Institute, CNR, Via Madonna del Piano 10, I-50019 Sesto Fiorentino (FI), Italy; ⁶Technical University in Zvolen, T.G. Masaryka 24, SK-96053 Zvolen, Slovakia; ⁷Centre for Energy Technology Brandenburg (CEBra), Bioenergy Research, 03046 Cottbus, Germany E-mail: donatella.paffetti@unifi.it

BMC Proceedings 2011, **5**(Suppl 7):P171

**Background:** The scenario of a changing environment, widely termed as global change is mainly caused by human activities. Oil and carbon