<figure><img src="image_45.png" /><figcaption>**Figure 1(abstract P97)** Results of infections and infestations on *Eucalyptus* species and clones. A) *E. nitens* challenged with *P. cinnamomi*, B) *E. grandis* clones challenged with *C. austroafricana* and C) *E. grandis* clones infested with *L. invasa*. Control, unchallenged plant material is indicated on the left and challenged plant material is indicated on the right of the respective panels.</figcaption></figure>

The high basal expression of *PR-3* in the GU hybrid may indicate that the MeJA and ET pathway is activated in this genotype. *PR-3* genes are chitinases, enzymes that are able to hydrolyze chitin, a component of fungal cell walls [6]. Further expression profiling of the diagnostic marker genes for the two main defense pathways during *C. austroafricana* challenge suggests that the SA pathway is important for defense against the pathogen in the tolerant interaction.

**Conclusions:** We have established important pathosystems between *Eucalyptus* and a fungal pathogen, *Eucalyptus* and an oomycete pathogen and between *Eucalyptus* and an insect pest. Genes diagnostic of the main defense signaling pathways have been identified and are being exploited to determine which pathways are activated in tree-pest/pathogen interactions. It is expected that transcriptome sequencing of each of these interactions will not only reveal the suite of genes important for defense against specific pathogens and pests, but the overlap of the responses at the molecular level, would be informative for broad spectrum resistance. These genes are potential future candidates for genetic improvement of disease tolerance in eucalypts.

References

1. Sturrock R, Frankel S, Brown A: Climate change and forest diseases. *Plant Pathol* 2011, **60**:133-149.

2. Pieterse C, Leon-Reyes A, van der Ent S: Networking by small-molecule hormones in plant immunity. *Nat Chem Biol* 2009, **5**:308-316.

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

4. Glazebrook J: Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic Pathogens. *Annu Rev Phytopathol* 2005, **43**:205-227.

5. Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees. *Plant Mol Biol Rep* 1993, **11**:113-116.

6. Chang M-M, Horovitz D, Culley D: Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding, fungal infection and the elicitor chitosan. *Plant Mol Biol* 1995, **28**:105-111.

P98

Identification of insect-specific target genes for development of RNAi based control of the *Eucalyptus* gall pest *Leptocybe invasa* Fisher & La Salle (Hymenoptera: Eulophidae)

Mathish Nambiar-Veetil*, Manoharan Sangeetha, K S Sowmiya Rani, V Aravinthakumar, R K Selvakesavan, Aiyar Balasubramanian, R Venkatachalam, Abraham Sanu Mary, John Prasanth Jacob, N Krishna Kumar Institute of Forest Genetics and Tree Breeding, Coimbatore, India
E-mail: nvmathish@yahoo.com
BMC Proceedings 2011, **5**(Suppl 7):P98

**Background:** *Eucalyptus* is grown in around 3.9 Mha in India. *Leptocybe invasa* Fisher & La Salle (Hymenoptera: Eulophidae) has emerged as a serious pest in *Eucalyptus* causing considerable loss of quality planting materials besides loss in productivity and quality of timber. Current strategies for its control include selected deployment of *Eucalyptus* clones tolerant to the pest resulting in several productive *Eucalyptus* genetic

<figure><img src="image_46.png" /><figcaption>**Figure 2(abstract P97)** Relative expression levels of the putative *Eucalyptus* PR-3 ortholog in various tissues of *E. grandis* x *E. urophylla* (data from Eucspesso [3]).</figcaption></figure>
resources, not being considered for plantation programmes, as in the
case of the widely planted *Eucalyptus* clone, ITC 10, and ITC 271. The
securely ensconced grub within the gall for a period of around 4 months
post oviposition makes it refractory to pesticide applications. This feature,
however, makes plant- incorporated protectants, via RNAi approaches a
potential strategy for engineering resistance [1-3]. However, application of
RNAi technology requires determination of sequence information of
insect-specific genes so that off-target effects in plants as well as human
beings are avoided.

**Methods:** Grubs were collected from the galls of *E. camaldulensis* clones, APNP 1.1 and ITC 351, infested with *L. invasa* from the nursery of the Institute of Forest Genetics and Tree Breeding, Coimbatore, India. Genomic DNA was isolated from the grubs homogenized in liquid nitrogen using a modified CTAB protocol [4]. RNA was isolated from the grubs using Qiagen RNeasy plant mini kit, followed by cDNA synthesis using SmartScribe reverse transcriptase.

Grubs were also directly used for PCR amplification circumventing DNA
isolation. Furthermore, to enable multiple PCR analysis, a single grub
was collected into 50 µl of sterile distilled water, vigorously vortexed for
2 min followed by brief denaturation at 95°C for 10 min and
centrifugation at 10,000 rpm for 10 min. The supernatant was used for
PCR analysis.

The PCR mix consisted either 100 ng genomic DNA or 100 ng cDNA or a
single grub or 2 µl of supernatant from denatured grub, in 1X PCR buffer,
0.8 mM dNTPs, 1 µM of each primer, 2.5 U Pfu polymerase (Fermentas).
The PCR conditions used were initial denaturation at 94°C for 5 min, cycle
denaturation at 94°C for 40s; annealing at 60°C for 40s; extension at 72°C
for 2 min for 30 cycles, and a final extension of 72°C for 5 min. In case of

grub PCR, initial denaturation was for 10 min at 95°C. The PCR products
were resolved on a 1.0% Agarose gel in 1X TAE. When multiple bands were
obtained, the products were gel eluted and either directly sequenced or
cloned into pGEMTeasy vector prior to sequencing. Sequencing was done
using the ABI PRISM 3130 XL Genetic Analyzer using Big Dye Terminator
version 3.1" Cycle sequencing kit through the commercial sequencing
services available at Chromous Biotech India Pvt Ltd.

**Results and discussion:** Chitin synthase gene was partially amplified using the degenerate primer sets, CSF3 (5'-TGYGCGACHA TGTGGCACGARAC-3')/ CSR1 (5'-GTCCTCSCCYT GRTCGTAYTGCAC-3') and CSF1 (5'-YTGGAY GGMGACATMGAYTTC-3')/ CSR1 (5'-GTCCTCSCCYTGRTCGTAYTGCAC-3') [5] from genomic DNA and cDNA. CSF3/R1 primers yielded multiple bands of which the expected 1KB size amplicon was sequenced. CSF1/R1 primers yielded an amplicon of 370 bp, which overlapped with the sequence from CSF3/R1. The sequence data obtained were aligned, annotated, and a sequence of 624 bp was submitted to NCBI (Accession number: JF772551/ to be published). The sequence comprising 2 exons and 2 introns, corresponded to 138 amino acids on conceptual translation. BLAST analysis of the nucleotide sequence showed 85% homology to *Nasonia vitripennis* chitin synthase 1 and 76% to *Apis mellifera* krotzkopf verkehrt (chitin synthase1) sequences. Furthermore, a rapid method of PCR amplification directly from grubs was developed. The amplicons generated using the primers CSF1/CSR1 yielded the same sequence, indicating its utility for rapid PCR screening of *L. invasa* grubs. In order to identify housekeeping genes for use as reference genes during quantitative PCR analysis, Elongation factor-1 alpha (EF-1 alpha) was partially amplified using primers EF1- (5'-GGTATCGACAAA CGT ACCATCG-3') /EF1-R (5'- AATCGAGCACAGG TGTGTAACC-3') and the sequence data of 534 bp was submitted to NCBI

<figure><img src="image_47.png" /></figure>
(Accession number: JF772552/ to be published). Blast analysis of this sequence showed 91% similarity to *N. vitripennis* EF-1 alpha.

**Conclusion:** Basic techniques like nucleic acid isolation and PCR amplification were adapted for this less studied insect pest. A rapid method for PCR amplification directly from the insect grub was developed for use in *L. invasa*. Our efforts have unraveled the partial gene sequence of *L. invasa* chitin synthase, a processive enzyme involved in chitin biosynthesis during different stages of insect development. The sequence of chitin synthase and EF-1 alpha represent the first genome sequence information for *L. invasa*. Chitin synthase offers a potential target for RNAi based pest control owing to its crucial role in growth and development of insect. Following full length sequence determination of *L. invasa* chitin synthase gene, RNAi target regions will be chosen for development of RNAi constructs. EF-1 alpha will be used as the reference gene in RT-PCR studies to quantitate the effect of RNAi on the transcript levels of the target gene. The effect of *Eucalyptus*-expressed dsRNA molecules cognate for chitin synthase, will be evaluated on the growth and development of *L. invasa*. Towards this end, protocols have been optimized for generation of transgenic *Eucalyptus* [6].

## References

1. Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J: *Control of coleopteran insect pests through RNA interference*. *Nat Biotechnol* 2007, 25(11):1322-6.

2. Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY: Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. *Nat Biotechnol* 2007, 25(11):1307-13.

3. Mao YB, Tao XY, Xue XY, Wang LJ, Chen XY: Cotton plants expressing CYP6AE14 double-stranded RNA show enhanced resistance to bollworms. *Transgenic Res* 2010, DOI10.1007/s11248-010-9450-1.

4. Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD: Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles. *PLoS one* 2010, 5(8):e11963.

5. Kumar N.S, Tang B, Chen X, Tian H, Zhang W: Molecular cloning, expression pattern and comparative analysis of chitin synthase gene B in Spodoptera exigua. *Comp Biochem Physiol B* 2008, 149:447-453.

6. Mathish NV, Lalitha S, Sudha N, Brindha D, Prashant K, Balasubramanian A, Sumathi R, Karthikeyan C, Sharadha N, Shanthi A, Sivakumar V, Yasodha R, Suryaprasak M, Franche C, Gherbi H, Swistoonoff S, Florence A, Hocher V, Bogusz D, Blumwald E, Gurumurthi K: Optimization of genetic transformation methods in Eucalyptus: towards understanding and enhancing salt tolerance. *National Conference on Frontiers in Plant Molecular Biology* Bharathidasan University, Tiruchirappalli 2010.

## P99

### Gene expression responses of black spruce (*Picea mariana*) to global climate change conditions

Om Rajora¹, Jinhong Kim¹, John Major², John Malcolm²

¹Faculty of Forestry and Environmental Management, University of New Brunswick, 28 Dineen Drive, Fredericton, NB E3B 5A3, Canada; ²Natural Resources Canada, Atlantic Forestry Centre, 1350 Regent Street, Fredericton, NB E3B 5P7, Canada

E-mail: Om.Rajora@unb.ca

BMC Proceedings 2011, 5(Suppl 7):P99

Global climate change conditions (elevated CO₂ and atmospheric temperatures) are subjecting our forests, especially Boreal and temperate forests, to significant abiotic stresses, such as drought. This can affect health, productivity and fitness of our forests. Therefore, it is imperative to understand genomic and eco-physiological responses of forest trees to global climate change. We are addressing this aspect in black spruce (*Picea mariana*) - a transcontinental, ecologically and economically important tree species of the North American Boreal forest. Our objective was to determine gene expression and physiological responses and their inter-relationships in black spruce to elevated CO₂, drought and co-stressed conditions.

We have used NGS whole transcriptome sequencing, cDNA-AFLP and qPCR analyses to identify, annotate and characterize genes expressed differentially in response to elevated CO₂, drought and combined elevated CO₂ and drought conditions in black spruce using the cloned

material. Photosynthetic rate and stomatal conductance were measured simultaneously with tissue collection for RNA extraction. Thousands of transcripts (genes) showed differential expression (no expression, up-regulation or down-regulation) in response to elevated CO₂, drought and/or their combined conditions, with over 1600 genes from several pathways showing >10-folds gene expression differences between control and treated plants. A number of genes showed 100 to 500 folds up or down regulation in response to elevated CO₂, drought or their combined conditions. Responses to each treatment at the gene expression and physiological levels were correlated well among different genotypes. We will present these results which contribute significantly to our understanding of tree's responses to global climate change.

## P100

### Oleoresin yield and carbon stocks in tapped subtropical *Pinus elliottii* forests

Kelly Cristine da Silva Rodrigues-Corrêa¹, Tanise Luisa Sausen², Fernando S Rocha¹, Arthur Germano Fett-Neto³*

¹Department of Botany, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil; ²Center for Biotechnology, Federal University of Rio Grande do Sul, P.O. Box 15005, 91501-970 Porto Alegre, RS, Brazil; ³Department of Botany and Center for Biotechnology, Federal University of Rio Grande do Sul, P.O. Box 15005, 91501-970 Porto Alegre, RS, Brazil

E-mail: fettneto@cbiu.ufrgs.br

BMC Proceedings 2011, 5(Suppl 7):P100

**Background:** Low-cost methods to mitigate the increasing levels of carbon dioxide in the atmosphere and their implications on global climate change have received considerable attention in the last years [1,2]. Afforestation is an important alternative to reduce the rise in atmospheric CO₂ concentration due to the system's ability to fix carbon in forest biomass and soil [3]. Several studies have been performed to estimate carbon sequestration in temperate, tropical, and mediterranean forests ecosystems [4,5]. However, there are no reports related to carbon balance in pine forests used for oleoresin tapping grown under subtropical climate. The main goal of this research is to estimate the effect of resin tapping on C sequestration by *Pinus elliottii* forests.

**Material and methods: Resin tapping operation:** In June 2009, 90 slash pine (*Pinus elliottii* Engelm.) trees of a 14 year-old forest were selected based on a previously determined DBH interval (between 23.48 ±1.12 and 22.77 ±0.88). Since the beginning of the essay, pine trees have been biweekly stimulated to produce oleoresin [6]. Three treatments were evaluated for pine biomass increase and oleoresin yield: bark streak (mechanical wound), paste (mechanical wound + chemical stimulation) and control (intact trees). At the end of each season, the released oleoresin was collected and weighed in a digital balance. The results shown below were obtained between Spring 2009 and Winter 2010.

**Biomass production and carbon accumulation:** In November 2010, fifteen pine trees (5 intact tree control, 5 mechanically wounded, and 5 paste stimulated tapped trees) were felled and weighed in the field to estimate the above and belowground total fresh biomass of trees. Sub-samples of each part of trees were collected and dried in an oven at 105° C until reaching constant dry weight. The carbon content per unit dry weight present in the aboveground and belowground biomass was estimated using a carbon content of 50% [7]. Data were analyzed for differences between resin treatments (bark streak vs. paste) by comparison of means by ANOVA and Tukey test (Systat Software Inc., Richmond, CA USA); significance was set at P £0.05).

**Results and conclusions:** The seasonal oleoresin production did not show the same pattern previously observed [8]. The highest oleoresin yield was observed in Spring (data not shown). Statistical differences between oleoresin yields of paste-treated and control (bark streak) trees were observed (Figure 1). These results are in agreement with the fact that besides genetic traits, physiological status, season and environmental conditions [9-11], inducible oleoresin biosynthesis consistently responds to exogenous chemical stimuli [6,8].

The production of aboveground biomass expressed on a dry weight basis was significantly higher than that of belowground biomass. This was observed for all treatments, and no significant differences were detected in biomass production and partition for tapped (both plain wound and chemical stimulation) or control trees (Figure 2). The aboveground biomass
<figure><img src="image_48.png" /><figcaption>**Figure 1 (abstract P100)** Annual production of oleoresin by pine trees. Standard errors of the means are indicated on top of bars. Bars with different letters indicate significant difference by a Tukey test (P ≤ 0.05). Each mean was calculated with 30 individual trees.</figcaption></figure>

showed a carbon accumulation between 221.3 and 235.9 Mg C ha⁻<fnref n="1" /> and in the roots, the carbon accumulation is in the range between 35.7 and 48.3 Mg C ha⁻<fnref n="1" />.

These results indicate that carbon stored in aboveground biomass appeared to represent the main carbon pool of tree biomass and that resin tapping had minor impact on C allocation in wood biomass, considering the time frame and stand age examined. Further studies and sampling times are ongoing in order to better characterize carbon stocks in subtropical slash pine plantations and to elucidate the contribution of oleoresin production in carbon stocks and its relation with C in wood biomass.

**Acknowledgements:** Funding for this investigation was provided by a consortium of forest companies (Trevo Florestal, Irani/Habitasul Florestal, Florestal Palmares) and the Brazilian agencies National Council for Scientific and Technological Development (CNPq), National Committee for

<figure><img src="image_49.png" /><figcaption>**Figure 2 (abstract P100)** Aboveground and belowground biomass among the treatments (control, bark streak and paste). Standard errors of the means are indicated on top of bars. Each mean was calculated with five individual trees. Identical letters indicate no significant difference by a Tukey test (P ≤ 0.05).</figcaption></figure>

Improvement of Higher Level Education Personnel (CAPES) and Rio Grande do Sul State Foundation for Research Support (Fapergs).

## References

1. Montagnini F, Nair PKR: Carbon Sequestration: An under exploited environmental benefit of agroforestry systems. *Agroforestry Systems* 2004, 61:281-295.

2. Winner WE, Thomas SC, Berry JA, Bond BJ, Cooper CE, Hinckley TM, Ehleringer JR, Fessenden JE, Lamb B, McCarthy S, McDowell NG, Phillips N, Williams M: Canopy Carbon Gain and Water Use Analysis of Old-growth Conifers in the Pacific Northwest. *Ecosystems* 2004, 7:482-497.

3. Climate Change: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. UNEP, New York 2007.

4. Návar J: Allometric equations for tree species and carbon stocks for forests of northwestern México. *Foret Ecology and Management* 2009, 257(2):427-434.

5. Redondo-Brenes A: Growth, carbon sequestration, and management of native tree plantations in humid regions of Costa Rica. *New Forests* 2007, 34:253-268.

6. Rodrigues KCS, Azevedo PCN, Sobreiro LE, Pelissari P, Fett-Neto AG: Oleoresin yield of *Pinus elliottii* plantations in a subtropical climate: effect of tree diameter, wound shape and concentration of active adjuvants in resin stimulating paste. *Industrial Crops and Products* 2008, 27:322-327.

7. Pettersen RC: The chemical composition of wood. *The Chemistry of Wood* Advances in Chemistry Series 207, American Chemical Society, Washington, DC, USA: Rowel, R.M. 1984, 57-126.

8. Rodrigues KCS, Fett-Neto AG: Oleoresin yield of *Pinus elliottii* in a subtropical climate: Seasonal variation and effect of auxin and salicylic acid-based stimulant paste. *Industrial Crops and Products* 2009, 30(2):316-320.

9. Steele CL, Lewinsohn E, Croteau R: Induced oleoresin biosynthesis in grand fir as a defense against bark beetles: (monoterpenes/turpentine/ diterpenoid resin acids/rosin/stress effects). *Proc. Natl. Acad. Sci USA* 1995, 92:4164-4168.

10. Lewinsohn E, Gijzen M, Muzika RM, Barton K, Croteau R: Oleoresinosis in Grand Fir (*Abies Grandis*) saplings and mature trees 1: modulation of this wound response by light and water stresses. *Plant Physiology* 1993, 101:1021-1028.

11. Roberds JH, Strom BL, Hain FP, Gwaze DP, McKeand SE, Lott LH: Estimates of genetic parameters for oleoresin and growth traits in juvenile loblolly pine. *Canadian Journal of Forest Research* 2003, 33:2469-2476.

# P101
## Tree-insect interaction - defence response against herbivorous insects

Hilke Schroeder<fnref n="1" />*, Andrea Ghirardo², Joerg-Peter Schnitzler², Matthias Fladung<fnref n="1" />

**Background:** The defoliation of oaks is an urgent problem for forestry in Central Europe. During the last outbreak of the green oak leaf roller (* Tortrix viridana* ) in 2003-2005, we observed fundamental differences in the defoliation level of individual *Quercus robur* trees in Germany. Some of the trees seem to be somehow "tolerant" (T oaks) against the insects grub while some seem to be conspicuously "susceptible" (S oaks). Within this study we aim to identify the underlying molecular and biochemical mechanisms in oaks responsible for the behavioural preference of *T. viridana*. By means of combined behavioural experiments and biochemical as well as molecular analysis of preformed and induced defence mechanisms in T and S oak phenotypes, we will identify the metabolic/chemical basis of the observed differences as prerequisite for the selection of candidate genes differentially expressed in tolerant and susceptible trees, respectively, after insects feeding.

**Methods:** For this purpose we until now grafted each four of the identified T and S oaks, and used them for our further experiments. The methods used at this time are as following:

<footnote n="1">¹Johann Heinrich von Thünen Institut (vTI), Institute for Forest Genetics, 22927 Grosshansdorf, Germany; ²Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Biochemical Plant Pathology, 85764 Neuherberg, Germany</footnote>

<footnote>E-mail: hilke.schroeder@vti.bund.de</footnote>

<footnote>BMC Proceedings 2011, **5**(Suppl 7):P101</footnote>
1. Establishment of a "bioassay": Larvae of the green oak leaf roller have been tested for feeding preferences on T or S oaks in a feeding choice experiment. Furthermore first olfactometer experiments with adult females were performed and the developmental performance of the larvae has been measured.

2. Preliminary analysis of emission pattern of plant volatile organic compounds (VOC) during the bioassay on T and S oaks has been done.

3. Biochemical analysis of phenolic compounds (e.g. tannins, soluble phenolic substances) and constitutive and inducible emissions of plant volatiles in T and S oaks during relevant stages of leaf ontogenesis and insect feeding and oviposition are ongoing.

4. Transcriptom sequencing analysis of T and S oaks: Overall gene expression differences between the T and S oaks have been tested using biochemically defined leaf material harvested after the bioassay by next generation sequencing analysis of the complete mRNA (transcriptom). Candidate genes involved in the defence response of oaks will be identified and compared to differences in constitutive and induced patterns of phenolic compounds and VOC emissions.

**Results:** We will present preliminary results of the following aspects of our study:

1. Feeding choice experiments with larvae of *Tortrix viridana*: The larvae had the choice between a leaf from T-oak and a leaf from S-oak. After 24h, the amount of feeding at each leaf was documented. We found a significant preference of the larvae for the leaves from S-oaks.

2. Olfactometer experiments with adult females: Adult paired moth had the choice between a grafted T- and S-oak in an olfactometer. After 30 minutes the decision of the moth was documented. A highly significant number of females chose the S-oaks instead of the T-oaks.

3. Performance experiment: Larvae had been fed with leaves of either S- or T-oaks only. The larvae fed with T-leaves needed much more leaf material to end up with the same weight of pupae than the larvae fed with S-leaves.

4. VOCs: In an extensive experiment the volatile substances emitted by T and S-oaks during feeding of larvae of *T. viridana* were measured online. We found clear differences in the amount of e.g. sesquiterpenes emitted by S- and T-oaks. Furthermore the S-oaks seem to emit attractants which leads to a higher amount of larvae feeding on them.

**Conclusions:** We started the project with an observation, thus, we first identified phenotypes. Now, concerning the behavioural and biochemical results, we conclude that there are other factors than only environmental ones which lead to tolerant and susceptible oaks. There are physiological differences between S- and T-oaks and the identification of the candidate genes responsible for these differences is ongoing.

This approach will give us an insight into the functional genomics of *Q. robusta* relating to the feeding of herbivorous insects. The question still remains unsolved whether the *Tortrix*-oak interaction is highly specific, or basics of the "*Tortrix* tolerance" can be transferred to other host-pathogen interactions. With the identification of molecular and biochemical markers of "*Tortrix* tolerance" in oaks we can contribute to decision support in sustainable forest management.

## P102
### Low temperature stress and changes in the lignin content and composition in *Eucalyptus globulus*

Jullyana Moura-Sobczak<fnref n="1" />*, UIara Souza<fnref n="1" />, Eduardo Kiyota<fnref n="1" />, Paulo Mazzafera²

It is known that many abiotic stresses, such as mineral deficiency, drought, UV-B radiation, wind and low temperatures, alter the quantity and composition of lignin in several species [1]. The aim of this work was to verify if the cold stress may cause changes in the quantity and composition of lignin in *E. globulus*. Additionally, we analyzed whether these changes can also be beneficial from the industrial point of view, that is, if this would allow a better extractability of lignin in this species, which is a desirable feature in the manufacture of paper. During the day, all plants were kept in a greenhouse at room temperature but at night, half of plants was transferred to growth chamber at 12 °C and half to

25 °C. These conditions were kept during 20 days. The stems of plants from the two groups were collected and analyzed for total lignin with thioglycolic acid [2] and submitted to thioacidolysis and GC-MS analysis for the determination of the monomeric lignin composition [3]. Part of the material was used to determine the digestibility of cellulose [4].

It was observed that cold reduced the accumulation of lignin in *E. globulus*. GC-MS analysis showed that the proportion of the S/G was reduced in plants subjected to low temperatures and it was also observed a lower digestibility of cellulose in these plants, indicating that this lignin could be more difficult to be removed in industrial processes of papermaking.

These results have been related with gene expression studies for the enzymes of the lignin biosynthesis pathway and may contribute to understand the processes controlling lignin deposition in eucalyptus.

**References**

1. Moura JCMS, Bonine CAV, Viana JOF, Dornelas MC, Mazzafera P: Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants. *J Integr Plant Biol* 2010, 52:360-376.

2. Lange BM, Lapierre C, Sandermann HJR: Elicitor-induced spruce stress lignin. *Plant Physiol* 1995, 108:1277-1287.

3. Rolando C, Monties B, Lapierre C: Thioacidolysis. *Methods in Lignin Chemistry* Berlin: Springer-Verlag: Lin S, Dence C 1992, 334-349.

4. Chen F, Dixon RA: Lignin modification improves fermentable sugar yields for biofuel production. *Nat Biotechnol* 2007, 25:759-761.

## P103
### Drought stress and changes in the lignin content and composition in *Eucalyptus*

Jullyana Moura-Sobczak, UIara Souza*, Paulo Mazzafera

1. Departamento de Fisiologia Vegetal. Instituto de Biologia - Unicamp. CEP:

13083-970 Campinas, SP, Brazil

E-mail: uiarakrs@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P103

It is known that many abiotic stresses, such as mineral deficiency, drought, UV-B radiation, wind and low temperatures, alter the quantity and composition of lignin in several species [1]. The aim of this work was to verify if the drought stress may cause changes in the quantity and composition of lignin in *Eucalyptus globulus* Labill and in the hybrids *E. urograndis* (*E. urophylla* x *E. grandis*) and *E. uroglobulus* (*E. globulus* x *E. urograndis*). In the experiments the plants were divided in three groups (control, drought and drought recovered.) The control plants were irrigated daily. The plants from the group "drought" were not irrigated and were collected when wilt symptom was observed. The plants from the group "drought recovered" were irrigated when wilt was observed and were collected after recovery.

Samples of basal and apical regions of the stem were collected and analyzed for total lignin with thioglycolic acid [2] and analyzed by GC-MS to determine lignin monomeric composition [3].

*E. urograndis* subjected to drought decreased the amount of lignin in the stem apical regions and increased lignin in the basal region. *E. globulus* showed opposite behavior in apical regions and showed no significant changes in the basal regions. *E. uroglobulus* showed a pattern similar to *E. urograndis* in apical regions and similar to *E. globulus* in basal regions.

Although *E. urograndis* and *E. uroglobulus* reduced lignin and *E. globulus* increased in the apical part of the stem, it was observed that these different adjustments of lignin deposition eventually result in an increased proportion of S/G in both species. Moreover, when the amount of lignin is increased in the basal regions of *E. urograndis* there is a decrease in the proportion S/G.

Increasing the proportion S/G, either by increasing the amount of lignin-rich syringyl units or reduction of coniferyl units can be an important aspect in the adaptation of both species to drought stress.

**References**

1. Moura JCMS, Bonine CAV, Viana JOF, Dornelas MC, Mazzafera P: Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants. *J Integr Plant Biol* 2010, 52(4):360-376.

2. Lange BM, Lapierre C, Sandermann HJR: Elicitor-induced spruce stress lignin. *Plant Physiol* 1995, 108:1277-1287.

3. Rolando C, Monties B, Lapierre C: Thioacidolysis. *Methods in Lignin Chemistry* Berlin: Springer-Verlag: Lin S, Dence C 1992, 334-349.

<footnote n="1">¹. Departamento de Fisiologia Vegetal. Instituto de Biologia - Unicamp. CEP: 13083-970 Campinas, SP, Brazil; ². Departamento de Fisiologia Vegetal.</footnote>

<footnote>Instituto de Biologia - Unicamp. CEP: 13083-970 Campinas, SP, Brazil</footnote>

<footnote>E-mail: jucbio@gmail.com</footnote>

<footnote>BMC Proceedings 2011, 5(Suppl 7):P102</footnote>
# S6. WOOD FORMATION

## P104

### A novel approach to increase cell wall saccharification for efficient biofuel production

Miron Abramson¹*, Oded Shoseyov², Ziv Shani³

¹The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Rehovot, Israel; Futuragene Ltd., Rehovot, Israel; ²The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Rehovot, Israel;

³Futuragene Ltd., Rehovot, Israel

E-mail: miron@futuragene.com

BMC Proceedings 2011, 5(Suppl 7):P104

The conversion of biomass to biofuels requires heat, pressure and acid treatments to overcome the recalcitrance of the cell wall and remove lignin that hinders access to cellulose. Economic production of biofuels from lignocellulosic biomass and efficient sugar release depends on the ability to reverse and overcome this natural recalcitrance of the plant cell wall.

Most of the current ideas and methods to generate lignocellulose feedstock which is more amenable to saccharification, are limited to the modification or down-regulation of the lignin biosynthesis. This often results in dwarfing, xylem vessel collapse or reduced fitness to stress. In order to overcome these limitations, we present here a novel approach for cell wall remodeling. By introducing new trans-genes from algae, fungi and viruses we create "Trojan Horses": polymer pockets of enhanced solubility within the cell wall, thus increasing cellulose accessibility to solvents and enzymes. Two different methodologies are presented. The first includes transgenic introduction of soluble polysaccharides such as **hyaluronan** into the cell wall during its development, leading to its intercalation with the cellulose microfibrils. Upon processing, these soluble intercalated polysaccharides are washed away, resulting in highly porous cell walls with increased accessibility to hydrolytic enzymes. The second methodology involves modification of the cellulose by expression of recombinant **cellobiose dehydrogenase (CDH)** in plant cell walls, leading to disruption of the cellulose microcrystalline lattice and thus increasing hydrolytic enzymes accessibility. Both methodologies result in transgenic plants that exhibit **significantly enhanced cellulose hydrolysis**, while maintaining their physiological and structural integrity.

This novel concept provides new prospects for more economical production of liquid biofuel to substitute fossil fuels.

## P105

### Characterising the role of the *Eucalyptus grandis* SND2 promoter in secondary cell wall biosynthesis

Jonathan Botha¹*, Desre Pinard¹, Nicky Creux¹, Steven Hussey¹, Christine Maritz-Olivier¹, Antanas Spokevicius², Gerd Bossinger², Eshchar Mizrachi¹, Alexander Myburg¹

¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; ²Department of Forest and Ecosystem Science, Melbourne School of Forest and Ecosystem Science, University of Melbourne, Australia

E-mail: Jonathan.Botha@fabi.up.ac.za

BMC Proceedings 2011, 5(Suppl 7):P105

**Background:** NAC and MYB transcription factors (TFs) have been shown to play prominent roles in the regulation of plant developmental processes. Two *Arabidopsis thaliana* NAC domain TFs (AtSND2, AtSND3) and one MYB domain TF (AtMYB103) were shown to be downstream targets of two master regulators of xylem fibre cell development, NST1 and SND1 [1,2]. These TFs were able to induce the expression of a GUS reporter gene under the control of the AtCesA8 promoter [3], indicating that they may be involved in the regulation of cellulose biosynthesis in the secondary cell walls of *A. thaliana* xylem fibres. It is hypothesized that putative orthologs of these TFs will also play important roles in regulating fibre secondary cell wall biosynthesis in woody plants such as *Eucalyptus grandis*. The transcriptional network regulating wood fibre development is uncharacterized in *E. grandis*, therefore it would be beneficial to identify upstream components of this transcriptional

network in *E. grandis*. In this ongoing study we aim to identify TFs which bind to the promoter of the putative ortholog of AtSND2 in *E. grandis* (EgSND2) and possibly also to the promoters of the orthologs of AtSND3 and AtMYB103. In parallel, we are characterizing the detailed heterologous expression patterns of the EgSND2, EgSND3 and MYB103 promoter regions in *A. thaliana* plants. This work forms part of an effort to elucidate the transcriptional network regulating wood fibre development in *E. grandis*.

**Methods:** A reverse BLAST approach was used to identify candidate orthologs of AtSND2, AtSND3 and AtMYB103 in *Eucalyptus*, *Populus* and *Vitis*. In *silico* cis-element analysis was performed on the promoter regions of the putative orthologs to identify previously characterised and novel cis-elements. The 1.5 kb regions upstream of the translational start site (TSS) of EgSND2, EgSND3 and EgMYB103 were isolated from *E. grandis* genomic DNA. The amplified fragments were cloned into pMDC162, a GUS reporter vector, and introduced into *A. thaliana* Col-O plants for heterologous GUS expression analysis. The same reporter constructs were also transformed directly into the vascular cambium of potted *E. grandis* plants to determine endogenous promoter activity by Induced Somatic Sector Analysis (ISSA, [4]). Qualitative GUS reporter analyses were performed on the EgSND2promoter::GUS, EgSND3promoter::GUS and EgMYB103promoter::GUS constructs in *Arabidopsis* plants at 1, 3 and 6 weeks. A 500 bp truncation upstream of the TSS of EgSND2 was generated and cloned into the pHIS2.1 vector along with the 1.5 kb EgSND2 promoter sequence for use in yeast one-hybrid (Y1-H) screening. Candidate proteins which bind to the cloned promoter sequences will be identified using Y1-H analysis and further characterised.

**Results and discussion:** A number of previously described (e.g. [5]) and novel cis-elements were present in the three cloned *Eucalyptus* promoters (EgSND2, EgSND3 and EgMYB103) suggesting regulation by an overlapping set of upstream transcription factors. The 1, 3 and 6-week GUS analyses of the EgSND2promoter::GUS and EgSND3promoter::GUS constructs revealed strong GUS expression in vascular tissues, but the GUS expression was not specific to vascular tissues as reported for the endogenous AtSND2 and AtSND3 genes [3]. This suggests that the 1.5 kb region upstream of the translational start site may not be sufficient for fibre-specific expression in a heterologous system. Similarly, the EgMYB103promoter::GUS construct was expressed in stems and leaves, in contrast to strong stem specificity reported by Zhong et al., [3] for the *Arabidopsis* ortholog. ISSA (ongoing) results of endogenous expression in *Eucalyptus* may clarify the possible regulatory divergence in these promoter sequences. We hope to soon identify and functionally annotate a number of protein candidates binding to the EgSND2 promoter using Y1-H analysis.

**Conclusions:** The EgSND2, EgSND3 and EgMYB103 promoters were found to contain common cis-regulatory elements, which suggests at least partial co-regulation in *E. grandis*. The 1.5 kb upstream regions of EgSND2, EgSND3 and EgMYB103 induced strong heterologous GUS expression in vascular and non-vascular tissues of *A. thaliana* suggesting either that these promoter sequences have functionally diverged in *Arabidopsis* and *Eucalyptus*, or that the 1.5 kb upstream regions alone are not sufficient to induce fibre-specific expression as previously reported in *Arabidopsis*.

### References

1. Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M: NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. *Plant Cell* 2007, 19:270-280.

2. Zhong R, Demura T, Ye ZH: SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. *Plant Cell* 2006, 18:3158-3170.

3. Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH: A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. *Plant Cell* 2008, 20:2763-2782.

4. Van Beveren KS, Spokevicius AV, Tibbits J, Wang Q, Bossinger G: Transformation of cambial tissue in vivo provides an efficient means for induced somatic sector analysis and gene testing in stems of woody plant species. *Functional Plant Biology* 2006, 33:629-638.

5. Creux NM, Ranik M, Berger DK, Myburg AA: Comparative analysis of orthologous cellulose synthase promoters from Arabidopsis, Populus and Eucalyptus: evidence of conserved regulatory elements in angiosperms. *New Phytol* 2008, 179:722-737.
P106

Effects of nitrogen fertilization on global xylem transcript profiling of *Eucalyptus urophylla* x *grandis* evaluated by RNA-seq technology

Eduardo Camargo¹*, Leandro Costa¹, Marçal Soler², Marcela Salazar¹,
Jorge Lepikson¹, Danieli Gonçalves¹, Wesley Marques³, Marcelo Carazzolle¹,
Yves Martinez², Jacqueline Grima-Pettenati², Gonçalo Pereira¹

¹Laboratório de Genómica e Expressão, IB, UNICAMP, Campinas, São Paulo,
Brazil; ²Génomique Fonctionnelle de l'Eucalyptus, LRSV, UPS/CNRS, Castanet-
Tolosan, France; ³Federative Research 3450, Castanet-Tolosan, France
E-mail: lealcamargo@gmail.com

*BMC Proceedings* 2011, **5**(Suppl 7):P106

**Background:** Eucalyptus species are the most widely planted hardwood trees in the world representing more than 4.75 million ha in Brazil. Their high productivity, valuable wood properties and wide adaptability could allow sustainable and cost-efficient production of lignocellulosic bioenergy. The main limitation to this objective is wood recalcitrance to degradation which is linked to the structure and composition of lignified secondary cell walls. Lignin, for example, impairs the accessibility of cellulose during kraft pulping as well as during saccharification, a key step of bioethanol production.

The application of nitrogen fertilizers is one strategy to increase growth rates and productivity since nitrogen is one of the most limiting nutrient for tree growth and carbon sequestration. However, the effects of nitrogen availability on wood properties and related gene expression are poorly understood.

In poplar, it was recently reported that N fertilization increased aerial biomass, while in wood, fibre morphology and secondary cell wall structure and composition were modified. An increase in cellulose coupled with a decrease in lignin was observed and the mRNA profiles evaluated by microarray showed that nitrogen and tension wood have overlapping effects [1]. Moreover, a highly significant genetic correlation was observed between plant growth and lignin/cellulose composition. Quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators [2].

In order to get an insight on the regulation of nitrogen availability on wood formation in Eucalyptus, we have studied the effects of nitrogen fertilization on xylem transcriptome profiles using RNA-seq technology.

**Methods:** An experimental system was set up in which rooted cuttings of *Eucalyptus urophylla* x *grandis* were fertilized during 30 days with three different amounts of N (limiting, -N; adequate, CT; excess, +N). For the treatment with excess of N fertilization, we used two different nutrient solutions with different concentration of NO₃⁻ and NH₄⁺.

Histochemical analyses were performed on stem transverse sections (80 µm thick) obtained using a Vibratome (LEICA VT 1000S). The Weisner reagent (phloroglucinol- HCl) was used in order to detect lignified cell walls, and calcofluor reagent to evaluate the cellulose content by fluorescence. The samples were observed in confocal microscopy (SP2-AOBS, Leica) and under bright-field microscopy (DM IRBE, Leica) coupled with a CCD camera (DFC 300 FX, Leica).

The construction of the RNA-seq libraries and sequencing were performed accordingly to Illumina's protocols. Prior to analyze the RNA-seq data, we have done an assembly of Eucalyptus ESTs (GENOLYPTUS and NCBI). The 53,412 unigenes produced were automatically annotated using BLAST (e-value cutoff of 1e-5) against different sequence databases.

The RNA-seq reads were aligned against the assembled unigenes using the SOAP2 aligner [3] configured to allow up two mismatches, discard sequences with "N"s and return all optimal alignments. In order to perform the differential expression analysis between libraries, a normalization and statistics pipeline were applied using DEG-seq software [4] considering 99% of confidence rate (cut-off value of 0.01).

**Results and discussion:** The intensity of staining with phloroglucinol was higher in stem sections from samples grown under -N treatment as compared to control, whereas it was lower in both +N treatments. This suggests that lignin biosynthesis is decreased in presence of excess of nitrogen fertilizer. We could also observe in the +N samples, an increase in cellulose staining intensity with the calcofluor reagent specially noticeable when comparing with -N samples suggesting that nitrogen also influence cellulose biosynthesis.

The RNA-seq analysis generated 123,121,154 sequence reads after filtering. Of the total reads, about 86 millions matched either to a unique

(47,9%) or to multiple (21,8%) EST locations. Each nitrogen treatment was represented by at least 28.8 million reads, a tag density sufficient for quantitative analysis of gene expression.

The sequence reads were aligned on the new *Eucalyptus* EST assembly resulting in 36,125 unigenes expressed (15,293 contigs and 20,832 singlets). After statistics analysis, we determined 14,400 differentially expressed genes, that for a preliminary analysis were divided in two scenarios: the genes down-regulated in -N and up-regulated in both +N treatments (8,967 genes), and the genes up-regulated in -N and down-regulated in both +N treatments (5,433 genes).

To facilitate the global analysis for each scenario, gene ontology (GO) classification (http://www.geneontology.org) was performed and showed differences in some biological process categories. For instance, we observed that genes involved in the biosynthesis of cell wall main components (lignin, cellulose and hemicelluloses) were differentially expressed between the treatments. For example, some genes of the lignin biosynthetic pathway were up-regulated in the treatment with less nitrogen (-N) and down-regulated in both +N treatments.

**Conclusions:** This first analysis in Eucalyptus allowed us to show an effect of nitrogen fertilization on cell wall composition both at the histological and at the gene expression level. We believe that further experiments completed by chemical analysis of wood samples will give more insights in the mechanisms of nitrogen fertilization on wood formation. This knowledge will be important to address new demands on better suited biomass quality for industrial applications.

References

1. Pitre FE, Lafarguette F, Boyle B, Pavy N, Caron S, Dallaire N, Poulin PL, Ouellet M, Morency MJ, Wiebe N, Lim EL, Urbain A, Mouille G, Cooke JEK, Mackay JJ: High nitrogen fertilization and stem leaning have overlapping effects on wood formation in poplar but invoke largely distinct molecular pathways. *Tree Physiol* 2010, 30:1273-1289.

2. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CR, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M: Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels. *New Phytol* 2009, 182:878-890.

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

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

P107

In silico and functional characterization of the promoter of a *Eucalyptus*
secondary cell wall associated cellulose synthase gene (*EgCesA1*)

Nicky Creux¹*, Minique De Castro², Martin Ranik³, Antanas Spokevicius⁴,
Gerd Bossinger⁵, Christine Maritz-Olivier², Zander Myburg¹

¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; ²Agricultural Research Council Biotechnology Platform, Onderstepoort, South Africa; ³Department of Forest Ecosystems and Society, Oregon State University, USA; ⁴Department of Forest and Ecosystem Science, Melbourne School of Forest and Ecosystem Science, University of Melbourne, Australia; ⁵Department of Genetics, University of Pretoria, South Africa

E-mail: nicky.creux@fabi.up.ac.za

BMC Proceedings 2011, **5**(Suppl 7):P107

**Background:** Cellulose is an important biopolymer produced by all plants and is used in a number of different industries, including for pulp and paper production. Cellulose is deposited into the plant cell wall by a large membrane-bound protein complex, which is composed of different cellulose synthase (CESA) proteins. The cellulose content and pattern of deposition in plant cell walls is highly variable depending on the function of the cell. All plant cells have a thin primary cell wall, but a number of plant cell types, including xylem cells, also deposit a secondary cell wall to give these tissues mechanical strength required to perform their function. Different cellulose synthase (CESA) genes have been shown to be involved in the deposition of primary and secondary walls. In *Arabidopsis*, three *CesA* (*AtCesA4*, 7 and 8) genes have consistently been associated with cells depositing secondary cell walls, while a different set of *CesA* genes have been shown to function during primary cell wall formation [Reviewed in 1]. These findings have been mirrored by studies of *CesA* gene orthologs in
Populus and *Eucalyptus* [2-4]. While there have been a number of studies on CesA genes and their functions, much less is known about the regulation of these genes. In a previous study, we investigated the promoters of CesA genes involved in primary and secondary cell wall formation by performing a phylogenetic footprinting analysis to identify cis-elements conserved in the promoters from orthologous *Arabidopsis*, *Populus* and *Eucalyptus* cellulose synthase genes [5]. We identified a number of putative cis-regulatory elements that may play a role in the regulation of cellulose biosynthesis during primary and secondary cell wall formation. In the current study our aim is to further validate the cis-elements identified in the CesA gene promoters by investigating their conservation across different *Eucalyptus* species and to determine the regulatory function of these promoter regions and the proteins which bind to them.

**Methods:** A number of different methods are being employed to investigate the regulatory functions associated with the *Eucalyptus* CesA promoters. Firstly, to validate the cis-elements previously identified, we cloned and sequenced the promoters of six CesA genes from 13 different *Eucalyptus* species. The promoter sequences were analysed on the nucleotide diversity level. The cis-elements identified in the previous study were mapped onto the cloned promoter sequences and analysed for conservation in sequence and position. Next, we studied the possible roles of promoter regions harbouring conserved cis-elements in spatio-temporal regulation of CesA genes. We tested regions of the *Eucalyptus grandis* CesA1 (*EgCesA1*) promoter for involvement in spatio-temporal regulation by cloning the full-length (2 kb) promoter and a series of truncates thereof upstream of the β-glucuronidase (GUS) reporter gene. These constructs were used to transform *Arabidopsis* (floral dipping) and *Eucalyptus* (Induced Somatic Sector Analysis, [6]). The GUS expression patterns were compared to the pattern produced by the 2 kb *EgCesA1* promoter. Finally, promoter regions identified as functionally active and harbouring conserved elements of interest are being used to screen a *Eucalyptus* immature xylem cDNA expression library for yeast-1-hybrid interactions to identify proteins which interact with these regions.

**Results and discussion:** Studying the diversity of CesA promoter sequences and cis-elements in 13 *Eucalyptus* species provided us with valuable insight into the relative conservation of specific promoters regions and the cis-elements within these regions. We found that the overall nucleotide diversity of the promoter sets varied greatly from promoter to promoter, but we could identify regions in the promoters that were as conserved as coding regions. We found that in many cases these localized decreases in nucleotide diversity corresponded to clusters of conserved cis-elements which were identified previously [5]. This was particularly noticeable at the transcriptional start site for most genes and in this region we noticed a repeat element in all of the promoters investigated. Some elements were also shown to be specific to either the primary or secondary cell wall associated promoters. The cis-element information obtained from this study was used to create seven truncates of the *EgCesA1* promoter. Using GUS expression analysis in *Arabidopsis* we identified a number of repression and activation sites within the promoter. We also observed a loss of leaf (vein) expression 800 bp upstream. One of the repeat elements fused to the 5'UTR greatly enhanced overall GUS expression in a non-specific way. In a *Eucalyptus* background (ISSA), six of the seven truncates showed xylem-specific expression, but the 5'UTR and repeat element fusion showed GUS expression in phloem and xylem. These regions have been used to construct bait vectors for Yeast-1-hybrid screening which is still ongoing.

**Conclusion:** In this study we addressed three main objectives, (1) investigate the evolution of the *Eucalyptus* CesA promoters and cis-elements associated with primary and secondary cell wall formation, (2) investigate the expression patterns of truncated versions of the *EgCesA1* promoter using the GUS reporter system and (3) test the *EgCesA1* promoter regions affecting gene expression in a yeast-1-hybrid assay to identify possible regulators of this gene. We have identified regions in the promoter that were conserved and corresponded to previously identified cis-elements. Using this information we produced seven promoter truncates and discovered several regions and cis-elements in the *EgCesA1* promoter which affect GUS expression patterns. These results will aid in understanding and elucidating transcriptional networks regulating xylogenesis in woody genera such as *Eucalyptus*.

## References

1. Taylor NG: Cellulose biosynthesis and deposition in higher plants. *New Phytol* 2008, 178(2):239-252.

2. Samuga A, Joshi CP: A new cellulose synthase gene (*PtrCesA2*) from aspen xylem is orthologous to *Arabidopsis* AtCesA7 (irx3) gene associated with secondary cell wall synthesis. *Gene* 2002, 296(1-2):37-44.

3. Ranik M, Creux NM, Myburg AA: Within-tree transcriptome profiling in wood-forming tissues of a fast-growing *Eucalyptus* tree. *Tree Physiol* 2006, 26(3):365-375.

4. Kumar M, Thammannagowda S, Bulone V, Chiang V, Han K-H, Joshi CP, Mansfield SD, Mellerowicz E, Sundberg B, Teeri T, *et al*: An update on the nomenclature for the cellulose synthase genes in *Populus*. *Trends Plant Sci* 2009, 14(5):248-254.

5. Creux NM, Ranik M, Berger DK, Myburg AA: Comparative analysis of orthologous cellulose synthase promoters from *Arabidopsis*, *Populus* and *Eucalyptus*: evidence of conserved regulatory elements in angiosperms. *New Phytol* 2008, 179(3):722-737.

6. Van Beveren KS, Spokevicius AV, Tibbits J, Wang Q, Bossinger G: Transformation of cambial tissue in vivo provides an efficient means for induced somatic sector analysis and gene testing in stems of woody plant species. *Functional Plant Biol* 2006, 33(7):629-638.

## P108

### Generation and analysis of expressed sequence tags (ESTs) from cambium tissue cDNA libraries of contrasting genotypes of *Eucalyptus globulus* Labill

Juan Pedro Elissetche*, Alexis Salas-Burgos, Renan Garcia, Carola Iturra, Regis Teixeira, Jaime Rodriguez, Sofia Valenzuela
Genomica Forestal SA, Barrio Universitario s/n, Centro de Biotecnologia, Universidad de Concepción, Concepción, 403000, Chile
E-mail: jelisset@udec.cl
*BMC Proceedings* 2011, **5**(Suppl 7):P108

In the present study, reported the generation and analysis of ESTs of cDNA libraries from cambium tissue of secondary xylem obtained of two genotypes of *Eucalyptus globulus* contrasting in wood density and pulp yield. The sequences were blasted and annotated to compare with genome and ESTs database of other plant species. The goal of study was to determinate wich genes was differentially expressed and compare levels of transcript, validated by qRT-PCR, in each genotype involved in wood formation, to explain the differences founding in wood traits of contrasting genotypes.Sequences obtained was 450,000 ESTs of which approximately 21,000 sequences showed homology with genes of different vascular plants, mainly *Vitis vinifera*, *Populus* sp, *Eucalyptus* sp, *Ricinus* sp. Moreover, it was determined that 265 genes differentially expressed in both genotypes, and 41 genes were directly involved in wood formation process (xylogenesis). Of the 41 differentially expressed genes could be determined that mainly correspond genes involved in lignin biosynthesis pathway which HCT, C3H, CAD, PAL, COMT and F5H and lignin polymerization like laccase and peroxidase. Otherwise we found genes involved in carbohydrate biosynthesis (cellulose and hemicellulose) among which Sussy, UDP glucose dehydrogenase, UDP-mannose dehydrogenase and β-xylosidase. Also we described genes involved in morphological characteristics of fiber such FLA, XTH and transcription factors such MYB and LIM related to fiber length, microfibrillar angle and extensibility of cell wall. Finally some of these genes were validated by the qRT-PCR technique to determine the level of transcripts in each genotype.

## P109

### *Eucalyptus* transcriptome analysis revealed molecular chaperones highly expressed in xylem

Danieli Gonçalves<fnref n="1" />*, Jorge Lepikson-Neto<fnref n="2" />, Marcela Salazar<fnref n="2" />, Leandro Nascimento<fnref n="2" />, Eduardo Camargo<fnref n="2" />, Wesley Marques<fnref n="2" />, Gonçalo Pereira<fnref n="2" />, Carlos Ramos<fnref n="1" />

E-mail: danieligoncalves@yahoo.com.br

*BMC Proceedings* 2011, **5**(Suppl 7):P109

**Background:** Plant development is very plastic, being coupled to environmental cues. As sessile organisms, plants must be able to respond

<footnote n="1">¹Laboratório de Bioquímica de Proteínas, Dep. de Química Orgânica - Instituto de Química, Universidade Estadual de Campinas, UNICAMP, Brazil;</footnote>

<footnote n="2">²Laboratório de Genômica e Expressão, Dep. de Genética, Evolução e Bioagentes - Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Brazil</footnote>
<figure><img src="image_50.png" /><figcaption>**Figure 1(abstract P109)** Molecular chaperones up-regulated in *E. globulus* xylem in comparison to *E.grandis* xylem.</figcaption></figure>

rapidly to environmental stresses such as changes in temperature and salinity, heavy metals and water deficit. Efficient stress response systems are prerequisites for plant survival and productivity [1].

Molecular chaperones (or Heat Shock Proteins – HSP) compose a ubiquitous class of proteins involved in cellular protein quality control (PQC) and homeostasis. They play a critical role in folding and degradation of polypeptides, and therefore, in maintenance and

modulation of cellular pathways, which are dependent of function (correct folding) and availability (stability and degradation) of involved proteins, under normal and stress conditions [2].

Genetics and proteomics studies of wood formation have highlighted some chaperones up-regulated in xylem of *Eucalyptus*, *Pinus* and *Populus* species, stating that they may play an important role in cell wall formation and xylem development [3,4].

<figure><img src="image_51.png" /><figcaption>**Figure 2(abstract P109)** Molecular chaperones, co-chaperones and Heat shock transcription factors are up-regulated in *E. urograndis* naringenin-chalcone (black bars) and naringenin-supplemented (gray bars) in comparison to control group.</figcaption></figure>
Different species of *Eucalyptus* are known for their superior performance in growth, wood quality and resistance to different types of stress [5]. Such characteristics are probably driven by distinct gene expression coordination in xylogenesis. *Eucalyptus grandis* is one of the most planted species in the world due to its rapid growth, wide adaptability and wood quality. *Eucalyptus globulus* wood has higher S/G ratio which provides high yields in cellulose extraction [6].

Lignin extraction consumes large quantities of chemicals and energy, and many efforts have been made to improve this process by modifying lignin content or composition in trees, in order to reduce lignin content or make it easier to extract. Results have been achieved by supplementation and genetic modification [7,8].

This study aims to identify chaperones possibly involved in wood formation and quality of wood for pulp and paper industries.

**Methods:** The RNA-Seq reads were produced from two xylem libraries for comparison between species (*Eucalyptus globulus* and *E. grandis*), and from two libraries for evaluating flavonoids supplementation (*E. urograndis* supplemented with naringenin and naringenin-chalcone).

Reads were aligned against the assembled unigenes using SOAP2 aligner [9] configured to allow up two mismatches, discard sequences with "N's and return all optimal alignments. To perform the differential expression analysis between libraries, a normalization and statistics pipeline were applied using DEG-seq software [10] (confidence rate: 99%; cut-off: 0.01). RNA-Seq libraries and Gbrowse are available at http://bioinfo03.ibi.unicamp.br/eucalyptus/.

**Results and discussion:** In this study, we identified chaperones differentially expressed between *Eucalyptus* species. RNA-seq analysis revealed chaperones as smHsp, Hsp40, Hsp70, Hsp90 and Hsp100 between 1 and 3 fold up-regulated in xylem of *E. globulus* (Figure 1).

*Eucalyptus urograndis* naringenin-chalcone or naringenin supplemented also presented molecular chaperones highly expressed (Figure 2). Recent findings reported that supplementation with these flavonoids can inhibit lignin biosynthesis, and change lignin content and composition [10].

SmHsp family is active in a wide range of environmental stresses, including heat, cold, drought, salinity and oxidative stress [11]. Gion et al [4] noted smHsp accumulation in wood forming tissues, and suggested a role in extending the cell wall thickening phase during xylogenesis.

Hsp70 family acts in protein refolding, translocation, and facilitating the degradation of unstable proteins, directing them to lysosomes or proteasomes [12].

Hsp90 family has a select group of substrate proteins as polymerases and kinases [13]. In *A. thaliana* some kinases are required for optimal cell elongation, which is important for plant growth and vascular system formation [14].

We have successfully identified chaperones with higher expression on *E. globulus* xylem and in *E. urograndis* flavonoids-supplemented plants, which provide evidences to link xylogenesis and chaperone expression. Those findings are crucial on helping to elucidate the role of chaperones on plant development, stress response and wood formation.

**References**

1. Neumann D, Nover L, Partier R, Scharf KD: Heat shock and other stress response systems of plants. *Biol. Zbl* 1989, 108:1-156.

2. Tiroli-Cepeda AO, Ramos CHI: An Overview of the Role of Molecular Chaperones in Protein Homeostasis. *Protein Pept. Lett* 2011, 18(2):101-9.

3. Le Provost G, Paiva J, Pot D, Brach J, Plomion C: Seasonal variation in transcript accumulation in wood forming tissues of maritime pine (Pinus pinaster Ait.) with emphasis on a cell wall Glycine Rich Protein. *Planta* 2003, 217:820-830.

4. Gion JM, Lalanne C, Le Provost G, Ferry-Dumazet H, Paiva J, Chaumeil P, Frigerio JM, Brach J, Barré A, de Daruvar A, Claverol S, Bonneu M, Sommerer N, Negroni L, Plomion C: The proteome of maritime pine wood forming tissue. *Proteomics* 2005, 5:3731-3751.

5. Grattapaglia D, Kirst M: Eucalyptus applied genomics: from gene sequences to breeding tools. *New Phytol* 2008, 179:911-929.

6. Silva JC: Eucalipto: a madeira do futuro. *Revista da Madeira* Curitiba 2001, 59:114.

7. Baucher M, Halpin C, Petit-Conil M, Boerjan W: Lignin: genetic engineering and impact on pulping. *Crit Rev Biochem Mol Biol* 2003, 38:305-350.

8. Chen W, Yun M, Deng F, Yogo Y: The rates of maize growth and lignin biosynthesis change after root-applied chalcone. *Weed Biology and Management* 2005, 5:118-122.

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

10. 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.

11. Vierling E: The roles of heat shock proteins in plants. *Annu. Rev. Plant Physiol. Plant Mol. Biol* 1991, 42:579-620.

12. Hartl FU: Molecular chaperones in cellular protein folding. *Nature* 1996, 381:571-580.

13. Gava LM, Ramos CHI: Human 90kDa heat shock protein Hsp90 as a target for cancer therapeutics. *2009*, 3:10-21.

14. Guo H, Li L, Ye H, Yu X, Algreen A, Yin Y: Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana. *PNAS* 2009, 106(18):7648-7653.

P110

Master regulators of wood formation in *Eucalyptus*

Hua Wang¹, Marcal Soler¹, Hong Yu¹, Eduardo Leal Oliveira Camargo², Heléne San Clemente¹, Bruno Savelli¹, Nathalie Ladouce¹, Jorge Paiva³, Jacqueline Grima-Pettenati¹*

¹LRSV, UMR 5546 Université Toulouse III /CNRS, BP 42617, 31326 Castanet-Tolosan, France; ²LRSV, UMR 5546 Université Toulouse III /CNRS, BP 42617, 31326 Castanet-Tolosan, France//LGE, IB, UNICAMP, Campinas, Sao Paulo, Brazil;

³ICT/MCTES - Palácio Burnay - Rua da Junqueira, 30, 1349-007 Lisboa, Portugal
E-mail: grima@lrsv.ups-tlse.fr

BMC Proceedings 2011, 5(Suppl 7):P110

With the current global focus on bioenergy, forest plantations are increasingly becoming important sources for second generation biofuel, where the whole plant lignocellulosic biomass is to be mobilized. The lignocellulosic biomass is mainly composed of secondary walls (SW) possessing unique characteristics (biochemical composition and tridimensional association of polymers), which govern the intrinsic properties of wood. They especially contain high amounts of lignins, hydrophobic phenolic polymers which constitute an obstacle to the optimal utilization of plant species in paper industry and for saccharification prior to bioethanol production. Among perennial species, *Eucalyptus* species grow very fast and produce high yields of lignocellulosic biomass. They represent the main industrial plantations in the worldland one of the most appealing lignocellulosic feedstock for bioenergy production. Dissection of the molecular switches controlling the coordinated lignin biosynthetic genes is therefore of utmost importance to understand the molecular mechanisms underlying tissue specific deposition of lignin and be able to improve secondary cell wall properties.

With the objective of improving Eucalyptus wood quality to better-fit industrial applications, we are focusing our efforts towards the identification and functional characterisation of regulatory genes controlling the biosynthesis of the cell wall polymers (mainly lignins).

We performed a precise mapping and functional characterization of the cis-regulatory elements contained in the promoters of two genes encoding key and consecutive steps of the lignin biosynthetic pathway i.e. Cinnamoyl CoA reductase (CCR) and Cinnamyl Alcohol dehydrogenase (CAD) (Rahantamala *et al*, 2010). Our results supported a major role for the MYB transcription factors (TF) consensus sites in the control of the coordinated expression of these two genes. The functional analysis of two MYB factors (EgMYB1 and EgMYB2) preferentially expressed in *Eucalyptus* xylem revealed that they are able to bind specifically to these promoters and regulate transcription *in vivo*. EgMYB1 behaves as a repressor whereas EgMYB2 is an activator (Goicoechea *et al*, 2005, Legay *et al*, 2010) of the lignin biosynthetic genes but also of the secondary wall biosynthesis. Indeed, both MYBs were shown recently to be master genes regulating the entire secondary wall biosynthetic program including cellulose, xylan and lignin genes (Zhong *et al*, 2010; Legay *et al* 2010). The presence of both positive and negative regulators in *Eucalyptus* xylem offers the possibility of a combinatorial control of gene expression that could provide the necessary flexibility to ensure tight temporal and spatial regulation of lignin biosynthesis or secondary cell wall.

To address this question and get a deeper insight the complex regulation of the SW formation in *Eucalyptus*, we are now studying the regulation of these two MYBs including fine spatial and temporal expression,
identification of their direct targets genes and of their protein partners. We have constructed a yeast-two-hybrid library from *Eucalyptus* xylem that will also be instrumental for deciphering the interactants of landmark genes for the International *Eucalyptus* community. Thanks to the recent release of the *E. grandis* genome (*Eucalyptus grandis* Genome Project 2010, http://www.phytozome.net/eucalyptus), we have performed a genome-wide survey of the large R2R3-MYB superfamily. The phylogenetic comparison of this family with *Arabidopsis*, rice, poplar and grapevine showed a marked expansion of some clusters putatively involved in wood-related processes. Some R2R3 MYB genes seem to be specific of woody plants. The spatiotemporal expression patterns of members of such clusters are currently being studied. Although Auxin is known as a key regulator of cambium activity and wood formation, the Auxin response mediators [Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors] extensively characterized in model plants, are still largely uncharacterized in tree species. We have identified 23 *Aux/IAA* and 17 *ARF* in the *E. grandis* genome. Comparative phylogenetic analysis revealed that several *Aux/IAA* and *ARF* subgroups have differentially expanded or contracted amongst the three dicotyledonous plants studied (*Arabidopsis*, *Populus* and *Eucalyptus*). Expression analysis and EST database surveys are currently underway to explore the transcript levels of each member in the different organs and tissues of *Eucalyptus* at key developmental stages as well as in response to hormonal treatments and to environmental stresses. Further functional genomics studies conducted on new candidate transcription factors, their regulation under the developmental or environmental stimuli will help identifying major factors underpinning the physicochemical properties of cell walls, the recalcitrance of which remains a key scientific challenge for establishing highly efficient, sustainably produced, second-generation biofuels.

## P111
### Partial suppression of a strongly expressed tonoplast sucrose transporter affects water use and carbon partitioning in Populus

Scott Harding¹, Christopher Frost, Raja Payyavula, Kate HC Tay, Chung-Jui Tsai
University of Georgia, USA

E-mail: sharding@uga.edu

BMC Proceedings 2011, 5(Suppl 7):P111

Sucrose export from source organs, and its subsequent distribution among differentiating organs in wood-forming stems and elsewhere depends on the activity of sucrose transporters (SUTs). There are no comprehensive reports on SUT function in temperate tree species valued for their lignocellulosic biomass. To begin to address this gap, the SUT gene family was characterized and functionally analyzed in transgenic *P. tremula* x *alba*. The *Populus* SUT family features the three major groups characteristic of other dicots. In general, functionally distinct SUTs fall into different phylogenetic groups. Group-1 *PtaSUT3* transcripts localize to leaf vascular traces and stem developing xylem; Group-4 *PtaSUT4* to leaf spongy mesophyll, stem developing xylem, cambium and phloem; Group-2 *PtaSUT5/6* to all leaf cells, stem developing xylem and phloem fibers. The SUT4 ortholog of *Populus* differs from that of other model plants in encoding a vacuolar transporter that is unusually well expressed in source leaves compared to Group-1 and 2 SUT genes. SUT4-RNAi transgenic plants demonstrated a shift of biomass allocation from stem to leaf in both nitrogen (N)-replete and N-limited plants. In those plants, sucrose exhibited a complex pattern of hyper-accumulation in exporting leaves and vascular tissues of the stem, and decreased accrual in the shoot tip and sink leaves. RNAi silencing of SUT4 reduced water uptake during drought simulation without significantly affecting overall shoot biomass accumulation.

## P112
### Time influence in the enzymatic saccharification of cellulose pulp samples

Cristiane V Helm¹*, Washington L E Magalhães¹, Edson A de Lima¹,
Patrícia R Silva¹, Kleber Hoffmann², Amanda Higa², Dayanne Mendes²
¹Embrapa Forestry, Brazilian Agricultural Research Corporation (Embrapa),
Colombo, Paraná, 83411-000, Brazil; ²Federal University of Paraná, Curitiba,
Paraná, 80060-000, Brazil

E-mail: cristiane@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P112

**Background:** With growing concern in obtaining clean and renewable energy, many works in bioenergy has been developed in order to obtain substantial gains for the environment.

Due to the large environmental impact associated to the use of fossil fuels, ethanol production from lignocellulosic materia lhas been widely explored, aiming environmental, economic and social benefits.

Lignocellulosic biomass, composed primarily of cellulose, hemicellulose, and lignin, can be submitted to enzymatic hydrolysis in order to produce reducing sugars, which in turn can be converted into ethanol. However, for the whole process to become economically feasible, optimization of the conditions is necessary [1].

To find innovative solutions for the use of lignocellulosic wastes, the aim of this study was to evaluate the ability of converting cellulose bleached pulp into reducing sugars from enzymatic hydrolysis, at different exposure times.

**Methods:** Cellulose pulp samples were weighed in the amount of 0.2 g and transferred to Falcon tubes, in which were added 40 mL of 50 mM sodium citrate buffer with pH 4.8. For hydrolysis, 0.05 mL of cellulase (Celluclast 1.5 L) and 0.05 mL of cellobiase (Novozyme 188) enzymes were transferred to each tube. The Falcon tubes were incubated in shaker at 50±1 °C and 250 rpm for 24, 48, and 72 hours. Subsequently, reducing sugars were quantified through the dinitrosalicylic acid (DNS) method [2].

**Results and conclusions:** The results were very similar for all incubation times. The average reducing sugar concentrations were 4.15 g/L, 4.14 g/L and 4.16 g/L, for 24, 48, and 72 hours, respectively. The composition of substrate was: 98% cellulose, 1.7% humidity and 0.3% ashes. Cellulose conversion into sugar was calculated considering that cellulose was converted mainly into glucose, in a proportion of 1.11 g glucose/g cellulose. The conversion percentages obtained were: 76.3%, 76.1% and 76.5%, for 24, 48 and 72 hours, respectively. These results show that the hydrolysis incubation time higher than 24 hours does not improve the conversion of cellulose into glucose. One possible explanation would be the fact that the enzymes mainly hydrolyze amorphous or less ordered crystalline cellulose. Therefore, remains just highly crystalline cellulose molecules, hindering the access of the enzyme. In other words, this highly crystalline cellulose prevents the total conversion, even over long periods of time. X-ray diffraction (XDR) measurements will be done to check this hypothesis.

**Acknowledgements:** We would like to gratefully acknowledge the Brazilian agency CNPq for the generous provision of a research fellowship to WLEM, and Embrapa Forestry for supporting this research project.

### References

1. Hamelinck C, Hooijdonk G, Faaij A: Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term. *Biomass and Bioenergy* 2005, 28:384-410.

2. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. *Analytical Biochemistry* New York 1959, 31:426-428.

3. Wood TM, Garcia-Campayo V: Enzymology of cellulose degradation. *Biodegradation* 1990, 1:147-161.

## P113
### The EnergyPoplar project

Magnus Hertzberg
SweTree Technologies AB, Umeå, 904 03, Sweden
E-mail: Magnus.Hertzberg@SweTree.com
BMC Proceedings 2011, 5(Suppl 7):P113

Energy Poplar (Enhancing Poplar Traits for Energy Applications) is an EC Seventh Framework Programme project aimed at further improving poplar trees as an energy crop. The work is directed to understand and improve traits such as yield and wood properties coupled to Bioethanol production. The project also addresses environmental and economical sustainability questions.

The final goal of ENERGYPOPLAR is to develop poplar as a bioenergy short rotation coppice crop, suitable for large-scale deployment in Europe in areas unlikely to be used for food agricultural production. All will be placed in an environmental framework to ensure environmental sustainability with respect to land use, inputs and soil status.

World primary energy consumption increased by 2.4% in 2007. With the worlds growing energy consumption, the development and use of renewable, sustainable liquid bio fuels has become a strategic priority for
the EU. Bio fuels can minimize energy import dependence, reduce greenhouse gas emissions and assist rural and agricultural development. Bio ethanol can be produced from energy crops that do not compete with food crops for land use and can be directly used by current transportation vehicles. This alcohol can be produced from biomass feedstock and in particular from cellulose, a sugar based polymer present in the cell wall of plants. Such crops are known as 'second generation' bio fuel crops.

To develop a new bio ethanol industry competitive with fossil fuels, the quality of the biomass feedstock, the methods to produce ethanol from cellulose and the yield per hectare must be improved. Energy trees must also support an environmentally sustainable agriculture that uses less agrochemicals, develops rural economies and spares natural forests from agricultural expansion.

Poplar is an economically and ecologically attractive energy crop because it displays a wide range of growth habits and can grow on marginal lands unsuited for food crops with reduced input costs and optimized land management. Additionally, poplar is a commercial crop and the model organism for hardwood tree genomics and physiology.
http://WWW.Energypoplar.eu for more information.

P114

The role of SND2 in the regulation of *Arabidopsis* fibre secondary cell wall formation

Steven Hussey¹*, Eshchar Mizrachi¹, David Berger², Alexander Myburg¹

¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; ²Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa

E-mail: steven.hussey@fabi.up.ac.za

BMC Proceedings 2011, 5(Suppl 7):P114

**Background:** Transcription factors (TFs) play important roles in the regulation of secondary cell wall (SCW) biosynthesis in herbaceous and woody plants. In *Arabidopsis*, the onset of SCW deposition is initiated by a nexus of NAC, MYB, homeodomain and several other families of TFs, which function in a transcriptional network regulating SCW biosynthetic genes. NAC family members SND1/NST1 and VND6/VND7 have been identified as functionally redundant master regulators of SCW formation in fibres and vessels, respectively [1,2]. *Arabidopsis* plants overexpressing SND2, an indirect target of fibre master regulator SND1, exhibited increased SCW thickness in inflorescence stem fibres, whilst dominant repression lines exhibited a decrease in fibre SCW thickness associated with a reduction in glucose and xylose cell wall sugar content [3]. The ability of SND2 to transactivate the CesA8 promoter [3] suggested that SND2 may regulate cellulose biosynthetic genes during fibre SCW formation. The evaluation of this hypothesis necessitates the identification of all downstream genes potentially regulated by SND2, and the analysis of SCW chemistry and morphology in overexpression lines. The aim of this ongoing study is to further elucidate the role of SND2 in fibre SCW formation through microarray analysis of over-expression lines and by independent confirmation of the effect of SND2 overexpression on fibre SCW chemistry and morphology in *Arabidopsis* plants.

**Methods:** We generated 2x35S::SND2 *Arabidopsis thaliana* Col-0 plants, screened them for CesA8 upregulation and phenotypically assessed several homozygous (T4) transgenic lines. Inflorescence stem fibre SCW thickness was measured from light and scanning electron micrographs, and the cell wall monosaccharide and Klason lignin composition of stems was determined relative to the wild type. We performed microarray analysis of the inflorescence stem transcriptomes of wild type and transgenic *Arabidopsis* plants using the Agilent 4x44k transcriptome array and confirmed the expression profiles of differentially expressed genes with RT-qPCR.

**Results:** Transgenic *Arabidopsis* (T4) lines showed no significant external phenotype, and we were unable to reproduce the increased fibre SCW thickness phenotype reported by Zhong *et al.*[3]. We identified a single homozygous line with CesA8 upregulation and moderate SND2 overexpression. Whole-transcriptome analysis of the line revealed the upregulation of several TFs and genes associated with SCW biosynthesis, which were reproducibly upregulated in an

independent trial. We additionally observed possible ectopic artifacts and gene dosage effects associated with excessive constitutive expression of the SND2 gene. Chemical analysis revealed only minor changes in SCW monosaccharides, despite the upregulation of SCW biosynthetic genes.

**Conclusions:** Our results implicate SND2 in the regulation of cellulosic and non-cellulosic components of fibre cell walls, and we provide a model for the position of SND2 in the transcriptional network regulating fibre SCW formation.

**References**

1. Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T: Transcription switches for protoxylem and metaxylem vessel formation. *Genes and Development* 2005, 19:1855-1860.

2. Zhong R, Richardson EA, Ye Z-H: Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of *Arabidopsis*. *Planta* 2007, 225:1603-1611.

3. Zhong R, Lee C, Zhou J, McCarthy RL, Ye Z-H: A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in *Arabidopsis*. *The Plant Cell* 2008, 20:2763-2782.

P115

Wood quality-related gene expressions of *Eucalyptus globulus* grown in a greenhouse

Negishi Naoki, Kazuya Nanto, Kazunori Hayashi, Shinichi Onogi, Akiyoshi Kawaoka²

Agrи-Biotechnology Research Laboratory, Nippon Paper Industries Co.Ltd
E-mail: akawaoka@np-g.com

BMC Proceedings 2011, 5(Suppl 7):P115

Eucalyptus species constitute the most widely planted hardwood trees in temperate and subtropical regions. Their wood is used as a raw material for the production of cellulose. Eucalyptus species have fast growth rates and the ability to adapt to a broad range of geographic locations. Most importantly, Eucalyptus has been listed as one of the candidate biomass energy crops [1]. *Eucalyptus globulus* is the main hardwood species grown in pulpwood plantations in temperate regions of the world.

In this study, we focused on four kinds of key genes, 4-coumarate-CoA ligase (4CL), LIM domain transcription factor (LIM) [2], coniferaldehyde 5-hydroxylase (CAld5H) and the three catalytic units of cellulose synthase (CesA1, CesA2 and CesA3) influencing wood quality. We investigated correlation between relative expression levels of these genes and wood qualities.

We have cloned the genes encoding LIM, 4CL, CAld5H and the cellulose synthase (CesA1, CesA2 and CesA3) from *E. globulus* by the method of cDNA library screening (Fig.1). A cDNA library was constructed using mRNA purified from stems of four-month old *E. globulus* grown in the greenhouse. The expression levels of LIM in basal stems of ten independent *E. globulus* lines showed similar patterns to those of 4CL, indicating that the LIM may control 4CL expression. We investigated the correlation between gene expression levels and wood qualities such as Klason lignin (KL) content, syringyl/guaiacyl (S/G) ratio and holocellulose (HC) content. Expression of the LIM and 4CL were positively correlated with KL content. A highly significant positive correlation was observed between CAld5H expression and S/G ratio. Furthermore, a ratio of the sum of the expression levels of three CesA1, CesA2 and CesA3 to 4CL showed positive correlation with a ratio of HC/KL content that positively correlated to the chemically extracted fiber content in this woody plant. Overall, our results provide a strong foundation for manipulating candidate genes such as LIM, 4CL, CAld5H and CesA towards the production of desirable wood qualities in an extremely important biomass plant, the Eucalyptus species.

**Acknowledgements:** We thank Toshiyuki Takano of Kyoto University, Jacqueline Grima-Pettenati of UMR CNRS-UPS, Philip Uren and Kenji Yoshinaga of BTP for technical support.

**References**

1. Li X, Weng J-K, Chapple C: Improvement of biomass through lignin modification. *Plant J* 2008, 54:569-581.

2. Kawaoka A, Nanto K, Ishii K, Ebinuma H: Reduction of lignin content by suppression of expression of the LIM domain transcription factor in Eucalyptus camaldulensis. *Silvae Genet* 2006, 55:269-277.
P116

Pre-treatment of eucalypts biomass towards enzymatic saccharification

Washington Luiz Magalhães¹*, Cristiane Helm¹, Patricia Silva¹, Edson Lima¹, Kleber Hoffman², Amanda Higa², Tielidy Lima²

¹Embrapa Forestry, Brazil; ²UFPR, Brazil

E-mail: wmagalha@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P116

**Background:** There are a few possible ways to produce ethanol from lignocellulosic biomass, for instance, thermochemical and acid hydrolysis. However, enzymatic hydrolysis of carbohydrates is considered the greenest process for saccharification, followed by sugar fermentation into ethanol.

The main challenge of the enzymatic saccharification process is that cellulose is not exposed to the enzyme action in the lignin matrix. The cellulose molecules are arranged in semi-crystalline nanofibrils immersed in lignin matrix with hemicelluloses (polyoses) and extractives between them acting as coupling agents. These nanofibrils are placed together to form helical microfibrils inside the cell wall. Thus, a pre-treatment is necessary to make room for the enzymes to reach the cellulose fibril surfaces in order for the whole process to become economically feasible. There are many pre-treatments proposed in specialized literature, but their efficiencies are dependent on the biomass composition [1-4]. Moreover, these treatments have to address some constraints such as the recyclability of the chemicals used, low consumption of energy, and sustainability concerns.

We devised a future possibility of a cellulose pulp mill to be transformed into a biorefinary, where besides cellulose pulp, ethanol could also be produced. In the Brazilian pulp mill industry, the process most commonly used is the Kraft process, so the digestion with green and white liquors can be adapted for pre-treatment towards enzymatic saccharification. Also, the industry had already tackled recycling of the black liquor – obtained after wood chips digestion –, recuperating thermal energy by burning lignin and recovering the green liquor.

This work is part of our research to evaluate some modifications on the green liquor digestion towards enzymatic saccharification. We evaluated efficiencies of some pre-treatments with green liquor through enzymatic hydrolysis for holocellulose saccharification.

**Methods:** A wood log from a 7-year old tree of *Eucalyptus benthamii* was cut into discs. They were cut into 30° wedges and were then grounded using a Willey mill and sifted to be between 20 and 40 Tyler mesh.

Composition of the *E. benthamii* wood obtained by NBR standards was: 3% of total extractives; 28.4% of Klason lignin; 0.03% of ash and 3.5 of syringyl:guaiacyl molar ratio.

Table 1 shows the characterization of the green liquor acquired from Iguaçu Celulose Papel S.A.

The pre-treatment conditions used in this study were two levels of temperature – 120 and 180 °C – and three levels of reaction time – 1, 2, and 3 hours. Cylindrical stainless steel reactors (Parr, USA) with volume capacity of 50 mL were used. The reactors were heated in an aluminum dry block furnace (Marconi, Brazil) with a temperature and time controller. The ratio of solid wood and green liquor was 1:4 (g.mL⁻¹). Typical values were 4 g of dry biomass in 16 mL of green liquor per reactor.

The pre-treated biomass was digested using Falcon tubes with enzymes in a buffer solution of sodium citrate (pH of 5.0) in an incubator (Tecnal TE-420, Brazil) at 50 °C and 250 rpm during 24 hours. The enzymes used were cellobiase from *Aspergillus niger* (Novozyme 188) and cellulase from *Trichoderma reesi* (ATCC 26921), acquired from Sigma Aldrich, USA. Typically, the proportion was 0.2 g of dry biomass, 40 ml of buffer solution, 46 mL of cellobiase and 90 mL of cellulase (both used as received). After saccharification, the hydrolyzate was filtered and the reducing sugars were quantified through the dinitrosalicylic acid (DNS) method.

**Results:** Table 2 shows the solid mass loss after treatment, the total reducing sugar after enzymatic hydrolysis, and the global yield of saccharification.

As can readily be seen on Table 2, temperature plays a crucial role in exposing cellulose for enzymatic hydrolysis, as does the length of cooking. For lowest temperatures, (120 °C) increasing time causes increased sugar release and conversion yield. On the other hand, for the highest temperature level (180 °C), the time of cooking plays a more complex role. The treatment at 180 °C after 1 hour reaches the highest sugar conversion based on holocellulose content of untreated biomass.

The above results can be explained by the following suppositions. Increasing treatment time to 2 and 3 hours results in hemicellulose and amorphous cellulose losses, which decreased conversions yield. However,

<table><caption>Table 1(abstract P116) Characterization of the industrial green liquor used in this work</caption><thead><tr><th>Analysis of green liquor</th><th>Content (g.L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Content of sodium sulfide (X) in Na<sub>2</sub>S</td><td>14.59</td></tr><tr><td>Content of sodium sulfite (Y) in Na<sub>2</sub>SO<sub>3</sub></td><td>13.9</td></tr><tr><td>Content of sodium thiosulfate (Z) in Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub></td><td>3.16</td></tr><tr><td>Total alkali (concentration of alkaline constituents: OH<sup>-</sup>+HS<sup>-</sup>+HCO<sub>3</sub><sup>-</sup>) expressed in NaOH</td><td>114.1</td></tr><tr><td>Active alkali (sum of concentrations of hydroxyl and hydro-sulfite ions including ions formed by hydrolysis of sulfides: OH<sup>-</sup>+HS<sup>-</sup>) expressed in NaOH</td><td>43.3</td></tr><tr><td>Effective alkali (concentration of hydroxyl ions including those formed from sulfides by hydrolysis: OH<sup>-</sup>) expressed in NaOH</td><td>32.5</td></tr><tr><td>Sulfidity</td><td>33.7%</td></tr></tbody></table>

<table><caption>Table 2(abstract P116) Mass loss after pre-treatment, total reducing sugar released after enzymatic digestion, and the yield of conversion based on holocellulose content in the untreated biomass</caption><thead><tr><th>Treatment</th><th>Mass loss (%)</th><th>Total reducing sugar (kg.kg<sup>-1</sup> of treated biomass)</th><th>Yield of sugar conversion based on holocellulose of untreated biomass (%)</th></tr></thead><tbody><tr><td>untreated</td><td>0.0</td><td>0.04</td><td>5.6</td></tr><tr><td>120°C, 1h</td><td>26.5</td><td>0.10</td><td>10.6</td></tr><tr><td>120°C, 2h</td><td>26.4</td><td>0.13</td><td>14.1</td></tr><tr><td>120°C, 3h</td><td>34.3</td><td>0.16</td><td>15.6</td></tr><tr><td>180°C, 1h</td><td>47.3</td><td>0.92</td><td>70.7</td></tr><tr><td>180°C, 2h</td><td>52.5</td><td>0.78</td><td>53.9</td></tr><tr><td>180°C, 3h</td><td>50.8</td><td>0.91</td><td>65.7</td></tr></tbody></table>
when the treatment time was increased to 3 hours, besides amorphous holocellulose loss, there was also the occurrence of crystalline modification of part of the crystalline cellulose type I into amorphous and cellulose type II. Both amorphous and crystalline type II cellulose are enzymatic digested easier than crystalline type I cellulose. This could explain why sugar conversion yield after 2 hours of treatment at 180 °C are smaller than after 3 hours. XRD and chemical characterizations of the pre-treated biomass have to be done in order to corroborate with the above suppositions.

**Conclusion:** Simple modifications of the Kraft process can be considered as an alternative route for pre-treatment of woody biomass towards enzymatic saccharification.

**Acknowledgements:** We would like to gratefully acknowledge the Brazilian agency CNPq for the generous provision of a research fellowship to WLEM and also the English language revision by Miss Bia Carneiro.

### References

1. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY: Coordinated development of leading biomass pretreatment technologies. *Bioresour Technol* 2005, **96**:1959-1966.

2. Perez JA, Ballesteros I, Ballesteros M, Saez F, Negro M, Manzanares P: Optimizing liquid hot water pretreatment conditions to enhance sugar recovery from wheat straw or fuel-ethanol production. *Fuel* 2008, **87**:3640-3647.

3. Monavari S, Galbe M, Zacchi G: Impact of impregnation time and chip size on sugar yield in pretreatment of softwood for ethanol production. *Bioresour Technol* 2009, **100**:6312-6316.

4. Virkajärv I, Niemelä MV, Hasanen A, Teir A: Cellulosic ethanol via biochemical processing poses a challenge for developers and implementors. *BioResources* 2009, **4**:1718-1735.

## P117

### Evaluation of the enzymatic digestibility of paper industry byproducts

Patrícia Silva¹, Washington Luiz Magalhães¹, Cristiane Helm¹*, Edson Lima¹, Dayanne Mendes², Tielidy Lima²

¹Embrapa Forestry, Brazil; ²Federal University of Paraná – UFPR, Brazil

E-mail: cristiane@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P117

**Background:** Researches concerning renewable energy sources and wastes recycling are strategic to ensure energetic and environmental sustainability of the planet. In this scenario, biofuels (including bioethanol) have attracted increasing attention.

Most ethanol is currently produced from sugar- and starch-based feedstocks, such as sugar cane in Brazil and corn in the U.S.A. However, lignocellulosic biomass also presents great potential for being used as an alternative raw material. Unlike ordinary feedstocks, sugars from cellulosic biomass are not readily available for fermentation and conversion to ethanol. A preliminary pretreatment step is necessary in order to remove the lignin that involves the structures of cellulose and hemicellulose, acting as a barrier. In the following step, fermentable sugars can be released by enzymatic hydrolysis of polysaccharides. Finally, monosaccharides can be converted into ethanol by anaerobic fermentation [1,2].

This process allows the use of forest wastes to produce ethanol. Besides adding value to these byproducts, this action could contribute to reducing environmental problems related to waste disposal.

Several solid byproducts from pulp and paper industry have a high conversion potential into ethanol [3-7], since they have high carbohydrate content and susceptibility to hydrolysis process. This is due to the fact they have already undergone a previous treatment of the fibers. One example is the sludge generated in wastewater treatment of paper recycling industries, which is composed primarily of cellulose and ash. Although studies have been conducted focusing recycling and reusing, currently its primary destination has been the disposal in landfills, causing high costs and requiring large storage areas. As this product usually has high content of moisture, the production of wet sludge can reach one ton per ton of paper produced [8]. Therefore, suitable utilization of such waste would result in significant economic benefits to the pulp and paper sector.

The aim of this study was to evaluate the enzymatic digestibility of byproducts generated by paper industry, including samples of recycled paper sludge and bleached and unbleached pulps.

**Methods:** The "recycled paper sludge" was a pressed solid material resulting from the wastewater treatment process of a paper recycling mill. Two samples obtained from Kraft pulping process were also used: an unbleached pulp and a bleached one. All materials were ground in a blender before use. Water and ash contents were determined gravimetrically, according to international standards.

Samples were suspended in 0.05 M sodium citrate buffer, pH 4.8, to obtain an initial solids concentration of about 5 g.L⁻¹ (dry basis). A mixture of two commercial enzyme preparations was added: cellulase 2.25 mL.L⁻¹ (Celluclast 1.5 L), and cellobiase 1.13 mL.L⁻¹ (Novozyme 188). Samples were incubated at 50 °C in an orbital shaker at 250 rpm for 24 h. This hydrolysis time was selected based on a recent study that has demonstrated little effect of time on the process yield over 24 hours [9]. Tests were performed in triplicate for each sample and the concentrations of reducing sugars were estimated by the dinitrosalicylic acid method.

**Results and conclusions:** Recycled paper sludge presented high contents of moisture (66.2%) and ash (20.6%, wet basis). Inorganic fraction probably includes additives such as kaolin, calcium carbonate and titanium dioxide, as well as metals from printing inks. Moisture and ash fractions were around 7.0% and 0.3% for bleached pulp and 22.5% and 1.1% for unbleached pulp.

Cellulose conversions estimated from the hydrolysis trials were: 82.1% for the bleached pulp, 69.5% for the sludge and 27.2% for the unbleached pulp. As expected, the sample that was not subjected to the bleaching process showed lower susceptibility to hydrolysis, probably due to its higher residual lignin content that hinders the enzyme action. The other materials showed relatively high saccharification efficiencies because they have already been "pretreated" during the production process itself. Although paper sludge have presented lower conversion than the bleached pulp, the presence of inorganic compounds seemed to cause no significant inhibition of the saccharification process.

Therefore, the present study suggests the technical feasibility of using the materials tested as a source of monosaccharides for ethanol production, without requiring an additional step of pretreatment. Special interest is focused in the use of sludge from the paper recycling process, adding value to this byproduct and minimizing environmental impacts.

**Acknowledgements:** We would like to gratefully acknowledge: the Brazilian agency CNPq for the generous provision of a research fellowship to WLEM, and Embrapa Forestry for supporting this research project.

### References

1. Hamelinck C, Hooijdonk G, Faaij A: Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term. *Biomass and Bioenergy* 2005, **28**:384-410.

2. Taherzadeh MJ, Karimi K: Enzyme-based hydrolysis processes for ethanol from lignocellulosic materials: a review. *BioResources* 2007, **2**:707-738.

3. Lark N, Xia Y, Qin C, Gong C: Production of ethanol from recycled paper sludge using cellulase and yeast, *Kluveromyces marxianus*. *Biomass and Bioenergy* 1997, **12**:135-143.

4. Ballesteros M, Oliva JM, Manzanares P, Negro MJ, Ballesteros I: Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis. *World J Microbiol Biotechnol* 2002, **18**:559-561.

5. Fan Z, South C, Lyford K, Munsie J, Walsum PVan, Lynd LR: Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor. *Bioprocess Biosyst Eng* 2003, **26**:93-101.

6. Marques S, Alves L, Roseiro J, Girio F: Conversion of recycled paper sludge to ethanol by SHF and SSF using *Pichia stipitis*. *Biomass and Bioenergy* 2008, **32**:400-406.

7. Zhang J, Lynd LR: Ethanol production from paper sludge by simultaneous saccharification and co-fermentation using recombinant xylose-fermenting microorganisms. *Biotechnol Bioeng* 2010, **107**:235-44.

8. Foelkel C: Resíduos Sólidos Industriais do Processo de Fabricação de Celulose e Papel de Eucalipto. Parte 03: Lodos & Lodos. *Eucalyptus OnLine Book*; 2010, 1-191[http://www.eucalyptus.com.br/eucaliptos/PT20_LODOS.pdf].

9. Helm CV, Magalhães WLE, Lima EA, Silva PR, Hoffmann K, Higa A, Mendes D: Time influence in the enzymatic saccharification of cellulose pulp samples (submitted paper). *UFRO Tree Biotechnology* 2011.
P118

mRNA sequencing of *Eucalyptus urograndis* trees supplemented with flavonoids shows changes on metabolic process and decrease of lignification

Jorge Lepikson-Neto¹*, Leandro Nascimento¹, Maria Carolina Scatollin¹, Wesley Marques¹, Marcela Salazar¹, Eduardo Camargo¹, Ramon Vidal², Danieli Gonçalves¹, Goncalo Pereira¹

¹Laboratório de Genômica e Proteômica State University of Campinas- UNICAMP, Brazil; ²Laboratório Nacional de Biociências – CNPEM/ABTLuS, Brazil

E-mail: lepiksonneto@gmail.com

*BMC Proceedings* 2011, **5**(Suppl 7):P118

**Background:** The flavonoids, naringenin-chalcone and narigenin, are intermediates in phenylpropanoid metabolism in plants, occupying the central position as primary intermediates in flavonoid biosynthesis and are synthesized by *chalcone synthase* (CHS) and *chalcone isomerase* (CHI) respectively.[1] It has been reported that supplementation of narigenin-chalcone and narigenin can inhibit the activity of 4CL in vitro, and suppress the growth and reduce lignin content in gramineous plants, while 4CL suppression affected plant phenotype and resulted on dwarfed trees on *Pinus* [2], and its down-regulation promoted enhanced growth phenotypes on transgenic aspens trees [3], also CHS expression controls flavonoid synthesis and reduced size phenotype on arabidopsis. [4] Eucalyptus is the main source of biomass for pulp and paper industries therefore it's imperative to study the influence of flavonoid supplementation on Eucalyptus and what kind of overall impact it can have on plant development, especially wood formation and gene expression.

**Methods:** Plant Material: 1 month *Eucalyptus* plantlets were divided into 3 groups with 25-30 individuals per group. Groups were given normal root induced nutritive solutions (Group 1) and added narigenin-chalcone (group 2) and narigenin (group 3). Treatment lasted 5 months with regular supplementation. Samples were then collected and properly stored.

**Histochemical analyses:** Stem samples were harvested and fixed in FAA for at least 24h. Sections were double stained with 1% alcoholic Safranin O and 1% aqueous Astra Blue. Hand sectioned tissue samples from the same material were stained with Mâule reagent: 1% KMnO4 for 15 min, 2% HCl for 5 min, and 2N NH4OH solution. Sections were observed with an Olympus BX51 microscope under white light, and the images were obtained with DP-72 digital camera and Image Pro Plus 6.3 software.

**mRNA Sequencing:** RNA was extracted from xylem tissue according to [5] and mRNA libraries were prepared following instructions from the Illumina mRNA-Seq Prep Kit. Three distinct libraries were generated: *E.urograndis* plantets supplemented with narigenin-chalcone; narigenin; and control solution, containing 34,157,958 (65,75% mapped); 33,743,449 (64,80% mapped); 32,076,198 (65,09% mapped) reads, (reads size approximately 36pb).

**Assembling and annotation:** We assembled 167,271 ESTs (130,290 from Genolyptus and 36,981 from NCBI) from several species and tissues using the program CAP3. [6] The assembly produced 53,412 unigenes (18,098 contigs and 35,314 singlets).

All unigenes were automatic annotated using the BLAST [7], including: non-redundant (NR) database of NCBI, pfam [8]. Moreover, we performed a functional annotation using the BLAST2GO software. [9]

The RNA-Seq reads were aligned against the assembled unigenes using the SOAP2 aligner [10] In order to perform the differential expression analysis between libraries, a normalization and statistical pipeline were applied using DEG-seq software [11] considering 99% of confidence rate (cut-off of 0.01).

**Results and discussion:** Dual staining with Astra-blue and safranin-O histochemical analysis show, that both groups of plants treated with flavonoids exhibit a much greater blue stain, typical of celluloses content, when compared to control plants, while exhibiting a lesser amount of the red/purple stain related to phenolics compounds. The Mâule color reaction which provides an effective method for the detection of syringyl units, show an increase of S lignin content on both group of plants supplemented with narigenin and narigenin-chalcone. mRNA sequencing shows drastic changes on genes related with flavonoid sinhtesys, annotated contigs for those genes presented a much lower expression than control groups. Almost all

genes from the phenylpropanoidpathway presented lower expression on treated groups, with genes at the beginning of the pathway presenting the most noteworthy reduction, while genes at the end of the pathway presented higher expression on treated groups which is interesting as it can suggest a shift from guaiacyl to syringyl monolignols increasing lignin solubility. Gene Onthology annotation shows significant differences among a number of metabolic process, including cell growth, cell division and response to stress. Among the most expressed genes were found heat-shock proteins, dehydrins and many no-hits. It is clear that supplementation with flavonoids alter gene expression especially regarding lignin biosynthesis.

References

1. Boland M, Wong E: Purification and kinetic properties of chalcone-flavanone isomerase from soya bean. *Eur J Biochem* 1975, 50(2):383-389.

2. Wagner A, Ralph J, Akiyama T, Flint H, Phillips L, Torr K, Nanayakkara N, Te Kiri L: Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in *Pinus radiata*. *Proc Natl Acad Sci USA* 2007, 104(28):11856-61.

3. Hu WJ, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai CJ, Chiang VL: Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. *Nat Biotechnol* 1999, 17:808-812.

4. Besseau S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M: Flavonoid accumulation in *Arabidopsis* repressed in lignin synthesis affects auxin transport and plant growth. *Plant Cell* 2007, 19:148-162.

5. Le Provost G, Herrera R, Paiva J, Chaumeil P, Salin F, Plomion C: A micromethod for high throughput RNA extraction in forest trees. *Biological Research* 2007, 40:291-297.

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

7. 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.

8. 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.

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

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. 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.

P119

The transcriptome of a *Populus* pseudo-backcross identifies genes and pathways co-expressed with monolignol biosynthesis

Evandro Novaes¹*, Carolina Boaventura-Novaes², Alexandre Coelho¹, Matias Kirst³

¹Escola de Agronomia e Engenharia de Alimentos, Universidade Federal de Goiás, Caixa Postal 131, CEP 74690-900, Goiânia, Brazil; ²Programa de Pós-Graduação em Genética e Melhoramento de Plantas, Universidade Federal de Goiás, Caixa Postal 131, CEP 74690-900, Goiânia, Brazil; ³School of Forest Resources and Conservation, University of Florida, PO Box 110401, Gainesville, USA

E-mail: novaes@agro.ufg.br

*BMC Proceedings* 2011, **5**(Suppl 7):P119

**Background:** Lignin is the main impediment for efficient cellulose extraction and ethanol production from woody tissues. Even though, the biosynthesis of the lignin constituents - the monolignols - is well characterized, little is known about the genetic control of the natural variation in lignin content and composition. The few association studies performed to date in forest species only uncover 5-20% of the heritable variation of quantitative traits such as wood composition. The "missing heritability" can be explained in part by the low resolution of these pioneering association studies in forest species. However, as observed in genome-wide association studies with humans, a large proportion of the "missing heritability" is likely to occur due to other factors, such as the
abundance of rare alleles observed in forest species and complex epistatic interaction between genetic elements. Causal rare alleles and genetic interactions remain undetectable with current statistical methods. In order to shed light on possible interactions between lignin biosynthesis genes and other pathways, we analyzed the transcriptome of 181 genotypes of a pseudo-backcross family of *Populus*. The analyses allowed identification of genes and pathways that were highly co-expressed with genes involved in the biosynthesis of monolignols. Correlations at the transcript level offer initial evidence of possible interactions between genetic elements for the production of monolignols.

**Methods:** A previously published microarray data [1] was utilized. Briefly, this microarray contains a gene expression probe for every gene annotated in the genome of *Populus trichocarpa* (v1.1.). Microarray chips were hybridized with cDNA synthesized from xylem tissue of 181 genotypes from a pseudo-backcross pedigree of *Populus trichocarpa* x *P. deltoides*. This family was also analyzed for lignin content on xylem tissue [2]. Genetic differences in gene expression allowed us to correlate the transcript abundance of 23 previously identified xylem-specific monolignol biosynthetic-genes [3] against the cDNA levels of all annotated genes of *Populus*. Correlation was estimated based on Spearman's rank method in R. The top correlated genes (r > 0.75, p-value <0.001) were clustered with the 23 monolignol biosynthetic-genes based on a "Modulated Modularity Clustering" method [4]. The genes clustered with the 23 monolignol biosynthetic-genes were GO annotated based on the BlastP best hit against the *Arabidopsis* gene models. A Fisher's exact test was applied to identify GO terms enriched within these clusters.

**Results:** Utilizing a correlation threshold of 0.75 (p-value < 0.001), 1369 genes were correlated with at least one of the 23 monolignol biosynthetic-genes. These genes were clustered together with the lignin phenotypic trait and 78 genes that were correlated with lignin (r > 0.45). The 23 monolignol genes were clustered in four groups, containing a total of 936 genes (Figure 1). Surprisingly, the lignin trait itself did not cluster with any

other gene, and the vast majority (75%) of the top 78 lignin-correlated genes were grouped on clusters 12 and 15. Clusters 14 and 16 did not have lignin-correlated genes. We analyzed the GO annotation among genes within the four monolignol-related clusters. A Fisher's exact test was utilized to find GO terms enriched within these clusters when compared to all annotated genes in the genome of *Populus*. Sixty-nine GO terms were significantly enriched (FDR < 0.01) among genes clustered with the 23 monolignol genes: 11 terms are from the ontology Cellular Component (CC), 26 from Molecular Function (MF) and 32 from Biological Process (BP). As expected, most of these MF and BP terms (>65%) are related to lignin, cellulose and hemicellulose biogenesis. This high specificity indicates that even the genes with unknown MF and BP (260 of the 936 monolignol clustered genes) might be involved in cell-wall biogenesis. Enriched GO terms not directly related to cell-wall biogenesis include "drought-recovery", "salicylic acid catabolism", "response to cadmium ion" and "response to zinc ion". In addition to the significantly enriched GO terms, we also constructed a co-expression network including the core, most highly correlated genes (r > 0.85) within each of the four identified, monolignol biosynthetic clusters. These networks, representing the most likely interacting genes, will be presented.

**Conclusion:** This work presents a set of approximately 900 genes highly co-expressed with xylem-specific monolignol biosynthesis genes. Many of these genes are currently annotated with unknown function, or are not known to be involved in cell-wall biogenesis. We offer initial evidence towards a role or interaction of these genes in the biosynthesis of lignin and possibly other cell-wall components.

**References**

1. Drost DR, Benedict CI, Berg A, Novaes E, Novaes CRDB, Yu Q, Miles BL, Dervinis C, Maia JM, Yap J, Miles B, Kirst M: Diversification in the genetic architecture of gene expression and transcriptional networks in organ differentiation of *Populus*. *Proceedings of the National Academy of Sciences of the United States of America* 2010, 107(18):8492-8497.

<figure><img src="image_52.png" /></figure>
2. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M: Quantitative genetic analysis of biomass and wood chemistry of *Populus* under different nitrogen levels. *New Phytologist* 2009, **182**(4):878-890.

3. Shi R, Sun YH, Li QZ, Heber S, Sederoff R, Chiang VL: Towards a systems approach for lignin biosynthesis in *Populus trichocarpa*: transcript abundance and specificity of the monolignol biosynthetic genes. *Plant and Cell Physiology* 2010, **51**(1):144-163.

4. Stone EA, Ayroles JF: Modulated Modularity Clustering as an Exploratory Tool for Functional Genomic Inference. *PLoS Genet* 2009, **5**(e1000479.

P120

Regulatory network in lignin biosynthetic pathway through small RNAs in *Acacia mangium*: implications to the pulp and paper industry

Seong Siang Ong*, Ratnam Wickneswari

School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

E-mail: ong.siang@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P120

**Background:** Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component of wood, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is a costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the monolignol biosynthesis pathway genes [1-3]. Non-coding RNAs represent an emerging class of riboregulators, which are processed to shorter miRNAs or siRNAs. The current paradigm indicated that plant system use small RNAs (miRNAs and siRNAs) as guide for post-transcriptional gene silencing and epigenetic regulation. Although miRNAs and siRNAs result from different biogenesis pathways but both interact with target transcripts for direct cleavage or translation repression, effectively shutting down that genes' functions. However, much less is known about the mechanism of gene regulation governed by these small RNAs in lignin biosynthesis pathway in *A. mangium*.

**Methods:** Total RNA was isolated from secondary xylem tissue with contrasting lignin content using mirVana microRNA Isolation Kit (Cat. AM1561, Ambion, Austin, TX, USA) following manufacturer's protocol. Thin cookies were first ground in a blender and then further ground to fine powder using mortar and pestle. Integrity of the isolated Total RNA was analyzed using Bioanalyser 2100 (Agilent Technology, Palo Alto, USA) and only Total RNA with RIN value above 7 was selected for library construction. Library construction, sequencing and bioinformatics pipeline analysis was done by Gene Pool Sequencing Centre, United Kingdom. Small RNA libraries were generated using DGE small RNA Sample Preparation Kit (Cat. # FC-102-1009; Illumina, San Diego, CA, USA). Illumina sequencing libraries were prepared using the 'long' Illumina protocol according to the manufacturer's direction and two libraries were sequenced on an Illumina GA-II following manufacturer's instructions. After masking of adapter sequences and removal of contaminated reads, the clean reads were filtered and the

<table><caption>**Table 1(abstract P120) Total Counts of the four selected highly conserved miRNA families isolated from secondary xylem of low lignin A. mangium 54 (Am54) and high lignin A. mangium 48 (Am48)**</caption><thead><tr><th>miRNA family</th><th>AM54</th><th>Am48</th><th>Predicted Target</th></tr></thead><tbody><tr><td>amg-miR159</td><td>3158</td><td>1008</td><td>MYB Trcription Factor</td></tr><tr><td>amg-miR168</td><td>79040</td><td>33447</td><td>Agronuate</td></tr><tr><td>amg-miR172</td><td>32743</td><td>18204</td><td>APETALA 2-LIKE transcription factor</td></tr><tr><td>amg-miR394</td><td>2037</td><td>226</td><td>F-box</td></tr></tbody></table>

<figure><img src="image_53.png" /><figcaption>**Figure 1(abstract P120)** Relative normalized expression of four selected conserved miRNAs in secondary xylem tissue of *A. mangium* Tension Wood (TW) using real time PCR technology. 5.8S rRNA was selected as references genes.</figcaption></figure>

resulting oligos were totalled. Clustering based on relative lengths from 7 nt to 35 nt were done using in house perl scripts. The targets were extracted by in house python script and annotated using BLAST to GO database. Only highly conserved miRNAs with strong differences in their expression level between high and low lignin secondary xylem were selected for validation using IQ5 real time - PCR technology (BioRad, Hercules, USA) (Table 1).

**Results:** A total of 14,582,383 reads were generated in Am54 and 10,281,313 reads in Am48. We have identified several conserved and novel small RNAs that may serve as an important regulatory role during secondary wall formation. Majority of these small RNAs emerged as critical regulators for normal growth and developmental processes in *A. mangium*. Only a few small RNAs were postulated to play an important role during epigenetic silencing. We found that the expression level of these miRNAs belong to four different families was up regulated in tension wood (Fig. 1). Tension wood is composed almost entirely of cellulose while compression wood is rich in lignin. We will further investigate the effects of over expression of these four highly conserved miRNAs in tension wood on the expression level of monolignol biosynthetic pathway genes.

**Conclusions:** From the sequence results, we concluded that *A. mangium* small RNAs consist of a set of 14 highly conserved miRNAs families found in plant miRNA database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. Out of these 14 highly conserved miRNAs families, only four miRNAs families were selected for validation in compression wood and tension wood and their total relative counts between Am54 and Am48 are shown (Table 1). Although these four miRNAs belong to different families, all of them were up regulated in tension wood, a region composed entirely of cellulose. The results obtained can be used to better understand the roles of small RNAs and for the development of a gene constructs for silencing of specific genes involved in monolignol biosynthesis with minimal effect on plant fitness and viability.

**Acknowledgements:** Special appreciation to IUFRO Tree Biotechnology Conference 2011 organizing committee for awarding Ong Seong Siang a Student Travel Grant to attend this conference. The author wish to thank Gene Pool Sequencing Centre for small RNA library preparation, sequencing and bioinformatics analysis. This work was funded by Ministry of Science, Technology and Innovation, Government of Malaysia through eScience grant 05-01-02-SF1020.

**References**

1. Baima S, Possenti M, Matteucci A, Wisman E, Altamura MM, Ruberti I, Morelli G: The *Arabidopsis* ATHB-8 HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristems. *Plant Physiol* 2001, **126**:643-655.

2. Kim J, Jung JH, Reyes JL, Kim YS, Kim SY, Chung KS, Kim JA, Lee M, Lee Y, Narry Kim V, Chua NH, Park CM: MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular development in *Arabidopsis* inflorescence stems. *Plant J* 2005, **42**(1):84-94.

3. Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL: Novel and Mechanical Stress Responsive MicroRNAs in *Populus trichocarpa* That Are Absent from *Arabidopsis*. *Plant Cell* 2005, **17**:2186-2203.
P121

Toward identifying molecules responsible for the peculiar properties of
the G-layer in tension wood fibres

Fernanda Guedes, Miyuki Takeuchi, Françoise Laurans, Gilles Pilate*
INRA, UR588 Amélioration, Génétique et Physiologie Forestières, F-45075
Orléans cedex 2, France
E-mail: pilate@orleans.inra.fr
BMC Proceedings 2011, 5(Suppl 7):P121

**Background:** Due to its peculiar properties, tension wood formation constitutes a remarkable adaptation mechanism, that makes possible for the tree to reorientate its axes (stem and branches) in response to environnemental cues. In poplar, tension wood fibres harbour an extra cell wall layer, the G-layer, responsible for the peculiar mechanical properties of tension wood. This G-layer is very thick, most likely devoid of lignins and strongly enriched in highly cristalline cellulose. In addition, cellulose microfibril orientation is almost parallel to the fibre axis.

We aim to identify molecular actors responsible for the tensioning of
cellulose microfibrils and we choose as candidate, molecules containing
complex carbohydrates, such as pectin and the glycosylated part of
arabinogalactan proteins. Indeed, a wide array of different carbohydrates
has been recently evidenced in the G-layer, suggesting the occurrence of
complex polysaccharides other than cellulose within this layer (1, 2).

**Material and methods:** As a first step, we realized a comparative study between tension and opposite wood fibres using immunochemistry. A number of antibodies raised against different polysaccharide epitopes were assessed.

**Results:** The study revealed important differences in the distribution of the labeling with the kind of wood, the cellular type and within a single fibre between the different cell-wall layers. When using AX1 antibody directed against arabinoxylans, the secondary cell wall layers exhibit a very strong labeling whereas G-layers were completely devoid of labeling (3). With LMS antibodies (directed against β-(1-4)galactans, opposite wood is mainly labeled at the primary wall (Figure 1A), whereas in mature tension wood the G-layer is strongly labeled (Figure 1B) as already observed by (4). With JIM14 antibody directed against cell surface arabinogalactan-proteins, a uniform but moderate labeling was visible on the middle lamella and primary cell wall of fibers, ray-cells and vessels from both opposite and tension wood. In addition, a strong labeling appears at the inner surface of the G-layer (5). The labeling of antibodies directed against the protein moiety of poplar fasciclin-like arabinogalactan proteins are also detected in the G-layer forming fibres, and mainly at the inner surface of G-layers whereas this labeling is hardly present on primary walls which were labelled with JIM14. With CCRC-M7 antibodies directed against RhamnoGalacturonan I, the labeling is restricted to the G-layer of young tension wood fibres and more specifically to the innerside of the G-layer.

**Conclusion:** Our results strongly suggest the involvement of pectin and arabinogalactan proteins in the building of the G-layer.

**Acknowledgements:** The PhD thesis of Fernanda TP Guedes is funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES.

References

1. Nishikubo N, Awano T, Banasiak A, Bourquin V, Ibatullin F, Funada R, Brumer H, Teeri T, Hayashi T, Sundberg B, Mellerowicz E: Xyloglucan endo-transglycosylase (XET) functions in gelatinous layers of tension wood fibers in poplar - a glimpse into the mechanism of the balancing act of trees. *Plant Cell Physiol* 2007, 48:843-855.

2. Kaku T, Serada S, Baba K, Tanaka F, Hayashi T: Proteomic analysis of the G-layer in poplar tension wood. *J Wood Sci* 2009, 55:250-257.

3. Decou R, Lhernould S, Laurans F, Sulpice E, Leplé JC, Déjardin A, Pilate G, Costa G: Cloning and expression analysis of a wood-associated xylosidase gene(PtaBXL1) in poplar tension wood. *Phytochemistry* 2009, 70:163-172.

4. Arend M: Immunolocalization of (1,4)-beta-galactan in tension wood fibers of poplar. *Tree Physiol* 2008, 28:1263-1267.

5. Lafarguette F, Leplé J-C, Déjardin A, Laurans F, Costa G, Lesage-Descauses M-C, Pilate G: Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood. *New Phytol* 2004, 164:107-121.

P122

Regulation of biomass growth and carbon partitioning in poplar –
molecular characterization of a candidate gene

Cintia Ribeiro¹*, Evandro Novaes², Christopher Dervinis³, Matias Kirst³
¹Plant Molecular and Cellular Biology Graduate Program, University of Florida, Gainesville, FL, 32611, USA; ²Escola de Agronomia e Eng. de Alimentos, Federal University of Goias, Goias, Brazil; ³School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611, USA
E-mail: leiteribeiro@ufl.edu
BMC Proceedings 2011, 5(Suppl 7):P122

Improvement of plant feedstock for bioenergy production can be achieved by modifying wood chemical properties and increasing biomass productivity. We previously identified a candidate gene for carbon partitioning and growth on chromosome 13 (cpg13) of poplar. Cpg13 was identified as the regulator of carbon partition and growth within a QTL interval that explains 56% of the variation in cellulose to lignin ratio, as well as 20-25% of the heritable variation in biomass. Putative homologues of cpg13 in Arabidopsis are annotated as proteins of unknown function; therefore, the functional characterization of cpg13 is essential. At present, evidence of the functional role of cpg13 is being obtained by the analysis of poplar transgenic plants transformed with RNAi, overexpression and GFP-fused – cpg13 constructs. Preliminary data indicates that down-regulating cpg13 positively impacts growth, while a negative effect is detected in lines overexpressing cpg13. Transgenic poplar 35S::cpg13:GFP shows localization in cell wall, consistent with *in silico* predictions. Comparative genomics indicate moderate similarity with methyltransferase. Analysis of the impact of differentially regulating cpg13 on lignin and cellulose is currently under way in the mutants. Furthermore, attempts to purify the cpg13 protein are in progress, to define its role through a series of biochemical function assays.

A

<figure><img src="image_54.png" /></figure>

B

<figure><img src="image_55.png" /><figcaption>**Figure 1 (abstract P121)** A: in opposite wood, LMS labeling is restricted to the primary cell wall. B: strong labeling of the G-layer in mature tension wood fibres.</figcaption></figure>
P123

Formation of woody biomass is regulated by class III HD Zip genes

Marcel Robischon
Albert Ludwigs Universität Freiburg
E-mail: marcel.robischon@web.de
*BMC Proceedings* 2011, **5**(Suppl 7):P123

Secondary growth and the development of woody tissue is a key process in the formation of woody biomass. The gene family of Class III HDZip genes has been shown in the herbaceous Arabidopsis model to play a central role in regulating polarity and vascular development. While Arabidopsis is a poor model for investigating processes of wood formation, in this project all poplar Class III HDZip genes were cloned and expressed in hybrid aspen as a tree model system. To circumvent an endogenous regulation mechanism involving microRNAs the sequences were also mutated to render them microRNA resistant. Lines expressing the mutated Poplar ortholog of the Arabidopsis Revoluta gene (Populus Revoluta PRE) show a spectacular phenotype with stunted growth, radialized and rolled leaves, and a double and at times triplication or quadruple layer of the xylem, suggesting the formation of multiple layers of cambium. ClassIII HDZip genes have thus been shown to be crucial for the formation of lignified tissue in trees.

P124

Transgenic aspen and birch trees for Russian plantation forests

Konstantin Shestibratov*, Vadim Lebedev, Alexey Podrezov, Margarita Salmova
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS
E-mail: schestibratov@rambler.ru
*BMC Proceedings* 2011, **5**(Suppl 7):P124

**Background:** Aspen(Populus tremula) and birch (Betula pubescens) are the fast-growing trees successfully used for the aim of plantation forestry. These species have the great potential in Russia to meeting the need for paper, timber and other wood-based products. However, enhanced growth rate, decreased lignin content and herbicide resistance are the

required properties of new trees for plantation forestry. Breeding of forest trees is a slow process due to the long generation intervals typical of most forest trees and because many traits can only be properly assessed at rotation age. Genetic modification is an alternative method that can be used for new trees creation.

**Methods:** Genetic transformation experiments were carried out using vectors pBI-4CL, pBI-Xeg, pGS and pBIBar and supervirulent *Agrobacterium tumefaciens* strain CBE21. pBI-4CL plasmid contains the expression cassette harboring inverted fragments of 4-coumarat-CoA-ligase gene (GeneBank AY043494). pBI-Xeg vector was constructed using cDNA of xyloglucanase gene cloned from *Penicillium canescens*. pGS and pBIBar vectors contains pine glutamine synthetase gene (GS) and phosphinothricin acetyltransferase gene (bar). For transcription control of expression 35S promoter used in all cassettes harboring the gene of interest.

Internodes from *in vitro* aspen plants (Populus tremula) and leaves from *in vitro* birch plants (Betula pubescens) cultivating on WPM medium were used as an explants for *Agrobacterium*-mediated transformation [1,2]. Transformed shoots selected on the modified MS medium with 25 (aspen) or 50 (birch) mg/l kanamycin.

To detect DNA fragments from binary vectors transformants were analysed by PCR. Expression of recombinant genes was detected by RT-PCR. Lignin content determination performed by using the Klason method.

**Results and conclusions:** As a result of two *Agrobacterium*-mediated transformation experiments with pGS vector 56 and 27 transgenic lines of birch and aspen have been produced respectively. Presence of recombinant glutamine synthetase GS gene confirmed by PCR in 52 birch and 23 aspen lines. GS gene expression at the RNA level demonstrated RT-PCR analysis (Figure 1).

Most of GS lines have resistance to sublethal dose of phosphinotricin as well as in the in vitro and greenhouse conditions. Comparative biometric analysis of GS plants in a greenhouse makes it possible to select 7 and 3 superior lines of birch and aspen respectively. Growth rate enhancement of selected lines was 20-25%.

Genetic transformation of aspen plants for 4-coumarat-CoA-ligase gene down-regulation and xyloglucanase overexpression has been performed

<figure><img src="image_56.png" /><figcaption>Table 1(abstract P124) Lignin content of extract-free wood from control and transgenic aspen plants</figcaption></figure>

<table><thead><tr><td>Line</td><td>Acid soluble lignin, %</td><td>Acid insoluble lignin, %</td><td>Total lignin, %</td><td>Lignin reduction, %</td></tr></thead><tbody><tr><td>Pt (control)</td><td>21,0</td><td>3,4</td><td>24,4</td><td>-</td></tr><tr><td>PtXIII4CL1c</td><td>21,4</td><td>3,0</td><td>24,3</td><td>0,2</td></tr><tr><td>PtXIII4CL2c</td><td>15,2</td><td>3,6</td><td>18,7</td><td>23,2</td></tr><tr><td>PtXIII4CL3a</td><td>22,5</td><td>3,0</td><td>25,5</td><td>-4,7</td></tr><tr><td>PtXIII4CL3c</td><td>17,9</td><td>3,7</td><td>21,7</td><td>11,1</td></tr><tr><td>PtXIII4CL4a</td><td>18,5</td><td>3,2</td><td>21,7</td><td>11,1</td></tr><tr><td>PtXIII4CL4c</td><td>17,4</td><td>3,8</td><td>21,2</td><td>13,0</td></tr></tbody></table>
<figure><img src="image_57.png" /><figcaption>**Figure 2(abstract P124)** Stem wood coloration of control (right) and transgenic 4CL plants (left).</figcaption></figure>

using pBI-4CL and pBI-Xeg binary vectors respectively. 11 transgenic lines with inverted fragments of 4CL gene and 15 lines harboring Xeg1 gene have been produced. DNA fragments of 4CL RNAi-construct were detected in all lines. 14 out of 15 transformants produced with pBI-Xeg vector harboring Xeg1 gene. Chemical analysis of stem wood of 4CL lines demonstrated decreased lignin content (Table 1). Lignin reduction from 11 to 23% was achieved. Line PtXIII4CL2cdemonstrated minimum lignin content 18,7%. Color modification of stem wood was observed for 8 out of 11 4CL lines (Figure 2).

Aspen and birch transgenic plants with *bar* gene coding resistance against herbicide phosphinotricin were also created. Transformation experiments were carried out using vector pBIBar. 19 aspen and 9 birch lines were selected as a result of transformation. Presence of *bar* gene was confirmed in all lines. Herbicide resistance was observed at 16 and 7 lines of aspen and birch respectively. Lines tested in a greenhouse demonstrated normal phenotype without somaclonal variations and may be recommended for the field trials.

To combine several new properties in one plant the co-transformation method was developed. This method allows transferring recombinant DNA fragments from three T-DNAs of two plasmids independently [3]. Efficiency of co-transformation was up to 100%. Currently we attempt to apply this method for the production of superior aspen and birch plants with enhanced growth rate, decreased lignin content and herbicide resistance at the same time.

**References**

1. Shestibratov KA, Bulatova IV, Novikov PS: In vitro response of transgenic aspen containing glutamine synthetase gene GSI to the sublethal dose of phosphinothricin. *Appl. Biochem. Micro* 2010, 46(8):763-768.

2. Shadrina TE, Schestibratov KA: Efficient shoot regeneration from leaf explants of *Betula pendula*, *B. pubescens* and *B. nigra*. *Acta Hort* (ISHS) 2010, 885:349-353.

3. Lebedev VG, Schestibratov KA, Shadrina TE, Bulatova IV, Abramochkin DG, Miroshnikov AI: Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one *Agrobacterium tumefaciens* strain. *Russ. J. Genet* 2010, 46(11):1282-1289.

P125

Selection of reference gene in *Eucalyptus camaldulensis* for real-time
qRT-PCR

Boby Unnikrishnan*, Gurumurthy Demlapura Shankaranarayan, Navin Sharma
Division of Plant Molecular Biology, ITC R&D Centre, Bangalore 560058, India
E-mail: bobyunni@rediffmail.com

BMC Proceedings 2011, 5(Suppl 7):P125

**Background:** Real time quantitative reverse transcription PCR (real-time qRT-PCR) is an established technique for quantification of mRNA and has been extensively used for gene regulation studies in plants. However, there are inherent challenges associated with the technique such as variability of RNA and extraction protocols, different rates of reverse transcription and PCR efficiencies. This demands an accurate method of normalization to obtain reproducible results. Among the various methods, normalization to a reference house keeping gene is the most commonly used method. If inappropriate reference genes are used for normalization, the experimental results can vary significantly leading to false results [1]. *Eucalyptus camaldulensis* Dehnh. is a widely distributed tree species used for planting in arid and semi-arid areas. The wood is composed of mainly cellulose and lignin and the pathway involved in lignin formation is fairly understood. The lignin biosynthetic genes viz Ferulate 5 Hydroxylase (F5H), 4 Coumarate CoA Ligase (4CL), Cinnamoyl CoA Reductase (CCR) and Cinnamoyl Alcohol Dehydrogenase (CAD) are highly conserved across the tree species and have been utilized as targets for manipulating lignin
content. The present study describes validation of a reference gene in various tissue types of eucalyptus and its subsequent use for the expression analysis of lignin biosynthetic genes.

**Materials and methods:** Samples were collected from three different tissues of *E. camaldulensis* namely xylem, young leaf and mature leaves and three developmental stages viz one, one and half and three years. The samples were named as X (1-3) for all the xylem samples coming from three developmental stages and Y(1-3) and M(1-3) for young leaves and mature leaves respectively (Figure 1). Total RNA was extracted with RNAqueous Kit (Ambion, Austin TX, USA) following manufactures protocol. cDNA was synthesized from 1 µg of RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) with random hexamer primers. Primers for house keeping genes and lignin biosynthetic genes were designed based on the EST data from Eucalyptus using Primer Express 3.0 Software (Applied Biosystems). The nucleotide sequence of CAD, CCR, 4CL and F5H from *E. camaldulensis* were deposited in NCBI database (HR309064, HR309065, HR309066, HR309067). The real time PCR efficiency of each primer pair was determined with the slope of a linear regression model and all PCRs displayed efficiencies between 90 and 95%. Comparative C~T~ method was followed for quantification of lignin biosynthetic genes with 18S rRNAs as the internal control as suggested by Schmittgen and Livak [2]. The stability analysis of each reference gene was carried out by the method given by Brunner *et al* [3].

**Results:** The choice of internal control is usually straight forward especially for analysis of samples from same tissues. However, when samples are derived from different tissues or developmental ages, as it is seen in our study, required validated reference gene/ genes. Six different housekeeping genes were chosen for the analysis which included NADP isocitrate dehydrogenase, 18S ribosomal RNA, Histone H4, 60S ribosomal protein L7, Translational initiation factor (TIF) and Glyceraldehyde 3 phosphate dehydrogenase. Among the genes used in the study, 18S rRNA showed the least C~T~ indicating higher abundance while the highest C~T~ was observed for TIF. Further, 18S rRNA showed maximum stability when C~T~ was plotted against samples (Figure 1). A gene with low stability index is considered to have highest stability [3]. In the present study with *E. camaldulensis*, 18S rRNA had the lowest stability index while GAPDH showed maximum variation among the samples.

Expression analysis of four lignin biosynthetic genes *viz* CCR, CAD, F5H and 4CL were carried out in different samples by using 18S rRNA as the reference gene. Lignin analysis of samples showed higher lignin content in three-year-old samples estimated by klason lignin analysis compared to one-year old. All genes under study showed increased expression in three year old samples except for CAD gene. The results also showed increased amount of F5H transcripts in older tissues indicating higher syringyl units in older tissue. Chen *et al* [4] reported syringyl type lignin content and

S/G ratio increased from younger internode to older internode while G and H decreased in parallel.

As the expression profile showed variations in different age samples, further experiment was under taken to evaluate the expression levels of these genes in same age trees (Three year old) in correlation with lignin content. Based on klason lignin content, trees with lignin content of 26± 0.3 and 24± 0.2% were chosen for the studies. All genes under study *viz*4CL, CAD, CCR and F5H showed increase in expression in high lignin sample in comparison with low lignin.

**Conclusions:** Among the house keeping genes under study, 18SrRNA exhibited lowest stability index and we are suggesting it as a reference gene for expression studies in Eucalyptus. The reduction in CAD transcript level and higher levels of F5H in older tissues indicated a possible shift towards an increased syringyl lignin where as when analysis was done at same age, expression levels of all selected lignin biosynthetic genes were high in high lignin tree. The study opens up the possibility for using these genes as candidate genes for the selection of desired genotypes.

**Acknowledgements:** The first author would like to thank Ramadevi P for lignin analysis and Girisha R in helping in statistical analysis.

**References**

1. Dhelda K, Hugget JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A: The implications of using an inappropriate reference gene for real time reverse transcription PCR data normalization. *Anal Biochem* 2005, 344:141-143.

2. Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C~T~ method. *Nature Prot* 2008, 3:1101-8.

3. Brunner MA, Yakovlev IA, Strauss SH: Validating internal controls for Quantitative plant gene expression studies. *BMC Plant Biol* 2004, 4:14.

4. Chen L, Auh C, Chen F, Cheng X, Aljoe H, Dixon RA, Wang Z: Lignin deposition and associated changes in anatomy, enzyme activity, gene expression and ruminal degradability in stems of tall fescue at different developmental stages. *J Agric Food Chem* 2002, 50:5558-5565.

P126
Genomic of *Eucalyptus globulus* for pulp and biofuels

Sofia Valenzuela¹*, Renán García¹, Juan Elissetche², Jaime Rodríguez¹,
Regis Mendonca¹, Claudio Balocchi³

¹Universidad de Concepción, Brazil; ²Genómica Forestal, Brazil; ³Bioforest S.A., Brazil

E-mail: sofvalen@udec.cl

BMC Proceedings 2011, **5**(Suppl 7):P126

*Eucalyptus* is a species of great interest for the pulp and paper industry. Worldwide there are 20 million hectares planted and within Chile a total of 700.000 ha, being the main species grown for short-fibre pulp *Eucalyptus*

<figure><img src="image_58.png" /><figcaption>**Figure 1(abstract P125)** Distribution of average C~T~ values of various house keeping genes under study.</figcaption></figure>
**globulus**, which is extensively used for pulping due to its fast growth, high pulp yield, and good fiber properties. Genetic programs of this specie have been oriented in improving commercial traits as volume, growth and form and recently traits as pulp ability have been included. Understanding the genomics of wood formation and identifying genes that are responsible for the traits of interest is a major challenge. An increase in 2% of pulp yield in trees can translate in a large economical gain, but most importantly there will be less pressure for land. There are two ways of reaching this goal, one through the use of genetic engineering and the second by genetic improvement, in both cases it is important to understand and identify the main genes involved in conferring the traits of interest. To identify these genes, different studies have been carried out, mainly by using EST libraries, finding a large number of sequences. Although there is large information regarding the enzymes and genes involved in the lignin pathway, little information is available regarding other metabolic pathways as cellulose and hemicellulose, as well as genes responsible for traits as density, cell wall, among others. In this study a detailed phenotypic characterization of 100 different genotypes was made, determining pulp content, lignin, cellulose, hemicellulose and Syringyl/ Guaiacyl (S/G) ratio, among others. By employing NIR models a larger number of *E. globulus* clones were characterized for the same properties. From the 300 genotypes studied, "contrasting genotypes", with high (good) and with low (bad) density and pulp yield were selected. It was found that clones with high density and pulp yield had high glucan content, lignin rich in S units, high β-O-4 linkages and low lignin, and xylan content. Out of these contrasting genotypes, one of each, low and high pulp yield and density, were further studied to find candidate genes involved in pulp ability. An EST library was made for each genotype, which was sequenced by the 454 platform, giving a total of 21,000 sequences, out of which 250 were differentially expressed. Due to the low amount of Eucalyptus sequences available, 28% of these sequences blasted to ESTs from wine grape, 22% to poplar and less than 5% to eucalypts. From these sequences, genes involved in lignin pathway, cellulose biosynthesis as well a transcription factors were identified. F5H was one of the main genes studied, which was characterized at a biochemical and expression level.

**Acknowledgments:** Funding for this research was provided by Genomica Forestal (INNOVA-Chile, CORFO) and FONDEF 03i1103.

# S7. PROPAGATION AND IN VITRO TECHNOLOGIES

## P127

**Somatic embryogenesis for mass propagation of elite Spruce families: effect of storage time on somatic embryogenesis initiation**

Iftikhar Ahmad, Sofie Johansson, Gisele Andrade*, Beata Dedicova, Ulrika Egertsdotter

Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, 90187, Sweden

E-mail: Gisele.Andrade@slu.se

BMC Proceedings 2011, 5(Suppl 7):P127

**Background:** Somatic embryogenesis (SE) is the only clonal propagation method that has potential for large scale production of elite conifer plants from the breeding programs. Methods that support bioreactor-based methods for SE propagation are developed [1,2]. Samples of somatic embryos can be stored indefinitely under liquid nitrogen for future plant production. Somatic embryo cultures are also studied as model systems for conifer embryo development to address fundamental research questions, or used as material for genetic transformation to study gene function in conifers.

In addition to utilizing SE for masspropagation of known elite clones previously tested in field tests, the SE technology offers an opportunity to directly capture and increase the value of small samples of elite seeds from the breeding programs. Furthermore, by direct masspropagation of families through SE, the value of the elite seed is increased; however without the cost of clonal testing. This is arguably an alternative approach to the traditional approach of only utilizing clonal field-tested material for SE masspropagation [3]. The aim of this project was to investigate the effect from seed storage time on the rate of somatic embryo initiation for the purpose of optimizing the use for SE over time

of small valuable seed samples. This was done by isolating ZE from seeds of Norway spruce that had been stored for various times, and were collected from different parts of Sweden.

**Material and methods: Plant material:** Nineteen batches of Norway spruce (*Picea abies*) seeds from commercial seed orchards in southern, middle and northern parts of Sweden were provided by the forest companies supporting the project.

**Initiation of Somatic Embryogenesis:** The spruce seeds were sterilized with 95% ethanol followed by 30% (v/v) commercial bleach and Tween 20. The bleach was discarded and the seeds was rinsed three times with sterile distilled water and left to imbibe overnight at room temperature. After imbibition, ZE were dissected from the female gametophyte under a dissecting microscope and cultured on half-strength LP medium supplemented with 10 µM 2, 4-Dichlorophenoxyacetic acid and 4.4 µM Benzyladenine for SE initiation. In total 90 ZE were isolated from each seed batch. The SE initiation was monitored on weekly basis.

**Maturation of Somatic Embryos:** One cell line per seed batch was tested for embryo differentiation from pro-embryogenic masses (PEMs) on DKM containing no plant growth regulators (PGRs) and maturation on DKM supplemented with 30 µM Abscisic acid.

**Results:** In total we tested 19 seed batches of Norway spruce where 90 ZE were isolated per seed batch and placed on ½ LP medium containing PGRs for SE initiation. Three weeks after isolation of ZE, callus formation was observed. Embryogenic callus is composed of PEMs that have a white and translucent appearance (Fig. 1) and are mostly produced from the hypocotyl region of the ZE. When whitish callus reached a size of 5x5 mm, it was isolated from primary explants and placed on proliferation medium for continuous growth.

All 19 seed batches showed SE initiation however at different frequencies (Fig. 2). The initiation frequency did not vary notably between the seeds from different parts of Sweden. There was also no difference in initiation frequency related to the time in storage. The seeds tested had been collected between 1984-2007; the highest initiation frequency was observed in seed batch FP 444 collected in 1992 and the lowest initiation rate was observed in seed batch Saleby collected in 2006.

One of the initiated and established cell lines from each seed batch was subjected to maturation medium to examine whether the cultures of

<figure><img src="image_59.png" /><figcaption>**Figure 1(abstract P127)** SE initiation from zygotic embryo of Norway spruce. ZE extracted from spruce seeds produced PEMs after 3-4 weeks in culture.</figcaption></figure>
<figure><img src="image_60.png" /><figcaption>**Figure 2(abstract P127)** SE initiation rates from seeds from different seed batches and collection years. The SE initiation rate for each seed batch is shown in percentage of seeds tested (90 for each batch) that produced PEMs that could be isolated and cultured. Only one cell line per seed was recorded.</figcaption></figure>

PEMs could produce mature somatic embryos. We observed that 11 out of 19 tested cell lines produced mature somatic embryos (data not shown). Since only one cell line from each seed batch was tested for maturation, we cannot exclude that the remaining 8 seed batches were capable of producing mature somatic embryos. However, similar to the initiation process, the maturation stage did not appear to be related to the storage time and the geographical origin of the seed.

**Conclusion:** We have demonstrated that it is possible to propagate small batches of Norway spruce seeds stored for up to 25 years through somatic embryogenesis. All initiated cell lines established cultures of PEMs and most cell lines tested produced mature somatic embryos. Thus we conclude that SE can provide a promising method for amplifying small valuable batches of elite seeds even if the seeds have been stored for up to 25 years.

**Acknowledgements:** We thank the Swedish Research Council (VR) and the Swedish Governmental Agency for Innovation Systems (VINNOVA) for financial support. We are grateful to Sveaskog, Bergvik, Holmen and Södra Skogsägarna for supplying the seeds and Edward Businge for his contribution during the experimental part of this project. Iftikhar Ahmad and Sofie Johansson contributed equally to the work.

**References**

1. Sun H, Aidun C, Egertsdotter U: Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos. *Biotechnology and Bioengineering* 2009, 105:588-599.

2. Sun H, Aidun CKA, Egertsdotter U: Possible Effect From Shear Stress on Maturation of Somatic Embryos of Norway Spruce (*Picea abies*). *Biotechnology and Bioengineering* 2010, 108:1089-1099.

3. Ratnaparkhe SM, Egertsdotter EMU, Flinn BS: Identification and characterization of a matrix metalloproteinase (*Pta1-MMP*) expressed

during Loblolly pine (*Pinus taeda*) seed development, germination completion, and early seedling establishment. *Planta* 2009, 230:339-354.

4. Lindgren D: A way to utilize the advantages of clonal forestry for Norway spruce. *Working Papers of the Finnish Forest Research Institute* 2008, 114:8-15.

P128

**Somatic Embryogenesis as a tool for forest tree improvement: a case-study in *Eucalyptus globulus***

Gisele Andrade¹*, Ravi Shah¹, Sofie Johansson¹, Gloria Pinto², Ulrika Egertsdotter¹

¹Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, 90187, Sweden; ²Centre for Environmental and Marine Studies (CESAM) & Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal

E-mail: Gisele.Andrade@slu.se

BMC Proceedings 2011, 5(Suppl 7):P128

**Background:** Somatic embryogenesis (SE) technology is expected to play a significant role in future forest tree improvement programs. Methods for efficient SE propagation in softwood species based on bioreactors are underway [1]. Furthermore, genetic transformation through SE is also the most promising approach to generate and propagate elite recalcitrant genotypes of forest trees [2], or clonal propagation of selected endangered high-value forest trees [3,4].

Somatic embryogenesis induction was first reported for the genus *Eucalyptus* in 1980. Mass production of SE plants however remains
difficult although procedures designed for plant regeneration have been reported for *E. citriodora*, *E. grandis*, *E. tereticornis* [5]. Recent data on *E. globulus* are promising and show the potential of this technology for application in clonal forestry [6,7].

A service platform for routine transformation of forest trees has been established at the Berzelii Center at Umeå Plant Science Centre using Norway spruce as the primary model species for gene function analyses. Other target species for future studies include hardwood species such as Poplar and Eucalyptus. We present here a brief summary of a case study for introduction of a recalcitrant forest species to the platform as demonstrated by the establishment of the SE technology for *E. globulus* following protocol previously established in Portugal [6,7] and preliminary results aiming to improve the induction of SE.

**Material and methods:** Half-sib seeds of *E. globulus* collected from trees in Portugal (Altri Florestal Breeding Program) were used in this study. Seeds were surface-disinfected with a mixture of 1:1 absolute ethanol: hydrogen peroxide 30% (v/v) for 15 minutes. Two sources of explants were tested: (a) cotyledons and hypocotyls isolated from 15 day-old *in vitro* seedlings germinated on standard MS medium and (b) zygotic embryos (ZE) isolated after removal of the seed coat. Briefly, using a standard protocol [7], explants were inoculated on induction medium (MS<sub>3NAA</sub>) consisting of MS with 3 mg/l NAA, 30 g/l sucrose, 2.5 g/l Gelrite, pH 5.8. Cultures were incubated in the dark at 24°C for 25 days. After this period, explants were transferred to the same MS medium without NAA (expression medium MS<sub>WH</sub>) under the same conditions.

To evaluate the effect of factors indicated from previous studies as essential for the process of SE induction, we then tested the effect of media composition using only ZE as explants: induction and expression using MS salts but containing other vitamins (MS, B5 and RP) and 5 days induction on MS containing 5 mg/l NAA or 2 mg/l 2,4-D and the remaining 20 days on MS<sub>3NAA</sub>.

Embryogenic potential was analyzed under the microscope and the results expressed as the percentage of explants showing somatic embryos. Callus production and root formation were also scored.

**Results:** Adopting a standard protocol, the source of explants affected the SE induction as previously reported for other species: no embryogenic response was observed and only callus and root formation occurred when cotyledons and/or hypocotyls was used as source of explants (data not shown); 5-20% of the explants showed formation of globular somatic embryos after transferring to expression medium within 5-8 weeks after induction when ZE was used. The present study included the same OP families previously tested in Portugal. The induction rates rated in a similar way to the previous study, confirming the importance of the genotype.

Preliminaries results from the evaluation of factors affecting SE inductions show that root and root hair formation was observed on most of the explants in all tested treatments. Adding different vitamins to the MS medium promote the same embryogenic response. When ZE were cultured for short period on medium containing 2,4-D or 5 mg/l of NAA, almost 100% of the explants showed callus formation. However, the embryogenic response was similar to the control treatment indicating that concentration/induction period should be adjusted and/or other PGRs combinations tested in the future.

Individual or small clusters of globular embryos were isolated and transferred to MS<sub>3NAA</sub> for capture of embryogenic cultures. The most developed somatic embryos isolated displaying a shoot and tap root were transferred to MS<sub>WH</sub> for plant conversion.

**Conclusion:** The data reported here show the reproducibility of the previously published protocol using different genotypes of *E.globulus*. The results also indicate the importance of genotype over culture condition for the induction success. New experiments are in progress aiming to increase the induction of SE and establish an efficient protocol that can form the basis for genetic transformation of hardwood SE cultures.

**Acknowledgements:** We thank the Swedish Research Council (VR) and the Swedish Governmental Agency for Innovation Systems (VINNOVA) for financial support. We are grateful to Lucinda Neves and Clara Araujo at Altri Florestal Breeding Program for supplying the seed batches. Ravi J. Shah is the primary technical research assistant for this project.

## References

1. Sun H, Aidun CKA, Egertsdotter U: Possible effect from shear stress on maturation of somatic embryos of Norway spruce (*Picea abies*). *Biotechnology and Bioengineering* 2010, 108:1089-1099.

2. Andrade GM, Nairn CJ, Le HT, Merkle SA: Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation. *Plant Cell Rep* 2010, 28:1385-1397.

3. Merkle SA, Andrade GM, Nairn CJ, Powell WA, Maynard CA: Restoration of threatened species: a noble cause for transgenic trees. *Tree Genetics & Genomes* 2007, 3:11-118.

4. Pijut PM, Lawson SS, Michler CH: Biotechnology efforts for preserving and enhancing temperature hardwood tree biodiversity, health and productivity. *In vitro Cell. Dev. Biol-Plant* 2011, 47:123-147.

5. Pinto G, Park YS, Loureiro J, Neves L, Araújo C, Silva S, Santos C: Somatic embryogenesis in Eucalyptus- an update to 2009. *Applications of plant biotechnology: in vitro propagation, plant transformation and secondary metabolite production* India: Aavishkar Publishers: Kumar A, Sopoy SK, 1 2010, 531-542.

6. Pinto G, Santos C, Neves L, Araujo C: Somatic embryogenesis and plant regeneration in *Eucalyptus globulus* Labill. *Plant Cell Rep* 2002, 21:208-213.

7. Pinto G, Park Y-S, Neves L, Araujo C, Santos C: Genetic control of somatic embryogenesis in *Eucalyptus globules* Labill. *Plant Cell Rep* 2008, 27:1093-1101.

## P129

### Practical testing of Scots pine cutting propagation - a joint Metla-Skogforsk-Silava project

Karl-Anders Högberg¹, Jörgen Hajek², Arnis Gailis³, Niina Stenvall⁴, Inga Zolina³, Satu Teivonen⁴, Tuija Aronen⁵⁺

¹Skogforsk, Ekebo, Sweden; ²Skogforsk, Sävar, Sweden; ³Silava, Latvia; ⁴Finnish Forest Research Institute, Haapastensyrjä, Finland; ⁵Finnish Forest Research Institute, Punkaharju, Finland

E-mail: tuija.aronen@metla.fi

BMC Proceedings 2011, 5(Suppl 7):P129

Testing of candidates as clones would greatly benefit breeding of Scots pine (*Pinus sylvestris* L.), but has not been applied because of vegetative propagation of the species is difficult. With a common interest in Scots pine breeding, forest research institutions from Sweden, Finland, and Latvia (Skogforsk, Metla, and Silava, respectively) joined in a collaborative project to develop pine cutting propagation for breeding purposes. The main objective of this effort was to find protocols for sufficient shoot production, and at the same time, maintain a high rooting response. Secondly, the aim was to increase the knowledge on the influence of different rooting agents, watering regimes and substrates on the rooting of cuttings.

From Finland and Latvia, respectively, 15 full-sib families from parents with high breeding values were included. From Sweden, 15 families from each of two research stations, one northern and one southern, were included. These sets of the families were used locally. Another collection of five families was shared among the participants, which means that 5 families extra were included at each location. All donor plants were pruned according to the same principles. However, the timing of the pruning varied in the five propagation models tested:

A. One-year-old donor plants pruned to yield 2x winter cuttings / 4-year method

B. One-year-old donor plants pruned to yield winter cuttings and late summer cuttings/ 3-year method

C. "Turbo line" i.e. one-year-old donor plants pruned 2 x cuttings in one year/ 2-year method

D. One-year-old donor plants pruned to yield 2x late summer cuttings/ 3-year method

E. Two-year-old donor plants pruned to yield winter cuttings/ 4-year method

According to the results, the average production of cuttings with propagation models including two harvests in consecutive years on the same donor plant can be predicted to be 10-15 cuttings per a donor plant, with a substantial variation among families and donor plants. Even though rooting responses above 50% can be achieved, this could not be repeated for a large number of propagations. The generally low and erratic rooting response leads to conclusion that the project was unsuccessful in developing improved and reliable protocols for Scots pine cutting propagation. However, a propagation model enabling two harvests on a 1-year-old donor plant within the same year is a promising option but needs further studies.

More detailed information on the project and its results is available at: http://www.metla.fi/julkaisut/workingpapers/2011/mwp198.htm
P130

Gene expression patterns associated with developmental transitions
during somatic embryogenesis in pine

Elena Carneros*, Inmaculada Hernández, Silvia P Solana, Carmen Díaz-Sala,
Dolores Abarca

Department of Plant Biology, University of Alcalá, Alcalá de Henares, Madrid,
28871, Spain

E-mail: elena.carneros@uah.es

BMC Proceedings 2011, 5(Suppl 7):P130

The low regeneration capacity of forest species is one of the major limitations for vegetative propagation [1]. The molecular mechanisms that determine the efficiency of clonal propagation programs via either adventitious organogenesis or somatic embryogenesis have not been established. For clonal propagation via somatic embryogenesis, the success of the process depends on an initial reprogramming step and on further developmental transitions involved in the maturation of somatic embryos [2]. The identification of candidate genes involved in the regulation of key steps of the regeneration processes is essential to generate tools and strategies to improve the success of clonal propagation programs in forest species.

The aim of this work is to identify new candidate genes potentially involved in the regulation of developmental transitions in somatic embryogenesis in pine. For that purpose, samples of embryogenic tissue from *Pinus radiata* D. Don at different stages of development were used: proliferative tissue (after 7 and 14 days at the last transference to fresh proliferation medium), somatic embryos at the beginning of differentiation and somatic embryos at cotyledonary stage [3,4].

Large-scale expression analysis using a microarray containing an EST collection enriched in auxin-induced genes, and several tissue-specific cDNA libraries from meristematic and embryonic tissues, were used for the identification of phase-specific candidate genes. Genes related to auxin signaling, regulation of gene expression, signal transduction, proliferation and embryo development were selected for further analysis. The expression of these candidate genes was confirmed by QRT-PCR. The information obtained from this work will open new ways of research on molecular mechanisms involved in developmental processes in conifers.

This work was funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

References

1. Abarca D, Díaz-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.

2. Abarca D, Díaz-Sala C: Reprogramming adult cells during organ regeneration in forest species. *Plant Signal Behav* 2009, 4(8):793-795.

3. Walter C, Find JI, Grace LJ: Somatic embryogenesis and genetic transformation in *Pinus radiata*. *Protocol for Somatic Embryogenesis in Woody Plants* The Netherlands: Springer: Jain SM & Gupta PK 2005, 11-24.

4. Carneros E, Celestino C, Klimaszewska K, Park YS, Toribio M, Bonga JM: Plant regeneration in Stone pine (*Pinus pinea* L.) by somatic embryogenesis. *Plant Cell Tiss Organ Cult* 2009, **98**:165-178.

P131

Development of micro-propagation and mini cutting protocol for fast
growing Melia, Dalbergia and *Eucalyptus* clones for pulpwood and bio-
energy plantations

Senniappan Chinnaraj*, C Malimuthu

S.V. Subrahmanyam Tamil Nadu Newsprint and Papers Ltd, India

E-mail: chinnaraj.s@tnpl.co.in

BMC Proceedings 2011, 5(Suppl 7):P131

Per capita paper consumption is only 9.2 kg in India, which is much lower when compared to other neighboring developing economies, such as, China (42 kg) & Indonesia (23 kg). Currently Indian economy is growing by 7.5% per annum. Along with economical growth, the paper demand is also expected to grow and cross 20 million tonnes per annum by 2020 from current 10 million tonnes. Availability & quality of raw material, non availability of land for pulp wood plantations, alternate use of pulp wood for other uses like bio-energy and regulations to increase green energy use in mill operations would become major challenge for Indian paper industry's survival and growth. To face these changes effectively, paper industries in India initiated many social forestry models and brought 4,00,000 hectors under pulp wood plantations. This has helped pulp wood availability and reduced the fibrous raw material shortage to some level but it is not enough to meet the growth.

Tamil Nadu Newsprint and Paper Limited (TNPL) is state owned integrated pulp and paper industry situated in southern most state of India and produce around 4,00,000 tons of paper per annum. TNPL has 300 Tonnes per day (TPD) hardwood fibreline along with 550 TPD bagasse fibre line to meet its pulp requirement. TNPL had setup biotechnology and bio-energy research facility under the existing R & D Department to work on identification, selection and multiplication of improved pulpwood clones of *Eucalyptus* and other alternate fast growing hardwood species through modern biotechnological methods to meet pulpwood and also bio-energy need. The plant tissue culture facility has one million plants per annum capacity and optimized protocols for many improved *Eucalyptus* pulp wood clones to use as clonal mother plants at Clonal Production and Research Centre of Plantation Department using mini cutting process. TNPL clonal production facility has capacity of 15 million seedlings per annum and already covered 52,000 acres of pulp wood plantation and plan to cover additional 15,000 acres every year.

<table><caption>Table 1(abstract P131) Results of wood pulping and pulp properties of selected pulpwood and bio-energy clones</caption>
  <thead>
    <tr>
      <th>Parameters</th>
      <th>Units</th>
      <th>Melia dubia</th>
      <th>Dalbergia sissoo</th>
      <th>Eucalyptus urograndis</th>
      <th>Eucalyptus tereticornis</th>
      <th>Eucalyptus camaldulensis</th>
      <th>Reference</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td>Bulk density</td>
      <td>kg/m3</td>
      <td>136</td>
      <td>230</td>
      <td>184</td>
      <td>213</td>
      <td>220</td>
      <td>225</td>
    </tr>
    <tr>
      <td>Basic density</td>
      <td>kg/m3</td>
      <td>318</td>
      <td>547</td>
      <td>402</td>
      <td>502</td>
      <td>520</td>
      <td>510</td>
    </tr>
    <tr>
      <td>Chemical addition</td>
      <td>%</td>
      <td>15.0</td>
      <td>15.0</td>
      <td>15.0</td>
      <td>15.0</td>
      <td>15.0</td>
      <td>15.0</td>
    </tr>
    <tr>
      <td>Screen rejects</td>
      <td>%</td>
      <td>0.8</td>
      <td>0.7</td>
      <td>0.2</td>
      <td>0.2</td>
      <td>0.8</td>
      <td>0.8</td>
    </tr>
    <tr>
      <td>Screened pulp yield</td>
      <td>%</td>
      <td>46.5</td>
      <td>47.0</td>
      <td>46.6</td>
      <td>47.3</td>
      <td>47.5</td>
      <td>44.2</td>
    </tr>
    <tr>
      <td>Kappa Number</td>
      <td></td>
      <td>21.6</td>
      <td>18.5</td>
      <td>20.4</td>
      <td>19.1</td>
      <td>19.7</td>
      <td>25.3</td>
    </tr>
    <tr>
      <td>Brightness</td>
      <td>% ISO</td>
      <td>27.8</td>
      <td>30.7</td>
      <td>29.1</td>
      <td>34.9</td>
      <td>30.5</td>
      <td>24.2</td>
    </tr>
    <tr>
      <td>Unbleached Strength properties at 300ml CSF</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Tensile index</td>
      <td>Nm/g</td>
      <td>94.2</td>
      <td>79.0</td>
      <td>98.0</td>
      <td>92.0</td>
      <td>95.0</td>
      <td>74.0</td>
    </tr>
    <tr>
      <td>Tear index</td>
      <td>mN.<br>m2/g</td>
      <td>11.9</td>
      <td>9.3</td>
      <td>8.7</td>
      <td>10.3</td>
      <td>9.5</td>
      <td>8.2</td>
    </tr>
    <tr>
      <td>Burst index</td>
      <td>kPa.<br>m2/g</td>
      <td>6.4</td>
      <td>5.6</td>
      <td>6.9</td>
      <td>6.5</td>
      <td>5.6</td>
      <td>5.1</td>
    </tr>
  </tbody>
</table>
As a part ongoing research activity, one Melia dubia clone for bio-energy/ pulpwood and one Dalbergia sissoo clone and three clones of eucalyptus, (each one from E. urograndis, E. tereticornis, and E. camaldulensis), were identified based on its biomass productivity, as well as, pulp quality and yield. Micro-propagation protocol was optimized for all the above five clones with modified MS medium with various concentrations of cytokinin and auxin. The seedlings produced using micro-propagation were used as mother plants for clonal mini garden in the concrete sand beds where the nutrient requirements where optimized for further multiplication using mini cutting process. The results of above study is presented and discussed in this paper.

Wood and pulp properties of all selected Melia dubia, Dalbergia sissoo, E. urograndis, E. tereticornis, and E. camaldulensis clones are presented in the Table 1 along with reference Eucalyptus pulp wood used in the mill. All the selected clones show high yield and strength properties. However, Melia dubia is not preferred as pulpwood due to low bulk density which would result in less through-put in fibreline. But it can be exploited for bio-energy applications especially for biomass gasification to generate producer gas and use in Lime Kiln to replace fuel oil and also for other wood product applications. All the selected clones give approximately minimum 2.5% more pulp yield with improved pulp properties when compared reference pulpwood currently used in the mill. Higher pulp yield would definitely give better economy and environmental performance of mill operations. For example, 1% yield increase would result in annual savings of INR 35.0 million in pulp wood cost for mill of our size i.e. 300 TPD. Therefore, for 2.5% yield increase would result in annual saving of around INR 87.5 million apart from chemical saving and other environmental benefits.

To take all the selected clones from lab to land in a short period, micro-propagation and mini cutting protocols were optimized. The results are

presented in the Table 2. E. tereticornis clone found to perform well during initiation, elongation and hardening. On the other hand, E. urograndis found to perform well during multiplication and gave maximum number of shoots per clump. All the Eucalyptus clones found to perform well during the rooting. Melia dubia and Dalbergia sissoo are found to perform poor in micro-propagation compared to Eucalyptus. Seedlings produced by micro-propagation are used for mini cutting experiments and results are presented in Table 2. E. urograndis produced more number of cuttings per plant per month followed by Dalbergia sissoo, E. tereticornis, E. camaldulensis and Melia dubia. Rooting and survival rate is also high for Eucalyptus clones when compared to Dalbergia sissoo and Melia dubia. Micro-propagation followed mini cutting protocol for propagation of all the above clones has been successfully adopted by mill for commercial production of quality seedling for mill's pulp wood and other plantation programme.

P132

Optimization of a plant regeneration and genetic transformation protocol for Eucalyptus clonal genotypes

Raj Deepika¹*, Adri Veale¹, Cathleen Ma², Steven H Strauss², Alexander A Myburg¹

¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; ²Department of Forest Ecosystems and Society, 321 Richardson Hall, Oregon State University, Corvallis, OR, 97330, USA

E-mail: raj.deepika@fabi.up.ac.za

BMC Proceedings 2011, 5(Suppl 7):P132

Background: Agrobacterium mediated gene transfer technology offers the potential to introduce novel, high-value traits into selected, elite

<table><caption>Table 2(abstract P131) Results of micro-propagation and mini cutting studies of selected pulpwood and bio-energy clones</caption><thead><tr><th>Parameters</th><th>Units</th><th>Melia dubia</th><th>Dalbergia sissoo</th><th>Eucalyptus urograndis</th><th>Eucalyptus tereticornis</th><th>Eucalyptus camaldulensis</th></tr></thead><tbody><tr><td><strong>Imitation</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Optimum Benzylaminopurine</td><td>mg/lit.</td><td>1.0</td><td>1.0</td><td>1.5</td><td>0.5</td><td>0.5</td></tr><tr><td>Initiation rate</td><td>%</td><td>56</td><td>36</td><td>48</td><td>84</td><td>64</td></tr><tr><td>Average shoot length after 30 days</td><td>cm</td><td>1.0</td><td>1.5</td><td>1.0</td><td>3.0</td><td>1.5</td></tr><tr><td><strong>Multiplication</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Optimum BAP</td><td>mg/lit.</td><td>0.25</td><td>0.15</td><td>0.15</td><td>0.15</td><td>0.15</td></tr><tr><td>Average Number of shoots per clump</td><td>no</td><td>2.0</td><td>3.0</td><td>32</td><td>24</td><td>15</td></tr><tr><td>Average shoot length</td><td>cm</td><td>3.0</td><td>2.5</td><td>0.5</td><td>1.5</td><td>1.0</td></tr><tr><td><strong>Elongation</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Optimum Auxin</td><td>mg/lit.</td><td>0.0</td><td>0.0</td><td>0.0</td><td>0.5 (IAA)</td><td>4.0 (NAA)</td></tr><tr><td>Average Number of shoots per clump</td><td>no</td><td>2.0</td><td>2.0</td><td>4.0</td><td>6.0</td><td>4.0</td></tr><tr><td>Average shoot length</td><td>cm</td><td>5.0</td><td>5.0</td><td>3.0</td><td>7.0</td><td>4.0</td></tr><tr><td><strong>Rooting</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Optimum IBA</td><td>mg/lit.</td><td>1.0</td><td>1.0</td><td>1.0</td><td>1.0</td><td>1.0</td></tr><tr><td>Average Number of roots per shoot</td><td>no</td><td>4.0</td><td>4.0</td><td>8.0</td><td>7.0</td><td>5.0</td></tr><tr><td>Average shoot length</td><td>cm</td><td>2.0</td><td>1.5</td><td>2.0</td><td>5.0</td><td>3.0</td></tr><tr><td>Rooting</td><td>%</td><td>90</td><td>40</td><td>100</td><td>100</td><td>100</td></tr><tr><td><strong>Hardening</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Survival</td><td>%</td><td>62</td><td>45</td><td>85</td><td>95</td><td>80</td></tr><tr><td><strong>Mini cutting</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of shoots per plant/ cutting</td><td>no</td><td>4.0</td><td>7.0</td><td>8.0</td><td>6.0</td><td>5.0</td></tr><tr><td>Rooting and survival</td><td>%</td><td>70</td><td>63</td><td>79</td><td>86</td><td>73</td></tr></tbody></table>
tree genotypes. Significant progress has been made in the regeneration and genetic transformation of *Eucalyptus* trees, but transformation efficiency has generally been low for eucalypts [1], especially for elite clonal genotypes. As a preliminary step towards the regeneration of explants derived from clonal material and the production of transgenic plants from commercially important *Eucalyptus* genotypes, there is a need to identify genotypes exhibiting high regeneration capacity. In this ongoing study, we are comparing eight *Eucalyptus* genotypes for callus induction and shoot regeneration potential. Browning of callus tissue and surrounding culture media is a common obstacle limiting regeneration of shoots in eucalypts. Hence, a second aim is to minimize the oxidation of phenolic compounds released from the wounded tissue and improve shoot regeneration rates.

**Methods:** Shoot buds collected from potted ramets of eight *Eucalyptus* clones (Sappi and Mondi, South Africa) were established under *in vitro* conditionson MS [2] basal medium containing BAP (Benzyl adenine purine). The established clones consisted of four *E. grandis* (G1, G2, G3 and G4), one *E. grandis* x *E. nitens* (GN1), two *E. grandis* x *E. camaldulensis* (GC1 and GC2) and one *E. grandis* x *E. urophylla* (GU1) genotype. Leaf explants were excised from *in vitro* shoot cultures and cultured on MS basal medium containing BAP, NAA (Naphthalene acetic acid) and TDZ (Thidiazuron). Browning and necrosis of the callus as well as surrounding culture media was observed in all cultures and PVP (polyvinylpyrrolidone) was therefore added to the media to reduce oxidation.

A standardised regeneration protocol was used for the optimization of *Agrobacterium* mediated genetic transformation of selected *Eucalyptus* clones. The *Agrobacterium tumefaciens* strain AGL1 containing pMDC162 with the *uidA* gene (β-glucuronidase; GUS) and *hpt* (hygromycin phosphotransferase) gene was used for transformation. To minimize extensive exudation of phenolic compounds from the wounded explants, the leaves were subjected to different wounding methods. Intact leaves were excised from *in vitro* shoot cultures and preconditioned for 24 hrs on callus induction medium followed by wounding with a surgical blade prior to *Agrobacterium* infection. The leaves were wounded in two different ways: (a) removing the edges of the leaf, making 8-10 small wounds and cutting the leaf transversally into two equal halves and (b) making 5-6 small wounds in the intact leaf without damaging the rest of the leaf. Alternatively, the leaves were wounded prior to 24 h preconditioning using the first wounding method (a). The explants were immersed in a bacterial solution (A<sub>600</sub> = 0.8) for 1 hr followed by blotting with sterile filter paper. The explants were co-cultivated for 24 hrs and stained for GUS activity after two days to determine the percentage of explants showing transient GUS expression.

**Results and discussion:** Callus initiation was observed at the cut edges and wounded regions of the leaf explants after 20 days of incubation in

<figure><img src="image_61.png" /><figcaption>**Figure 2(abstract P132)** Percentage of leaf explants exhibiting shoot bud induction after one month of culture on shoot regeneration medium containing PVP. Error bars indicate the standard error of the mean of three replicates.</figcaption></figure>

complete darkness at 25 ± 2°C. Shoot regeneration was also observed on the same medium after 30 days of culture. Callus induction percentages of 76% (GU1) to 100% (G3) and shoot regeneration percentages of 3.4% (G4) to 75% (G1) were obtained from the leaf explants (Figure 1). Tissue oxidation was observed in all of the cultures when exposed to light, which hampered shoot bud development and elongation considerably. Addition of PVP to the media resulted in reduction of tissue browning and increased shoot regeneration rate ranging from 6.7% (G4) to 86.7% (G1 and GN1) (Figure 2). Browning of the tissue and surrounding media occurs due to oxidation of the phenolic compounds by polyphenoloxidase, peroxidise or exposure to air which can be reduced by the addition of antioxidants such as PVP to the culture media [3]. Based on the modified protocol including the addition of PVP, *Eucalyptus grandis* (G1) and *E. grandis* x *E. nitens* (GN1) exhibited the highest shoot regeneration potential (86.7%) and may serve as suitable starting material for further genetic transformation studies.

Different wounding methods did not have a significant effect on transient GUS expression in leaf explants. However, good GUS activity was detected in the transformed callus induced from the leaf explants wounded prior to preconditioning.

**Conclusion:** We have achieved efficient and rapid callus induction, as well as shoot regeneration for selected *Eucalyptus* clones. The information presented here forms the basis for ongoing optimization of a protocol for the generation of transgenic plants of clonal *Eucalyptus* genotypes.

**References**

1. Dibax R, Deschamps C, Bespalhok Filho JC, Vieira LGE, Molinarí HBC, De Campos MKF, Quoirin M: *Organogenesis and *Agrobacterium tumefaciens** – mediated transformation of *Eucalyptus saligna* with P5CS gene. *Biol Plant* 2010, 54:6-12.

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

3. Laine E, David A: Regeneration of plant from leaf explants of micropropagated clonal *Eucalyptus grandis*. *Plant Cell Rep* 1994, 13:473-476.

<figure><img src="image_62.png" /><figcaption>**Figure 1(abstract P132)** Effect of genotype on percentage of callus induction and shoot regeneration after one month of culture on regeneration media without PVP. Callus induction rate= Number of explants inducing callus / Total number of leaf explants cultured x 100; Shoot regeneration rate= Number of calli inducing shoots/ Total number of leaf explants cultured x 100. Error bars indicate the standard error of the mean of five replicates.</figcaption></figure>

P133

Screening of genes associated with early stages of adventitious root formation from progenitor adult cells of pine

Dolores Abarca, Alberto Pizarro, Alicia Del Amo, Carmen Diaz-Sala*
Department of Plant Biology, University of Alcala, 28871 Alcalá de Henares,
Madrid. Spain
E-mail: carmen.diazsala@uah.es
BMC Proceedings 2011, **5**(Suppl 7):P133

**Background:** In plants, the possibility to regenerate adventitious roots, shoots or embryos directly from adult tissues has been known for more than 60 years and has been exploited in horticulture, agriculture and forestry [1]. However, little is known about the mechanisms that enable a
somatic differentiated cell to switch its fate into a multipotent, pluripotent or totipotent cell that can develop a root, shoot or embryo or repair damaged tissues [2]. Although apparent dedifferentiation and respecification of cells seems to occur, whether acquisition of competence to regenerate organs occurs, as in animal cells, through dedifferentiation, or whether it is via transdifferentiation or by the use of pre-existent totipotent, pluripotent or multipotent cells in adult tissues remains unknown. In either case, cell fate switches are characterized by remarkable changes in the pattern of gene expression, as cells switch from an expression pattern typical of a somatic cell to a new one directing a new developmental pathway [3]. Thus, determining the way by which cells reset their gene expression pattern, especially for the timetable and repertoire of gene expression characteristic of the earliest stages of normal development, is crucial to understand cellular plasticity. In forest species, a loss of regeneration capacity is associated with tree age and maturation, which makes forest species representative and reliable systems to study how cell fate becomes fixed during development and how plant cells can manage to retain developmental plasticity [4]. The decline in the capacity to regenerate roots from cuttings is one of the most dramatic effects of tree maturation and has been the subject of investigations on the basic nature of the process [5].

**Methods:** An experimental design based on the analysis of temporal and spatial expression of genes during the earliest steps of adventitious root formation, when cell dedifferentiation and reorganization takes place but before the onset of cell division, was set up in pine cuttings to provide additional clues to the process [6,7]. Two approaches were followed: 1. Expression screening using cDNA collections, and 2. Analysis of the expression pattern of candidate genes potentially involved in root meristem specification, such as genes encoding GRAS proteins, transcription factors involved not only in root patterning but also in the establishment of the quiescent centre identity and in the maintenance of the stem cell status of the surrounding initial cells during the embryonic pattern formation and postembryonic development.

**Results and conclusions:** For the first approach, the expression pattern of regulatory genes identified from cDNA collections enriched in auxin-responsive, meristem specific and regeneration capacity specific genes was analysed by cDNA microarray and by qRT-PCR. Gene clustering by functional classification and expression pattern revealed the existence of auxin-dependent and auxin-independent pathways potentially involved in the initial phases of regeneration. In addition, genes specifically expressed either in competent or in non-competent tissues were identified.

For the second approach, fifteen GRAS genes have been identified in the conifer genome using *in silico* analysis of conifer gene databases. Expression analysis in rooting competent and non-competent pine cuttings revealed different patterns of expression during adventitious root induction: 1. genes that were induced in the presence of exogenous auxin within the initial 24 of the root induction process in rooting competent cuttings only (*PrSCL1*), 2. genes that were induced within the initial 24 of the root induction process in rooting competent cuttings only, and whose expression was not dependent on the presence of exogenous auxin (*PrSHR*), 3. genes that were not induced during the adventitious rooting but their level of expression was associated with the developmental stage of the cutting (*PrSCR*), 4. genes that showed a transient short-term expression in both types of cuttings, and 5. genes that did not show an expression pattern related to neither developmental stage nor adventitious rooting competence. Spatial analysis of the expression of two of these genes, *PrSCL1* and *PrSHR*, during adventitious root formation showed a transient increase of mRNA levels asymmetrically localized to the cambial area and rooting competent cells during the earliest stages of adventitious root induction, before the resumption of cell division leading to the root formation pathway, but not in non-competent cuttings. These results suggest the presence of specific cellular signalling pathways or specific factors, perhaps distributed in a localized- or developmental-specific manner, in the tissues involved in rooting. Since auxin transport, accumulation and metabolism do not account for the difference in the rooting capacity of pine cuttings, other factors such as an asymmetric auxin distribution or other determination factors could be involved in the control of age dependent cellular plasticity. Auxin distribution has been analyzed in rooting competent and non-competent cuttings during the initial 24 h of the root induction process. Results showed an asymmetric auxin distribution localized to the cambial area and rooting competent cells in rooting competent cuttings. The spatiotemporal colocalization of auxin accumulation and GRAS gene

expression was observed in rooting competent cuttings only. This result suggests that signaling pathways involving GRAS transcription factors and auxin distribution could be related to the regulation of age-related changes in regeneration capacity.

**Acknowledgements:** This work has been funded by Spanish Ministry of Science and Innovation (AGL2008-05105-C02-01/FOR).

**References**

1. Vasil IK: A history of Plant Biotechnology: from the cell theory of Schleiden and Schwann to biotech crops. *Plant Cell Rep* 2008, 27:1423-1440.

2. Birnbaum KD, Sanchez Alvarado A: Slicing across kingdoms: regeneration in plants and animals. *Cell* 2008, 132:697-710.

3. Terranova R, Pereira CF, Du Roure C, Merkenschlager M, Fisher AG: Acquisition and extinction of gene expression programs are separable events in heterokaryon reprogramming. *J Cell Sci* 2006, 119:2065-2072.

4. Abarca D, Díaz-Sala C: Reprogramming adult cells during organ regeneration in forest species. *Plant Signaling and Behavior* 2009, 4:793-795.

5. Abarca D, Díaz-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.

6. Sánchez C, Vielba JM, Ferro E, Covelo G, Solé A, Abarca D, de Mier S.B, Díaz-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, Díaz-Sala C: Characterization and expression of a *Pinus radiata* putative ortholog to the Arabidopsis *SHORT-ROOT* gene. *Tree Physiol* 2008, 28:1629-1639.

P134

In vitro shoot organogenesis from *Eucalyptus* sp. leaf explants

Juliana Degenhardt-Goldbach¹*, Marguerite Quoirin², Surya Buss³,
Yohana de Oliveira⁴, Luziane Franciscon¹, Isabel Gerhardt¹

¹Embrapa Forestry, Brazilian Agricultural Research Corporation, Colombo,
Paraná, 83411-000, Brazil; ²UFPR, Curitiba, Paraná, Brazil; ³Graduating student
UFPR, Brazil; ⁴PhD student UFPR, Brazil
E-mail: juliana@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P134

**Background:** *In vitro* organogenesis is one of the key techniques associated with genetic transformation, as it determines the successful regeneration of transgenic plants after co-cultivation with bacteria. Therefore, the development of efficient regeneration protocols is the most critical step in developing genetic transformation. Protocols have been developed for several species of eucalyptus. In most of the studies cotyledon, hypocotyl and leaf segments of plants cultivated *in vitro* are used as explants [1]. Several eucalyptus functional genomics projects have used *Populus* species as a model for it counts with well established regeneration and transformation protocols. However, the use of *Eucalyptus* clones as model plants could be more adequate, if clones with high regeneration rates as those obtained for *Populus* could be found. The aim of this study was to evaluate several variables in the *in vitro* organogenesis from leaves of an *Eucalyptus* sp. clone maintained *in vitro* at Embrapa Forestry.

**Methods:** The experiments were performed at the Laboratory of Tissue Culture of Embrapa Forestry, Colombo, PR. Leaf explants were collected from *in vitro* grown plants maintained on MS medium [2] containing 30 g L⁻¹ sucrose, 0.88 µM BAP, 0.05 µM NAA, 0.5 g L⁻¹ and 7 PVP g L⁻¹ agar. The youngest leaves were cut longitudinally and placed with the adaxial side facing the media. In the first experiment, the effect of 0.5 µM thidiazuron (TDZ) was compared with different concentrations of zeatin (2.28, 4.56, 9.12 and 13.68 µM). The plant growth regulators were added to the basic medium, composed of MS salts with half of nitrogen concentration (N/2), supplemented with vitamins of Morel and Wetmore, 30 g L⁻¹ sucrose, 0.1 g L⁻¹ myoinositol, 0.1 µMNAA and 7 g L⁻¹ agar. The second experiment compared the effect of MS salts N/2, WPM [3] and B5 [4] on the same basic medium described above, containing 0.5 µM TDZ. In the third experiment the effect of different concentrations of TDZ (0.25, 0.5, 0.75, 1 and 2 µM) added to the basic medium, replacing the MS N/2 by WPM was evaluated. The explants were kept in growth chamber with