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# Advances of in vitro culture for horticultural breeding

Sergio Ochatt

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Sergio Ochatt. Advances of in vitro culture for horticultural breeding. Symposium of Culture in vitro and micropropagazione in ortoflorofrutticoltura, Agro Bio Frut. FRA., Jun 1989, Cesena, Italy. 321 p. hal-01600687

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CONVEGNO SU

# COLTURE IN VITRO E MICROPROPAGAZIONE

## in ORTOFLOROFRUTTICOLTURA

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Finito di stampare nel mese di aprile 1992
presso GRAFICHE MDM Litografia s.r.l.
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# Advances of *in vitro* culture for horticultural breeding

Summary review of Session 1, 2, 3 of ISHS Symposium on "*In Vitro* Culture and Horticultural Breeding". May 30 - June 3, 1989, Cesena, Italy

SERGIO J. OCHAT T

I.N.R.A., Station d'Amélioration des Espèces
Fruitières et Ornamentales,
49000 Beaucouzé, Angers, France.

## INTRODUCTION

Few tasks can entail more difficulty than having to describe, discuss and critically highlight the breakthroughs in work carried out by other researchers, whilst making justice, in a reduced space, to the quality of the results reported. Such a task becomes even more difficult if one has to deal with a wide array of subjects and a broad variety of objectives pursued in each of such reports. This paper, therefore, is aimed at summarizing, as concisely and comprehensively as possible, the 65 scientific reports communicated during the first three Sessions of the International Symposium on "*In Vitro* Culture and Horticultural Breeding", hosted by the University of Bologna, and held in Cesena, from May 31 to June 3, 1989, under the auspices of the International Society for Horticultural Science. For this purpose, the various reports will be summarized in groups, each corresponding to the respective Session where the reports were presented. As mentioned above, no summary can ever be fully comprehensive and the reader should, consequently, refer to the Symposium Proceedings, published as volume n. 280 of *Acta Horticulturae*, for further details.

## SESSION 1: CELL AND PROTOPLAST CULTURE

This Session comprised 5 oral communications and 8 poster presentations. The scarcity of reports in this Session shows that this is an area of biotechnology that clearly requires further input. It is noteworthy, though, that, probably for the first time ever, half of the results reported in this field (in all 6 papers) was concerned with woody species (trees and shrubs) and also that, after nearly three decades of basic protoplast research, it is now clear that the potential for economic gain through cell and protoplast manipulations will finally be fulfilled and the technology progressively accepted by plant breeders and seed companies.

### a. Cell culture

Only two groups presented results of experiments with cell and callus cultures as such. Forti et al, reported preliminary results on the induction and growth of callus and cell suspension cultures of sugarbeet. Working with 4 male-sterile (CMS) lines and their respective maintainer (ML) lines, these authors found a clear influence of the cytoplasmic genome on the cultural requirements for the efficient induction and growth of callus and cell suspension cultures and also on their subsequent capacity for plant regeneration. In this re-
spect, when a CMS line was compared with its ML line counterpart, differences in cultural requirements were not apparent between them. The reverse was true when comparisons were performed between different CMS-ML couples.

In the second report, Wetten and Cumbus examined the influence of light intensity on callus growth for tomato cv Sonatine, in an attempt to determine the optimum intensity for callus growth and plant regeneration with this cultivar.

## b. Protoplast Culture

Six of the ten reports on protoplast culture dealt with various applications of protoplast technology for the breeding of woody plant species.

Ochatt summarized the advances and breakthroughs, to date, on the use of protoplast-based strategies for top-fruit trees (Rosaceae). For this group of species, some exciting results have been reported in the last few years. It is now possible to regenerate whole trees from cultured protoplasts of the fruit tree rootstocks wild pear (Pyrus communis var pyraster L.), Colt cherry (Prunus avium x pseudocerasus), sour cherry (P. cerasus L.) clones CAB 4D and CAB 5H, and M.9 apple (Malus domestica Borkh.), and for the scion varieties Conference and Williams' Bon Chretien pears (Pyrus communis L.), Spartan apple (Malus domestica Borkh.) and kiwifruit (Actinidia chinensis var chinensis Planch.). Noteworthy in this respect, was the development of a new strategy for plant regeneration from callus, of several genotypes, with rhizogenesis as an intermediate step. It was also reported that electroporation of freshly isolated protoplasts (of Colt cherry, Conference pear, and of several other woody and herbaceous species) markedly enhances DNA synthesis, the onset of division of protoplast-derived cells, the subsequent growth of protoplast-calli, the shoot bud regeneration ability from such calli and the rooting capacity of the protoplast-derived shoots thus produced. Highly significant, in terms of a direct application for breeding, are somaclonal variation and somatic hybridisation. In this context, the production, via a direct recurrent selection strategy, of stable, salt and drought tolerant protoplast-derived trees of the cherry rootstock Colt was reported. In addition, results were summarized on the recovery of intergeneric (intersubfamilial) somatic hybrid fruit tree rootstocks, wild pear (+) Colt cherry (Pyrus communis var pyraster L., Pomoideae (+) Prunus avium x pseudocerasus, Prunoideae). Since these novel somatic hybrid rootstocks proved graft-compatible with species from both parental subfamilies, they could provide a basis for further studies on the cellular mechanisms underlying graft-compatibility in rosaceous top-fruit trees.

Patat-Ochatt and Power reported on the regeneration of partially differentiated calli from callus protoplasts of the apple scion, Bramley's Seedling, and of complete plants from leaf mesophyll protoplasts of MM. 106 apple rootstock. With the latter genotype, shoot bud regeneration ability was retained over an extended period (>12 months), whilst the overall quality and rootability of the protoplast-derived shoots was significantly improved by transferring the differentiated calli, with shoots still attached, from the regeneration medium, to a medium as used for the micropropagation of parental axenic shoots, but with an increased vitamin concentration.

Marino, reported on the proliferation of cell colonies from the, generally regarded as recalcitrant, cultured mesophyll protoplasts of grapevine.

As for forest tree species, Lineberger and colleagues reported very interesting results with protoplast-derived trees of elm (Ulmus X Pioneer), where protoclonal variation for an enhanced resistance to the dutch elm disease (Ceratocystis ulmi) was fixed in clones
through asexual propagation. This, in turn, shows that protoplast fusion and somatic hybridisation may not always be a sine qua non requisite for the fixation of valuable, newly acquired/selected horticultural traits.

In a clarifying lecture, Zelcer stated the differences between the strategies for production of symmetric hybrids (i.e. those involving the full genome) and asymmetric hybrids or cybrids (those involving the partial transfer of nuclear traits from a donor species, using the, so-called, donor-recipient scheme). The potential uses and applications of these strategies was exemplified with the production of cybrids of *Lycopersicon peruvianum* (+) *L. esculentum* and symmetric hybrids of *Solanum muricatum* (+) *Lycopersicon esculentum*.

Additional demonstration of efficient cybridizations was provided by Vardi and co-workers, with the recovery of cybrid trees between *Microcitrus* and *Citrus* species (e.g. sour orange, raugh lemon), that are now growing in the greenhouse for further evaluation.

For herbaceous species, Al-Atabee and Power, working with ornamental members of the typically recalcitrant *Compositae* family, reported on the production of callus from protoplasts of *Ageratum*, *Calendula* and *Tithonia* species, coupled with regeneration of complete protoplast-derived plants for *Rudbeckia purpurea* and *R. laciniata*, the latter including rhizogenesis as an intermediate step. In addition, De Donato et al successfully regenerated complete atrazine-resistant plants from protoplasts of similarly resistant biotypes of *Solanum nigrum* L., thereby showing the retention of useful traits following regeneration of plants via protoplast culture.

Amongst basic experiments using protoplasts, Bagni and Pistocchi examined the uptake of polyamines by cells, protoplasts and organelles of carrot and *Helianthus tuberosus*, whilst Lucretti et al employed a novel technique, fluorescence activated cell sorting, to assess the ploidy level (i.e. the nuclear DNA content), heterogeneity and viability as related to the cell wall regeneration capacity for protoplasts of several cultivars of potato and various *Citrus* lines.

Finally, also included in Session 1 was an intriguing talk by Herman. Based on a few, mostly unpublished examples suggesting an enhancing effect of a mild contamination, *in vitro*, on the subsequent *in vitro* growth and *ex vitro* weaning and for several species, he posed the question "Do we need to worry about bacterial contamination?". This question though still remains unanswered.

## SESSION 2: ORGANOGENESIS AND EMBRYOGENESIS

During this Session 13 oral communications and 28 poster presentations were reported, out of which 29 dealt with organogenesis and 20 with embryogenesis. The opening lecture for this Session addressed the genetic and physiological approaches for a successful organogenesis and embryogenesis (Nuti Ronchi), and was followed by a fully comprehensive review on the uses of tissue culture for the genetic improvement of grapevines (Mullins), where the breakthroughs and unsolved problems, to date, were highlighted.

### a. Organogenesis

In recent years, the induction of shoot buds from leaf discs is a technique that has gained considerable momentum, and many workers around the world are now developing
such a strategy with a view to its application for a wide array of objectives, including large-scale propagation, the induction and exploitation of somaclonal variation and the recovery of transgenic plants.

Against this background, 10 of the papers dealing with organogenesis reported during this Session were concerned with the regeneration of shoot buds from leaf discs, and in 6 out of them the experimental material used was apple leaves (Ancherani et al; Druart; Dufour; Fasolo and Predieri; Perfanova; Theiler-Hedtrich and Theiler-Hedtrich). In these reports, the conditions for culture, the hormonal composition of the medium, the culture atmosphere, the type of vessel used, the influence of light/dark during culture, the use of different cytokinins and caseine hydrolysate, the size of explants and their position on the medium and in the mother plant, were all assessed for their effects on the efficiency of shoot bud regeneration from the cultured leaf discs. In addition, a pre-dipping treatment of wounded leaves into hormonal solutions (Druart) or the irradiation of leaf discs with gamma rays (up to 500 rad) prior to culture determined a marked increase in the capacity for adventitious shoot bud regeneration from the apple leaf discs.

Regeneration was also reported from leaf discs of *Prunus canescens* (Druart), the dwarfing cherry rootstock *P. incisa x serrula* (Druart), *Lisianthus russellianus* (Ruffoni et al) and *Passiflora coerulea*, where ascorbic acid proved beneficial for the differentiation of adventitious shoot buds (Bennici et al).

Leaves were also used as a callus source wherefrom shoot bud regeneration was subsequently obtained, and two such reports, both for kiwifruit, were reported during the Symposium. Thus, Marino and Battistini examines the effect of media pH (5. 7-8.5) on callus growth and shoot regeneration abilities, and they found that at the pH optimum for callus growth (i.e. 5.7) shoot regeneration and survival were reduced, as compared to responses in this respect at higher pHs. In addition, Muleo and Morini examined the effects of culture in dark or under three different light qualities (blue, red and fluorescent) on the organogenic competence of kiwifruit leaf callus. Roots were regenerated earlier in culture when callus were maintained under red light, as compared to the other culture conditions assessed, whilst shoot bud regeneration occurred under red light only.

Several results were reported, dealing with growth and regeneration from callus of other explant origins, including both embryo/seedling and non-seedling explants as the callus source.

Amongst reports based on embryo/seedling-derived callus cultures, Rota and coworkers reported a protocol for efficient shoot bud regeneration from immature cotyledons of the bean, *Phaseolus coccineus*. Additionally, by linking culture of *Citrus aurantifolia* hypocotyls with a short-term (6 h) antimetabolite treatment (cycloheximide, actinomycin-D), Dua and Garg dramatically increased rhizogenesis from the cultured hypocotyl explants. Fari and Thury comparatively studied the shoot regeneration capacity of seedling explants (cotyledons, shoot tips, upper and basal hypocotyl sections), and they found that optimum responses were obtained on cotyledons, while a basipetal reduction of organogenic competence was apparent for the other explants examined.

Plant regeneration was also reported from callus cultures derived from mature embryos of *Alstroemeria* (Gonzalez-Benito and Alderson), whereas Leva and Falcone reported preliminary results on attempts at the *in vitro* propagation and introduction of organogenesis from cotyledons and leaves taken from very young seedlings of *Anacardium occidentale*.

Very encouraging results on the elusive field of induction of triploidy were reported by Kagan-Zur et al, who reported on the proliferation of callus from tomato endosperm.
Moreover, they succeeded to differentiate roots and eventually shoots from such calli and, although the shoots thus produced failed to survive, these results represent a significant step forward towards the production of triploid plants from endosperm explants.

Reports concerned with the induction of callus and the subsequent differentiation of shoots from them, using non-seedling explants other than leaves, included shoot regeneration from immature (12 days post-blooming) achenes of strawberry (Lis), the differentiation of dark-green nodules from cell suspension-derived callus and of adventitious buds from shoot segment-derived calli of carnation (Radojevic et al), whilst Ruffoni et al, also working with carnation, reported on the regeneration of shoot buds from meristem-derived callus. Finally, Mensuali-Sodi and colleagues reported on the regeneration of bulblets on scales taken from *in vitro* cultured bulblets of *Lilium speciosum* and *L. longiflorum*. They also examined the involvement of ethylene in regeneration competence and observed that ethylene levels were inversely correlated to rooting for *L. speciosum*, whilst no such relationship could be detected for *L. longiflorum*.

Other studies reported included the establishment of a *Ribes* germplasm collection *in vitro*, coupled with preliminary results on the long-term, low-termerature storage of such a collection (Brennan et al), the fiel evaluation (in terms of yield) of androgenetic lines of eggplant (Sanguineti et al), and several quite interesting observations on the implications of use of growth retardants (daminozide, ancymidol, paclobutrazol, uniconazole) in liquid cultures of *Gladiolus* in bioreactors (Ziv). All growth retardants examined increased the number of buds produced per explant, reduced leaf elongation and enhanced cormlet production, which could all aid in the scaling-up of cultures for micropropagation purposes. Care should be taken, however, since some growth retardants (paclobutrazol, uniconazole) may have a carry-over inhibiting effect on shoot development in soil. Results were also presented on the regulation of organogenesis (rooting and shoot branching) with jasmonic acid, for stem cuttings and meristems of potato, grapevine and bean plants cultured *in vitro* (Ravnikar et al) The authors found that 0.001 to 1 µM jasmonic acid significantly increased the length and fresh weight of plants and stimulated lateral root formation, while media with higher (to 100 µM) concentrations of jasmonic acid resulted in a shortening of main roots and coupled with an increased root diameter.

Rooting *in vitro* of microcuttings is still difficult for several important horticultural crops, and results were reported on the fate and relationship between basic peroxidases and the rooting of *in vitro*-produced shoots of apple (De Klerk et al), while Lankes and Zimmerman examined the impact of osmotic potential on shoot multiplication and the subsequent rooting of microcuttings for apple. Finally, giving a clear indication of the present importance of *in vitro* culture techniques, results were also presented on the growth of plants and tissue cultures on a stimulated lunar soil (Venketeswaran et al).

## b. Embryogenesis

Janick and Kim comprehensively reviewed the "state of the art", in terms of the induction of embryogenesis and the production of "synthetic seeds". The various cultural stage and their requirements were exemplified with carrot and celery as the model systems. Thus, the strategy for producing synthetic seeds consists, basically, of drying the embryos produced and coating them with polyethylene oxide. Attention was drawn to the size/developmental stage of embryoids, as this affects their subsequent survival. In this context, the use of sucrose gradients may help to separate germinating embryos from an
embryogenic suspension and, hence, enhancing embryo survival.

Wetzstein and colleagues critically examined the factors influencing somatic embryogenesis and plantlet regeneration in pecan (*Carya illinoensis*), and they reported that maximum embryogenesis occurred on zygotic embryos, placed into culture 15 weeks post-pollination. Germinability of the embryos produced was limited, but their conditioning (under a low temperature [4 °C] and with dissecession) prior to light culture in a half-strength Murashige and Skooh medium improved it.

A method of scaling-up somatic embryogenesis for celery was developed by Nadel et al. They reported that the addition of mannitol to the medium increased the number of embryos produced, enhanced their growth and produced more singular embryos. In addition, synchronization of the embryogenic suspensions was achieved by separation of the somatic embryos of the various sizes by differential sieving, and it resulted in a clear improvement of the conversion of embryos to plantlets. Likewise, abscisic acid was reported to induce high frequency somatic embryogenesis from leaves of the tropical tree, *Sapindus trifoliatus*, with high sucrose concentrations significantly improving the frequency of normal embryos produced (Krishnan et al).

Antonelli induced somatic embryogenesis on the surgace of immature cotyledons of almond, and obtained elongated embyoids. Convention of these to plantlets, though, took place only occassionally. Similarly, Curir and co-workers succeeded to induce somatic embryogenesis in immature zygotic embryos of *Genista monosperma* Lam., but all attempts at germination met with failure, while Piagnani and Eccher also obtained somatic embryos from chestnut cotyledons but reported only the initial stages of germination for such embryos.

On the other hand, the successful recovery of plants from somatic embryos was reported for *Prunus incisa x serrula* (Druart), peach (Scorza et al), orange (Starrantino and Caponetto), the winged bean, *Psophocarpus tetragonolobus* (Venketeswaran et al), *Alstroemeria* (Gonzalez-Benito and Alderson) and banana (Ma et al). Particular significance, amongst these reports, was attached to the results with peach, where somatic embryos derived from immature sexual embryos have continuously proliferated *in vitro* for over 3 years (Scorza et al). Also significant are two reports that were the only ones not to use a seedling explant as the source of embryogenic suspensions, i.e. the results for banana, where embryogenic suspensions were established from callus induced from sections of inflorescence tips (Ma et al), and those reported by Druart for the dwarfing cherry rootstock, *Prunus incisa x serrula*, where somatic embryos and embryogenetic calli were induced on the roots of *in vitro* propagated plants.

## SESSION 3: ANDRO/GYNOGENESIS

Only 10 papers were presented in this Session, but they included some of the most exciting results reported during the Symposium, in terms of their immediate agronomic/ horticultural value.

Zhang and co-workers showed results on the production of haploid apple trees from *in situ* parthenogenesis, induced by irradiated pollen, followed by rescue and *in vitro* culture of immature embryos. The haploid apple trees thus produced were successfully established in soil and are of direct applicability for breeding purposes (as homozygous parents, for the study of inheritance of horticultural traits and for mutation breeding). They could also serve as a basis for studies of cytology and cytogenetics and for genetic ma-
nipulations, such as somatic hybridisation and genetic transformation.

Hoefer and Hanke reported on the production of callus from anthers of sweet cherry, and coupled with the formation of embryos, without any callus phase, from anthers of apple.

For *Vitis*, Cersosimo et al obtained organogenic and embryogenic plants from cultured anthers. All the anther-derived plants, however, were diploid. Likewise, diploid plants only were occasionally obtained from cultured anthers of several *Citrus* species (Geraci and Starrantino).

For herbaceous species, 5 papers on androgenesis were communicated. Chiang et al examined the progeny of anther culture-derived 2-X cabbage plants, in terms of their fertility and meiosis, and they identified male-sterile and male-fertile plants amongst the first generation of such anther culture-derived individuals.

Vischi and colleagues studied the phenological stages of microsporogenesis and capitulum size in *Gerbera* and produced callus from cultured anthers with uninucleate microspores, while Fuller et al reported on the recovery of plants, via embryogenesis, from anther cultures of cauliflower. Haploidy of the regenerated cauliflower plants was not confirmed. Similarly, for strawberry, the production of anther callus-derived plants was reported, but haploidy of such plants was not confirmed (Svensson).

In the field of gynogenesis, only two papers were communicated, both of them very significant. In this respect, Zhang et al obtained embryos from apple ovules, while Tosca and colleagues successfully produced haploid plants from cultured ovules of 5 genotypes of *Gerbera jamesonii*.

## CONCLUDING REMARKS

A rapid overview of the experimental results reported during Session 1, 2 and 3 and, indeed, during the Symposium as a whole, clearly show the importance that the developed and newly emerging biotechnological approaches, based on *in vitro* culture, have for the breeding of horticultural crops. It also appeared that there are still areas of biotechnology where further input is required. This, however, will surely take place in the near future, since an ever increasing number of scientists and commercial organizations in the world are now investigating the uses and applications of *in vitro* culture in the context of the improvement of horticultural crop species.

## REFERENCES

### SESSION 1: CELL AND PROTOPLAST CULTURE

* AL-ATABEE J., POWER JB.: *Protoplast isolation and plant regeneration in ornamental Compositae*. Poster

* BAGNI N., PISTOCCHI R.: *Polyamine uptake in protoplasts and plant organelles*. Poster

* DE DONATO M., MOZZETTI C., PERUCCO E.: *Plant regeneration from protoplasts of Solanum nigrum L. atrazine-resistant. Evaluation of resistance in regenerants*. Poster.

* FORTI E., MANDOLINO G., RANALLI P.: *Callus and cell cultures in sugarbeet*. Poster

* HERMANN EB: *Non-axenic plant tissue culture. Possibilities and opportunities*. Contributed Paper

* LINEBERGER RD, STICKLEN MB, PIJUT PM, KROGGEL MA, FINK CVM, DOMIR SC: *Use of protoplast, cell and shoot tip culture in an elm germplasm improvement program*. Contributed Paper
LUCRETTI S., CHAPMAN JV, LISTER A., MORETTI F., DE GIACOMETTI F., IANNI PALARCHIO P.: Flow cytometric assessment of viability and differentiation in potato and Citrus cell suspension protoplasts. Poster

MARINO G.: Isolation and culture of mesophyll protoplasts of grape (Vitis vinifera), cv 'Trebbiano di Roma-gna". Poster

OCHATT S.J.: Protoplast technology and top-fruit tree breeding. Invited Paper

PATAT-OCHATT E.M., POWER J.B.: Advances in plant regeneration from apple protoplast. Poster

VARDI A., GONEN P., BLEICIIMAN S., FRYDMAN-SHANI A., GALUN E.: Citrus cybrids: transfer of Microcitrus organelles into Citrus cultivars. Cintributed Paper

WETTEN A., CUMBUS I.P.: Influence of light intensity on tomato callus growth. Poster

ZELCER A.: Cell engineering by protoplast fusion: implications for plant improvement. Invited Paper

SESSION 2: ORGANOGENESIS AND EMBRYOGENESIS

ANCHERANI M., PREDIERI S., ROSATI P.: Adventitious shoot formation from in vitro leaves of MM106 apple rootstock. Poster

ANGELINI ROTA R., GENGA A., ALLAVENA A.: Tissue cultures of bean (P. coccineus L.) and their applications to breeding. Poster

ANTONELLI M.: Somatic embryogenesis on immature almond cotyledons. Poster

ANTONELLI M., DRUART PH: The use of a brief 2,4-D pretreatment to induce leaf regeneration on Prunus canescences. Contributed Paper

BENNICI A., VESTRI F., SCHIFF S.: In vitro plantlet regeneration in Passiflora coerulea. Poster

BRENNAN R.M., MILLAM S., DAVIDSON D., WILSHIN A.: The establishment of an in vitro Ribes germplasm collection and preliminary investigations into long term low temperature germplasm storage for use in a Ribes genetics programme. Poster

CURIR P., RUFFONI B., MASSABÒ F., DAMIANO C.: Induction of somatic embryogenesis in Genista monosperma Lam. Poster

DE KLERK G.J., TER BRUGGE J., SMULDERS R., BENSCHOP M.: Basic peroxidases and rooting in microcuttings of Malus. Contributed Paper

DRUART PH: Effect of culture conditions and selected leaves on adventitious caulogenesis of Prunus canescens Bois GM 79 and Malus domestica Borkh. cv McIntosh "Wijeik". Poster

DRUART PH: Improvement of the somatic embryogenesis of the cherry dwarf rootstock INMIL/CM9 by the use of different sugars. Poster

DUA IS., GARG N.: Antimetabolite cycloheximide actinomycin-D-IAA induced rhizogenesis in Citrus aurantifolia and its in vitro monoeuvrability by nutritional and regulatory factors. Poster

DUFOUR M.: Adventitious shoots in apple: dramatic improvement of yield. Contributed Paper

FARI M., THURY Z.: Comparative studies on in vitro plant regeneration from seedling expants of hot pepper varieties (Capsicum annuum L.). Poster

FASOLO F., PREDIERI S.: Cultivar dependent responses to regeneration from leaf in apple. Contributed Paper

GONZALES BENITO E., ALDERSON PG: Regeneration from Alstroemeria callus. Poster

JANICK J., KIM YH: Synthetic seeds. Invited Paper

KAGAN-ZUR V., MILLS D., MIZRAHI Y.: Callus formation from tomato endosperm. Poster

LANKES C., ZIMMERMAN RH: Impact of osmotic potential on in vitro cultures of apple. Contributed paper

LEVA A., FALCONE AM: Propagation and organogenesis in vitro of Anacardium occidentale L. Poster

LIS EK: In vitro clonal propagation of strawberry from immature achenes. Poster

MA SS, HUANG PL, LIAUH YW, PUNG CL, HUANG IC, LU CC, HER JV: Somatic embryogenesis and plant regeneration from suspension cultures of banana. Poster

MARINO G., BATTISTINI S.: Leaf callus growth, shoot regeneration and somaclonal variation in Actinidia deliciosa: effect of media pH. Contributed Paper

MENSUALI SODI A., PANIZZA M., TOGNONI F.: Ethylene involvement in vitro in regeneation of Lily. Poster

MULEO R., MORINI S.: Effect of light quality on callus regeneration of Actinidia deliciosa. Poster

MULLINS MG: Tissue culture and the genetic improvement of grapevines: a review. Invited Paper

NADEL BL. ALTMAN A., ZIV M: Regulation of large scale embryogenesis in celery. Contributed Paper

NUTI RONCHI V.: Organogenesis and embryogenesis: genetic and physiological approaches. Invited Paper

PERFANOVA A.: Organogenesis from apple leaf tissues: Studies and application. Poster

PIAGNANI C., ECCHER T.: Somatic embryogenesis in chestnut (Castanea sp.). Poster

RADOJEVIC L., DJORDJEVIC N., PETROVIC J.: In vitro culture techniques for carnation cultivars breeding. Poster
RAVNIKAR M., GOGALA N., RODE J: *The regulation of organogenesis with jasmonic acid in vitro*. Poster

RUFFONI B., DAMIANO C., MASSABÒ F.: *Organogenesis and embryogenesis of Lisianthus russellianus Hook.* Contributed Paper

RUFFONI B., MASSABÒ F., CURIR P., DALLA GUIDA C.: *Adventive regeneration in cell colonies of car- nation*. Poster

SANGUINETI MC, TUBEROSA R., CONTI S.: *Field evaluation of androgenetic lines of eggplant*. Poster

SCORZA R., CORDTS J., MANTE S.: *Long-term somatic embryo production and plant regeneration from embryo-derived peach callus*. Poster

STARRANTINO A., CAPONNETTO P.: *Effect of cytokinins on embryogenic callus formation from undeveloped ovules of orange*. Poster

THEILER-HEDTRICH C., THEILER-HEDTRICH R.: *Influence of TDZ and BAP on adventitious shoot regeneration from leaves of Malus sp.*. Poster

UNNI KRISHAN, MEHTA AR, BHATT PN: *Abscisic acid induced high frequency embryogenesis from Sapindus trifoliatus leaves*. Contributed Paper

VENKETESWARAN S., HENNINGER DL, DIAS K., DIAS MADL, WEYERS UV: *Growth of plants and plant tissue cultures on similated lunar soil - implications for a lunar CELSS*. Contributed Paper

VENKETESWARAN S., DIAS MADL, WEYERS UV: *Organogenesis and somatic embryogenesis of winged bean callus Psophocarpus tetragonolobus (L.)DC*. Poster

WETZTEIN HY, AULT JR, MERKLE SA: *Factors influencing somatic embryogenesis and plant regeneration in Pecan, Carya illinoensis*. Contributed Paper

ZIV M.: *The effect of growth retardants on shoot proliferation and morphogenesis in liquid cultured plants*. Poster

## SESSION 3: ANDRO-AND GYNOGENESIS (HAPLOIDS)

CERSOSIMO A., ALTAMURA MM, CRESPAN M.: *Embryo-organogenesis plant regeneration from anther culture in Vitis sp.*. Contributed Paper

CHIANG MS, CHONG C., CRETE R., FRECHETTE S.: *Fertility and meiosis in anther culture-derived cab- bage 2x-plants*. Contributed Paper

FULLER MP., TURTON S., GROUT BWW: *Anther culture of winter heading cauliflower*. Poster

GERACI G., STARRANTINO A.: *Trials to regenerate haploid plants from in vitro cultures of Citrus anthers*. Contributed Paper

HOEFER M., HANKE V.: *Induction of androgenesis in vitro in apple and sweet cherry*. Poster

PUNDEVA R., ZAGORSKA N.: *Induced callusogenesis and embryogenesis in Capsicum anther cultures*. Poster

SVENSSON M.: *Anther culture of Fragaria x ananassa*. Poster

TOSCA A., LOMBARDI M., MARINONI L., CONTI L., FRANGI P.: *Genotype response to in vitro gynogenesis technique in Gerbera jamesonii*. Poster

VISCHI M., CIPRIANI G., MARCHETTI S., OLIVIERI AM: *Relationship between microsporogenesis and capitulum size in Gerbera and first results of anther culture*. Contributed Paper

ZHANG Y, LESPINASSE Y., CHEVREAU E: *In vitro research for haploidy in fruit trees: pratical results and prospects*. Invited Paper
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