4. TOWARDS A BETTER UNDERSTANDING OF GRAPE
4.1. Ontogenesis and differentiation of somatic embryogenesis
Various explants have been employed for obtaining somatic embryogenesis in grape (Table 13.1); however, the male flower is the most commonly used. Anther tissues, in- deed, proved to be highly competent for embryogenic callus initiation; besides, this or- gan could be interesting for the potential production of dihaploid plantiets when morpho- genesis may arise from pollen grains.
For many crop species, haploid production has been one of the most promising appli- cations of plant tissue culture, and a consistent amount of research has been developed starting from the 1960's; today, even considering the potential of dna-recombinant tech- nologies that address attention to genetic transformation, dihaploids are still a relevant objective of plant breeding (Guha-mukherjee, 1999). In the view of this latter application of somatic embryogenesis, several histological studies have been carried out for assess- ing the somatic or gametic origin of anther-derived plantlets in grapes.
In one case only, regeneration of putative haploid plants from grape anther cultures was reported, and refers to a local Chinese variety named Shengli (Zou and Li, 1981).
Here, within a various degree of ploidy levels (di- tri- and tetra-ploidy), haploid chromo- some sets (n = 19) were found in the root tip cells of regenerated plantlets. Recently, such a mixoploid population of cells was also found in anther-derived plantlets of V. lati/olia, a wild relative of grapevines (Salunkhe et aI., 1999). However, it has generally been re- ported that embryos regenerated from in vitro anther cultures derive from the diploid cells of the connective tissues, and are therefore of somatic origin.
In a study conducted on callus induction and further regeneration of embryogenic cal- lus and plantlets in two grape hybrids (Gloryvine and lS. 23-416), Rajasekaran and Mullins (1983) excluded any haploid origin of somatic embryogenesis obtained from anthers. Callus was developed from the connective tissues of the anther filaments or lo- culi. Identical results were obtained during anatomical studies using light microscopy in V. rupestris cv du Lot (Newton and Goussard, 1990), where the callus was observed to arise from the abaxial side or the lateral walls of the anther, the filament, and all connec- tive tissues between the locules. In the same genotype, endothecium resulted in the only anther tissue non-participating in callusing (Altamura et aI., 1992). Involvement of the endothecium, on the other hand, was described in V. vinifera cv Grenache noir, where
342 L. MARTINELLI and 1. GRJBAUDO
direct proembryo development was observed from these tissues (Faure 'et al., 1996a). In this latter genotype, somatic origin of embryogenesis was also proven, and microspore degeneration was documented during the callus culture progression (Faure et aI., 1996a).
Further evidence of a somatic origin of callusing was given by the work of Rajasekaran and Mullins (1983) when cariotipic characterization was conducted in the callus and bio- chemical and ampelographic parameters were further considered in the regenerated plant- lets. In the callus stage, a predominance of cells with 2C- and a consistent population with 4C- nuclear DNA levels were scored, while a low percentage of haploid sets of chromo- somes (5% and 1% of cells of the total after respectively 10 and 20 days of callus induc- tion) as well as of cells with 1 C-nuclear DNA (1.5% after 10 days of callus induction) were found. As for the plantlets, the identity of the isoenzyme banding patterns obtained in both anther-derived plants and original genotypes for indophenol oxidase, leucine amino pepti- dase and catechol oxidase was considered a proof of no involvement of pollen grains in the embryogenesis; besides, according to those authors, the high degree of somaclonal varia- tion occurring in the leaf shape of the Rl self crossed plants would exclude a homozygous diploid origin related to a doubling of a haploid nucleus.
Since regeneration of haploid plants from anthers has generally failed, microspores have been used as explants, as an attempt at completely excluding somatic anther tissues from the cultures. Production of haploid callus (Gresshoff and Doy, 1974) and the de- velopment of globular structures - however not producing viable embryos - was docu- mented from microspores of Vitis rupestris cv du Lot microspores (Altamura et al.,
1992) and, recently, of several grape genotypes (V. vinifera cv GrUner Veltliner and various Vitis interspecific hybrids) (Sefc et aI., 1997).
Generally, in most explants, somatic embryogenesis is indirect, i.e. it takes place after completion of callusing induction (Fig. 13.5); however a primary embryogenetic process arising directly from the mid vein of the leaves (Stamp and Meredith, 1988a), from the anther endothecium (Faure et al., 1996a) and from zygotic embryos as well (Stamp and Meredith, 1988b) was described.
Even within the large variability observed among the different genotypes, explants and protocols (Table 13.1), the shift from undifferentiated callus to embryogenic callus generally takes place after several months of induction: three months were reported for different V. vinifera cultivars (Krul and Worley, 1977; Vallania et aI., 1994) and up to 7 months in Vitis rupestris (Martinelli et aI., J993a). Once induced, however, the embryo- genic competence is retained for long periods, up to several years (Matsuta and Hiraba- yashi, 1989; Perl et aI., 1995; Tsolova and Atanassov, 1996; Torregrosa, 1998; L. Marti- nelli, E. Candioli, D. Costa, V. Poletti, and N. Rascio; L. Martinelli, I. Gribaudo, D. Ber- toldi, E. Candioli, and V. Poletti, unpublished).
Embryogenic callus is typically granular and white, and is composed of clusters of small isodiametric cells, interspersed with larger vacuolated cells (Gray and Mortensen, 1987); it is usually associated with a dark callus (Fig. 13.5) which, after a biochemical analysis, proved to be non viable, autolyic tissue (Gianazza et aI., 1992). The relation- ship between necrotic cells and somatic embryo initiation is not clear, and it has to be proven whether embryogenesis would be the cause or the result of cell lysis (Krul and
SOMA TIC EMBRYOGENESIS IN GRAPEVINE 343
Figure 13.5. Somatic embryogenesis from ovary cultures of Vilis vinifera cv Brachetto. Embryos develop asynchronously from the callus.
Worley, 1977; Newton and Goussard, 1990; Gray, 1992; Robacker, 1993).
In the callus, embryoid formation involves both internal ("deep genesis") and periph- eral layer ("superficial genesis") of single cells (Altamura et at., 1992). Embryogenic cells are easily identified by their prominent nuclei and nucleoli, by a small size and a dense cytoplasmic content (Krul and Worley, 1977; Newton and Goussard, 1990; Alta- mura et aI., 1992; Faure et al., 1996a and 1996b).
A strong correlation between the genesis of somatic and zygotic embryos has been well documented in Vitis rupestris cv du Lot (Altamura et aI., 1992) and in V vinifera cv Grenache noir (Faure et al., 1996a and 1996b): by the time of the first division of the proembryonic cell, the polarity is established with the formation of two cells, homolo- gous to the basal and the apical zygotic ones, giving rise respectively to the suspensor and to the sensu stricto embryonic cells; moreover, a further Asterad type development occurs, and subsequent progression of somatic embryos follows the canonic phases (globular, heart-shaped, torpedo), up to the conversion into plants.
4.2. Molecular markers for somatic embryogenesis characterization
Recently, in model plants, research aiming to understand molecular and genetic mecha- nisms leading to somatic embryogenesis has been developed (de Vries, 1998). However, basic studies on morphogenesis are very limited in the Vitis genus, principally because of the low success during in vitro cultures.
As already discussed, morphological development during somatic embryogenesis has been well documented in Vitis; however, there is a lack of publications reporting correla-
344 L. MARTINELLI and I. GRIBAUDO
tive differences at molecular level related to developmental steps. The knowledge of the biochemical changes occurring during morphogenesis would provide suitable markers for predicting the embryogenic potential of cultured explants and calli and for allowing an objective selection among cultures. The biochemical changes associated with morphogene- sis, from explant to plantlet, were analyzed in Vitis rupestris S.: the analysis of the electro- phoretic patterns of total proteins (Gianazza et al., 1992) and specific enzymes (acid phos- phatase, alcohol dehydrogenase, esterase) (Martinelli et al., 1993 b) proved to be effective approaches to the characterization of the main steps of somatic embryogenesis.
The developmental stages between primary callus and the embryogenic callus, and between embryo isolation from embryogenic callus and plantlet formation, were charac- terized, and appeared to be different from the point of view of the appearance of a stage-specific set of peptides. The comparison of the different stages leading from callus to embryogenic callus, to embryo development and to plantlet formation, revealed typi- cally different two-dimensional electrophoretic patterns. In particular, dramatically dif- ferent patterns were found between non-embryogenic and embryogenic calli (Fig. 13.6), suggesting a massive shift in gene expression involved in the arising of the embryogenic competence in callus. This last result also suggests that activation of many genes' expres- sion is necessary for embryogenesis during the callus culture, and therefore both the low frequency (4%) and the slow progression (up to 7 months) from callus to embryogenesis can be justified (Martinelli et aI., 1993a). In addition, during embryo development and piantlet formation, a simplification of the two-dimensional protein pattern occurred, sug- gesting a less radical change in gene expression; accordingly, these latter developmental steps progressed much faster.
Also, the obtained zimograms for ADH, AcP and EST observed were different either in the specific activity or in the number and isoelectric point of the expressed isoforms (Martinelli et aI., 1993b). The differences detected were always related to the subsequent steps of embryogenesis (i.e. the enzymatic changes observed in embryogenic callus were always retained by developing embryos). Moreover, typical isoenzyme level and types of embryo status were not retained in the callus differentiated from somatic embryos, suggesting differences in gene expression.
Polyamine content has been analysed for characterizing the principal steps from early- to torpedo embryos in V vinifera cv Grenache noir (Faure et aI., 1991). Signifi- cant changes in the free polyamine content and in the putrescine/spermidine ratio during the development of somatic embryos was assayed. It is noteworthy that polyamine levels (and in particular the putrescine component) were considerably higher in somatic than in zygotic embryos when comparing equivalent developmental stages.
Recently, identification of grapevine histone proteins has been carried out in the root- stock 41 B (Redon et af., 1999). The HI forms of the histone proteins have been consid- ered as a suitable marker for early stage somatic embryogenesis characterization (Redon et aI., 1996). In the yOlmg embryogenic clusters and in the globular embryos, five HI histone variants have been distinguished on the basis of molecular weight, and the relative ratio has been determined. Both qualitative and quantitative shifts of this component have been as- sayed during the further heart stage development. Since histone modifications have been re-
SOMATIC EMBRYOGENESIS IN GRAPEVINE 345
Figure 13.6. Two-dimensional protein pattern of callus from petiole (a) and embryogenic callus (b) of Vitis rupestris S. Run : first on a 4-10 non linear immobilized pH gradient and then at right angle on a 7.5 - 17.5% polyacrylamide gradient gel in presence of sodium dodecyl sulfate. Protein pattern was stained with silver nitrate (from: Martinelli e/ al., 1993, J. Plant Physio!. 141 :476-481).
lated to differentiation in plants (Chambers and Shaw, 1987; Wolffe, 1996), the authors propose such variation as an indication of major changes in gene expression related to embryo morphogenesis.
5. CONCLUSIONS
Since the 1970's, when first attempts were carried out for obtaining somatic embryo- genesis in the Vilis genus, interesting results have been reported for a quite large number of genotypes (Table 13.1). However, for a relevant number of agronomically important genotypes, this technique is still far from routine and efficiencies need to be improved.
The earliest phases of somatic induction from the plated explants seem to be the limit- ing step of the overall protocol, as activation of many genes could be necessary. Then, the later further developmental phases seem to proceed more efficiently; however, tera- tology and low conversion rate are important limiting aspects of an efficient protocol definition. In general, there is a need to define efficient protocols and replace the costly
"trial and error" procedures.
Several strategies have been assessed to overcoming problems affecting somatic em- bryogenesis in the Vilis genus, as discussed in this chapter. On the other hand, few basic studies aiming to understand - and thus control - tissue competence have been conducted in grape. A morphologic approach has often been adopted; however, we believe that efforts should be addressed to develop new strategies for better understanding of the key steps of regeneration and possibly to identify molecular markers and sets of specific marker genes.
346 L. MARTINELLI and 1. GRIBAUOO ABBREVIATIONS
2,4-0: 2,4-dichlorophenoxyacetic acid; 2,4,5-T: 2,4,5-trichlorophenoxyacetic acid;
ABA: abscicic acid; AcP: acid phosphatase; ADH: alcohol dehydrogenase; BA: 6- benzyJadenine (6-benzylaminopurine); CPPU: N-(2-chloro-4':pyridyl)-N' -phenyJurea;
EST: esterase; GA3: gibberellic acid; IAA: indole-3-acetic acid; IASP: indoJe-3-aspartic acid; IBA: indole-3-butyric acid; KT30: N-(2-chloro-4-pyridyl)-N'-phenylurea; NAA: 2- naphtaleneacetic acid; NOA: 2-naphthoxyacetic acid; TDZ: N-(l,2,3-thidiazol-5-yl)-N'- phenyJurea (thidiazuron).
ACKNOWLEDGMENTS
Authors wish to thank Richard Cirami for his valuable help in critically discussing the manuscript.
REFERENCES
Altamura, M.M., Cersosimo, A, Majoli, e., and M. Crespan (1992) Histological study of embryogenesis and organogenesis from anthers of Vitis rupestris du Lot cultured in vitro. Protoplasma 171: 134-141.
Altamura, M.M., Cersosimo A, Majoli e., Crespan M., and F. Capitani (1993) Formation of secondary em- bryoids obtained from anthers of Vitis rupestris Scheele cultured in vitro. Giorn. Bot. Ital. 127:297-299.
Ammirato, P.Y. (1983) Embryogenesis. In: Techniques for Propagation and Breeding, Evans D.A, Sharp W.R., Ammirato P.V., Yamada Y. (Eds). Handbook of Plant Cell Cultures, Vol. T, pp. 82-123.
Bouquet A, Piganeau, B., and M. Lamaison (1982) Influence du genotype sur la production de cals, d'em- bryoides ct de plantes enticres par culture d'antheres in vitro dans Ie genre Vitis. Comp!. Rend. Acad. Sci.
Paris 295:569-574.
Cao, Z. (1990) Grape: anther culture. In. Handbook of Plant Cell Culture, Vol. VI, Perennial crops, Zenghua Chen et of. (Eds). McGraw-Hili, New York, pp. 300-311
Chambers, S.A and B.R. Shaw (1987) Histone modifications accompanying the onset of developmental com- mitment. Dev. BioI. 124:523-531.
Choi, J.H. and Z.R. Sung (1989) Induction, commitment and progression of plant embryogenesis. In: Plant Biotechnology, Kung S., Arntzen CJ. (Eds). Butterworths, pp. 141-159.
Compton, M.E. and OJ. Gray (1996) Effects of sucrose and methylglyoxal bis-(guanylhydrazone) on control- ling grape somatic embryogenesis. Vitis 35(1): 1-6.
Coutos-Thevenot, P., Goebel-Tourand, I., Mauro, M.C., Jouanneau, J.P., Boulay, M., Deloire, A, and J. Guern (1992a) Somatic embryogenesis from grapevine cells. I-Improvement of embryo development by changes in culture conditions. Plant Cell Tiss. Org. Cult. 29:125-133.
Coutos-Thevenot, P., Maes, 0., Jouenne, T., Mauro, M.e., Boulay, M., Deloire, A, and 1. Guern (l992b) Extracellular protein patterns of grapcvine cell suspensions in embryogenic and non-embryogenic situa- tions. Plant Sci. 86:137-145
De Vries, S.e. (1998) Making embryos in plants. Trends in Plant Sci. 3: 451-452
Deloire, A and M.e. Mauro (1991) Amelioration de la vigne par la voie des biotechnologies: realites et pers- pectives. Rev. Cytol. BioI. Veget. Bot. 14: 265-269.
Dos Santos, A.P, Cutter, EG, and M.R. Davey (1983) Origin and development of somatic embryos in Medi- cago sativa L. (alfalfa). Protoplasm a 117: I 07-115.
Dubois, T, Guedrira, M., Dubois, .1., and 1. Vasseur (1991) Direct somatic embryogenesis in leaves of
SOMATIC EMBRYOGENESIS IN GRAPEVINE 347 Cichorzum: a histological and SEM study of early stages. Protoplasm a 162:120-127.
Emershad, R.L. and D.W. Ramming (1994) Somatic embryogenesis and plant development from immature zygotic cmbryos of seedless grapes (V. vinifera L.). Plant Cell Rep. 14:6-12.
Eynard, I. and G. Dalmasso (1990) Viticultura Moderna (loth edition). Hoepli PUbl., Milano.
Fallot, J., Tey-Rulh, P., Coutouly, P, Petitprez, M., Roustan, J.P., Philippe, I., and R. Tabacchi (1990) Cultures in vitro, etude de I'eutypiose et strategies de creation de somaclones de vigne tolerants. Les colloques de I'INRA 51 :151-159.
Faure, O. (1990) Embryons somatiques de Vitis rupestris et embryons'zygotiques de Vitis sp: morphologie, histologie, histochimie et developpement Can . .T. Bot 68:2305-23 I 5.
Faure, O. and 1. Aarrouf (1994) Metabolism of reserve products during development of somatic embryos and germination of zygotic embryos in grapevine. Plant Sci. 96: 167-1 78.
Faure, o. and A. Nougarcdc (1993) Nuclear DNA content of somatic and zygotic embryos of V. vinifera cv Grenache noir at the torpedo stage. Flow cytometry and in situ DNA microspectrophotometry. Proto- plasma 176:145-150.
Faure, 0., Aarrouf, l, and A Nougarede (l996a) Ontogenesis, differentiation and precocious germination in anther-derived somatic embryos of grapevine (V. vinifera L.): proembryogenesis. Ann. Bot. 78:23-8.
Faure, 0., Aarrouf, J., and A Nougarede (I 996b) Ontogenesis, differentiation and precocious germination in anther-derivcd somatic embryos of grapevine (V. vinifera L.): embryonic organogenesis. Ann. Bot.
78:29-37.
Faure, 0., Dewitte, W., Nougarede, A, and H. VanOnckelen (1998) Precociously germinating somatic em- bryos of V. vinifera have lower ABA and IAA levels than their germinating zygotic counterparts. Physiol.
Plant. 102:591-595.
Faure, 0., Mengoli, M., Nougarede, A, and N. Bagni (1991) Polyamine pattern and biosynthesis in zygotic and somatic embryo stages of V. vinifera. J. Plant Physiol. 138:545-549.
Favre, J .M. (1977) Premiers resultats concernant I'obtenition in vitro de neoformations caulinaires chez la Vigne. Ann. Arnel. Plantes 27:151-169.
Finkelstein, R.R. and M.L. Crouch (1984) Precociously germinating rapeseed embryos retain characteristics of embryogeny. Planta 162:125-131.
Franks, T., He, D.G., and M. Thomas (1998) Regeneration of transgenic V. vinifera L. Sultana plants: geno- typic and phenotypic analysis. Malec. Breed 4:321-333.
Gianazza, E., De Ponti, P., Scienza, A, Villa, P., and L. Martinelli (1992) Monitoring by two-dimensional electrophoresis somatic embryogenesis in leaf and petiole explants from Vitis. Electrophoresis 13 :203- 209.
Gocbel-Tourand, I., Mauro, M.C., Sossountazov, 1.., Miginiac, E., and A Deloire (1993) Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA, BAP and zeatin in stimulating plantlet development. Plant Cell Tiss. Org. Cult. 33:91-103.
Goussard, P.G. and 1. Wiid (1992) The elimination of fanleaf virus from grapevines using in vitro somatic embryogenesis combined with heat therapy. S. Afr. .T. Enol. Vitic. 13:81-83.
Goussard, P.G., Wiid, 1., and G.G.F. Kasdorf (1991) The effect of in vitro somatic embryogenesis in eliminat- ing fanlcafvirus and leafroll associated viruses from grapevines. S. Afr. 1. Enol. Vitic. 12:77-81.
Gray, DJ. (1987) Quiescence in monocotyledonous and dicotyledonous somatic embryos induced by dehydra- tion. HortSci. 22(5):810-814.
Gray, D.l (1989) Effects of dehydration and exogenous growth regulators on dormancy, quiescence and ger- mination of grape somatic embryos. In vitro Cell Devel. BioI. 5: 1173-1 178.
Gray, DJ. (1992) Somatic embryogenesis and plant regeneration from immature zygotic embryos of muscad- ine grapevine (Vilis rotundifolia) cultivars. Am. J. Bot. 79:542-546.
Gray, DJ. (1995) Somatic embryogenesis in grape. In: Somatic Embryogenesis in Woody Plants, Jain S., Gupta P. and R. Newton (Eds). Kluwer Acad. Pub!., NL, Va!. 2, pp. 191-217.
Gray, OJ. and M.E. Compton (1993) Grape somatic embryo dormancy and quiescence: potential of dehy- drated synthetic seeds for germplasm conservation. In: Synseeds, Applications of Synthetic Seeds to
348 L. MARTINELLI and J. GRIBAUDO Crop Improvement, Redenbaugh K. (Ed.). CRC Press Inc., pp. 367-379.
Gray, OJ. and c.P. Meredith (1992) Grape. In: Biotechnology of Perennial Fruit Crops. Hammerschlag F.A, Litz R.E. (Eds). CAB Intern. pp. 229-262.
Gray, DJ. and I.A Mortensen (1987) Initiation and maintenance of long term somatic embryogenesis from anthers and ovaries of Vilis longii "Microsperma". Plant Cell Tiss. Org. Cult. 9:73-80.
Gresshoff, P.M. and C.H. Doy (1974) Derivation of a haploid cell line from V. vinifera and the importance of the stage of meiotic development of anthers for haploid culture of this and other genera. Z. Pflanzen- physioJ. 73: 123-14 J.
Guha-Mukherjee, S. (1999) The discovery of haploid production by anther culture. Turning point article. In vitro Cell. Dev. BioI. Plant 35:357-360.
Harst, M. (1995) Development of a regeneration protocol for high frequency somatic embryogenesis from explants of grapevines (Vilis spp.).Vitis 34(1):27-29.
Hebert-Soule, D., Kikkert, I.R., and B.1. Reisch (1995) Phosphothricin stimulates somatic embryogenesis in grape (Vilis sp. 1.). Plant Cell Rep. 14:380-4.
Hirabayashi, T., Kozaki, I., and T. Akihama (1976) In vivo differentiation of shoots from anther callus in Vilis grape research. HortSei. 11:511-512.
Jayasankar, S., Gray, D.J., and R.E. Litz (1999) High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine. Plant Cell Rep. 18:533-537.
Konar, R.N., Thumas, E., and H.E. Street (1972) Origin and structure of embryoids arising from epidermal cells of the stem of Ranunculus scleratus 1. I. Cell Sci. 11:77-93.
Krochko, I.E., Bantroch, OJ., Greenwood, lS., and J.D. Bewley (1994) Seed storage proteins in developing somatic embryos in alfalfa: defects in accumulation compared to zygotic embryos. I. Exp. Bot. 45 :699- 708.
Krul, W.R. (1985) In vitro propagation of vine. USA Patent No. 4,532,733, Aug. 6, 1985.
Krul, W.R. and J.F. Worley (1977) Formation of adventitious embryos in callus cultures of "Seyval", a French hybrid grape. J. Am. Soc. Hort. Sci. 102(3):360-363.
Krul, E.R. and G.B. Muwbray (1984) Grapes. In: Handbook uf Plant Cell Cultures, Sharp W.R., Evans D., Ammirato P.V., Yamada Y (Eds). Vol. II, Crop Species, pp. 396-434.
Kuksova, V.B., Piven, N.M., and YY. Gleba (1997) Somaclonal variation and in vitro induced mutagenesis in grapevine. Plant Cell Tiss. Org. Cult. 49: 17-27.
Lebrun, 1. and M. Branchard (1986) Embryogenese "indefinie " chez Vitis sp. cultive in vitro. Proc. 3° Symp.
Intern. sur Physiologie de la Vigne, 24-27 June 1986, Bordeaux, France, pp. 38-41.
Maes, 0., Coutos-Thevenot, P., Jouenne, T., Boulay, M., and J. Guern (1997) Influence of extracellular pro- teins, proteases and protease inhibitors on grapevine somatic embryogenesis. Plant Cell Tiss. Org. Cult.
50:97-105.
Marchenko, AO. (1991) Induction of embryogenesis in primary calluses from grape stems and leaves.
Fiziologiya Rastenii 38(3):580-591.
Margosan, D.A, Emershad, R.1. and D. W. Ramming (1994) Origin of somatic embryos from embryo rescue cultures of seedless grapes (V. vinifera 1.). Proc. Intern. Symp. on Table Grape Production, 28-29 June 1994, Anaheim, California, pp. 133-135.
Martinelli, 1. (1997) Regeneration and Genetic Transformation in the Vilis Genus. Ph.D. thesis for the award of the degree of Doctor of Agricultural and Environmental Sciences at the Agricultural University of Wageningen, The Netherlands.
Martinelli, 1. and G. Mandolino (1994) Genetic transformation and regeneration of transgenic plants in grape- vine (Vilis rupestris S.). Theor. App!. Genet. 88:621-628.
Martinelli,1. and G. Mandolino (2001) Genetic transformation in Vilis. In: Biotechnology in Agriculture and Forestry, Ba.i<U y'P.S. (Ed.). Springer-Verlag, Berlin, Vol. 47, pp. 325-338.
Martinelli, 1., Bragagna, P., Poletti, V., and A Scienza (1993a) Somatic embryogenesis from leaf- and petiole- derived callus of Vilis rupestris. Plant Cell Rep. 12:207-210.
Martinelli, L., Scienza, A, Villa, P., De Ponti, P. and E. Gianazza (1993b) Enzyme markers for somatic em- bryogenesis in Vilis. J. Plant. Physio!. 141:476-481.