2. PROTOCOLS FOR SOMATIC EMBRYOGENESIS IN GRAPE
2.1. Induction and culture of embryogenic callus
The main protocols proposed for obtaining callusing and further differentiation of so- matic embryos are detailed in Table 13 .1. All the reported protocols led to plantlet re- generation.
Among the many factors involved in these processes, genotype is commonly consid- ered as one of the most relevant. However, in recent years, the range of grape genotypes that have been successfully subjected to somatic embryogenesis induction has widened, particularly in V. vinifera.
Concerning the explant types, anthers are still the most widely used and suitable or- gans for culture initiation, while for leaves and petioles protocol efficiencies could be improved. Most recently, protoplasts underwent somatic embryogenesis and regenerated plants; this was possible only when protoplasts were isolated from leaf-derived embryo- genic callus or embryos (Reustle et al., 1995; Zhu, et aI., 1997). Additionally, tendrils were proposed as an alternative explant (Salunkhe et aI., 1997).
The developmental stage of explants as well as their preconditioning may be of great importance; for example, it is a common practice to collect anthers at an early stage (un- inucleate microspores) and to chill them (4°C) for a few days.
As for the hormonal balance in the culture media, different combinations of a phenoxy-auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D) or 2-naphthoxyacetic acid (NOA) with 6-Benzyladenine (BA), or - less frequently - other cytokinins, have been employed for inducing embryogenic callus. For further culture of the embryogenic callus, the cited auxins are generally removed, decreased or substituted by other auxinic compounds. In some cases, however, these two morphological steps are obtained with the same growth regulator composition.
Refinements of the protocols have been frequently proposed, and can contribute to in- crease the fmal rates of success. For example, the type of solidifying agent has shown to significantly influence callus initiation and formation of embryogenic structures, with new gelling polymers being superior to agar (Perl et al., 1995; Torregrosa, 1998).
It has been reported that a low amount (0.5 mgTl) of the herbicide phosphinotricin significantly stimulated the production of somatic embryos in the Vitis hybrid Chancellor (Hebert-Soule et aI., 1995). In other studies, a low dose of (y-ray irradiation (10 Gy) seemed to speed up the embryogenetic process, from 3-4 months to 2 weeks (Kuksova et aI. , 1997).
Table 13.1. Main protocols for induction of somatic embryogenesis in Vitis, The first culture steps (from callus induction to embryo differen- tiation) are reported, Genotypes EXPLANTS INDUCTION OF EMBRYOGENIC CALLUS CULTURE OF EMBR YO-REFERE0ICES Media basal composition SolidlLiquid PGRs(~M) GmaC CALLUS (~1) VItlS interspecific hybrid SeyvaJ Leaves, petioles, internodes, MS S 2,4-D (4,5) + BA (OA) MS + I\AA (10,7) + BA Krul and Worley, 1977 young florets (OA) v. rinjfera and V. rupr::sfns (several Leaves (from In VIVO and In NI\ S NOA (5) + BA (0,9) Same culture conditions Stamp and Meredith, 1988a cvs) vi/ro plants), anthers V. vin?j"era (10 cvs) and V. x labruscana Leaves, ovaries, anthers Half strength MS S 2,4,5-T (10) + TDZ or CPPU Same basal composition + Nakano et al .. 1997 Delaware (10) 2,4-D(i) V. vim/era x V. mpesfrrs Gloryvine and Rajasekaran and Mullin:., 1979; others CYS, V. rupeMns, V. /OflKii, V. Anthers NN L 2,4-D (5) + BA (I) PGR-free liquid ~ villifera Grenache Mullins and Rajasekaran, 1980 V l'1f1{fera, V rupeMri5, V. l'iparia and Anthers NI\ L 2,4-D (4 5) + BA (1.1) Solid NI\ + 1AA (5 7) + BA Bouquet ef aI., J 982 ViliS interspecitic hybrids (22) Half strength MS + casein hydrolysate (250 mg)"l) + Same basal composition + V. viJ1?fera Cabernet-Sauvignon Anthers glutamine (100 mg}"l) + S 2,4-D (4.5) + BA (I) Mauro et aI" 1986 phenylalanine (10 mg)"!) NOA (0.5) + BA (I) + adenine (1 rug"!"l) V IOJJgii Mlcrosperma Anthers, ovaries MS S 2,4-D (5 + BA I) Same culture conditions Gray and Mortensen, 1987 V riparia Anthers MS -filtered Fe-Na S 2,4-D (5) + (0.9) PGR-free, solid modified Moszar and Sule, 1994 EDTA MS V ),1II~fera (4 cvs) Anthers MS S 2,4-D (9) + BA (0.9) Solid MS + NOA (10) + BA Perl el aI., 1995 (0.9)+ IASP (17) V vinifera Grenache nair Anthers NI\ L 2,4-D (45) + BA (1.1) PGR-free solid NN Faure el a/. , 1996a V vln?fera (several cvs) l<\nthers Modified MS S 2,4-D (5) + BA (I) Same culture conditions Torregrosa,1998 Same basal composition -I.- V vimfera Sultana Anthers Modified NI\ S 2,4-D (4.5) + BA (9) NOA (10) + 1AA (20) + BA Franks ef aI., 1998 (I) V lati/olia Anthers NN S 2,4-D (20) + BA (9) Same basal composition + Salunkhe et ai" 1999 NAA (10) + BA (9) BA (5) for 3 wks, then NOA (5) Liquid NI\ + NOA (5) + BA V vimfera Cabernet-Sauvignon Ovules NI\ L + BA (5) for 2 wk" then NOA Mullins and Srinivasan, 1976 (5) +BA(25) (2.5)
w w o r ~ ~ tTl r t: § 0.. :- Cl c: CO ;l> C o o
Genotypes INDUCTION OF EMBRYOGENIC CALLUS CULTURE OF EMBR YO-REFERENCES EXPLANTS Media basal composition SolidlLiquid PGRs (flM) GENIC CALLUS (~M) V. vinifera Koshusanjaku Leaves NN S 2,4-D (S-IO) + TDZ or Vitamin-, inositol-and glycine-Matsuta and Hirabayashi, KT30 (5-10) free solid NN + 2,4-D (I) 1989 V. vinifera Krymskaya Zhemchuzhina, Solid MS + NAA (10.7) + BA Leaves, internodes (from in (2.2-4.4) if callus from V monticoia, ViflS interspecific hybrids vitro plants) MS S 2,4-D (4.S) + BA (9-20) internodes; NAA (2.7-16) + Marchenko, 1991 (2 cvs) BA (22 2) if callus from leaves V. rllpes/ris Leaves, petioles MS S 2,4-D (4.S) + BA (OA-Solid MS or NN + IAA (S.7) + Martinelli el al., 1993a 44) or lBA (O.S) 2,4-D (9) + BA (4.4), V. rotundifolia Regale and Fry Leaves, petioles NN S then NAA (10.7) + BA PGR-free solid NN Robacker, 1993 (09) V. vimfera x V. rolUndijolia (2 cvs); Leaves (from in vitro plants) Modified MS S 2,4-D (S) + BA (1.1) Modified solid MS + IAA. (S) + Torregrosa el al., 1995 Vilis interspecific hybrid VMH 1 BA(I.1) Vilis interspecific hybrids SeyvaJ blanc Leaves (from in 'liilro plants) Modified NN + phenyla-S NOA (20) + BA (40) or Same culture conditions Harst, 1995 and Chancellor; V thunbergli lanine (2.S nuVI) TDZ(4) V. rupesfris du Lot Leaves ModifiedNN S 2,4-D (9) + BA (9) Modified solid MS + NOA (5) Tsolova and Atanassov, + BA(0.9) 1996 2,4-D (9) + BA (4.4) for V. villifera Podarok Magaracha Leaves (from in vitro plants) MS S 6 wks, then NAA (SA) + Solid MS + lAA (O.S) Kuksova et at., 1997 BA(44) V vmifera (3 cvs) Tendrils Modified NN S NAA(O.4)+BA(IO)+ Solid ER + BA (I) Salunkhe et af., 1997 GA3 (2.8) Protoplasts (isolated from leaf-Embedded V. vinifera Koshusanjaku ModifiedNN protoplasts in NAA (10.7) + BA (2.2) Same culture conditions Izhu el al., 1997 derived embryogenic callus) liquid culture Protoplasts (isolated from leaf-Embedded Vuts interspecific hybrids Seyval blanc derived embryos and embry-ModifiedNN protoplasts in NOA (20) + TDZ (4) PGR-free modified NN Reustle et al., 1995 aids) liquid culture V. vinifera (4 cvs); V ion Tii Zygotic embryos NN S NOA (5) + BA (0.9-4.S) Same culture conditions Stamp and Meredith, 1988b V rotundifolia (5 cvs) Zygotic embryos from in ovulo NN S NOA (S) + BA (0.9) PGR-free, solid modified MS Gray, 1992 embryo culture Abbrc"iatlOUS: cvs = cultivars; ER = Emershad and Ramming. 1994; MS:'" Murashige and Skoog, 1962, NN ... Nitsch and Nitsch, 1969; S "" solid medium; L == liqUid medium; PGRs = plant growth regulators, \Vks = weeks; for the other abbre- viations, see the "List of abbreviations" section.
[JJ o ~ ::j n tT:I ~ OJ :< o o ~ tT:I [JJ -[JJ Z o ~ rg < Z tT:I w w
332 L. MARTINELLI and I. GRIBAUDO 2.2. Long-term embryogenic cultures
During the subsequent subcultures, embryo development and their eventual conversion into plants may lead to strong reduction or loss of the primary embryogenic callus. Sev- eral strategies have been adopted in order to keep indefmitely the embryogenic compe- tence of the cultures and thus obtain a long-term source of somatic embryos. Subcultures have been performed in the presence of auxin, with (Gray and Mortensen, 1987; Stamp and Meredith, 1988a; Perl el aI., 1995) or without cytokinins (Matsuta and Hirabayashi, 1989; Martinelli et aI., 1993a), or on a growth regulator-free medium (Krul and Worley, 1977; Lebrun and Branchard, 1986; Gray and Mortensen, 1987). Both embryogenic cal- lus and single somatic embryos have been used for this purpose; the latter case will be discussed in the following section describing secondary embryogenesis. Embryogenic callus storage was performed adopting a combination of low temperature and silicone treatment, which allowed a reduction of the subculture frequency while calli retained their embryogenic ability (Moriguchi et aI., 1988).
The maintenance of embryogenic cultures has been reported to last even for several years (Gray, 1989; Torregrosa, 1998); in our experience, cultures of V. vinifera and Vilis rupestris are still retaining their embryogenic competence after respectively three and ten years (L. Martinelli, E. Candioli, D. Costa, V. Poletti, and N. Rascio, unpublished).
2.3. Somatic embryogenesis from embryonic tissues
Somatic and zygotic embryos exhibited a high competence in somatic embryogenesis regeneration. This aptitude has been successfully applied in genetic transformation ex- periments where whole- (Martinelli and Mandolino, 1994 and 2000; Martinelli et at., 1996 and 2000; Scorza et ai., 1996) or sections (Mullins et aI., 1990) of somatic em- bryos, and zygotic embryos (Scorza et aI., 1995) were induced to secondary embryo- genesis during co-culture with Agrobacterium tumefaciens. However, zygotic embryos are unsuitable materials for those breeding programs where cultivar integrity must be preserved. In fact, their genetic constitution is unknown and they can be quite different genotypically from the parental cultivars, because of the high heterozigosity of grape- vine. On the other hand, they can be usefully adopted as a model system for regeneration and genetic transformation studies (Stamp and Meredith, 1988b).
In somatic embryos, recurrent secondary embryogenesis has been described in various V. vinifera cultivars (Krul and Worley, 1977; Matsuta and Hirabayashi, 1989; ViJaplana and Mullins, 1989; Perl et aI., 1995; Kuksova et at., 1997), hybrids (Vilaplana and Mullins, 1989), and in different species, such as Vilis rupestris (Newton and Goussard, 1990; Alta- mura et aI., 1993; Martinelli et at., 1993a) and Vitis rotundifolia (Robacker, 1993). Both direct and indirect secondary embryogenesis were described. As for direct embryogenesis, it has been generally observed that somatic embryogenesis occurs more frequently on the root/shoot transition zone, and in the root region (Fig. 13.1), confIrming a gradient of em- bryogenic competence along the embryo tissues (Martinelli et at., 1993a).
SOMA TIC EMBRYOGENESlS IN GRAPEVINE 333
Figure 13.1. Secondary embryogenesis regeneration on a Vilis rupestris somatic embryo, as seen with the scanning electron microscope (Hitachi S-2300, tungsten filament at 25 kV): the forma- tions are typically produced on the root region.
For the embryogenesis induction, various growth regulator compositions have been ap- plied on isolated somatic embryos in solid medium: (i) both indole-3-acetic acid (lAA) and indole-3-butyric acid (lBA) proved optimal in Vitis rupestris S. (Martinelli et aI., 1993a);
(ii) a combination ofBA with indole-3-aspartic acid (IASP) and NOA was amenable in the V vinifera cv Superior seedless (Perl et aI., 1995); (iii) various combinations of BA and 2,4-D proved optimal in different grape genotypes (Mozsar and Viczian, 1996).
A genotype effect on the capability to regenerate secondary embryos was proven by Mozsar and Viczian (1996). In a study conducted on samples of anther derived somatic embryos of different genotypes (several cultivars of V vinifera and Vitis riparia, a clone of Vitis amurensis, and an interspecific rootstock) secondary embryogenesis was induced from torpedos and early cotyledonary stage somatic embryos, and efficiencies varied considerably in the different assessed genotypes.
Immature and mature entire zygotic embryos have been employed for somatic em- bryogenesis induction in both Euvitis (Stamp and Meredith, 1988b; Emershad and Ramming, 1994) and Muscadinia subgenera (Gray, 1992). In solid medium, BA alone (Emershad and Ramming, 1994) or in combination with NOA proved suitable (Stamp and Meredith, 1988b; Gray, 1992).
In both somatic and zygotic embryos, secondary embryogenesis was superficial, originating from the epidermal layer of the embryos (Altamura et aI., 1993; Margosan et aI., 1994). This could be the reason why the application of a co-culture of Agrobacte- rium tumefaciens with such embryogenic tissues resulted in a very efficient strategy for
334 L. MARTINELLI and I. GRIBAUDO genetic transformation.