Figure 12.1. Procedure for eliminating browning of explants upon 'establishment (from Goussard, 1984): a) thin layer of sterile distilled water added; explants remain adrift. b) explant growth after ,30, days of culture with 10 mg/l ZR. c) primary shoot elongation after 45 days of culture. d) shoot
elongation after 55 days of culture. Note axillary shoot growth (arrow).
286 L. TORREGROSA et a!.
are commonly classified into those using pre-existing meristems and those requiring neoformation of buds or meristem-like structures.
3.1. Nodal and meristem tip culture
The standard method uses explants bearing intact apical or axillary buds cultured on growth regulator-free media containing sucrose, macro and microelements and vitamins, and solidified with a gelJing agent, generally agarose or gelrite. Depending on genotype and environmental conditions, an axillary bud gives a single rooted plant growing at the rate of 2-4 nodes per month, which represents yearly multiplication rates of 104 to 106.
This technique has been the subject of many efforts to optimize culture conditions for the largest scale of Vitis genotypes (Galzy et a!., 1990; Torregrosa and Bouquet, 1995).
Due to its advantages (operational feasibility, genotype stability, ease of plantlet transfer to greenhouse) and despite a moderate multiplication yield, nodal culture re- mains a widely used technique. Meristem cultures have been specifically developed for virus sanitation using the smallest possible excisions; i.e. the apical dome and the first definable leaf or tendril primordia. Procedures are based on amending culture media with cytokinins at specific concentration levels to promote meristem elongation.
3.2. Axillary bud proliferation
Micropropagation output can be considerably increased by the use of cytokinins, a fam- ily of plant growth regulators with the property of overcoming the apical dominance of axillary buds. When cytokinins are applied to the culture, intense shoot proliferation is induced by the enhanced release of axillary buds. Cultures no longer have a defined plant form but appear as dense proliferative clusters of small root-free shoots (Goussard, 1982; Figure 12.2). Axillary bud proliferation is currently considered to be the most convenient and reliable regeneration technique for shoot mUltiplication in many plants, herbaceous species and woody, ornamental and fruit species, including grapevine (Hu and Wang, 1983). This procedure provides a powerful propagation system in which the number of regenerated shoots exponentially increases with the number of subcultures.
Yearly production can theoretically reach 108 buds per initial explant. Due to its poten- tial, many research groups have attempted to improve and adapt it to the broadest scale of Vitis genotypes (Table 12.1).
However, not all the genotypes behave perfectly and the [mal output is often lowered be- cause of vitrification, mineral or water supply deficiency and/or accumulation of phenolic compounds. The selection of appropriate growth regulators is of critical importance with the application of axillary branching for maximum production of uniform plants, especially when several cycles of multiplication are repeated. BAP proved highly effective for many genotypes. Among other cytokinins, ZR resulted in lower quantities of proliferating shoots but with morphological characteristics resembling those of the normal condition (Gous- sard, 1982, 1984 and 1987). Micropropagation induced with ZRproduces single-axed shoots, slim and uniform in size, bearing almost typical leaves and resulting in clumps oflow densi-
IN VITRO CULTURE AND PROP AGA TION OF GRAPEVINE 287
Figure 12.2. Enhanced release of axillary buds resulting in dense proliferative clusters of small root-free shoots (from Goussard, 1982).
ties (Goussard 1982, 1984; Figures 12.3 and 12.4). Therefore, for routine procedures the utilisation of ZR within different Vitis genotypes can be highly recommended.
3.3. Regenerative procedures
Production of buds, shoots and plants may also be obtained through regeneration, a neo- formation process using tissues without pre-existent meristematic structures. Two ways of regeneration are commonly recognized: adventitious organogenesis, which provides caulinary structures, and somatic embryogenesis, which provides embryo or embryo-like formations.
The organogenesis pathway uses the ability of competent tissues to form adventitious bud-like structures directly on the explant or indirectly from callus that develops on the cut surfaces. Induction media used for bud organogenesis contain high cytokinin and low auxin levels (generally 5 to 10 IlM BAP and 0.1 to 0.25 IlM NAA). In grapevine, bud neo- formation can be obtained from various types of organs (for reviews, see Gray and Meredith, 1992; Torregrosa, 1995): leaf primordia from fragmented apices (Barlass and Skene, 1980a;
Barlass et al., 1981), internodes (Rajasekaran and Mullins, 1981), leaves and petiole stubs (Tang and Mullins, 1990) and hypocotyls or cotyledons from somatic embryos (Vilaplana and Mullins, 1989). Direct organogenesis from in vitro established petioles and leaves has been reported, with a reasonable rate of success for several important grapevine genotypes (Cheng and Reisch, 1989; Stamp et aI., 1990; Torregrosa and Bou- quet, 1996).
288 1,. TORREGROSA et al.
Table 12.1. Studies on axillary bud proliferation in grapevine.
Species Genotype Studied factor Reference
V. vinifera Sylvaner BAP/KinINAA Jona and Webb (1978)
V vinilera 10 cvs BAP/GAl Silvestroni (J 981)
V. vinifera Chenin blanc BAP/ZR Goussard (1981; 1982)
Vilis hybrids 18 genotypes BAP/IAAlIBAINAA
V. vinifera 3 cvs MS strength/P04 Harris and Stevenson (J 982) Organic compounds/agar
Vitis hybrid Craciunel BAP/Kin/2iP/IBA
Novak and Juvova (1983)
V. vinifera 7 cvs MS strength
Vilis hybrid Rougeon BAPINAA
Chee and Pool (1982) Photoperiod
V. vinifera Limberger Culture vessel size Monette (1983) Vilis hybrids Marechal Foch BAPINAAlIBA
Li and Eaton (1984)
Cascade MS strength
Vitis hybrid Remaily seedless Vitamins
Chee and Pool (1985) Amino acids
Vilis hybrids 15 genotypes HAP /Kin/Pic1oram
V labrusca Alba Adenine
Reisch (1986)
V. labruscana 3 cvs MS strcngth
V. vinifera 3 cvs
Vilis hybrid Remaily seedless Light spectrum Chee (1986) Mn and KI
Vitis hybrid Remaily seedless Salt formulation Chee and Pool (1987)
V. rotundifolia Summit BAP/IBA Lee and Wetztein (1990)
Vitis hybrid 41B BAP/IBAIGAl
V. vinifera Kalecik Karasi MS strength Celik and Batur (1990) Vitamins
V. vinifera Barbera TDZ Gribaudo and Fronda (1991)
V. rotundifolia 4 cvs BAP/TDZ/Kin Sudarsono and Goldy(l991)
Initial nodal position
V. rotundifolia 3 cvs BAP/TDZ/KinlNAA Explant length Gray and Benton (1991)
Vitis hybrid 5BB MS strength
V. vinifera 3 cvs Vitamins Zlenko et al. (1995)
Mgand Ca
Vilis x Mus- 5 genotypes BAP
cadinia hybrids Fercal Salt formulation Torregrosa and Bouquet (1995) Vilis hybrid
V. vinifera Listan Negro BAP/2iPINAA
Molina et al. (1998) Darkness
Somatic embryogenesis can be induced with explants from various origins, using induc- tion media containing high auxin and more often low cytokinin levels (5 to 10 JlM NOA or 2,4-D plus I JlM BAP). In the last few years there has been a considerable increase in re- search aimed at developing protocols for grapevine regeneration via somatic embryogenesis.
For a comprehensive review ofthe choices of methodologies for induction and maintenance
IN VITRO CULTURE AND PROP AGA TION OF GRAPEVINE 289
Figure 12.3. Shoot proliferation with ZR during subculturing (from Goussard, 1984).
of embryogenic tissue culture, and germination of somatic embryos, see Gray and Mere- dith (1992), Torregrosa (1995) and Gribaudo and Martinelli (this issue). This technol- ogy, however, is still far from routine for many Vitis genotypes.