IN VITRO CULTURE AND PROPAGA nON OF GRAPEVINE 283 plant are critical, since the tissue must be physiologically competent to survive the initial culture and elicit the appropriate response.
2.1. Choice of explants
In vitro propagation can be obtained by shoot development from nodal cuttings (Galzy, 1969), shoot apices (Harris and Stevenson, 1979; Goussatd, 1981), and by adventitious shoot formation from fragmented apices (Barlass and Skene, 1978). There is some evi- dence that explant origin influences in vitro survival and shoot production; apical tips are commonly considered the best choice in explant type for general micropropagation pur- poses (Hu and Wang, 1983). In grapevine, however, Yu and Meredith (1986) reported that survival and shoot production potential were greater with explants from axillary buds than with those from terminal positions.
Other frequently noted factors are the environmental conditions and the physiological state of the mother plant. Yu and Meredith (1986) obtained better results in micropropa- gation with explants from shoots grown in shade than from those developed under full sun. This observation is consistent with that of Fanizza et al. (1984), who noted greater survival of greenhouse- and shadehouse-grown shoot tip explants than from field-grown grapevine. The vigour of the mother plant is also a factor in shoot tip culture (Hu and Wang, 1983). In grapevine, apical explants are generally obtained from rapidly expand- ing shoots (Barlass and Skene, 1978; Chee and Pool, 1982). Harris and Stevenson (1982) noted that explant survival was reduced when mother plants had been subjected to water or nutrient stress, but Yu and Meredith (1986) observed that explants from weak shoot tips had a higher survival rate than those from vigorous ones.
2.2. Handling of stock plants
Proper handling of source material before in vitro culture begins is of the utmost impor- tance in obtaining successful establishment and reducing further contamination, espe- cially with hardwood species (Hartmann and Kester, 1983). This includes keeping the material disease-free insofar as possible: no insects in the greenhouse, only water in the containers, keeping the material relatively dry, good disease prevention. The procuring of large quantities of elongating grapevine shoots bearing appropriate organs prior to excision and preparation of desirable explants was reported and is still in use (Goussard, 1981). This method, based on the collection of dormant canes from selected vines, cold storage at 3°C and the enhancement of bud-burst and shoot elongation under controlled indoor conditions, offers the advantage of availability of young tissues with reduced con- tamination at any time of the year. In addition, the subjection of canes (after removal from cold storage and prior to bud-burst) to hot-water treatments proved efficient for decontamination from most external pathogens (Goussard, 1977), from phytoplasma (Caudwell et aI., 1997) and front bacteria (Burr et ai., 1989).
284 L. TORREGROSA et al.
2.3. Production of sterile explants
Common surface disinfectants and procedures used prior to aseptic culture establishment were reviewed by Street (1977) and Hu and Wang (1983). In grapevine, explants are generally soaked with sodium or calcium hypochlorite (1 or 5% w/v respectively), with a wetting agent (Tween 20 at 0.01-0.1 % v/v) for 10-20 minutes and then rinsed three times for 5 minutes each with sterile water (Krul and Mowbray, 1984). The disinfectants tend to penetrate into the organs through cut surfaces, causing 1-5 mm of tissue bleaching.
During culture, injured tissues become brown in a few days, producing large amounts of phenolic compounds, which diffuse into the medium and hinder further explant devel- opment. This problem is generally avoided by removing 5 mm of the exposed ends.
2.4. Culture media and hormone requirements
Major functions of the medium are (i) to supply the basic nutritional requirements for continued growth of the isolated explants and subsequent propagules, and (ii) to manipu- late growth and development through the control of growth regulator balance. Compo- nents of the medium providing successful establishment of explants and subsequent growth ofpropagules vary with the kind of plant (de Fossard, 1976; Gamborg and Shy- luk, 1981; Hu and Wang, 1983). In general, they can be grouped into two main catego- ries: inorganic salts and organic compounds. The components commonly used to estab- lish grapevine explants in culture and sustain further growth of propagules consist of either that of Murashige and Skoog (1962), generally in diluted form, or that of Galzy et at. (1990).
In vitro development is commonly controlled by (i) the kind of growth regulator, (ii) the concentration and combination, and (iii) the sequence in which growth regulators are supplied. The primary classes are auxins and cytokinins; however gibberellins and ab- scisic acid are used in specific situations. The most important synthetic auxins include 1- naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 4-chlorophenoxyacetic acid (CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2-naphthoxyacetic acid (NOA) and in- dole-3-acetic acid (IAA). Main cytokinins include 6-Benzyladenine or ben- zylaminopurine (BAP), kinetin (Kin), 6-(2-isopentenyl) adenine (2iP), zeatin or zeatin riboside (ZR) and 3-(1,2,3,-thidiazol-5-yl)-I-phenylurea or thidiazuron (TDZ). Although advances have been made, more knowledge about growth regulator requirements at spe- cific concentrations and combinations, in particular for possible synergistic effects, is still needed for further progress.
2.5. Browning of explants
The browning and necrosis of explants during in vitro culture are usually attributed to the production of deleterious phenolic compounds. Like other woody species, grapevine tissues exhibit high levels of polyphenols and tannins (Harding and Roubelakis- Angelakis, 1994) and lipid peroxidation products (Benson and Roubelakis-Angelakis,
IN VITRO CULTURE AND PROP AGA TlON OF GRAPEVINE 285 1992). Oxidative stresses, which can be directly caused by various aspects of in vitro culture, may contribute to failure of the explant development (Benson and Roubelakis- Angelakis, 1994). Moreover, explants contain endogenous substances which exude from the cut surfaces into the medium, and are capable of inhibiting development and causing browning of uncontaminated excisions (de Fossard, 1976; Hartmann and Kester, 1983).
Interestingly, Yu and Meredith (1986) observed a strong negative correlation between in vitro survival and the pre-existing phenolic content of explants. During culture, such problems can be solved using liquid media containing antioxidants (Hartmann and Ke- ster, 1983), or by subjecting explants to a preselection substage. In grapevine, the addi- tion of a thin layer of sterile distilled water to the surface ,of the solidified medium was shown to successfully eliminate browning of the explant (Goussard, 1984; Fig. 12.1).