(iii) pretransplant/rooting of shoots; and (iv) acclimatization and transplantation of plant- lets to soil.
5.1. Stage I: In vitro culture establishment
The function of this stage is to establish a sterile explant in culture to accomplish sequential non-contaminated growth and development. Factors that affect the success of this stage (regardless of the desired type of regeneration) during in vitro culture of grapevine have been discussed previously. It is very important that any factor or condition leading to the lowering of success rates in this stage be avoided. Although conditions for successful in vitro propagation and regeneration of grapevine have been thoroughly investigated, some factors hampering the effectiveness of this stage are still experienced. These factors are applicable to more sophisticated types of regeneration like organogenesis and embryogene- sis. The culture medium is an overwhelming factor in the success of stages ii and iii.
5.2. Stage II: Regeneration and multiplication
The primary goal of stage II is to increase the number of propagules for subsequent root-
294 L. TORREGROSA et al.
ing to the plantlet stage. The ability of in vitro material to reform a rooted shoot depends on the pathway of propagation and regeneration. Except for the specific effects of in vitro confinement, no special difficulty is experienced with nodal culture producing en- tire plantlets. When apical dominance is suppressed by high cytokinin content, the shoots resulting from axillary branching and adventitious shoot organogenesis exhibit the same morphological characteristics. Moreover, Chee and Pool (1985) pointed out that within high-density shoot clusters produced by axillary branching, the occurrence of adventi- tious organogenesis cannot be ruled out. The main morphological characteristics of this material are related to the tendency to vitrification and to the deficiency of rooting.
A number of problems hinder practical application of regeneration-based techniques:
(i) due to the range of cultivar responses, the techniques cannot be generalised as routine procedures; (ii) productivity is often reduced by the low rooting ability of regenerated shoot tips or the poor germination capacity of somatic embryos; (iii) due to the risk of genetic instability, propagules obtained via the initiation of adventitious shoots (directly as well as from callus) or via embryogenesis are not recommended for large scale propagation. Therefore, only pre-existing meristems cultured on plant growth regulator- free media should be used for in vitro propagation of grapevine. Otherwise, owing to the problem of rejuvenation, it is safer to limit the number of in vitro propagation cycles. To reduce risk, it is advisable to periodically transfer plants to the greenhouse and check their phenotypic conformity.
5.3. Stage III: Pretransplantation
The aim of this stage is to prepare propagules for transplanting and establishment outside the artificial, closed environment of the culture vessel. In vitro structures vary in their abilities to be transferred to soil according to the method used in this stage. When cul- tures are conditioned in a high cytokinin medium to favour shoot proliferation or regen- eration, root initiation is inhibited. Root initiation and growth must then be induced, ei- ther in vitro or by treating the propagules as mini cuttings and rooting them directly un- der non-aseptic conditions in a potting mix. For grapevine, the majority of reports (Bar- lass and Skene, 1978; Goussard, 1981; Chee and Pool, 1988; Harris and Stevenson, 1982; Li and Eaton, 1984) favour the former procedure.
Rooting is influenced by several factors, of which the stage of growth of propagules and growth regulator requirements are of major importance. With the majority of plants an active state of elongation is preferred. Individuals are transferred to a rooting medium in vitro, either singly in test tubes or as batches (6-10) in larger culture vessels. Rooting usually occurs on media lacking cytokinin but containing auxin. Barlass and Skene (1978 and 1980b) achieved successful rooting of shoot tips on a medium lacking auxin, whereas the need for NAA was reported by Chee and Pool (1982 and 1988) and Lee and Wetzstein (1990). IBA and lAA proved to be effective in promoting rooting of grape- vine; 2,4-D did not (Grenan, 1979; Novak and Juvova, 1983). When applied to a large scale of genotypes, IBA proved superior to IAA, NAA and 2,4-D (Roubelakis-Angelakis and Zivanovitc, 1991).
IN VITRO CULTURE AND PROPAGATION OF GRAPEVINE 295 Since auxin stimulates root initiation but may inhibit subsequent root growth (Galzy, 1969), the appropriate concentration is of critical importance. Zlenko et al. (1995) and Novak and Juvova (1983) observed that the effects of auxins on rooting depend on the mineral composition of the nutrient media. Root initiation is not influenced by salt con- centration, but root growth is enhanced when the salt content of the rooting medium is reduced (Harris and Stevenson, 1979). A new culture medium with an enhanced mineral balance has been developed by Roubelakis-Angelakis and ãZivanovitc (1991) specifically to promote in vitro rhizogenesis of grapevine cuttings. Compared to the MS medium, this new medium proved more effective in inducing formation of roots in the absence of auxin or developing more roots in the presence of 3 to 5 ~M lBA.
Abnormal anatomy and physiology of leaves resulting from in vitro culture (Dami and Hughes, 1995) could explain difficulties in acclimatizing plantlets to greenhouse. An increase of gas exchanges for its effect on metabolism can be used to modify plantlet vigour and harden them in preparation for acclimatization (Thomas, 1999). Under suit- able light exposure, a CO2 enrichment of the air inside the vessel was shown to stimulate the vigour of plantlets (Fournioux and Bessis, 1993). Conversely, Lakso et al. (1986) showed that CO2 enrichment stimulates root growth and shoot development. When the CO2 level was raised from 350 ppm to 1200 ppm, the leaf area per plant and the rootshoot ratio doubled. These authors suggested that doubling the root:shoot ratio would be expected to enhance the water status of the plantlets. An increase in osmotic pressure of the medium was suggested as another way to reverse rejuvenation and to harden the plantlets (Dami and Hughes, 1995). These authors incorporated polyethylene glycol into the medium and obtained plants with more organized leaves, showing com- pact and normal-shaped palisade cells and higher chloroplast number.
5.4. Stage IV: Transplant to soil
The final stage of micropropagation involves the transfer of in vitro rooted plantlets from the aseptic environment to soil media, to function as independently growing plantlets. This stage causes problems in certain cases, such as wilting, rotting of roots and low taking per- centages, which could be linked to conditions prevailing during the preceding in vitro stages. In some cases leaves may appear green but are incapable of photosynthesis, making the plantlets dependent on the sucrose in the medium for their energy supply (see above).
Roots may lack root hairs, be non-functional and be prone to pathogen attacks.
There is a lack of knowledge about the biological behaviour of plantlets during accli- matization, when slowly growing plants are extremely sensitive to environmental condi- tions, especially to water stress. Different aspects of water resistance, such as cuticle development and osmotic adjustment, need the accumulation of compounds such as lip- ids, waxes, sugar and amino acids. These compounds require large amounts of carbon and energy, which would therefore not be available for growth (Lakso et al., 1986).
Studies on photosynthesis and gas exchange during this stage are of particular interest (Slavtechva and Dimitrova, 2000). Numerous recommendations and procedures have been identified as contributing to successful taking percentages of in vitro rooted plantlets. For
296 L. TORREGROSA et al.
example, Smith et al. (1992) improved the resistance of plantlets to wilting after transplant- ing by adding Img rl paclobutrazol to the rooting medium and using culture vessels that reduce the relative humidity from 100% to 94%. Despite successes achieved with the im- plementation of such procedures, in some cases there is a lack of well-defmed guidelines.
Investigations have resulted in the development of a technique which proved highly successful for rapid acclimatization of in vitro cultured grapevine plants involving minimal labour and inexpensive equipment (Goussard and Wiid, 1989). Vinelets cul- tured in vitro are removed from test tubes, washed with sterile water to remove agar and organic nutrients and transferred to shorter flat-bottomed tubes. Leaves near the root zone are removed to prevent contact with water or nutrient solutions. Roots are sub- merged in sterile water, which is replaced after 24 hours with standard inorganic nutrient solutions. The leaves should be at some distance from the top of the tubes, allowing shoot elongation to proceed from a test tube atmosphere to a lower "outside" humidity.
To augment humidity, the tubes are capped lightly with tinfoil. The inorganic solution is replaced every 48 hours to prevent interference by microorganisms. Rapid growth reac- tions occur under controlled conditions of light, photoperiod and temperature. Irrespective of cultivar differences, the formation and elongation of functional roots (containing root hairs) take place within a few days. The tinfoil coverings are gradually lifted by developing leaves with subsequent direct exposure of young leaves to a harsher and drier environment.
Following the emergence of at least two well-developed leaves above the top of the tubes, vine lets are transferred to pots containing standard potting media and remain uncovered.
The vine lets are routinely watered as required for container-grown plants.
Acclimatization of in vitro vine lets by immersion of root systems in standard nutrient solutions holds numerous advantages. Active development and growth of new functional roots and leaves enable the plantlets to be self-sufficient, thus increasing the chances of survival. Leaving the vine lets uncovered prevents overwet conditions that may result in attacks by various pathogens.
However, the use of in vitro propagation of grapevine for commercial purposes is considerably hampered, if not rendered impossible, by a major constraint. Since most vineyards are infested with phylloxera, grapevine propagation requires grafting; Vitis vinifera rooted plants produced by in vitro culture cannot be used. Furthermore, due to smaller size, micropropagated rootstock plants are difficult to use directly for establish- ment of vineyards. The green-grafting-rooting technique could be a powerful tool and a complementary method to the micropropagation of grapevine. But to follow viticultural practices and regulations, the rootstock cuttings have to be at least 20 em long and con- stituted of I or 2 nodes. To produce herbaceous cuttings satisfying these requirements, a separate step in the greenhouse under special growth conditions is required. This extra stage and the practice of green-grafting require technical skills that considerably increase the cost of propagation (Martin et al., 1987; Boubals, 1987). Consequently, these tech- niques are not currently used for commercial purposes but rather to rapidly propagate rare or improved genotypes.