NUEVOS CONCEPTOS DE CÉLULAS

Due to the limitations of heterozygosity and generation times (as discussed in 2.1.2), propagation

and regeneration of new plantlets in vitro from tissue culture material has become central to the

improvement of woody perennial crop plants. However, such species are often particularly

recalcitrant to adventitious bud formation. Mullins and Srinivasan (1976) were the first to report the successful culture of totipotent grapevine tissue, and the regeneration of whole plantlets from this tissue. These researchers found that ovules of the Cabernet Sauvignon variety, cultivated on media containing added auxin (β-naphtoxyacetic acid; NOA) and cytokinin (6-benzyl amino purine; BAP) could produce stable embryogenic callus (EC), from which plantlets could be germinated by altering phytohormone concentrations in the media.

In recent years, interest in the in vitro regeneration of grapevine plantlets has increased as transgenic

techniques have acquired a record of successful crop improvement. Many groups have attempted to regenerate plantlets from grapevine tissue. However, efforts have been frustrated by the variable degrees of success obtained with different grapevine varieties.

2.3.1

Issues of chimerism

Initial grapevine transformation experiments showed that introduction of foreign DNA via

Agrobacterium is possible. However, the use of shoot apex cultures as starting material produced

plant shoots that contained both transformed and non-transformed cells (BARIBAULT et al. 1990). The

chimerism of recovered plants is likely to be a result of regeneration from partially transformed material that is able to survive low levels of antibiotic selection. A stepwise increase in antibiotic concentration in regenerating plant media has been successful in limiting chimerism somewhat with

respect to transgenes following transformation by organogenesis (MEZZETTI et al. 2002). However, the

chimeric nature of meristem remains a hurdle to the regeneration of phenotypically homogenous vines from this tissue (see section 2.1.4)

2.3.2

Somatic embryo tissue culture

Regeneration of grapevines from embryo cultures does not preserve the periclinal chimerism of the

parent plant (FRANKS et al. 2002; BERTSCH et al. 2005). As a result, somatic embryos have been the

tissue type of choice for transformation experiments. Since the initial report by Mullins and Srinivasan, attempts have been made to initiate somatic embryo cultures from a host of grapevine varieties, as described in the following sections. While many researchers have tested a wide array of media types, explant types and growth conditions, still no single protocol has been achieved which is

material collection and even agar types have been reported as decisive factors in achieving the consistently low rates of successful somatic embryogenesis.

Explant material

Floral tissues have been almost exclusively used for initiation of somatic embryo cultures. Of these,

the excised anthers of unopened flowers are most common (KIKKERT et al. 1997; TORREGROSA 1998;

IOCCO et al. 2001).

While attempting to adapt the initiation of embryogenic callus (EC) from anthers to a variety of different cultivars, Perrin and colleagues (2004) found anthers cut from the calyx rather than plucked were twice as efficient for the initiation of EC, as initiation always begins at the cut site. The stage of flower development also contributes significantly to successful initiation, with most successful initiations being from stage II (6-8cm flower clusters with 1.5mm diameter buds and 0.8-1.0mm long, yellowish, translucent anthers) or stage III (9-10cm flower clusters with 1.5-2.0mm diameter buds

and 1.0mm long, yellowish, cloudy anthers) flowers (DHEKNEY et al. 2009).

Carimi and co-workers (2005) reported higher EC initiation efficiencies from stigma and style tissues than from anthers for three grapevine cultivars, although it should be noted that only one stigma- style unit is obtained per dissected flower, as opposed to five anthers. Kikkert and colleagues (2005) similarly found a 7-fold average increase in initiation efficiency when using ovaries in comparison

with anthers for the initiation of EC from twelve Vitis genotypes. Whole flowers have also been found

to be appropriate for EC initiation in four cultivars (GAMBINO et al. 2007).

A couple of research groups have reported the generation of EC from the young leaves of certain grapevine varieties, but this technique has not yet proved successful for any of the famous wine- making varieties (DAS et al. 2002; DHEKNEY et al. 2009).

Culture regimes

The success and efficiency of initiation of EC is dependant on both the nutrient and phytohormone concentrations of media used. Several cultivars have been successfully initiated on PIV medium,

which uses major elements according to Nitsch and Nitsch (1969) with 4.5μM 2,4-

Dichlorophenoxyacetic acid (2,4-D) and 8.9μM BAP (FRANKS et al. 1998; IOCCO et al. 2001). Torregrosa

(1998) reported successful initiation of EC from four grapevine varieties using C1P medium, which

contains major elements according to Murashige and Skoog (1962) at half-strength, 5μM 2,4-D and

1μM BAP. Both media types use microelements according to Murashige and Skoog. Using a three-

step series of media, with different phytohormone concentrations at each step, Perrin and colleagues (2004) successfully initiated EC from anthers of 19 grapevine genotypes on medium containing macro-and microelements according to Murashige and Skoog. Fifteen of these could be

maintained as established EC for at least 84 days. More recently, the generation of somatic embryos has been reported for many more grapevine varieties, but a single broadly applicable technique has still not been identified (DHEKNEY et al. 2009; OLAH et al. 2009).

While EC cultures are commonly maintained with periodic subculturing on the same solid media used for initiation, there is evidence that moving EC onto media with higher ammonium concentrations

favours proliferation (PERRIN et al. 2001). Long-term maintenance of EC as suspension cultures in

liquid medium has also been reported (MAURO et al. 1995). Ben Amar (2007) and co-workers showed

that establishment of liquid cultures from EC masses is improved by the use of medium pre- conditioned with previously established EC cultures or through the addition of arabinogalactan.