Vanilla produces numerous minute seeds that do not germinate under natural condi-tions. Tissue culture technique can be used to successfully germinate the seeds.
Protocols for seed and embryo culture of vanilla have been standardized (Gu et al., 1987; Knudson, 1950; Minoo et al., 1997; Withner, 1955).
Seed culture in different basal media indicated that vanilla seeds had no stringent nutritional requirements for the initiation of germination unlike some terrestrial orchids of temperate climate (Minoo et al., 1997). The germination of seeds began within four weeks of culture and the initial stages of germination were typical of most orchids, such as swelling of the embryo followed by rupturing of the seed testa, and the subsequent emergence of protocorms (Figure 5.3). Seeds germinated directly into plantlets in the medium supplemented with benzyladenine (BA) (0.5 mg L−1) alone, without any intervening callus phase, and could thus be utilized for the production of selfed progenies/seedlings. The addition of tryptone had a growth-promoting effect on the size and development of protocorm, irrespective of the basal medium to which it was added. In treatments with BA, most of the protocorms remained the same with the scale-like leaf primordial and developing into shoots, whereas treatment with auxin supplements showed the gradual disorganization of the protocorms into callus. Murashige and Skoog’s (MS) medium gave a better response than Knudson’s medium, for in vitro cultures of vanilla. The minimum germination (26%) was observed in MS medium at half strength and the maximum (85%) was recorded in full strength MS medium supplemented with 2 g L−1 tryptone (Minoo, 2002).
The requirement of cytokinin for germination is considered to be related to the utilization of lipids that constitute the primary storage material in most orchid seeds
and it has been observed that unless storage lipid is utilized, germination does not continue (De Pauw et al., 1995).
Vanilla, a cross-pollinated crop, is known to have many meiotic and postmei-otic chromosomal abnormalities (Ravindran, 1979). As a result, it is possible to get various cytotypes in the seed progenies. Culturing of seeds can thus give many genetically varied types. Studies on in vitro germination of vanilla seeds and the resultant progeny showed morphological and biochemical variations. Isozyme pro-fi les of superoxide dismutase (SOD) and peroxidase (PRX) were studied in selfed progenies of V. planifolia. The profi les clearly indicated differences among prog-enies as expressed by the presence or absence of specifi c bands. The maximum similarity that these progenies exhibited was 47.37%, indicating high segregation and level of heterozygosity existing in V. planifolia (Minoo et al., 1997). This heterozygosity was further confi rmed by AFLP analyses (Bory et al., 2008c). Thus, in vitro culture can be used for the germination of seeds and the selection of useful genotypes from segregating progenies that might be mass propagated for obtaining disease-free planting material.
MICROPROPAGATION
In vitro propagation of vanilla is essential to generate uniform, disease-free plantlets and for conserving the genetic resources. In vitro propagation using apical meristem has been standardized for the large-scale multiplication of disease-free and geneti-cally stable plants (Cervera and Madrigal, 1981; George and Ravishankar, 1997;
Kononowicz and Janick, 1984; Minoo, 2002; Minoo et al., 1997; Philip and Nainar, 1986; Rao et al., 1993b). In vitro propagation of V. tahitensis (Mathew et al., 2000) and endangered species of vanilla, such as V. wightiana, V. andamanica, V. aphylla, FIGURE 5.3 In vitro seed germination.
58 Vanilla and V. pilifera (Minoo et al., 2006b) have been standardized to protect these species from extinction.
Clonal propagation methods for the effi cient multiplication of V. planifolia by induction of multiple shoots from axillary bud explants (Figure 5.4) using semi-solid MS medium supplemented with BA (2 mg L−1) and α-naphthale neacetic acid (NAA, 1 mg L−1) have been reported (George and Ravishankar, 1997). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg L−1 and NAA at 0.5 mg L−1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots was obtained from a single axillary bud explant over a period of 134 days. The use of an intervening liquid medium was found to enhance the multiplication of shoots.
In another study (Minoo et al., 1997), the subculture of the explants onto prolif-eration MS media containing various levels of cytokinin (BA) and auxin (indole butyric acid, IBA) was evaluated (Table 5.1). The initiation of preexisting buds to grow in vitro could be induced in MS medium with low cytokinin. However, a com-bination of cytokinins and auxin promoted multiple shoot formation. The ideal medium for multiplication was MS supplemented with BA (1 mg L−1) and IBA (0.5 mg L−1). In this medium, an average of 15 multiple shoots were induced in 90 days of culture (Figure 5.4). Nodal segments gave a better response, with a mean of 15 shoots per culture compared to the shoot tips, which gave a mean of seven shoots per culture (Minoo, 2002). The culture media and conditions favorable to micro-propagation of V. planifolia were suitable for other related species, such as V. anda-manica, V. aphylla (Figure 5.5), and V. pilifera. The number of shoots induced in different species varied (Table 5.2). About 12–15 shoots/culture could be induced in V. planifolia, followed by V. aphylla (8–10 shoots). Among the species studied, the lowest multiplication rate was observed in V. pilifera. Elongated shoots from prolif-eration medium were rooted on MS growth regulator free medium containing 30 g L−1 sucrose (Figure 5.6). In vitro plantlets with well-developed roots were
FIGURE 5.4 Micropropagation of V. planifolia.
acclimated with a survival percentage of more than 70%. The root initiation on microcuttings started between four and six days after culture, reaching 100% of the cultures after two weeks, indicating that the optimal endogenous levels of plant growth regulators required for rooting were already present in the tissue/explants.
Janarthanam et al. (2005) and Kalimuthu et al. (2006) have devised simple and rapid protocols for the mass multiplication of V. planifolia. A commercially viable protocol for the mass propagation of V. tahitensis, another cultivated species of Vanilla, was standardized with a multiplication ratio of 1:4.7 over a culture period of 60–70 days (Mathew et al., 2000). Rao et al. (2000) have reported the occurrence and micropropagation of V. wightiana Lindl., an endangered species. Giridhar et al.
(2001) and Giridhar and Ravishankar (2004) have studied the effects of other addi-tives, namely, silver nitrate, thidiazuron, zeatin, coconut milk, and so on, on in vitro shoot multiplication and root formation in V. planifolia.
TABLE 5.1
Effect of Growth Regulators on Multiple Shoot and Root Induction from Shoot Explants of V. planifolia on MS Medium (Mean of 20 Replicates)
Growth Regulators
1.0 0.5 0.0 1.0 1 Branching
0.5 1.0 0.0 1.0 1 Velamen
BA = benzyladenine, IBA = indole-3-butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.
60 Vanilla
The conversion of root tips into shoots was observed in V. planifolia and V. aphylla when cultured on MS medium supplemented with BA (1.0 mg L−1) and IBA (0.5 mg L−1). These shoots developed into plantlets and were hardened and estab-lished in soil. The conversion of root meristem into shoots in in vitro cultures of vanilla was earlier reported (Philip and Nainar, 1988). These meristematic conver-sions without callus stage are assumed to minimize the chances of induced epige-netic changes. Earlier studies by Sreedhar et al. (2007) indicated no difference in the AFLP-banding patterns of any of the micropropagated samples for a particular primer, suggesting the absence of variation among the micropropagated plants.