Vanilla

FIGURE 5.3 In vitro seed germination.

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 profiles of superoxide dismutase (SOD) and peroxidase (PRX) were studied in selfed progenies of V. planifolia. The profiles clearly indicated differences among prog-enies as expressed by the presence or absence of specific 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 confirmed 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 genetically 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, and V. pilifera (Minoo et al., 2006b) have been standardized to protect these species from extinction.

Clonal propagation methods for the efficient 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 proliferation 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 combination 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 proliferation 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 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.

FIGURE 5.4 Micropropagation of V. planifolia.

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 additives, 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 Concentration				Multiple Shoots Frequency (%)	Average No. of Shoots/Culture ± SD	Roots Development/Culture
Kin	BA	NAA	IBA			No.	Type
0.5				0.0		—	—
1.0			0.0		—	—
	0.5			71 ± 3.45	4.18 ± 0.30	—	—
	1.0			20 ± 3.63	1.0	—	—
		0.5		0.0	1.0	1	Velamen
		1.0		0.0	1.0	1	Velamen
			0.5	0.0	1.0	1	Long roots
			1.0	0.0	1.0	1	Long roots
0.5	0.5			0.0	1.0	—	—
1.0	0.5			0.0	1.0	—	—
0.5	1.0		0.0	1.0	—	—
0.5		0.5		0.0	1.0	1	Velamen
1.0		0.5		0.0	1.0	1	Branching
0.5		1.0		0.0	1.0	1	Velamen
0.5			0.5	0.0	1.0	1	—
1.0			0.5	0.0	1.0	1	—
0.5			1.0	0.0	1.0	1	—
	0.5		0.5	0.0	1.0	1
	1.0		0.5	97 ± 6.5	15.15 ± 3.63	—	—
	0.5		1.0	65 ± 11.4	10.35 ± 3.45	—	—
					0.0	1.0	1	Healthy roots