| Growth Regulators | In Vitro Responses | |||
|---|---|---|---|---|
| V. planifolia | V. andamanica | V. aphylla | V. pilifera | |
| Kin S | ingle shoot | Single shoot | Single shoot | Single shoot |
| BA | Multiple shoots | Single shoot | Multiple shoots (3–4) | Multiple shoots (3–4) |
| NAA | Root induction | Root induction | Root induction | Root induction |
| BA + Kin | Single shoot | Single shoot | Single shoot | Single shoot |
| Kin + IBA | Single shoot | Single shoot | Single shoot | Single shoot |
| BA + IBA (1.0 + 0.5 mg L−1) | Multiple shoot induction (12–15 nos., in 10 days of culture) | Multiple shoot induction (5–7 in 90 days) | Multiple shoot induction (8–10 in 90 days) | Multiple shoot induction (2–4 in 120 days) |
| BA + NAA | Callusing and plant regeneration | Multiple shoots | Callusing and plant regeneration | Single shoot |
| Kin + NAA | Single shoot | Single shoot | Single shoot | Single shoot |
| Basal medium | Single shoot elongation and development of roots | Single shoot elongation and development of roots | Single shoot elongation and development of roots | Single shoot elongation and development of roots |
BA = benzyladenine, IBA = indole butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.
a All growth regulators were supplemented on MS basal medium at 0.5 to 1.0 mg L−1.
FIGURE 5.6 In vitro rooting in V. planifolia cultures.
Plant Regeneration through Callus Cultures
Continuous vegetative propagation and lack of sufficient variations in the gene pool hamper crop improvement programs. Introduction of somaclonal variation through callus cultures has been attempted to broaden the narrow genetic base. A callus induction and in vitro plant regeneration system has been optimized from both vegetative and reproductive tissues. The best results were obtained using vegetative tissues and over 80% callusing was achieved in MS medium supplemented with 1 mg L−1 BA and 0.5 mg L−1 NAA. Callus differentiated into shoots that could be multiplied successfully in 1:12 ratio in a combination of 1 mg L−1 BA and 0.5 mg L−1 IBA, when supplemented with MS medium (Table 5.3). In vitro rooting was induced with an efficiency of 100% in basal MS media devoid of any growth regulators. This ability of dedif-ferentiated tissue to regenerate is a crucial prerequisite for genetic transformation experiments. The protocol was successfully extended to the endangered wild species, V. aphylla, offering the potential of applying the protocol for mass multiplication as well as induction of variations in Vanilla species, in a limited time.
| Growth Regulators (mg L−1) | Callusing (%) | Shoot Regeneration (%) | No. of Shoots/Culture |
|---|---|---|---|
| 0 | 0 | 0 | 0 |
| NAA (0.5) | 80 | 0 | 0 |
| BA (1.0) | 0 | — | — |
| BA (1.0) + NAA (0.5) | 80 | 90 | 10 |
| BA (0.5) + NAA (1.0) | 0 | — | — |
| BA (1.0) + IBA (0.5) | 10 | 60 | 6 |
BA = benzyladenine, IBA = indole butyric acid, Kin = kinetin, NAA = α-naphthaleneacetic acid.
Reports on variability among callus-regenerated plants in vanilla are few. They concern successful plant regeneration from leaf- and seed-derived callus (Davidonis and Knorr, 1991; Davidonis et al., 1996; Janarthanam and Seshadri, 2008; Xju et al., 1987) and studies among indigenous collections of vanilla, through polyacrylamide electrophoretic (PAGE) studies (Rao et al., 1993a). A study comprising randomly selected callus-regenerated progenies showed variability in morphology and RAPD profiles (Figure 5.7) among the callus-regenerated plants in comparison with the control plant V. planifolia (Minoo, 2002). It showed that a significant amount of variability can be generated with this protocol and be utilized in vanilla improvement programs for developing variants with desirable agronomic characters.