Protocol development for micrpropagation of vanilla (Vanilla planifolia Andr.) Clone (Van.2/05)

Abstract

Vanilla (Vanilla planifolia Andr.) is an important spice, which is economically valued for its cured fragrant beans that make one of the most expensive spices in the world trade. Although the crop had been introduced to Ethiopia and proved to adapt well to the hot humid agroecologies, the country did not exploit its potentials for vanilla production due to shortage of planting material; hence, the crop is still under maintenance at the Tepi National Spices Research Center (TNSRC). The crop is commonly propagated through stem cuttings. However, the technique is not recommended in modern vanilla cultivation, since it arrests subsequent plant growth and development and serves as an ideal means for the spread of varied diseases, etc. Therefore, there is a need of using the modern tissue culture techniques to alleviate the above stated challenges of multiplication. However, no efficient in vitro protocol had so far been developed to propagate the available vanilla clone in Ethiopia, which has been introduced recently from Mauritius. The present study was carried out with the main objective of developing micropropagation protocol for the V. planifolia accession (Van.2/05). In the current study, the initial materials for stock plant establishment were obtained from TNSRC, and established in the greenhouse of the Plant Biotechnology Laboratory of JARC, where all the subsequent experiments were carried out. Nodal explants and Murashige and Skoog (1962), MS, basal medium were used exclusively throughout the experiments. In all cases, the experiments were laid out in Completely Randomized Design (CRD) with factorial treatment combinations and replicated three times, whereby each replicate has 2 explants for hormone involving experiments. In this study, statistically highly significant (p < 0.01) differences were recorded from the use of different concentrations of active chlorine in local bleach (Berekina® ) and varied levels of treatment durations on aseptic culture initiation. Therefore, the best result (72.23% rate of asepsis and survival) was obtained from the use of 20 minutes treatment of nodal explants using a solution Berekina® containing 5% active chlorine. With regard to shoot multiplication, the combined use of BAP and NAA revealed statistically very highly significant (p < 0.001) differences, whereby 2 mg l-1 BAP and 0.5 mg l-1 NAA proved to be the best providing the highest mean number (5.33) and length (4.9 cm) of shoots after five weeks of culture. Statistically very highly significant differences (p < 0.001) were also observed on in vitro rooting from the combined use of different MS basal medium strengths and IAA concentrations. Therefore, the best were recorded from the use of ½ MS basal medium added with 0.5 mg l-1 IAA for the number of roots and length, viz. 4.00 roots per plantlet with an average length of 6.1cm. Acclimatization of in vitro derived plantlets was also successful, whereby the average rate of ex-vitro survival was 83.4%. Therefore, the advent of this protocol could be of considerable value to relieve the problem of mass multiplication and enhance the expansion of vanilla cultivation in Ethiopia. And further studies on virus indexing, and cryopreservation should be considered using the protocol.