Optimizing in-vitro protocol and diversity studies of vanilla (vanilla spp.) from five counties in Kenya

Abstract

Vanilla production can be a source of income to the farmers in Kenya. In-vitro regeneration is a rapid mass multiplication of quality plantlets; however, this technology has not been exploited at large scale for vanilla production in Kenya. Morphological and genetic diversity is an important tool for crop improvement. Production of vanilla in Kenya is partly limited due to inadequate knowledge on vanilla diversity. The objectives of the study were to (i) determine the effect of kinetin (KN) and indole-3-acetic acid (IAA) concentration levels on stem nodal segments of vanilla in Kenya, (ii) characterize vanilla accessions in Kenya using phenotypic traits (iii) characterize vanilla accessions in Kenya using microsatellite DNA markers. Shooting and rooting were established using 0, 0.4, 0.8, 1.2, 1.6, 2.0 mg L-1 concentration levels of KN and IAA on stem nodal segments of vanilla. In-vitro treatments were laid in Completely Randomized Design with five replications. Data were collected on plants with shoots, shoots per plant, number of leaves, number of nodes, shoot length, number of plants with roots, roots per plant, root length, dead plants, dormant plants and contaminated media. Data was subjected to analysis of variance at p ≤ 0.05 level of significance using general linear model procedure of SAS version 9.1. Means were separated by Tukeys’ Honestly Significant Difference Test at 5%. Phenotypic traits were used to estimate the level of accessions variation. POPGENE version 1.32 was used to compute the genetic parameter, while Powermarker version 3.25 was used to determine molecular variance. Treatment with kinetin at 1.2 mg L-1 resulted in superior number of plants with shoots (4.2±0.2), shoots per plant (2.6±0.4), number of nodes (2.4±0.4) and shoot length (11.0±0.6cm). IAA 2.0mgL-1 resulted in the longest root length (4.6±0.6 cm). Cluster analysis for qualitative traits grouped accessions into two major clusters and five sub-clusters based on the county of origin. Cluster analysis for quantitative traits grouped accessions into three groups. Amplicons ranged from 1 and 4 with 27 (96.43%) alleles being observed. Effective alleles mean was 1.63. Gene diversity mean was 0.35, while the mean shannon information index and polymorphic information (PIC) content values were 0.5 and 0.35, respectively. The highest PIC recorded was 0.375. Kinetin at 1.2mgL-l and IAA at 2.0mgL-1 were considered the best treatments for rapid mass multiplication of vanilla and can be used at large scale production. Phenotypic traits failed to group vanilla accessions into respective counties. Genetic markers failed to reveal high polymorphism among the studied vanilla accessions.