Evaluation Of 2, 4-Dichlorophenoxy Acetic Acid and Naphthalene Acetic Acid Concentration On Callogenesis, Somaclonal Variation and Sugarcane Mosaic Virus Elimination in Sugarcane (Saccharum Officinarum L).

Abstract

Sugarcane, Saccharum officinarum (L.), is one of the most important crops in Kenya and has wide range of economic benefits. However, the industry has been facing several challenges including declining yields due to use of poor quality planting materials. The prolonged seed multiplication process of newly released varieties and local environmental conditions that do not favour production of flowers are some of the other major constraints to sugarcane production in Kenya. In vitro culture offers a practical and rapid method for mass propagation of disease-free clonal materials. Further, unintended variations observed during in vitro callus regeneration have proved quite promising in sugarcane improvement programs. The study therefore aimed at establishing the effect of 2, 4-D and NAA concentrations on the callogenesis, somaclonal variation and disease response of three varieties of sugarcane, namely; CO421, CO945 and N14. The effects of 2, 4-D and NAA concentrations on callus induction and shoot regeneration in three sugarcane varieties; CO945, CO421 and N14 were investigated in the study. Young leaf spindle explants were cultured on MS basal medium supplemented with 2, 4-D (0.0, 2.0, 2.5, 3.0, 3.5 and 4.0 mg L-1) and NAA (0.0 and 1.0 mg L-1). Experiments were laid out in the laboratory in completely randomized design replicated three times. Observations were recorded on percent callus formation, percent shoot formation and morphological characterization of callus. Data was subjected to ANOVA at 5% level of significance. Significantly higher callus production (93%) was observed at 3 mg L-12, 4-D. The presence of NAA tended to depress callusing and shoot production in sugarcane variety N14. The interaction between NAA and 2, 4-D had no significance on the parameters. Application of 2, 4-D at between 2.0 and 3.5 mg L-1 gave the highest % callus formation and shoot formation in all sugarcane varieties. Application of 2.5 and 3.5 mg L-1 2, 4-D was most effective for sugarcane callogenesis and regeneration for the three sugarcane varieties. NAA treatment in Callus formation media was not beneficial. The study reported the SCMV indexation of in vitro regenerated plants by infectivity assay method in sugarcane. The plants were developed by organogenesis through callus. For infectivity test sap of in vitro regenerated plants was inoculated on Sorghum one month after hardening. All the SCMV indexed plants were grown in green house and monitored for mosaic symptoms at weekly intervals. All 36 treatments showed some levels SCMV infection. In vitro regeneration of sugarcane through callus induction of young leaf spindles did not eliminate SCMV and was not therefore recommended for the multiplication of viral disease free sugarcane planting materials. Callogenesis is one of the tools in sugarcane tissue culture for generating agronomically significant variation. Tissue culture derived variations are known as somaclonal variation. The study was carried out to investigate the effect of MS media supplemented with various concentrations of NAA and 2, 4-D on somaclonal variation in sugarcane genotype CO421, CO945 and N14. Screening of somaclonal variants was done on four months old field grown in vitro culture generated plants. The treatments were laid out in split-split plot design. The morphological characters studied included; tillering capacity, diameter of the cane, internode length, leaf length and width. Analysis of variance was done using GenStat version 17.0 and pair wise comparison of means of phenotypic traits of all somaclones computed by calculating fisher’s least significant difference (LSD) at P≤0.05. Pearson’s correlation between all phenotypic traits and Turkey test of the selected clones were computed. Multivariate analysis of variance (MANOVA) done reveal the patterns of phenotypic diversity of quantitative traits studied. Means of each quantitative character were standardized before subjecting to the principal component analysis (PCA). The standardized data of 5 quantitative traits were then used as an input for the PCA biplot loading and cluster analysis. An agglomerative, hierarchical cluster classification technique with Average linkage strategy was performed. The results of the analysis of variance for the differences in morphological traits indicated that genotype, 2, 4-D and the various interactions had significant effect on the various morphological traits. Application of 2, 4-D to CFM led to somaclonal variation irrespective of the sugarcane genotype used. The observed variation however had no correlation to the hormonal concentration supplemented in the CFM. The dendrogram demonstrated variation among the somaclones based on morphological traits, could be a valuable source for sugarcane improvement program. Application of 2.5 and 3.5 mg L-1 2, 4-D was most effective for sugarcane callogenesis and regeneration for the three sugarcane varieties. In vitro regeneration of young leaf spindles of sugarcane through callus induction at various concentrations of 2, 4-D and NAA did not eliminate SCMV and was not therefore recommended for the multiplication of viral disease free sugarcane planting materials. Application of 2, 4-D to CFM led to somaclonal variation irrespective of the sugarcane genotype used. The observed variation however had no correlation to the hormonal concentration in callus formation media. The study therefore established that 2, 4-D concentration have an effect callogenesis, somaclonal variation and disease response of three varieties of sugarcane, namely; CO421, CO945 and N14 and could be useful in rapid multiplication of sugarcane planting materials and generation of useful variations for breeding programs.