In vitro micropropagation and genetic fidelity studies using SCoT and ISSR primers in Annona reticulata L.: an important medicinal plant

Abstract

An efficient and reproducible in vitro plant regeneration protocol has been developed for Annona reticulata L., a medicinally important plant of family Annonaceae. This is the first report of an efficient in vitro micropropagation and genetic fidelity evaluation of Annona reticulata using nodal segments as explants. Nodal explants were cultured on Murashige and Skoog (MS) medium augmented with various concentrations of cytokinins alone and in combination with auxins. MS medium supplemented with 2.5 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/l indole-3-butyricacid (IBA) combination has proven the best for optimum regeneration response (87.4%) and a maximum number of multiple shoots (6.33 ± 0.37). Rhyzogenesis occurred on half-strength MS medium supplemented with 4 mg/l IBA, resulted in the highest percentage of rooting (73%) and a maximum number of roots (4.65 ± 0.23) along with maximum root length (1.76 ± 0.08 cm). The well developed regenerates were shifted to the mixture of soil, sand and organic manure (2:1:1) in small plastic cups and acclimatized successfully. Genetic fidelity of the discussed etiquette was validated using two types of DNA fingerprinting techniques i.e. start codon targeted (SCoT), and inter simple sequence repeat (ISSR) analyses. Among the ten SCoT (SC) and ISSR primers used, an excellent amplification with scorable DNA bands was produced by SC9 and ISSR4 primers. The results showed that the regenerated plantlets are monomorphic and true-to-type with mother plant. This study provides important information about selection of suitable regeneration medium to improve multiple shoot induction and rooting of Annona reticulata. It can be used for commercial cultivation and for further genetic redevelopment investigation studies, also applied for large scale propagation of elite genotypes.