Saad Bin Javed

Two-way germination system of encapsulated clonal propagules of Vitex trifolia L.: an important medicinal plant

ABSTRACT In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system of Vitex trifolia, for easy transport of the propagules and efficient utilization of its in vitro regeneration system. Nodal segments harvested from 8-week-old in vitro cultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl2.2H2O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed by in vitro rooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants.

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.