Synthetic Seeds Production by Encapsulated Somatic Embryo of ‘Rough Lemon’ (Citrus Jambbhir) Rootstock and Assessment of Genetic Stability Using ISSR Analysis

Abstract

Simple protocol has been developed and standardized in vitro propagation methods by cultureimmature seeds and embryonic callus induction, encapsulation somatic embryos and generation and genetic stability of ‘Rough lemon’ (Citrus jambbhir)were evaluated. Immature seeds were cultured on MS mediumfree growth regulators, after seedling formation, it was divided into three parts (new leaves, stem, roots) and isolated cotyledon were cultured on MS media with 2,4, D at 0.75, 1.0 and 1.25 mg/l. to induce embryocalli..To maintain continuous embryogenesis the embryonic calli were transferred on MS supplemented with kin at 0.1, 0.2 and 0.3 mg/l. The somatic embryos were induced by transfer on MS media containing coconut water at 1, 2 and 3 % for 7 weeks.The encapsulation process was induced by using sodium Alginate at 2 and 4 % and gelling by CaNo₃ at 8 and 16 % for 30 and 45 min..Germination encapsulation somatic embryos on MS free hormone. Genetic stability of embryos derived plantlets was assessed using ISSR markers as compared to plantlets grown ex vitro. Results showed that the highest level of embryo genic callus induction 92% was achieved in MS +2,4-D at1.0 mg/l   the highest callus weight obtained 57g treated with 1.25 mg/l 2,4-D for cotyledon, The highest somatic count (57embryo/g) treated with (MS +0.3 mg/l kin)and The highest percentage of somatic embryo 90 %   by (MS + 0.2 mg/l kin) for cotyledon calli . The best growth and differentiation of somatic embryos was MS + 2 % coconut water for leaves and cotyledons 5.83 g for each. The suitable encapsulated somatic embryos with Na –Alginate 2% and solidified by CaCl₂ at 20% for 30 min. High Genetic stability of regenerated plantlets derived from encapsulated somatic embryos as compared with plantlets grown ex vitro by using ISSR markers.Synthetic seeds obtained from encapsulated somatic embryogenesis is a suitable method for micro propagation at any time and has the potential for use in the commercial propagation and useful in exchange of sterile material between laboratories, germplasm conservation of lime without genetic variation.