A study Towards an Efficient Protocol for Somatic Embryogenesis in Sri Lankan Citrus Species

Abstract

Higher production of Sri Lankan Citrus spp. is needed to reduce the import expenditure. Lack of elite planting material is one of the major barriers for intensive cultivation. Development of an efficient in vitro regeneration protocol is essential for elite planting material production and genetic improvement of Citrus. This study evaluated the effect of maltose concentration on local Citrus spp. for direct somatic embryogenesis followed by plant regeneration. Mature embryos of Naran (Citrus crenatifolia), Heennaran(Citrus reticulate) and Panidodam (Citrus sinensis) were used as explants. In experiment 1, cotyledons of Naran were placed in 100mg/l and 200mg/l of maltose with liquid MS to select the effective maltose level. Multiple som atic embryos formed from single Naran cotyledons after 5 days of culture. Significantly higher frequency of som atic embryogenesis was observed from 200mg/l maltose level. In experiment 2, Heennaranand panidodam cotyledons were placed in selected level of 200 mg/1 of maltose. Somatic embryos of Heennaran cotyledons developed into seedlings of different heights from 0.5 cm to 5 cm after 5 days of cotyledon culture. Percentage of first somatic embryogenesis from total explants was 87% . The secondary somatic embryos were formed in 30°/fr of cotyledons. In Panidodam, 78% and 69% of initial and secondary somatic embryos were formed, respectively. A significant difference could not be detected between two species on somatic embryogenesis on 200 mg/1 of maltose level in liquid MS culture. Possibility for genetic transformation of Citrus was also tested: Cotyledon explants were transformed by Agrobacterium bearing 35S: VlmybA2 for induction of anthocyanin pigments. Putative 35S: VlmybA2somatic embryos emerging from transformed cotyledons, produced purple dots after 1 week of culture indicating the potential of transformation. This study indicates the potential of this protocol for efficient somatic embryogenesis for future multiplication and crop improvement programmesthrough genetic transformation of Sri Lankan Citrus spp.