Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn't result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.

No abstract

Alfalfa (Medicago sativa) is one of the most important forage legume crops because of its mass production and high feeding value. It originated in Asia and is one of the most ancient plants cultivated throughout the world as a fodder. Despite the well-studied somatic embryogenesis of alfalfa, there is a lack of a long-term maintainable somatic embryogenic system. Every time an embryogenic callus culture must be started from new explants, which is laborious, costly and time consuming. In addition to this, endogenous microorganisms present in ex vitro explants of alfalfa can often cause contamination, reducing the efficiency of callus culture. An attempt was made to establish long-term continuous somatic embryogenesis system in alfalfa using cultivar Regen-SY. Nine somatic embryogenesis pathways were studied and evaluated for embryo yield, plant conversion rate and embryogenic sustainability. Somatic embryos passed through the same stages (globular, heart-shaped, torpedo and cotyledonary) as characteristic of the zygotic embryo and secondary somatic embryogenesis was also observed. B5H-B5 system showed the highest embryo yield and plant conversion rate whereas SH4K-BOi2Y system demonstrated the highest embryogenic sustainability and maintained the embryogenic potential even after six subculture cycles. Scanning electron microscopy was applied to study the morphology of the somatic embryos and secondary somatic embryogenesis. Therefore, long-term maintainable somatic embryogenesis system protocol was developed through this study, which will help to enhance and accelerate the alfalfa biotechnology research.

No abstract

No abstract