Abstract
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.
Highlights
Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study
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
The study showed that low concentration of cellulase (2%) and macerozyme (1.5%) are sufficient for the release of protoplasts in short incubation period (6 h)
Summary
Alfalfa (Medicago sativa) is one of the important leguminous forage crops be-. It originated in Asia and is cultivated across the world for fodder [1]. A single stand of alfalfa can fix about 300 pounds of nitrogen each year. This results in the increase nitrogen availability for the plants and increase in soil nitrogen fertility for subsequent crops in rotation [3]. Alfalfa hybrid Regen-SY was released in 1989 and it was produced using first generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars [5]
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