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.
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