Optimization of Isolation and Culture of Protoplasts in Alfalfa (Medicago sativa) Cultivar Regen-SY - American Journal of Plant Sciences

AJPS > Vol.10 No.7, July 2019

Optimization of Isolation and Culture of Protoplasts in Alfalfa (Medicago sativa) Cultivar Regen-SY ()

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DOI: 10.4236/ajps.2019.107086 1,555 Downloads 3,521 Views Citations

Author(s)

Ankush Sangra¹, Lubana Shahin¹, Sarwan K. Dhir^2*

Affiliation(s)

¹College of Agriculture, Family Sciences and Technology, Fort Valley State University, Fort Valley, GA, USA.
²Department of Plant Sciences, Center for Biotechnology, Fort Valley State University, Fort Valley, GA, USA.

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.

KEYWORDS

Alfalfa, Protoplast, Protoplast Isolation, Protoplast Culture, Optimization

Share and Cite:

Sangra, A. , Shahin, L. and Dhir, S. (2019) Optimization of Isolation and Culture of Protoplasts in Alfalfa (Medicago sativa) Cultivar Regen-SY. American Journal of Plant Sciences, 10, 1206-1219. doi: 10.4236/ajps.2019.107086.

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