Efficient Isolation of Protoplasts from Hypocotyl-Derived Callus of Brassica juncea Var. B9.

Protoplasts were isolated from 14 days-old hypocotyls-derived callus of Brassica juncea var. B9 using enzymatic tissue digestion. Callus was grown in artificial callus-inducing media i.e., MS basal added with growth hormones 2,4-D, NAA, and BAP at 1mg/ml concentrations. Cell wall degrading enzymes, Cellulase R10, Macerozyme, and Driselese were mixed in three different combinations (Set-1,2&3) in 0.6 M mannitol solution, and pH adjusted to 5.8. Callus tissue was digested under three different periods in all the enzyme sets and there was inconsistency in the number of protoplast yields. The highest protoplast yield i.e., 14.26 x 105 / gm tissue was recorded in Set-1 with 14 hours of incubation, and the lowest i.e., 5.22 x 105 / gm tissue was recorded in Set-3 with 18 hours of incubation. The protoplast yield was found consistent in the number in Set-2, and inconsistent in Set-1, and Set-3 against varied periods of incubation. The viability of the purified protoplasts was recorded at about 95.35% with a density of 20.32 x105 cells/ml/gm tissue. The hypocotyl-derived callus formation and protoplast isolation in Brassica juncea var. B9 was optimized in this study which may further be used in commercial crop breeding programs in the field of agricultural biotechnology.