Alfalfa cultivar Regen-SY germplasm (PI 537440) was obtained in the form of seeds from Western Regional PI Station through U.S. National Plant Germplasm System.

Seeds were surface sterilized using 70% ethyl alcohol for 30 s followed by 20% bleach (Clorox^®) treatment for 10 min. Seeds were rinsed with sterile distilled water for three times and then germinated on Murashige and Skoog (MS) basal medium (PhytoTechnology Laboratories, KS, USA) containing 3% sucrose and 0.7% agar (PhytoTechnology Laboratories).

Fully expanded dark leaves from plants of different age (2, 4, 6 and 8 weeks of subculture) were excised and 1 g of leaf tissue was weighed. Leaf tissues were provided incisions using sterile scalpel. Plant material was immediately transferred to deep petri dish (60 × 20 mm, Nunc Lab-Tek^®) containing 10 mL of enzyme (PhytoTechnology Laboratories) solution (Table 1). Plant material was incubated with enzyme solution for 2 - 8 h in the dark with gentle shaking (50 - 70 rpm) on a shaker (Brunswick C2 Platform shaker) for enzymatic digestion. Similarly, the enzyme solution consisting of different concentrations of cellulase (PhytoTechnology Laboratories) and macerozyme (PhytoTechnology Laboratories) along with various concentration of mannitol were tested to determine their

effect on protoplast isolation. Dark conditions were created by wrapping aluminum foil around the petri dish.

· After enzymatic incubation the digestion solution was passed through the nylon mesh of appropriate size (50 µm).

· Protoplasts were then washed three times with cell and protoplast washing solution (CPW) containing 0.7 M mannitol and centrifuged (HN-SII centrifuge, IEC, USA) at 500 - 2000 rpm for 10 min after each washing. The composition of CPW is given in Table 2.

· Purified protoplast suspension was then checked for protoplast yield and viability.

Protoplasts were quantified using hemocytometer. Viability of the protoplasts was estimated by Fluorescein diacetate (FDA) staining assay. About 10 µL of protoplast mix was pipetted on to a Neubauer Hemocytometer (Reichert, USA) with cover slips. Entire chamber was filled with protoplast suspension and filled hemocytometer slide was viewed under microscope of 40× magnification. FDA stains living (viable) protoplasts resulting in green fluorescence.

Percentage viability was determined by the formula given below:

Percentageviability = Numberofviablecellscounted Total   cellls   counted   ( Viableanddead ) × 100

The concentration of protoplasts per mL per gram of leaves was determined by formula:

Concentration = Averagenumberofcellsinonelargesquare weightofleavesmaterialused × dilutionfactor × 10 4

Fluorescein diacetate stock solution was prepared by dissolving 5 mg/mL FDA (Sigma) in acetone. Fluorescein diacetate working solution was prepared by taking

20 µL of working solution and mixing it with 1 mL of CPW solution. For staining, equal volume of FDA working solution and protoplast suspension was used.

· Protoplasts were cultured at different densities. i.e. 1 × 10⁴, 2 × 10⁴, 1 × 10⁵, 2 × 10⁵ per mL of liquid KP8 medium (modified from [18] , Table 3).

· The cultures were maintained in the dark for 5 d and then transferred to 25˚C ± 2˚C with 16 h light regime of 25 μE∙m⁻²∙s⁻¹ light radiation.

· A mixture of KP8:K8 (modified from [19] ) was used for the progressive replacement of the bathing medium after 7 (3:1), 14 (2:1), 21 (1:1) and 28 (0:1) days.

· Microscopic observations of the protoplast experiments were carried out using Olympus IX70 inverted fluorescent phase contrast microscope (Olympus, Japan).

Experimental data were statistically analyzed using analysis of variance (ANOVA). Treatment means were separated by using Tukey Kramer honestly significance difference (HSD) test at p ≤ 0.05. Data were analyzed using Shiny application (Web based software for ANOVA) [20] .

All the enzyme treatments shown in Table 2 were tried and out of these treatment, the treatments shown in the graph (Figure 1) showed considerable Digestion

of tissue within 8 h. One gram of leaf tissue was weighed and incisions were provided on the leaf surface with the help of sterile scalpel. Leaf tissue was incubated with the enzyme solution on C2 Platform shaker (New Brusnwick Scientific, Edison, NJ, USA) at 50 rpm. Some treatments showed completed digestion within 6 hours and some treatments didn’t result in complete tissue digestion even after 10 hours. All treatments were observed under microscope and yield and viability was recorded for only those treatments which digested more than 50% of tissue. The maximum yield and viability was shown by treatment consissting of 2% Cellulase + 1.5% macerozyme.

The effect of emzymolyisis time was determined by using best enzyme treatment and taking leaf tissue from the 4-week old plant. One gram of the leaf tissue was incubated with best enzyme treatment for the 2, 4, 6 and 8 h. Two-hour enzymolysis treatment resulted in the minimum protoplast yield and viabilty (Figure 2). The tissue was not completey digested and therefore protoplast yield was low after 2 h of enzymolysis treatment. Six-hour enzyme incubation resulted in the maximum yield (5.49 × 10⁶) and viability (89.5%) of protoplasts. After 6 h leaf tissue was completely digested and this treatment, spherical shaped green protoplasts were obtained. The digestion of the cell wall was confirmed by absence of fluorescence after staining with calcoflour white. Two types of protoplasts were obtained, i.e. small (20 - 30 µm) and large (30 - 40 µm). The protoplasts had dense cytoplasm with chloroplasts arranged in peripheral area. Eight hour incubation treatment also caused complete digestion of the tissue but due to the

prolonged exposure of the portoplasts to enzyme, there was shrinking and bursting of the protoplasts resulting in reduced yield and viability as compared to the 6 h treatment. Therefore through this experiment it was observed, exposing protoplasts after tissue digestion results in toxicity which reduce yield and viability of protoplasts.

The effect of plant age was determined by taking 1 g leaf tissue from 2, 4, 6 and 8-week old plants. Tissue was incubated under optimum conditions of enzymatic degradtion (6 h). Protoplast yield obtained was lowest for the 2-week old plant whereas protoplast yield was highest for the 4-week old plant and protoplast viability was highest for 4-week old plant followed by 2 and 6-week old plant. Eight week old plant resulted in lowest protoplast viability (Figure 3). This experiment shows that age of plant has a significant effect on the protoplast yield. In case of alfalfa Regen-SY, 4-week old plant resulted in optimum results. The low protoplasts yield of 2-week old plant could be because of the fact that alfalfa Regen-SY plants at the age of 2-week does not have fully expanded leaves and leaves are often folded towards inside due to which they are not able to make optimum contact with the enzyme solution. As 4-week old plant has fully expanded leaves and less enzyme resistant content as compared to the 6 and 8-week old plant, therefore it resulted in maximum yield and viabilty.

The effect of mannitol (osmoticum) concentration was determined by using best enzyme treatment on 4-week old tissue for 6 h in CPW solution containing different concentrations of mannitol (0.2 M, 0.5 M, 0.7 M, 1 M). As protoplast can rupture in hypertonic solution and collapse in hypotonic solution, optimizing osmoticum concentration becomes very important. Protoplast yield and viability was lowest at 0.2 M mannitol concentration causing protoplast to burst and rendering them inviable. The protoplast yield and viability was highest with 0.7 M mannitol concentraion. At this concentration, protoplast retained their shape and orientation. Using 1 M concentration of mannitol caused many protoplasts to fuse which resulted in the reduced yield and viability (Figure 4).

To determine the effect of the centrifugation speed, 1 g of leaf tissue was excised from the 4-week old plant and incubated with best enzyme treatment for 6 h in

CPW containing 0.7 M mannitol. After incubation protoplasts were purified by passing through 50 μm mesh. The protoplasts were then centirifuged at varying centrifugation speed (500, 1000, 1500 and 2000 rpm) in 15 mL centrifuge tubes for 10 min. It was observed that pellet was not completely formed at the 500 rpm and lot of plant material kept floating in the supernatant. It resulted in the lowest protoplast yield and viability (Figure 5). Optimum centrifugation speed was found to be 1000 rpm for 10 min which resulted in highest yield and viability. The reduction in yield and viability was observed at centrifugation speed over 1000 rpm. The protoplast obtained where spherical and retained their shape at 1000 rpm. Centrifugation speed of 2000 rpm resulted in rupturing of the protoplasts and loss of conformation as they were exposed to because of high centrifugation speed.

To study the effect of the shaker speed on the protoplast isolation, leaves from four week old plant were incubated with the best enzyme treatment at 6 hours of enzymolysis was used. Four experiments were set up at different shaker speed (50, 55, 60 and 65 rpm). The protoplast yield and viability was highest at 55 rpm speed and the protoplast yield and viability decreased over 55 rpm due to the more enzyme solution interaction with the leaf tissue (Figure 6). Shaker speed is one of the conditions that need to be optimized for the protoplast isolation. Too less shaker speed can result in increase in the incubation time which can cause enzyme toxicity making protoplasts inviable. Too high Shaker speed can cause disruption of chloroplast orientation in the protoplast as well as bursting of protoplasts. There was no significant difference in the protoplast yield and viability at different shaker speeds. Since 55 rpm resulted in maximum yield it was selected as optimum condition for the further experiment.

Based on the optimization experiments, the optimum conditions for the protoplast culture of alfalfa cultivar Regen-SY are given in Table 4.

Freshly isolated protoplasts using optimum conditions (Table 4) were cultured at four different densities 1 × 10⁴, 2 × 10⁴, 1 × 10⁵, 2 × 10⁵ in the liquid medium. Protoplasts only survived at the densities 1 × 10⁴ and 2 × 10⁴ and protoplasts at the remaining plating densities died. The plating efficiency was found to be 78.25% ± 5.40% and 75% ± 5.24% for 1 × 10⁴ and 2 × 10⁴ culture densities respectively.