Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design

A protocol was established for plant regeneration from leaf protoplasts of guava (Psidium guajava L.) using mixture-amount (concentration) experiments. A protoplast yield of 3.7 × 10⁶ (viability >90 %) was obtained when 1 g leaf strips were digested in a solution of ∼0.75 M osmoticum with 6 % (w/v) enzyme containing cellulase: macerase: hemicellulase as proportion of ∼0.4: 0.5: 0.1. Protoplasts developed the maximum number of microcalli using 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Maximum shoot formation (>12) via organogenesis from resulting calli was obtained using >3.4 mg l⁻¹ PGRs containing kinetin (K): 6-benzylaminopurine (BAP) at a ratio of 0.6:0.4. Shoots were rooted in medium containing indole-3-butyric acid and plantlets were successfully acclimatized. Results of polynomial response models revealed that: (1) Osmolarity was the primary determinant for protoplast yield and viability, irrespective of osmoticum type; (2) Most of the variation in protoplast yield was driven by macerase concentration; (3) Protoplast viability was driven mainly by cellulase concentration; (4) NAA was superior to BAP for callus induction, an antagonistic proportional effect was observed when they were blended; and (5) K was more effective than BAP in shoot regeneration, but due to synergistic blending the response was highest when both were present. Overall, guava was amenable to protoplast culture and the mixture-amount design effectively characterized this protoplast system.