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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Publications at this Location » Publication #318830

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

Author
item Niedz, Randall
item REZZAZADEH, RAMEZAN - University Of Queensland

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2015
Publication Date: 5/16/2015
Citation: Rezzazadeh, R., Niedz, R.P. 2015. Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design. Plant Cell, Tissue And Organ Culture. 122(3):585-604

Interpretive Summary: In vitro plant breeding is the use of plant cell, tissue, and organ culture methods for plant variety development. In vitro methods are used when the objective is impossible or difficult to achieve using conventional plant breeding and horticultural methods. The in vitro method of protoplast culture is required for the application of technologies such as somatic hybridization, electroporation, microprotoplast-mediated chromosome transfer, and organelle or DNA microinjection. Protoplast culture systems have been developed for a number of important fruit tree crops, but there are no reports for guava. Guava is an important fruit grown in tropical and sub-tropical climates. Further, there has been some interest in interplanting guava in citrus groves to repel the citrus psyllid which transmits the bacteria thought to cause the serious disease called huanglongbing (citrus greening). We report the results from a series of experiments designed to determine how to isolate, culture, and regenerate trees from individual protoplasts isolated from guava leaves. The result was a complete protocol for the isolation, culture, and regeneration of trees from protoplasts. The protocol is very efficient and well-suited for the application of protoplast-based in vitro methods for guava improvement.

Technical Abstract: 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 percent) was obtained when 1 g leaf strips were digested in a solution of approximately 0.75 M osmoticum with 6 percent (w/v) enzyme containing cellulase: macerase: hemicellulase as proportion of approximately 0.4: 0.5: 0.1. Protoplasts developed the maximum number of microcalli using 1.0 mg l-1 a-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 (IBA) 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) Naphthaleneacetic acid was superior to 6-benzylaminopurine for callus induction, an antagonistic proportional effect was observed when they were blended; and 5) K was more effective than 6-benzylaminopurine 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 was suitable to characterize this protoplast system.