Evaluation of the efficiency of somatic embryogenesis in guava (Psidium guajava L.)

Abstract

SummaryAn embryogenic protocol for plant regeneration of guava (Psidium guajava L.) was established using 10-week post-anthesis, zygotic embryo explants. Somatic embryogenesis was induced on Murashige and Skoog medium (MS) containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) by continuous treatment of the zygotic embryo explants. Somatic embryos appeared as globular structures at the end of the third week from culture initiation, and heart-shaped, cotyledonary-stage, and torpedo-stage embryos appeared within the next few weeks. The development of somatic embryos was asynchronous and showed five-to-seven discernible stages. Depending upon the response of the somatic embryos during their maturation, germination, acclimatisation, and encapsulation, they were grouped into one of three categories. The preferred type of somatic embryos (≥ 1.5 mm) were called the “elongated torpedo” (ET) category. The slightly less-preferred type of stomatic embryos (from 1.0 – 1.5 mm) were termed the “short torpedo” (ST) category. The least preferred types of somatic embryos, at the cotyledonary, heart-shaped, and/or globular stages of development (< 1.0 mm), were grouped into a third category designated “CHG”. The suitability and efficacy of various growth regulators and other treatments were assessed based on six different embryogenic parameters: (i) the frequency of embryogenesis; (ii) the intensity of embryogenesis, defined as the average number of somatic embryos produced per culture (“ANEPC”); (iii) the frequency of ET somatic embryos; (iv) the frequency of ST somatic embryos; (v) the frequency of CHG somatic embryos; and (vi) the overall efficiency of embryogenesis, defined as the potential of a treament to produce somatic embryos at the ET or ST stages, or at both stages of development, that could be converted into plantlets. In the present report, we found that 0.01 mg l–1 2,4-D gave the maximum frequency and intensity of embryogenesis. But the highest frequencies of ET and ST somatic embryos were produced on MS medium containing 3% (w/v) sucrose and 0.001 mg l–1 2,4-D, while CHG embryos were produced at the highest frequency on the same medium, but containing 0.5 mg l–1 2,4-D. It was difficult to calculate the most effective concentration of 2,4-D for somatic embryogenesis based on parameters (i) – (v) above. Hence, quantitative estimations of the efficiency of embryogenesis (sixth parameter) were imperative in order to analyse the potential of the different treatments. The highest efficiency of somatic embryogenesis was achieved by continuous treatment of 10-week post-anthesis, zygotic embryo explants with 0.01 mg l–1 2,4-D on full-strength MS agar medium containing 3% (w/v) sucrose. These somatic embryos matured normally on the same medium, and germinated well both on half-strength solid and in half-strength liquid MS medium containing 3% (w/v) sucrose. They grew in full-strength liquid MS medium with 3% (w/v) sucrose and showed maximum survival upon transfer to soil and hardening. Evaluations of the efficiency of somatic embryogenesis in guava, based on the six parameters defined above, have also helped us to understand and evaluate processes for high efficiency micropropagation in other species.