Study on the optimization of amino acids on proliferation of calli and somatic embryogenesis in Litchi chinensis‘Feizixiao’

Abstract：【Objective】Clonal propagation is an essential tool in biotechnology and breeding programs. Among the available clonal propagation methods, somatic embryogenesis is the most attractive technique for the large-scale propagation of plants and the long-term conservation of different embryogenic cell lines without changing their initial characteristics. Additionally, somatic embryogenesis is an ideal system for cell biology and molecular studies, including the study of the cell cycle, cell division, cell differentiation, and for understanding different genetic and epigenetic mechanisms for gene expression and phenotypic variation in plants. However, for many species, in vitro clonal propagation is currently difficult or inefficient. The low efficiency of somatic embryo induction is among the major problems hampering efficient regeneration and limiting practical applications. Therefore, high-quality somatic embryo production is one of the most important factors for using somatic embryogenesis protocols in commercial propagation and breeding programs. Litchi (Litchi chinensis Sonn.) is one of the treasure fruits cultivated in the south of China. Breeding elite litchi varieties with resistance to diseases is very impor-tant to the litchi industry. Many studies on the somatic embryogenesis of litchi have been reported. However, there has been no detailed report on the the effect of amino acids on callus proliferation and somatic embryo induction in litchi. The study aimed to investigate the effects of amino acids on the efficiency of somatic embryogenesis in Feizixiao litchi and apply these findings to improve the protocol for the induction of somatic embryos.【Methods】The calli Feizixiao litchi were used as materials, the effects of different amino acids on callus proliferation, somatic embryogenesis and regeneration were studied using an L9(3⁴ ) orthogonal experiment.【Results】The results showed that the effects of glutamine (Gln) and γ-aminobutyric acid (GABA) on the callus morphology and proliferation rate were significant (p＜0.01), while the effect of alanine (Ala) was not significant. The effects of the three amino acids on the proliferation rate of callus were in the order of Gln＞GABA＞Ala. The method of the callus proliferation was established. The optimal medium for the callus proliferation was: MS + 1 mg·L^-1 2,4-D + 0.1 g·L^-1 GABA + 0.05 g · L^{- 1} Ala. After 20 days of culture, the proliferation rate of callus was 14.04 times as many as that of the control. Compared with the control group, the boundary between embryogenic callus and non-embryogenic calli in the amino acid treatment was clear and easy to distinguish.The embryogenic calli were granular and light yellow, while the non-embryonic calli were pale, heavily water stained, and was located at the top or edge and was easy to pick out. The callus cells treated with amino acids were elliptical with a dense cytoplasm and were mostly dividing. The callus cells on the control medium were divided into clumps. The effects of the Gln, GABA, and Ala on somatic embryogenesis and germination were significant (p＜0.01). The effects of the three amino acids on the induction of somatic embryos and germination were in the order of Gln＞GABA＞Ala. The optimal treatment regimen was YA5: MS + 1 mg ·L^-1 2,4-D + 0.3 g ·L^-1 GABA + 0.2 g ·L^-1 Gln. The optimized program used for somatic embryo induction produced 477 embryos per gram of calli, and most of the somatic embryos were deformed curved monocotyledon embryos, while the smaller somatic embryos were dicotyledonous or polycotyledon embryos. The second medium for inducting somatic embryos was YA6 (MS + 1 mg·L^-1 2,4-D + 0.3 g·L^-1 GABA + 0.4 g·L^-1 Gln + 0.05 g·L^-1 Ala) with the induction number of 461 embryos per gram of calli, and the forms of the somatic embryos on YA6 were different, including globular embryos, clumped embryos, dicotyledonous embryos, and polycotyledon embryos. The best medium for inducing somatic embryos was the YA6 treatment, through which 77 regenerations were obtained per gram of callus, followed by the YA11 (MS + 1 mg·L^-1 2, 4-D + 0.3 g·L^-1 GABA + 0.4 g·L^-1 Gln + 0.1 g·L^{- 1} Ala) and YA5 treatments with 62 and 60 regenerations per gram of calli, respectively. The somatic embryo greening and germination in the amino acid treatments occurred later than that in the control. The regeneration appeared in the form of multiplying bud spots, the stems was bright green, the stem tips were bright white and bright green, and the somatic bodies were mostly light green with some red color. In the control, the somatic embryos were larger and thicker, and the stems were about 1.5 cm in length. The effects of the Gln, GABA, and Ala on the somatic embryogenesis and germination were significant (p＜0.01). The effects of the three amino acids on the induction of somatic embryos were in the order of GABA＞Gln＞Ala. The effects of the three amino acids on the regeneration were in the order of Gln＞GABA＞Ala. The optimal treatment regimen (YAE6) for the somatic embryogenesis and germination was as follows: MS + 1 mg·L^-1 2,4-D + 0.3 g·L^-1 GABA + 0.4 g·L^-1 Gln + 0.05 g·L^-1 Ala with 270 embryos and 72 regenerations per gram of calli.【Conclusion】The optimal amino acid treatment regimen for the callus proliferation was the same as that for the embryo induction, but the number of somatic embryos and germinations during the course of the callus proliferation were higher than that during the course of the somatic embryogenesis stage, indicating that the mechanism of thepromotion of the somatic embryogenesis by amino acids might be related to the regulation of the embryogenic callus.