Effects of exogenous polyamine application on callus proliferation and somatic embryogenesis in Litchi chinensis‘Feizixiao’

Abstract:【Objective】Plant regeneration in vitro is the basis for transgenic breeding and rapid propaga- tion of litchi (Litchi chinensis Sonn.). Components in the medium are the main factors that affect the ef- ficiency of litchi regeneration in vitro. At present, the research on the medium components mainly focus- es on cytokinin and auxin, and very few studies have been made on polyamines. In this experiment, the study on the effects of polyamines on callus proliferation and somatic embryo induction can provide a stronger basis for determining the function of polyamines in in vitro regeneration of litchi, and open up a new way to establish efficient plant regeneration systems and transgenic breeding of litchi.【Methods】The effects of polyamines and their inhibitors on callus proliferation and somatic embryo induction were studied using a single factor experiment. 【Results】When polyamines and their inhibitors were added into the proliferation medium, they were all found to promote callus proliferation, and the differences were significant. The color state of the callus and the non-embryonic callus on the medium were obvious. The granules of the embryonic callus were tiny, obvious and light yellow in color. Cell division was vigorous, and there were many cell masses. The non-embryonic callus grew at the top of the plant, and it was pale and flabby. The somatic embryo induction and germination rates of callus among samples grown in polyamine and those grown in the presence of its inhibitor differed among different concentration treatments. The somatic embryo induction and germination rates of callus from polyamine and its inhibitor medium were lower than the control. The number of creamy embryos that appeared at low- concentration polyamine medium (below 20 mg · L-1) was far smaller than those that appeared at higher concentrations, and the somatic embryos were small, about 0.3 cm. Somatic embryos took longer to turn green and germinated at a lower rate than those grown at higher concentrations. The roots were shorter than those of plants grown at higher concentrations, about 0.5 cm, and they browned gradually in the presence of lamplight. The inhibition of Put was obvious, and the rooting and stem-regenerating happened slowly. The somatic embryo induction and germination rates increased significantly with the increasing concentrations of polyamine inhibitor. The germination rate was relatively consistent, and most of them differentiated into cotyledon embryos. Regenerated plants had stems with short internodes. They were stouter and greener than non-regenerated plants. When the concentration of Cha was 200 mg · L-1, the number of somatic embryos reached 960 Pcs · g-1 (FW), and the germination rate was 29.41%. At this time, most of the somatic embryos were still bright and milky white with full state. Al- though they did turn green only slowly, they all put out roots and stems and germinated. When the con- centration of D- Arg was 700 mg · L-1, the germination rate of somatic embryos was the highest (32.35%), and the number of somatic embryos was only 450 Pcs·g-1 (FW). Most of the embryo bodies were partly pink, the stem segment was about 1.2 cm, the taproots were 1.5 cm long, and there were ti- ny roots. When polyamines and their inhibitors were added to the somatic embryo induction medium, polyamines and their inhibitors had different effects on somatic embryo induction and germination rates. Most of polyamines, all Cha and high concentration of D-Arg treatments had inhibitory effects, while low concentration of D-Arg and Spd had promoting effects. When Spd concentration was 20 mg·L-1, the number and germination rate of creamy embryos were highest, achieving 590 Pcs · g- 1 (FW) and 30.3%, respectively. The somatic embryos were bright and creamy in texture, the cotyledon tip was pink, the stem length was 1-1.5 cm, and the root was 3.5 cm. Smaller somatic embryos (0.4 cm) could also germinate【. Conclusion】All three kinds of polyamines were found to promote callus proliferation, maintain callus embryogenesis, and inhibit pro-embryogenesis. High concentration of D-Arg and Cha promoted the development of pro-embryogenesis in callus. The low concentration of Spd promoted so- matic embryo differentiation of the callus. Spd played an important role in the early stage of somatic embryo, which inhibited the differentiation of embryonic cells into proembryos. Spm inhibited the formation of proembryo and somatic embryo from embryogenic cells, but it was beneficial to the callus proliferation. Put inhibited the formation of proembryo and somatic embryo in embryogenic cell differentiation. The low concentration of Put had a slight promoting effect at the initial stage of somatic em bryo. Therefore, the effect of low concentration of Put on somatic embryogenesis can be considered, regardless of Put proliferation. Considering the effect of low concentration of Spd on somatic embryogen- esis, Spm may have the opposite effect.