Somatic embryogenesis and plantlet regeneration of disease-resistant Pinus massoniana Lamb.

doi:10.3969/j.issn.1000-2006.201806005

CHEN Tingting,YE Jianren,WU Xiaoqin,SHEN Liyuan,ZHU Lihua

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2019, Vol. 43 ›› Issue (03) : 1-8.

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JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2019, Vol. 43 ›› Issue (03) : 1-8. DOI: 10.3969/j.issn.1000-2006.201806005

Somatic embryogenesis and plantlet regeneration of disease-resistant Pinus massoniana Lamb.

CHEN Tingting,YE Jianren^*,WU Xiaoqin,SHEN Liyuan,ZHU Lihua

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【Objective】Pinus massoniana Lamb., a local tree species, was severely damaged with the epidemic spread of pine wilt disease. We explored and optimized somatic embryogenesis(SE)and plant regeneration conditions for disease-resistant P. massoniana Lamb. to enable rapid mass propagation.【Method】 Immature zygotic embryos of disease-resistant P. massoniana Lamb. were used to study the effect of hormone combinations on embryogenic callus induction by tissue culture method. We also used different culture methods to find the optimal method to propagate embryonic callus. Upon screening for various plant hormone concentration, we reported the optimal hormone combination for root development. Finally, we studied the differentiation of callus and plant regeneration.【Result】① The different plant hormones played a substantial role in inducing callus tissue. The optimal SE induction hormone medium was LP + 2 mg/L 2,4-D + 1 mg/L 6-BA and the highest induction rate of SE was up to 27.8%. We established 115 embryonic cell lines in 2015, and 6 months later 22 stable embryonic cell lines were obtained by proliferation. ② During multiplication of embryonal callus, suspension cultivation resulted in more embryonic calluses and a higher synchronization in a short period of time compared to those observed by solid proliferation cultivation method. The multiplication rate reached 3.88 after 7 days of suspension cultivation.③There was significant difference in the differentiation ability of the callus tissue cultured using different culture methods. The differentiation in suspention culture-solid was better than that in solid culture-solid. After culture, the cell had better differentiation ability and the induction of somatic embryos could reach 600 in each culture dish. In contrast, the cells could only differentiate a small number of somatic embryos and most of those somatic embryos were malformed. Somatic embryo germination was accomplished after 30 days using high quality somatic embryos and the highest germination rate achieved was 89.6%. ④In germination culture, many somatic embryos could not develop root successfully. Our study demonstrated that NAA and IBA were beneficial for root formation, and 1.0 mg/L IBA + 0.2 mg/L NAA was the optimal hormone combination for root formation. Strengthening the shoots before transplantation can improve the survival rate. Brassinolide could promote the growth of somatic embryos at an optimal concentration of 0.01 mg/L. Somatic embryos that were cultivated with brassinolide showed a healthy dark green color and well developed roots with more branch roots compared to those cultivated without brassinolide. ⑤The regenerated plants were transplanted into a matrix with the farmland to soil volume ratio of 2:1. During the process of transplantation, the survival rates of regenerated plants of different size were significantly different. The survival rate of regenerated plants was low, when small transplants were used. Conversely, the survival rate of regenerated plants was high when large transplants were used. However, the regenerated plants had a low degree of lignification and they were more prone to lodging. When the regenerated plants grew up to about 5 cm, it could be transplanted and the highest survival rate achieved was 90.3%. 【Conclusion】This study successfully established a plant regeneration system via somatic embryogenesis of P. massoniana Lamb.. The optimal SE induction hormone medium was LP + 2.0 mg/L 2,4-D + 1.0 mg/L 6-BA. Cell suspension culture for differentiation of somatic embryos was better than solid culture. WPM + 1.0 mg/L IBA + 0.2 mg/L NAA was the best combination for root formation. Regenerated plant length of 5 cm were suitable for transplanting.

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CHEN Tingting,YE Jianren,WU Xiaoqin,SHEN Liyuan,ZHU Lihua. Somatic embryogenesis and plantlet regeneration of disease-resistant Pinus massoniana Lamb.[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2019, 43(03): 1-8 https://doi.org/10.3969/j.issn.1000-2006.201806005

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