抗松材线虫病马尾松体胚发生与植株再生条件的优化
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摘要
【目的】为使抗松材线虫病马尾松(Pinus massoniana Lamb.)快速大规模繁殖,对抗性马尾松体细胞胚胎发生和植株再生条件进行优化。【方法】以抗病马尾松未成熟合子胚为材料,探讨胚性愈伤组织的诱导、增殖与维持、分化成熟以及萌发的合适培养条件,并对体细胞胚进行生根诱导,对再生植株进行壮苗培养以及驯化移栽。【结果】以LP为基本培养基,添加2.0 mg/L 2,4-D(二氯苯氧乙酸)和1.0 mg/L 6-BA(6-苄氨基腺嘌呤),抗性马尾松胚性愈伤组织诱导率最高达27.8%。在胚性愈伤组织增殖维持过程中,悬浮培养中的增殖系数和同步化程度都显著高于固体培养,且经过悬浮培养的细胞系分化能力更强。萌发过程中只有少量能顺利分化出根系,未能顺利分化出根的体胚于添加适量IBA和NAA的生根培养基中能够更好地生根。壮苗培养过程中,添加0.01 mg/L油菜素内酯有助于体胚苗的生长。体胚苗在驯化移栽时不同大小的苗移栽成活率相差较大,5 cm左右的体胚苗成活率最高达90.3%。【结论】生长素2,4-D和6-BA组合(2.0 mg/L和1.0 mg/L)对抗性马尾松胚性愈伤组织诱导率较好;悬浮培养后的细胞系更利于诱导体胚,适宜浓度的IBA和NAA(1.0 mg/L和0.2 mg/L)能够促进体胚苗根系生长。在壮苗培养基中添加适量油菜素内酯能够促进抗性马尾松再生植株的生长,待苗长至5 cm左右移栽较好。
【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.
【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.
引文
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[9] 高芳,沈海龙,刘春苹,等.红松成熟胚愈伤组织诱导外植体选择及培养条件优化[J].南京林业大学学报(自然科学版),2017,41(3):43-50.DOI:10.3969/j.issn.1000-2006.201605034.GAO F,SHEN H L,LIU C P,et al.Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis[J].Journal of Nanjing Forestry University (Natural Sciences Edition),2017,41(3):43-50.
[10] NUNES S,MARUM L,FARINHA N,et al.Somatic embryogenesis of hybrid Pinus elliottii var.elliottii × P.caribaea var.hondurensis and ploidy assessment of somatic plants[J].Plant Cell,Tissue and Organ Culture (PCTOC),2018,132(1):71-84.DOI:10.1007/s11240-017-1311-7.
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[13] 施季森,高燕,席梦利,等.诱导马尾松未成熟种子胚性胚柄细胞团再生植株的方法:CN101849505A[P].2010-10-06.SHI J S,GAO Y,XI M L,et al.Methods for inducing regeneration of plantlets from immature embryo embryos of Pinus massoniana:CN101849505A[P].2010-10-06.
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