[1]陈婷婷,叶建仁*,吴小芹,等.抗松材线虫病马尾松体胚发生与植株再生条件的优化[J].南京林业大学学报(自然科学版),2019,43(03):001-8.[doi:10.3969/j.issn.1000-2006.201806005]
 CHEN Tingting,YE Jianren*,WU Xiaoqin,et al.Somatic embryogenesis and plantlet regeneration of disease-resistant Pinus massoniana Lamb.[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(03):001-8.[doi:10.3969/j.issn.1000-2006.201806005]
点击复制

抗松材线虫病马尾松体胚发生与植株再生条件的优化
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
43
期数:
2019年03期
页码:
001-8
栏目:
研究论文
出版日期:
2019-05-15

文章信息/Info

Title:
Somatic embryogenesis and plantlet regeneration of disease-resistant Pinus massoniana Lamb.
文章编号:
1000-2006(2019)03-0001-08
作者:
陈婷婷叶建仁*吴小芹沈李元朱丽华
南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏省有害生物入侵预防与控制重点实验室,江苏 南京 210037
Author(s):
CHEN TingtingYE Jianren*WU XiaoqinSHEN LiyuanZHU Lihua
Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Nanjing Forestry University, Nanjing 210037, China
关键词:
松材线虫病 抗病马尾松 体胚发生 植株再生 悬浮培养 生根
Keywords:
pine wilt disease disease-resistant Pinus massoniana Lamb. somatic embryogenesis regenerated plants suspension cultivation rooting
分类号:
S791.24; S722.3
DOI:
10.3969/j.issn.1000-2006.201806005
文献标志码:
A
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。
Abstract:
【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.

参考文献/References:

[1] 陈碧华, 梁一池, KRYSTYNA K, 等. 马尾松成熟胚愈伤组织的增长研究[J]. 林业科技开发, 2010, 24(6): 114-116. DOI:10.3969/j.issn.1000-8101.2010.06.032.
CHEN B H, LIANG Y C, KRYSTYNA K, et al. Study on the callus growth started from mature embryo of Pinus massoniana[J]. China Forestry Science and Technology, 2010, 24(6): 114-116.
[2] 吴小芹, 张艺, 谢小桃, 等. 外生菌根菌对抗性黑松和赤松不同家系生长影响[J]. 林业科技开发, 2009, 23(1): 19-22. DOI:10.3969/j.issn.1000-8101.2009.01.005.
WU X Q, ZHANG Y, XIE X T, et al. Effect of ectomycorrhizal fungi(EMF)on growth of the Pinus densiflora and P. thunbergii family trees resistant Bursaphelenchus xylophilus[J]. China Forestry Science and Technology, 2009, 23(1): 19-22.
[3] 徐六一, 章健, 高景斌, 等. 安徽省松材线虫病抗性育种研究进展[J]. 安徽林业科技, 2013, 39(2): 8-10,14. DOI:10.3969/j.issn.2095-0152.2013.02.002.
XU L Y, ZHANG J, GAO J B, et al. Research progress on resistance breeding to pinewood nematodiasis in Anhui Province[J]. Anhui Forestry Science and Technology, 2013, 39(2): 8-10,14.
[4] 张锴, 梁军, 严冬辉, 等. 中国松材线虫病研究[J]. 世界林业研究, 2010, 23(3): 59-63. DOI:10.13348/j.cnki.sjlyyj.2010.03.008.
ZHANG K, LIANG J, YAN D H, et al. Research advances of pine wood nematode disease in China[J]. World Forestry Research, 2010, 23(3): 59-63.
[5] 韩兵, 朴春根, 汪来发, 等. 中国松材线虫病的发生现状及治理对策[J]. 中国农学通报, 2007, 23(2): 146-150. DOI:10.3969/j.issn.1000-6850.2007.02.036.
HAN B, PIAO C G, WANG L F, et al. Development status of pinewood nematode disease and its management strategies in China[J]. Chinese Agricultural Science Bulletin, 2007, 23(2): 146-150.
[6] OH M J, NA H R, CHOI H K, et al. High frequency plant regeneration system for Nymphoides coreana via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures[J]. Plant Biotechnology Reports, 2010, 4(2): 125-128. DOI:10.1007/s11816-010-0126-3.
[7] GUPTA P K, PULLMAN G, TIMMIS R, et al. Forestry in the 21st century: the biotechnology of somatic embryogenesis[J]. Journal of Endocrinology, 1980, 123(3):441-444.
[8] 季孔庶, 王潘潘, 王金铃, 等. 松科树种的离体培养研究进展[J]. 南京林业大学学报(自然科学版), 2015, 39(1): 142-148. DOI:10.3969/j.issn.1000-2006.2015.01.026.
JI K S, WANG P P, WANG J L, et al. Review on in vitro culture of tree species in Pinaceae[J]. Journal of Nanjing Forestry University(Natural Sciences Edition), 2015, 39(1): 142-148.
[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.
[11] PULLMAN G S, OLSON K, FISCHER T, et al. Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation[J]. New Forests, 2016, 47(3): 453-480. DOI:10.1007/s11056-016-9525-9.
[12] 黄健秋, 卫志明, 许智宏. 马尾松成熟合子胚的体细胞胚胎发生和植株再生[J]. 植物学报, 1995, 37(4): 289-294,338.
HUANG J Q, WEI Z M, XU Z H. Somatic embryogenesis and plantlet regeneration from callus of mature zygotic embryos of masson pine[J]. Journal of Integrative Plant Biology, 1995, 37(4): 289-294,338.
[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.
[14] 杨模华, 张冬林, 杨艳, 等. 马尾松幼胚离体培养器官发生植株再生研究[J]. 中南林业科技大学学报, 2011, 31(3): 90-96. DOI:10.3969/j.issn.1673-923X.2011.03.019.
YANG M H, ZHANG D L, YANG Y, et al. Micropropagation in immature embryos of Pinus massoniana in vitro[J]. Journal of Central South University of Forestry & Technology, 2011, 31(3): 90-96.
[15] 张婷玉.抗松材线虫病马尾松幼胚体细胞胚胎发生研究[D]. 南京:南京林业大学, 2015.
ZHANG T Y. Studies on somatic embryogenesis from immature zygotic embryos of disease-resistant Pinus massoniana Lamb.[D]. Nanjing: Nanjing Forestry University, 2015.
[16] 李清清, 叶建仁, 朱丽华, 等. 黑松未成熟胚的体细胞胚胎发生和植株再生[J]. 林业科学, 2012, 48(12): 39-44.DOI:10.11707/j.1001-7488.20121206.
LI Q Q, YE J R, ZHU L H, et al. Somatic embryogenesis and plantlet regeneration from immature zygotic embryos of Pinus thunbergii[J]. Scientia Silvae Sinicae, 2012, 48(12): 39-44.
[17] 史昆, 杨模华, 李志辉, 等. 马尾松胚性细胞悬浮增殖培养体系的建立[J]. 中南林业科技大学学报, 2014, 34(1): 64-68. DOI:10.14067/j.cnki.1673-923x.2014.01.019.
SHI K, YANG M H, LI Z H, et al. Establishment of embryoge-nic suspension cultures in somatic embryogenesis of Pinus massoniana Lamb.[J]. Journal of Central South University of Forestry & Technology, 2014, 34(1): 64-68.
[18] 袁澍, 贾勇炯, 林宏辉. 诱导植物体细胞胚发生的几个生理因素[J]. 植物生理学通讯, 2003, 39(5): 508-512. DOI:10.13592/j.cnki.ppj.2003.05.041.
YUAN S, JIA Y J, LIN H H. Several physiological factors inducing somatic embryogenesis of plant[J]. Plant Physiology Communications, 2003, 39(5): 508-512.
[19] 翟晓巧, 程斐, 朱延林. 二乔刺槐愈伤组织超低温保存及适宜降温方法[J]. 林业科学, 2009, 45(10): 49-54. DOI:10.3321/j.issn:1001-7488.2009.10.009.
ZHAI X Q, CHENG F, ZHU Y L. Methods of lowering temperature for cryopreservation of calli of Robinia bella-rosea[J]. Scientia Silvae Sinicae, 2009, 45(10): 49-54.
[20] GUPTA P K, TIMMIS R. Mass propagation of conifer trees in liquid cultures progress towards commercialization[J]. Plant Cell, Tissue and Organ Culture, 2005, 81(3): 339-346. DOI:10.1007/s11240-004-6654-1.
[21] 张建伟, 王军辉, 马建伟. 粗枝云杉胚性愈伤组织增殖后期的体细胞胚发生方式转变[J]. 植物生理学报, 2014, 50(2): 197-202. DOI:10.13592/j.cnki.ppj.2014.02.016.
ZHANG J W, WANG J H, MA J W. The way change of somatic embryogenesis at the late stage of embryogenic callus proliferation of Picea asperata Mast[J]. Plant Physiology Journal, 2014, 50(2): 197-202.
[22] 郭奕明, 杨映根, 郭毅, 等. 落叶松体细胞的胚胎发生[J]. 植物生理学通讯, 2003, 39(5): 531-535. DOI:10.13592/j.cnki.ppj.2003.05.045.
GUO Y M, YANG Y G, GUO Y, et al. Somatic embryogenesis of larch[J]. Plant Physiology Communications, 2003, 39(5): 531-535.
[23] 齐力旺, 韩一凡, 韩素英, 等. 麦芽糖、NAA及ABA对华北落叶松体细胞胚成熟及生根的影响[J]. 林业科学, 2004, 40(1): 52-57. DOI:10.3321/j.issn:1001-7488.2004.01.009.
QI L W, HAN Y F, HAN S Y, et al. Effects of maltose, NAA and ABA on somatic maturation and radicle rooting of Larix principis-rupprechtii[J]. Scientia Silvae Sinicae, 2004, 40(1): 52-57.
[24] 张彩云, 朱丽华, 谈家金, 等. 抗松针褐斑病湿地松体细胞胚胎发生与植株再生[J]. 东北林业大学学报, 2016, 44(6): 17-22. DOI:10.3969/j.issn.1000-5382.2016.06.006.
ZHANG C Y, ZHU L H, TAN J J, et al. Somatic embryogenesis and plantlet regeneration of disease-resistant slash pine(Pinus elliottii Engelm.)to brown spot needle blight[J]. Journal of Northeast Forestry University, 2016, 44(6): 17-22.
[25] 陆燕元. 美国花旗松组织培养及再生系统建立的研究[D]. 杨凌: 西北农林科技大学, 2005.
LU Y Y. Study on the technique of tissue culture and regeneration of douglas-fir[D]. Yangling: Northwest A & F University, 2005.
[26] DESLAURIERS S D,LARSEN P B. FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls[J]. Molecular Plant,2010,3:626-640. DOI: 10.1093/mp/ssq015.
[27] 刘冬云, 史宝胜, 李银华, 等. 不同碳源及PP333、GA3对山丹组培苗鳞茎增大的影响[J]. 河北农业大学学报, 2005, 28(2): 32-35. DOI:10.3969/j.issn.1000-1573.2005.02.008.
LIU D Y, SHI B S, LI Y H, et al. The bulbs augmentation influenced by the carbon resources PP333 and GA3 during the low lily tissue culture[J]. Journal of Agricultural University of Hebei, 2005, 28(2): 32-35.
[28] 赵雪松, 王倩, 闫青地, 等. 油菜素内酯对水稻根系发育的调控作用[J]. 中国细胞生物学学报, 2016, 38(10): 1191-1198.
ZHAO X S, WANG Q, YAN Q D, et al. Function of brassinolide in the regulation of root development in rice[J]. Chinese Journal of Cell Biology, 2016, 38(10): 1191-1198.

相似文献/References:

[1]熊大斌,马阅,解春霞,等.火炬松自然条件下感染松材线虫病初报[J].南京林业大学学报(自然科学版),2009,33(06):159.[doi:10.3969/j.issn.1000-2006.2009.06.037]
 XIONG Da bin,MA Yue,XIE Chun xia,et al.Preliminary reports on Pinus taeda L.infected by Bursaphelenchus xylophilus in natural environment[J].Journal of Nanjing Forestry University(Natural Science Edition),2009,33(03):159.[doi:10.3969/j.issn.1000-2006.2009.06.037]
[2]陈玉惠,赵宁,宁夏玲,等.思茅松健康材和松材线虫病病死材的电导率及pH[J].南京林业大学学报(自然科学版),2010,34(05):059.[doi:10.3969/j.jssn.1000-2006.2010.05.013]
 CHEN Yu hui,ZHAO Ning,NING Xia ling,et al.The difference of conductivity and pH value between healthy and pine wilt diseased woods of Pinus kesiya var.langbianensis[J].Journal of Nanjing Forestry University(Natural Science Edition),2010,34(03):059.[doi:10.3969/j.jssn.1000-2006.2010.05.013]
[3]唐颖,吴小芹,叶建仁*.黑松组培再生植株的感病性及生长性状评价[J].南京林业大学学报(自然科学版),2011,35(02):031.[doi:10.3969/j.jssn.1000-2006.2011.02.006]
 TANG Ying,WU Xiaoqin,YE Jianren*.Susceptibility and evaluation of growth traits about the tissue of Pinus thunbergii[J].Journal of Nanjing Forestry University(Natural Science Edition),2011,35(03):031.[doi:10.3969/j.jssn.1000-2006.2011.02.006]
[4]秦莉,吴小芹*,王张丽.抗松材线虫病赤松组培苗的生根与移栽研究[J].南京林业大学学报(自然科学版),2011,35(03):017.[doi:10.3969/j.jssn.1000-2006.2011.03.018]
 QIN Li,WU Xiaoqin*,WANG Zhangli.Study on rooting and transplanting of the tissuecultured plantlets from Pinus densiflora with good resistance to pine wilt disease[J].Journal of Nanjing Forestry University(Natural Science Edition),2011,35(03):017.[doi:10.3969/j.jssn.1000-2006.2011.03.018]
[5]秦莉,吴小芹*.激素和光照对抗松材线虫病赤松丛生芽离体保存的影响[J].南京林业大学学报(自然科学版),2011,35(05):031.[doi:10.3969/j.jssn.1000-2006.2011.05.007]
 QIN Li,WU Xiaoqin*.Effects of hormone and light on in vitro preservation of axillary buds of Pinus densiflora resistant to pine wilt disease[J].Journal of Nanjing Forestry University(Natural Science Edition),2011,35(03):031.[doi:10.3969/j.jssn.1000-2006.2011.05.007]
[6]赵桂华,陈啸寅,吴玉柱,等.松材线虫病木中真菌的研究[J].南京林业大学学报(自然科学版),2006,30(02):079.[doi:10.3969/j.jssn.1000-2006.2006.02.019]
 ZHAO Gui-hua,CHEN Xiao-yin,WU Yu-zhu,et al.Study on the Fungi from the Diseased Pine Wood Nematode[J].Journal of Nanjing Forestry University(Natural Science Edition),2006,30(03):079.[doi:10.3969/j.jssn.1000-2006.2006.02.019]
[7]王敏敏,叶建仁*,王云华.引诱剂防治松材线虫病及其配套技术[J].南京林业大学学报(自然科学版),2006,30(04):129.[doi:10.3969/j.jssn.1000-2006.2006.04.030]
 WANG Min-min,YE Jian-ren*,WANG Yun-hua.Aplication of Attractant for Controlling Pine Wilt Disease and Interrelated Techniques[J].Journal of Nanjing Forestry University(Natural Science Edition),2006,30(03):129.[doi:10.3969/j.jssn.1000-2006.2006.04.030]
[8]徐福元,葛明宏,汪企明,等.马尾松种源对松材线虫病的抗性[J].南京林业大学学报(自然科学版),1998,22(02):029.[doi:10.3969/j.jssn.1000-2006.1998.02.007]
 Xu Fuyuan Ge Minghong Wang Qiming Zhang Pei Zhu Kegong(Forestry Research Institute of Jiangsu Province Nanjing ) (Nanjing Forestry University ) Zhao Julin Xu Dong Wang Mingming(Agroforest Bureau of Nanjing).STUDIES ON THE MASSON PINE PROVENANCES RESISTANCE TOPINE WOOD NEMATODE (PWN) DISEASE IN CHINA[J].Journal of Nanjing Forestry University(Natural Science Edition),1998,22(03):029.[doi:10.3969/j.jssn.1000-2006.1998.02.007]
[9]来燕学,周岳松,周永平,等.触破式微胶囊剂在飞防松材线虫病中的应用[J].南京林业大学学报(自然科学版),2000,24(02):075.[doi:10.3969/j.jssn.1000-2006.2000.02.022]
 Lai Yanxue Zhou Yuesong ?Zhou Yongpin\ Qi Xinkang\ Wang Zufei(Forest and Plant Quarantine Station of Ningbo City\ Ningbo\ 000).Application of Contacted breaking Release Microcapsules by Aerial Spraying for Pine Wilt Disease Controlling[J].Journal of Nanjing Forestry University(Natural Science Edition),2000,24(03):075.[doi:10.3969/j.jssn.1000-2006.2000.02.022]
[10]郭道森,赵博光,李周直.松材线虫病致病机理的研究进展[J].南京林业大学学报(自然科学版),2000,24(04):064.[doi:10.3969/j.jssn.1000-2006.2000.04.019]
 GUO Dao sen,ZHAO Bo guang,LI Zhou zhi.The Pathogenic Mechanism of Pine Wilt Disease Caused by Bursaphelenchus xylophilus[J].Journal of Nanjing Forestry University(Natural Science Edition),2000,24(03):064.[doi:10.3969/j.jssn.1000-2006.2000.04.019]

备注/Memo

备注/Memo:
收稿日期:2018-06-05 修回日期:2019-01-03
基金项目:江苏省林业三新工程项目(LYSX[2016]47); 江苏高校优势学科建设工程资助项目(PAPD)。
第一作者:陈婷婷(154353079@qq.com)。*通信作者:叶建仁(jrye@njfu.edu.cn),教授,ORCID(0000-0001-5711-0516)。
更新日期/Last Update: 2019-05-15