Synchronization regulation of somatic embryogenesis in Black Locust

doi:10.3969/j.issn.1000-1565.2020.03.009

Abstract

Abstract: In order to establish a high frequency and synchronous somatic embryo culture system, immature embryos of Black Locust(Robinia pseudoacacia L.)were used as explants to study the synchronization of somatic embryogenesis induction. Somatic embryogenesis synchronization system was established by suspension culture, osmotic pressure choice and exogenous hormone adjustment and low temperature processing method. The results showed that 1)callus stayed in low temperature(4 ℃)for 7 days showed- DOI:10.3969/j.issn.1000-1565.2020.03.009刺槐体细胞胚发生的同步化调控习洋¹,孙宇涵²,李云²(1.国家知识产权局专利局专利审查协作北京中心医药生物部,北京 100160;2.北京林业大学生物科学与技术学院,北京 100083)摘要:为了建立一个高频率、同步化发生的体细胞胚培养系统,以二倍体刺槐为材料,对影响刺槐体细胞胚同步化发生的培养基渗透压、培养基中添加的植物生长调节剂、低温等培养环境条件以及悬浮培养结合看护培养方法进行了研究,建立了刺槐体细胞胚同步发生体系.结果表明:1)4 ℃低温条件下处理7 d的胚性愈伤组织,最有利于刺槐体细胞胚的同步化诱导,同步化率可达37.3%;2)培养基中蔗糖质量浓度为50 g/L最适宜调控刺槐的体细胞胚同步化发生,同步化率可达35.0%;3)培养基中的2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)质量浓度为0.1 mg/L时最适宜体细胞胚同步化的调控,体细胞胚发生的同步化率可达43.6%,同时对畸形胚的发生有较好的控制作用;4)将悬浮培养的胚性细胞用50目筛(孔径0.28 mm)过滤,将留在筛上的细胞团接种到铺有滤纸的半固体体细胞胚诱导培养基上(培养基为MS(Murashige & Skoog)+NAA(naphthylacetic acid)0.5 mg/L+ BA(benzyladenine)0.5 mg/L+2-吗啉乙磺酸(MES)500 mg/L+谷氨酰胺 500 mg/L+水解酪蛋白500 mg/L+蔗糖 30 g/L+琼脂3 g/L),培养21 d后,球形胚大量发生,占到总胚状体数目的67.3%.本结果可为其他植物种类的体细胞胚同步化调控研究提供参考.关键词:刺槐;体细胞胚;同步化中图分类号:S792.27 文献标志码:A 文章编号:1000-1565(2020)03-0283-08Synchronization regulation of somatic embryogenesis in Black LocustXI Yang¹, SUN Yuhan², LI Yun²(1.The Pharmaceutical and Biological Invention Examination Department, Patent Examination Cooperation(Beijing)Center of the Patent Office, CNIPA, Beijing 100160, China; 2.College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China)Abstract: In order to establish a high frequency and synchronous somatic embryo culture system, immature embryos of Black Locust(Robinia pseudoacacia L.)were used as explants to study the synchronization of somatic embryogenesis induction. Somatic embryogenesis synchronization system was established by suspension culture, osmotic pressure choice and exogenous hormone adjustment and low temperature processing method. The results showed that 1)callus stayed in low temperature(4 ℃)for 7 days showed- 收稿日期:2019-11-09 基金项目:国家自然科学基金资助项目( 31170629) 第一作者:习洋(1986—),女,河北石家庄人,国家知识产权局助理研究员,博士,主要从事与生物技术相关的林木遗传研究及医药生物发明专利审查.E-mail: judyxi2011@163.com 通信作者:李云(1963—),男,河北蔚县人,北京林业大学教授,主要从事与生物技术相关的林木遗传研究.E-mail:yunli63@163.com第3期习洋等:刺槐体细胞胚发生的同步化调控the highest synchronization rate, which reached 37.3%;2)somatic embryogenesis medium which had sucrose 50 g/L showed the highest synchronization rate, which reached 35.0%;3)somatic embryogenesis medium which had 2,4-D 0.1 mg/L effectivelly controlled the occurrence of abnormal embryo and showed the highest synchronization rate, which reached 43.6%;4)cells from embryogenic cell suspension were cultured on the semi-solid medium, which was MS+NAA 0.5 mg/L+BA 0.5 mg/L+MES 500 mg/L+glutamine 500 mg/L+hydrolyzed casein 500 mg/L+sucrose 30 g/L+agar 3 g/L, for 21 days and formed a lot of globular-stage embryo at frequency of 67.3%.This study can provide theoretical support for somatic embryo synchronization regulation in other plant species.

Key words: Robinia pseudoacacia, somatic embryo, synchronization

CLC Number:

S792.27

XI Yang, SUN Yuhan, LI Yun. Synchronization regulation of somatic embryogenesis in Black Locust[J]. Journal of Hebei University (Natural Science Edition), 2020, 40(3): 283-290.

References

[1] 张宇, 沈海龙. 植物体细胞胚同步化发生的控制[J]. 植物生理学通讯, 2007, 43(3): 583-587. DOI: 10.13592/j.cnki.ppj.2007.03.024.
[2] OKAMURA S, MIYASAKA K, NISHI A. Synchronization of carrot cell culture by starvation and cold treatment[J]. Exp Cell Res, 1973, 78(2): 467-470. DOI: 10.1016/0014-4827(73)90094-3.
[3] 陈春玲, 赖钟雄. 龙眼胚性愈伤组织体胚发生同步化调控及组织细胞学观察[J]. 福建农林大学学报(自然科学版), 2002, 31(2): 192-194. DOI: 10.13323/j.cnki.j.fafu(nat.sci.).2002.02.014.
[4] 习洋, 胡瑞阳, 王欢, 等. 刺槐未成熟合子胚的体细胞胚胎发生和植株再生[J]. 林业科学,2012,48(1):60-69.
[5] 冯玥, 习洋, 陈串, 等. 刺槐胚性细胞悬浮体系的建立[J]. 东北林业大学学报, 2014, 42(12): 80-84. DOI: 10.13759/j.cnki.dlxb.20141104.001.
[6] 徐元红, 朱四易. 伊贝母与平贝母胚状体诱导条件的比较[J]. 陕西师范大学学报(自然科学版), 1998, 26(2): 119-120. DOI: 10.15983/j.cnki.jsnu.1998.02.036
[7] MAHESHWARI S C, TYAGI A K, MALHOTRA K, et al. Induction of haploidy from pollen grains in angiosperms - the current status[J]. Theor Appl Genet, 1980, 58(5): 193-206. DOI: 10.1007/BF00288437.
[8] BOMAL C, TREMBLAY F. Effect of desiccation to low moisture content on germination, synchronization of root emergence, and plantlet regeneration of black spruce somatic embryos[J]. Plant Cell Tissue and Organ Culture, 1999,56(3): 193-200. DOI: 10.1023/A:1006201414616.
[9] 刘选明, 周朴华, 余平. 杂交水稻体细胞胚诱导与同步化的研究[J]. 作物学报, 1994,20(4):465-471.
[10] 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.
[11] 李凤荣, 张自立. 蚕豆根尖细胞分裂同步化的方法[J]. 遗传, 1990, 12(2): 40-42. DOI: 10.16288/j.yczz.1990.02.016.
[12] 周爱文, 柯铁, 梅兴国, 等. 低温诱导红豆杉细胞同步化的研究[J]. 武汉化工学院学报, 2001,23(1): 12-14.
[13] 梅兴国, 周爱文, 柯铁,等. 红豆杉细胞同步化的研究[J]. 生物技术, 2001,11(3): 9-11. DOI: 10.16519/j.cnki.1004-311x.2001.03.004.
[14] 毛春娜, 张爱民, 薛建平, 等. 低温处理对半夏悬浮培养细胞同步化的影响[J]. 中国中药杂志, 2011, 36(8): 959-962.
[15] DANSO K E, FORD-LOYD B V. Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation[J]. Plant Cell Rep. 2004, 22(9):623-631. DOI:10.1007/s00299-003-0727-1.
[16] 杨映根, 桂耀林, 唐巍,等. 青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究[J]. 植物学报, 1994, 36(12): 934-939, 981.
[17] MERKLE S A, DEAN J F. Forest tree biotechnology[J]. Curr Opin Biotechnol, 2000, 11(3): 298-302. DOI: 10.1016/s0958-1669(00)00099-9.
[18] 王宏伟, 史振声, 邢志远. 玉米幼胚组织培养褐化发生因素的研究[J]. 安徽农业科学, 2006, 34(18): 4665-4666. DOI: 10.13989/j.cnki.0517-6611.2006.18.084.
[19] 张涛. 芸芥体细胞胚胎发生的组织细胞学研究[J]. 园艺学报, 2007, 34(1): 131-134. DOI: 10.16420/j.issn.0513-353x.2007.01.026.
[20] EVANS D A, SHARP W R, FLICK C E. Plant tissue culture-methods and applications in agriculture[M]. New York: Academic Press, 1981.
[21] 崔国栋. 林木悬浮细胞系建立过程中的影响因素[J]. 山西林业科技, 2010, 39(4): 35-37.
[22] FINER J J. Plant regeneration from somatic embryogenic suspension cultures of cotton(Gossypium hirsutum L.)[J]. Plant Cell Reports, 1988, 7(6):399-402.
[23] SAKHANOKHO H F, ZIPF A, RAJASEKARAN K, et al. Somatic embryo initiation and germination in diploid cotton(Gossypium arboreum L.)[J]. In Vitro Cellular & Developmental Biology - Plant, 2004, 40(2): 177-181. DOI: 10.1079/ivp2003497.
[24] FINER J J. Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflower(Helianthus annuus L.)on a high sucrose-containing medium[J]. Plant Cell Rep, 1987, 6(5): 372-374. DOI: 10.1007/BF00269564.
[25] TORRES A C, ZE N M, CANTLIFFE D J. Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato[J]. In Vitro CellDevBiol-Plant, 2001, 37(2): 262-267. DOI:10.1007/s11627-001-0047-5.
[26] NADEL B L, ALTMAN A, LIV M. Regulation of somatic embryogenesis in celery cell suspension 2. Early detection of embryogenic potential and the induction of synchronzed cell cultures[J]. Plant Cell Tissue and Organ Culture, 1990, 20: 119-124.
[27] KUMAR M, TULI R. Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis[J]. In Vitro Cell Dev Biol - Plant, 2004, 40(3): 294-298. DOI: 10.1079/ivp2004531.
[28] TONON G, BERARDI G, ROSSI C, et al. Synchronized somatic embryo development in embryogenic suspensions of Fraxinus angustifolia[J]. In Vitro Cell DevBiol-Plant, 2001, 37(4): 462-465. DOI: 10.1007/s11627-001-0081-3.
[29] OSUGA K, MASUDA H, KOMAMINE A. Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and application in plant development[J]. Methods Cell Sci, 1999, 21(2/3): 129-140. DOI: 10.1023/a:1009884806166.