响应面优化长白落叶松体胚形成早期培养条件的研究

doi:10.7525/j.issn.1673-5102.2017.04.018

摘要/Abstract

摘要： 以长白落叶松胚性愈伤组织为材料，通过Box-Behnken Design设计试验，建立以体胚发生量为响应值的响应面模型，优化长白落叶松体胚形成早期的培养条件。结果表明：相对离子浓度（P=0.007）、肌醇浓度（P=0.000）、培养周期（P=0.000）、肌醇浓度与培养周期的交互作用（P=0.007）对长白落叶松体细胞胚胎发生量的影响极为显著。长白落叶松体胚形成早期的最适培养条件为：相对离子浓度26.767 7% BM、肌醇浓度10.454 5 g·L^-1、培养周期12.656 6 d，预测体细胞胚胎的发生量为337.04 个·g^-1。为便于实际操作，验证试验将培养条件调整为：离子浓度25% BM、肌醇浓度10 g·L^-1、培养周期13 d，体胚平均发生量为336.46 个·g^-1，与预测值336.29 个·g^-1基本一致，体胚萌发率与生根率分别达100%和83.33±；5.78%，与前期试验相比，长白落叶松体胚发生的数量和质量均有显著提高。

关键词: 落叶松属, 长白落叶松, 响应面优化, 体胚发生, 胚胎形成早期

Abstract: With the larch embryogenic callus, through the Design Box-Behnken design test, we established a response surface model based on the somatic embryogenesis. The culture conditions of somatic embryo early maturation was optimized in Larix olgensis. The relative ion concentration(P=0.007), the inositol concentration(P=0.000), the culture period(P=0.000) and the interaction between inositol content and culture period(P=0.007) effect on the number of somatic embryos of L.olgensis were extremely significant. Cultivation of Larix somatic embryo maturation was 337.04 ind·g^-1 at the early stage of the most optimum conditions for relative ion content of 26.767 7% BM basic culture medium, inositol 10.454 5 g·L^-1, culture time 12.656 6 d, and the number of somatic embryos average. In order to facilitate the practical operation, the culture condition was changed to the relative ion concentration of 25% BM, inositol 10 g·L^-1, incubation period of 13 d, then the average number of somatic embryos was 336.46 ind·g^-1, that was consistent with the predicted value 336.29 ind·g^-1. Compared with previous test, the quantity and quality of L.olgensis somatic embryos were increased significantly.

Key words: Larix, Larix olgensis, response surface optimization, somatic embryogenesis, early embryo formation

中图分类号:

宋跃, 李淑娟, 白晓明, 张含国. 响应面优化长白落叶松体胚形成早期培养条件的研究[J]. 植物研究, 2017, 37(4): 613-620.

SONG Yue, LI Shu-Juan, BAI Xiao-Ming, ZHANG Han-Guo. Response Surface Optimization of Culture Conditions for the Formation of Early Embryos in Larix olgensis[J]. Bulletin of Botanical Research, 2017, 37(4): 613-620.

参考文献

1. 王伟达,李成浩,杨静莉,等. 杂种落叶松未成熟胚的体细胞胚发生和植株再生[J]. 林业科学,2009,45(8):34-38.Wang W D,Li C H,Yang J L,et al. Somatic embryogenesis and plantlet regeneration from immature zygotic embryos of hybrid larch[J]. Scientia Silvae Sinicae,2009,45(8):34-38.
2. 杨书文. 落叶松的遗传改良[M]. 哈尔滨:东北林业大学出版社,1994.Yang S W. Genetic improvement of larch[M]. Harbin:Northeast Forestry University Press,1994.
3. 刘足根,朱教君,袁小兰,等. 辽东山区长白落叶松(Larix olgensis)种子雨和种子库[J]. 生态学报,2007,27(2):579-587.Liu Z G,Zhu J J,Yuan X L,et al. On seed rain and soil seed bank of Larix olgensis in montane regions of eastern Liaoning Province,China[J]. Acta Ecologica Sinica,2007,27(2):579-587.
4. 宋跃,甄诚,张含国,等. 长白落叶松胚性愈伤组织诱导及体细胞胚胎发生[J]. 林业科学,2016,52(10):45-54.Song Y,Zhen C,Zhang H G,et al. Embryogenic callus induction and somatic embryogenesis from immature zygotic embryos of Larix olgensis[J]. Scientia Silvae Sinicae,2016,52(10):45-54.
5. Von Aderkas P,Klimaszewska K,Bonga J M. Diploid and haploid embryogenesis in Larix leptolepis,L. decidua,and their reciprocal hybrids[J]. Canadian Journal of Forest Research,1990,20(1):9-14.
6. 吕守芳,张守攻,齐力旺,等. 日本落叶松体细胞胚胎发生的研究[J]. 林业科学,2005,41(2):48-52.Lü S F,Zhang S G,Qi L W,et al. Somatic embryogenesis from immature embryos of Larix kaempferi[J]. Scientia Silvae Sinicae,2005,41(2):48-52.
7. 汪小雄,杨映根. 日本落叶松体细胞胚胎发生的研究[J]. 安徽农业科学,2010,38(4):2118-2121.Wang X X,Yang Y G. Study on the somatic embryogenesis of Larix leptolepis[J]. Journal of Anhui Agricultural Sciences,2010,38(4):2118-2121.
8. 齐力旺. 华北落叶松体细胞胚胎发生与遗传转化系统建立的研究[D]. 北京:中国林业科学研究院,2000.Qi L W. Studies on the somatic embryogenesis and establishment of experimental system in Larix principis-Rupprechtii[D]. Beijing:Chinese Academy of Forestry,2000.
9. Klimaszewska K. Plantlet development from immature zygotic embryos of hybrid larch through somatic embryogenesis[J]. Plant Science,1989,63(1):95-103.
10. Lelu M A,Bastien C,Klimaszewska K,et al. An improved method for somatic plantlet production in hybrid larch(Larix×leptoeuropaea):Part 1. Somatic embryo maturation[J]. Plant Cell,Tissue and Organ Culture,1994,36(1):107-115.
11. 王伟达,李成浩,杨静莉,等. 不同植物生长调节物质对杂种落叶松胚性愈伤组织增殖的影响[J]. 东北林业大学学报,2008,36(9):5-7.Wang W D,Li C H,Yang J L,et al. Effect of different plant growth regulators on embryogenic callus proliferation of hybrid larix[J]. Journal of Northeast Forestry University,2008,36(9):5-7.
12. 中国林业科学研究院林业研究所. 一种提高针叶树种体细胞胚胎发生数量与质量的培养基:中国,CN1733906A[P]. 2006-02-15.INST Forestry CAF. Medium for improving quantity and quality of conifer somatic embryogenesis:Chinese,CN1733906A[P]. 2006-02-15.
13. 中国林业科学研究院林业研究所. 一种改良针叶树种胚性愈伤组织质量的培养基:中国,CN1733905A[P]. 2006-02-15.INST Forestry CAF. Culture medium for improving conifer embryogenic callus quality:Chinese,CN1733905A[P]. 2006-02-15.
14. 罗猛,宋卓悦,胡娇阳,等. 超声法提取山里红叶总黄酮及其抗氧化活性研究[J]. 植物研究,2015,35(4):632-637.Luo M,Song Z Y,Hu J Y,et al. Optimization of ultrasonic-assisted extraction of total flavonoids from Crataegus pinnatifida leaves and its antioxidant activities[J]. Bulletin of Botanical Research,2015,35(4):632-637.
15. 郝学财,余晓斌,刘志钰,等. 响应面方法在优化微生物培养基中的应用[J]. 食品研究与开发,2006,27(1):38-41.Hao X C,Yu X B,Liu Z Y,et al. The application of response surface methodology in optimization of microbial media[J]. Food Research and Development,2006,27(1):38-41.
16. 东北林业大学. 一种提高长白落叶松体细胞胚发生和植株再生效率的过渡培养基及培养方法:中国,CN105409767A[P]. 2016-03-23.Northeast Forestry University. Transitional culture medium improving somatic embryogenesis and plant regeneration efficiency of Larix olgensis and culture method:Chinese,CN105409767A[P]. 2016-03-23.
17. 李淑娟,詹亚光,杨传平,等. 基于响应面法的白蜡属花粉离体萌发培养基优化[J]. 植物学报,2009,44(2):223-229.Li S J,Zhan Y G,Yang C P,et al. Response surface methodology to determine the optimal medium for pollen germination for ash[J]. Chinese Bulletin of Botany,2009,44(2):223-229.
18. Amenaghawon N A,Nwaru K I,Aisien F A,et al. Application of box-behnken design for the optimization of citric acid production from corn starch using Aspergillus niger[J]. British Biotechnology Journal,2013,3(3):236-245.
19. 王化,周丽萍,李梦莎,等. 微波辅助提取暴马丁香中紫丁香苷和橄榄苦苷的工艺研究[J]. 植物研究,2016,36(1):141-145.Wang H,Zhou L P,Li M S,et al. Optimization of microwave-assisted extraction of syringin and oleuropein from Syringa reticulata[J]. Bulletin of Botanical Research,2016,36(1):141-145.
20. 刘燕,陈小银,杨丽丽,等. 响应面优化菊芋菊糖的提取工艺研究[J]. 植物研究,2016,36(4):627-633.Liu Y,Chen X Y,Yang L L,et al. Extraction process optimization of inulin from Helianthus tuberosus L. by response surface methodology[J]. Bulletin of Botanical Research,2016,36(4):627-633.
21. 林建原,季丽红. 响应面优化银杏叶中黄酮的提取工艺[J]. 中国食品学报,2013,13(2):83-90.Lin J Y,Ji L H. Optimization of flavonoids from Ginkgo biloba using respone surface analysis[J]. Journal of Chinese Institute of Food Science and Technology,2013,13(2):83-90.
22. 崔凯荣,戴若兰. 植物体细胞胚发生的分子生物学[M]. 北京:科学出版社,2000.Cui K R,Dai R L. Molecular biology of plant somatic embryogenesis[M]. Beijing:Science Press,2000.
23. 汪小雄,卢龙斗,郝怀庆,等. 松杉类植物体细胞胚发育机理的研究进展[J]. 西北植物学报,2006,26(9):1965-1972.Wang X X,Lu L D,Hao H Q,et al. Research advances about developmental mechanism of somatic embryos in conifers[J]. Acta Botanica Boreali-Occidentalia Sinica,2006,26(9):1965-1972.
24. Singh H. Embryology of gymnosperms[M]. Berlin:Borntrager,1978.
25. Filonova L H,Bozhkov P V,Von Arnold S. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking[J]. Journal of Experimental Botany,2000,51(343):249-264.
26. Filonova L H,Bozhkov P V,Brukhin V B,et al. Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm,Norway spruce[J]. Journal of Cell Science,2000,113(24):4399-4411.
27. 向太和,钟华鑫,梁海曼,等. 水稻胚性悬浮细胞系建立过程中的生理生化变化[J]. 作物学报,1995,21(2):223-119.Xiang T H,Zhong H X,Liang H M,et al. Physiological and biochemical changes during the establishment of embryogenic suspension cell lines of rice(Oryza sativa L.)[J]. Acta Agronomica Sinica,1995,21(2):223-119.
28. Irvine R F,Schell M J. Back in the water:the return of the inositol phosphates[J]. Nature Reviews Molecular Cell Biology,2001,2(5):327-338.
29. Hall P J,Bandurski R S. [³H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue[J]. Plant Physiology,1986,80(2):374-377.
30. Peskan T,Westermann M,Oelmüller R. Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants[J]. European Journal of Biochemistry,2000,267(24):6989-6995.
31. Tiede A,Bastisch I,Schubert J P,et al. Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes[J]. Biological Chemistry,1999,380(5):503-523.
32. Borner G H H,Sherrier D J,Weimar T,et al. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts[J]. Plant Physiology,2005,137(1):104-116.
33. Luo Y,Qin G,Zhang J,et al. D-myo-inositol-3-phosphate affects phosphatidylinositol-mediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis[J]. The Plant Cell,2011,23(4):1352-1372.
34. 王静. 利用胚性愈伤组织进行落叶松遗传转化体系的研究[D]. 北京:中国林业科学研究院,2007.Wang J. Studies on the transformation of Larix based on the system of somatic embryogenesis[D]. Beijing:Chinese Academy of Forestry,2007.

[1]	周雪燕, 王璧莹, 郝雪峰, 胡兴国, 吴江涛, 郎凯, 胡钦波, 赵曦阳. 长白落叶松半同胞家系生长和木材性状遗传变异与联合选择[J]. 植物研究, 2022, 42(3): 383-393.
[2]	孙彤彤, 王忠娟, 张秀娟, 杨诗涵, 陈小强, 张莹. 响应面法优化提取虎耳草总酚及其抑制五步蛇毒PLA₂酶活性的相关性[J]. 植物研究, 2022, 42(2): 259-267.
[3]	杨宇宁, 董昊, 董实伟, 王乃锐, 宋跃, 张含国, 李淑娟. 长白落叶松转录因子LobHLH34克隆及表达分析[J]. 植物研究, 2022, 42(1): 112-120.
[4]	张丹丹, 李翔, 王璧莹, 王喜和, 孙权, 吴蕴洋, 李平扬, 李德尧, 李玉磊, 赵曦阳. 长白落叶松种子园亲本无性系生长性状变异研究[J]. 植物研究, 2022, 42(1): 130-137.
[5]	彭春雪, 崔雪梅, 沈海龙. 暴马丁香成熟胚愈伤组织和体胚诱导及其生理状态解析[J]. 植物研究, 2021, 41(4): 557-563.
[6]	王芳, 蒋路平, 张秦徽, 陆志民, 杨雨春, 张建秋, 赵曦阳. 不同地点51个长白落叶松无性系苗期生长变异和遗传稳定性分析[J]. 植物研究, 2021, 41(3): 336-343.
[7]	刘婷岩, 郝龙飞, 王续富, 闫海霞, 白淑兰. 氮沉降及菌根真菌对长白落叶松苗木根系构型及根际酶活性的影响[J]. 植物研究, 2021, 41(1): 145-151.
[8]	张磊, 熊欢欢, 曹庆, 赵佳丽, 张含国. 瞬时遗传转化长白落叶松NAC基因植株抗旱性的研究[J]. 植物研究, 2020, 40(3): 394-400.
[9]	白晓明, 董实伟, 杨宇宁, 宋跃, 张含国, 李淑娟. 长白落叶松过氧化氢酶LoCAT1基因克隆及表达分析[J]. 植物研究, 2019, 39(4): 539-546.
[10]	彭春雪, 崔雪梅, 刘春苹, 杨玲, 王秋水, 沈海龙. 3种抗褐化剂影响水曲柳体胚发生的生理分析[J]. 植物研究, 2019, 39(2): 252-258.
[11]	郑密, 赵国辉, 孙晓阳, 张秦徽, 赵昕, 董利虎, 李凤日, 赵曦阳. 长白落叶松无性系抗寒性比较研究[J]. 植物研究, 2018, 38(5): 682-687.
[12]	张鑫鑫, 夏辉, 赵昕, 张莹, 李光岩, 张磊, 孙晓阳, 韩冬荟, 赵曦阳. 长白落叶松种子园亲本生长与结实性状综合评价[J]. 植物研究, 2017, 37(6): 933-940.
[13]	杨玲, 刘虹男, 张冬严, 魏骋, 沈海龙. 生长调节剂和渗透调节物质对水曲柳体胚发生的影响[J]. 植物研究, 2017, 37(5): 682-689.
[14]	王艳红1,2；贾庆斌1；张磊1；张含国1*. 长白落叶松4CL基因单核苷酸多态性及其与木材材性的关联分析[J]. 植物研究, 2016, 36(2): 242-251.
[15]	黄振1,2；李赟1,2；李媛1,2；王沛琦1,2；康向阳1,2*. ‘哲引3号’×‘北京杨’杂交子代悬浮细胞系的建立和植株再生[J]. 植物研究, 2015, 35(5): 696-703.