我们的网站为什么显示成这样?

可能因为您的浏览器不支持样式,您可以更新您的浏览器到最新版本,以获取对此功能的支持,访问下面的网站,获取关于浏览器的信息:

|Table of Contents|

红松成熟胚愈伤组织诱导外植体选择及培养条件优化(PDF/HTML)

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

Issue:
2017年03期
Page:
43-50
Column:
研究论文
publishdate:
2017-05-31

Article Info:/Info

Title:
Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis
Article ID:
1000-2006(2017)03-0043-08
Author(s):
GAO Fang SHEN Hailong* LIU Chunping WANG Yi ZHANG Peng YANG Ling
State Key Laboratory of Tree Genetics and Breeding, School of Forestry, Northeast Forestry University, Harbin 150040, China
Keywords:
Pinus koraiensis callus mature embryo explants hormone combinations culture condition
Classification number :
S722.3; S791.247
DOI:
10.3969/j.issn.1000-2006.201605034
Document Code:
-
Abstract:
【Object】 Through optimizing culture conditions and determining suitable explant sources, our goal was to resolve the problems of low induction rates, poor growth and high browning rates in callus induction from mature Pinus koraiensis embryos. Our study could provide insights into establishing an effective system of somatic embryogenesis. 【Methods】We used eight different parts of mature P. koraiensis seeds(cotyledon slices from naked embryo, hypocotyl slices from naked embryo, radicle slices from naked embryo, intact naked embryo, intact kernel with embryo and megagametophyte, kernel with 0.5 cm slices at the ovule hole end, half kernel with the ovule hole end, and two-third longitudinally kernel slices of intact embryo)as explants. To find the optimal conditions for callus induction, we individually cultured these explants in five types of basic media(DCR, GLH, MSG, LM and EM), supplemented with sucrose at three different concentrations(30, 35 and 40 g/L)and nine combinations of 6-BA(1, 1.5 and 2 mg/L)and NAA(1, 2 and 4 mg/L). 【Results】 ① Of the five tested media, the DCR medium supported the highest callus induction rate and the optimum callus growth status, with 27.03% callus browning rate, which was significantly lower than those in LM and EM media, but not significantly different from those in GLH and MSG media. The callus derived from the hypocotyl of the intact embryo explant was semi-transparent and white in color with a tiny protuberance on the surface, and exhibited strong growth capability. ② DCR medium supplemented with 35 g/L sucrose led to the highest callus induction rate(92.01%)and the lowest browning rate(17.01%)among the three tested sucrose concentrations. Furthermore, the callus exhibited a more healthy growth status and stronger proliferation capability under this condition than with the other two sucrose concentrations. ③ DCR medium with 35 g/L sucrose, 2.0 mg/L NAA and 1.5 mg/L 6-BA led to the highest callus induction rate, lowest browning rate, and optimum growth status among the nine tested combinations of plant growth regulators. NAA exhibited a stronger influence on callus induction rate than 6-BA, but they displayed comparable effects on callus browning rates. ④ In addition, under these optimized culture conditions of DCR with 35 g/L sucrose, 2.0 mg/L NAA, and 1.5 mg/L 6-BA, the sliced hypocotyl explant of naked P. koraiensis mature embryo showed higher callus induction rate(98.00%), lower browning rate(20.00%), and stronger proliferation capacity than the other seven explants, and the morphological and histological characteristics of the induced callus also conformed to those of embryogenic calli. On the other hand, sliced cotyledon from naked embryo showed a relatively low induction rate(56.92%)and high browning rate(47.69%)without any embryogenic callus morphological and histological characteristics. Sliced radicle from naked embryos and intact naked embryos exhibited both high callus induction rates(88.57% and 100%, respectively)and significant browning rates(67.14% and 91.43%, respectively). The other explants, including intact kernel with embryo and megagametophyte, kernel with 0.5 cm slices on the ovule hole end, half kernel with ovule hole end, and longitudinal two-third slices of the kernels of instant embryos showed high callus induction rates(>80%)and markedly low browning rates(<5%), but the callus originated from a megagametophyte that could not proliferate in subculture. 【Conclusion】 The optimal combination of culture conditions and explant type for callus induction from mature seeds of P. koraiensis was the use of DCR medium supplemented with 35 g/L sucrose, 2.0 mg/L NAA and 1.5 mg/L 6-BA to culture sliced hypocotyls of naked mature embryos.

References

[1] OLIVIER M. Micropropagation and production of forest trees[C]//PACK Y S, BONGA J, MOON H K. Vegetative propagation of forest trees. Korea: Suwon, 2016: 32-55.
[2] ELHITI M, STASOLLA C. The use of zygotic embryos as explants for in vitro propagation: an overview[J]. Methods Mol Biol, 2011, 710: 229-255. DOI:10.1007/978-1-61737-988-8_17.
[3] LELU-WALTER M A, THOMPSON D, HARVENGT L, et al. Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction[J]. Tree Genetics & Genomes, 2013, 9(4): 883-899. DOI:10.1007/s11295-013-0620-1.
[4] 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.1000-2006.2015.01.001. 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.
[5] SALAJ T, MATUSOVA R, SALAJ J. Conifer somatic embryogenesis-an efficient plant regeneration system for theoretical studies and mass propagation[J]. Dendrobiology, 2015, 74: 69-76. DOI:10.12657/denbio.074.007.
[6] 席梦利, 施季森. 杉木成熟合子胚器官发生和体胚发生[J]. 林业科学, 2006, 42(9):29-33. DOI:10.3321/j.issn:1001-7488.2006.09.006. XI M L, SHI J S. Organogenesis and somatic embryogenesis from mature zygotic embryos of Cunninghamia lanceolata.[J] Scientia Silvae Sinicae, 2006, 42(9):29-33.
[7] ADERKAS P V, KONG L, PRIOR N A.In vitro techniques for conifer embryogenesis[C]// PACK Y S, BONGA J, MOON H K. Vegetative Propagation of Forest Trees. Korea: Suwon, 2016.
[8] TRONTIN J F, TEYSSIER C, MOREL A, et al.Prospects for new variety deployment through somatic embryogenesis in maritime pine[C]// PACK Y S, BONGA J, MOON H K. Vegetative propagation of forest trees. Korea: Suwon,2016: 572-606.
[9] KIM Y W, SHIN H N, MOON H K. Somatic embryogenesis in rigitaeda pine(Pinus rigida × P. taeda)[C]// PACK Y S, BONGA J, MOON H K. Vegetative propagation of forest trees. Korea: Suwon, 2016:657-673.
[10] 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.
[11] PULLMAN GS, BUCALO K. Pine somatic embryogenesis using zygotic embryos as explants[J]. Methods Mol Biol, 2011, 710: 267-291. DOI:10.1007/978-1-61737-988-8_19.
[12] KUBE M, DRáNá N, KONRáDOVá H, et al. Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply[J]. In Vitro Cellular & Developmental Biology-Plant, 2014, 50(1): 45-57. DOI:10.1007/s11627-013-9589-6.
[13] KIM Y W, MOON K M, KIM J A. Initiation of embryogenic suspensor masses and somatic embryogenesis in Japanese red pine(Pinus densiflora)[C]//PACK Y S, BONGA J, MOON H K. Vegetative propagation of forest trees. Korea: Suwon, 2016: 639-656.
[14] SALAM A M A, CHOWDHURY K, BAKRY A A E. Effect of sugar types, culture age, concentrations of 2,4-D and sucrose on somatic embryogenesis of Cymbopogon schoenanthus subsp. proximus[J]. Plant Tissue Culture & Biotechnology, 2015, 25(1):7-7. DOI:10.3329/ptcb.v25i1.24125.
[15] CRIOLLO H, PEREA M, TORIBIO M, et al. Effect of the combination of NAA, kinetin and sucrose on the induction of somatic embryogenesis in lulo(Solanum quitoense Lam.)[J]. Agronomía Colombiana, 2014, 32(2):170-179. DOI:10.15446/agron.colomb.v32n2.43861.
[16] TANG W, GUO Z C, YANG F O. Plant regeneration from embryogenic cultures initiated from mature Loblolly pine zygotic embryos[J].In Vitro Cell Developmental Biology-Plant, 2001, 37(3): 558-563. DOI:10.1007/s11627-001-0097-8.
[17] TANG W, NEWTON R J. Plant regeneration from callus cultures derived from mature zygotic embryos in white pine(Pinus strobus L.)[J]. Plant Cell Reports, 2005, 24(1): 1-9. DOI:10.1007/s00299-005-0914-3.
[18] 唐巍, 欧阳藩, 郭仲琛. 火炬松胚性愈伤组织诱导和植株再生的研究[J]. 林业科学, 1998, 34(3): 115-119. DOI:10.3321/j.issn:1001-7488.1998.03.017. TANG W, OUYANG F, GUO Z C. Studies on embryogenic callus induction and plant regeneration in loblolly pine[J]. Scientia Silvae Sinicae, 1998, 34(3): 115-119. DOI:10.3321/j.issn:1001-7488.1998.03.017.
[19] 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. LI Q Q, YE J R, ZHU L H, et al. Somatic embryogenesis and plantlet regeneration from immature zygotic of Pinus thunbergii[J]. Scientia Silvae Sinicae, 2012, 48(12):39-44.
[20] 梁艳,沈海龙,高美玲,等. 红松种子发育过程中内源激素含量的动态变化[J]. 林业科学, 2016: 52(3), 105-111. DOI:10.11707/j.1001-7488.20160213. LIANG Y, SHEN H L, GAO M L, et al. Content dynamics of endogenous hormones in different seed developmental stages of Korean pine[J]. Scientia Silvae Sinicae, 2016, 52(3):105-111.
[21] 李正理,张新英. 红松后胚离体培养的研究 I. 不同培养方式下幼苗生长的比较观察[J]. 植物学报, 1962, 10(2): 103-112. LI Z L, ZHANG X Y. Studies on the embryos of Pinus koraiensis grown in vitro I. growth of the seedlings under various experimental conditions[J]. Acta Botanica Sinica, 1962, 10(2): 103-112.
[22] 李正理,张新英. 红松后胚离体培养的研究 II. 具雌配子体与离体后胚培养的比较观察[J]. 植物学报, 1962, 10(3): 179-186. LI Z L, ZHANG X Y. Studies on the embryos of Pinus koraiensis grown in vitro Ⅱ. Comparative growth of embryos with and without megagametophyte under various cultured conditions[J]. Acta Botanica Sinica, 1962, 10(3): 179-186.
[23] 刘玉喜,孙洪年,陆志华,等. 红松成熟胚离体培养的研究[J]. 东北林业大学学报, 1991, 19(S1): 148-153. LIU Y X, SUN H N, LU Z H, et al. Culture in vitro of the mature embryo of Pinus koraiensis[J]. Journal of Northeast Forestry University, 1991, 19(S1): 148-153.
[24] 刘玉喜,唐翠,詹亚光. 红松成熟胚离体营养器官发生组织学研究[J]. 植物研究, 1992, 12(2): 189-194. LIU Y X, TANG C, ZHAN Y G. Study on histogenesis and organogenesis of the in vitro development from Pinus koraiensis embryos[J]. Bulletn of Botanical Research, 1992, 12(2): 189-194.
[25] BOZHKOV P V, AHN I S, PARK Y G. Two alternative pathways of somatic embryo origin from polyembryonic mature stored seeds of Pinus koraiensis Sieb et Zucc[J]. Canadian Journal of Botany. 1997, 75(2): 509-512. DOI:10.1139/b97-055.
[26] 王高. 红松体细胞胚胎发生及超低温保存技术研究[D]. 上海:上海交通大学. 2009. WANG G. Somatic embryogenesis and cryopreservation of Pinus koraiensis Sieb. et Zucc[D]. Shanghai:Shanghai Jiao Tong University, 2009.
[27] 申晓辉, 蒋湘宁, YILL S P, 等, 红松体细胞胚胎培养技术体系的建立[J]. 成都大学学报(自然科学版), 2005, 24(1): 11-14. DOI:10.3969/j.issn.1004-5422.2005.01.004. SHEN X H, JIANG X N, YILL S P, et al. Somatic embryogenisis(SE)in Korean pine(Pinus koraiensis Sieb. et Zucc)[J]. Journal of Chengdu University(Natural Science), 2005, 24(1): 11-14.
[28] 曹焱. 红松合子胚愈伤组织诱导及不定芽发生的研究[D]. 哈尔滨:东北林业大学, 2009. CAO Y. Callus induetion and shoot regenerationin zygotic embryos of Pinus koraiensis[D]. Harbin: Northeast Forestry University, 2009.
[29] 王祎, 赵彤彤, 杨魏, 等. 红松成熟合子胚愈伤组织诱导的适宜培养条件[J]. 森林工程, 2015(2): 5-7, 13. WANG Y, ZHAO T T, YANG W, et al. Proper condition for callus induction from mature zygotic embryo explants of Pinus koraiensis[J]. Forest Engineering, 2015(2): 5-7, 13.
[30] 刘宝光,李成浩,张含国. 红皮云杉胚性愈伤组织保持与增殖阶段影响因子的筛选与分析[J]. 东北林业大学学报, 2010, 38(7): 56-60. DOI:10.3969/j.issn.1000-5382.2010.07.019. LIU B G, LI C H, ZHANG H G. Screening of influence factors on maintenance and proliferation of embryogenic callus of Picea koraiensis[J]. Journal of Northeast Forestry University, 2010, 38(7): 56-60.
[31] 史昆,杨模华,李志辉,等. 蔗糖与肌醇对马尾松胚性细胞系增殖的影响[J]. 植物生理学报, 2014, 50(6): 785-791. SHI K, YANG M H, LI Z H, et al. Effects of sucrose and myo-inositol on proliferation of embryogenic cell lines in Pinus massoniana[J]. Plant Physiology Journal, 2014, 50(6): 785-791.
[32] 于大德, 肖宁, 王企珂, 等. 云南松胚性愈伤组织诱导及增殖[J]. 西北植物学报, 2011, 31(10):2009-2123. YU D D, XIAO N, WANG Q K, et al. Induction and proliferation of embryo callus from Pinus yunnanensis Franch[J]. Northwest Journal of Botany, 2011, 31(10):2009-2123.
[33] 沈海龙. 植物组织培养[M]. 北京:中国林业出版社. 2005, 37-115.
[34] SAHA P, RAYCHAUDHURI S S, SUDARSHAN M, et al. Analysis of trace elements during different developmental stages of somatic embryogenesis in Plantago ovata Forssk using energy dispersive X-ray fluorescence[J]. Biological Trace Element Research, 2010, 135(1):283-94. DOI:10.1007/s12011-009-8497-4.
[35] 蔡琼, 岳素青, 姬爱国. 植物组培过程褐变现象及抗褐技术研究进展[J]. 黑龙江生态工程职业学院学报, 2012(1):32-33. CAI Q, YUE S Q, JI A G. Research progress on browning phenomenon and anti-browning technique during plant tissue culture process[J]. Journal of Heilongjiang Ecological Engineering Institute, 2012(1):32-33.
[36] 赵晓敏, 沈海龙, 杨玲, 等. 兴安落叶松胚性愈伤组织诱导影响因子的研究[J]. 植物研究, 2007, 27(5): 538-543. DOI:10.3969/j.issn.1673-5102.2007.05.008. ZHAO X M, SHEN H L, YANG L, et al. Factors affecting induction of embryogenic callus of Larix gmelinii[J]. Bulletin of Botanical Research, 2007, 27(5): 538-543.
[37] TRET’IAKOVA I N, VOROSHILOVA E V. Embryo initiation from Pinus sibirica megagametophytes in in vitro culture[J]. Russian Journal of Developmental Biology, 2014, 45(2):93-100. DOI:10.1134/s1062360414020064.
[38] TRET’VAKOVA I N, SHUVAEV D N. Somatic embryogenesis in Pinus pumila and productivity of embryogenic lines during long-term cultivation in vitro[J]. Russian Journal of Developmental Biology, 2015, 46(5):276-285. DOI:10.1134/s1062360415050070.
[39] ZHANG C X, LI Q, KONG L. Induction, development and maturation of somatic embryos in Bunge’s pine(Pinus bungeana Zucc. ex Endl.)[J]. Plant Cell Tissue & Organ Culture, 2007, 91(3):273-280. DOI:10.1007/s11240-007-9294-4.
[40] HUMáNEZ A, BLASCO M, BRISA C, et al. Somatic embryogenesis from different tissues of Spanish populations of maritime pine[J]. Plant Cell Tissue & Organ Culture, 2012, 111(3):373-383. DOI:10.1007/s11240-012-0203-0.
[41] PULLMAN G S, JOHNSON S, TASSEL S V, et al. Somatic embryogenesis in loblolly pine(Pinus taeda)and Douglas fir(Pseudotsuga menziesii)and Douglas fir(improving culture initiation and growth with MES pH buffer, biotin, and folic acid[J]. Plant Cell Tissue & Organ Culture, 2005, 80(80):91-103. DOI:10.1007/s11240-004-9099-7.

Last Update: 2017-05-20