云杉未成熟合子胚诱导体细胞胚胎发生

doi:10.11707/j.1001-7488.20140406

摘要/Abstract

摘要：

以云杉未成熟合子胚为外植体，研究球果采集日期、基因型、植物生长素和细胞分裂素对愈伤组织诱导的影响，同时对影响体细胞胚分化和成熟的关键因素进行探讨。结果表明：合适的胚性愈伤组织诱导和增殖培养基为1/2 LM + 2，4-D 2.2 mg·L^-1 + 6-BA 1.1 mg·L^-1 + 蔗糖 10 g·L^-1+ 凝胶2 g·L^-1+ 酶解酪蛋白1 g·L^-1；合适的分化培养基为1/2 LM + ABA 24 mg·L^-1 + PEG4000 50 g·L^-1 + 蔗糖30 g·L^-1 + 活性炭1 g·L^-1+ 凝胶4 g·L^-1 + 水解酪蛋白1 g·L^-1；合适的萌发培养基为1/4 LM + 蔗糖20 g·L^-1 + 活性炭1 g·L^-1+ 凝胶4 g·L^-1+ 水解酪蛋白1 g·L^-1。以上培养基中均附加谷氨酰胺0.5 g·L^-1，pH5.8。

关键词: 云杉, 体细胞胚胎发生, 胚性愈伤组织, 成熟体细胞胚

Abstract:

Immature zygotic embryos of Picea asperata were used as explants to investigate effects of cone collection dates, genotypes, different concentration of auxin and cytokinin on the induction of embryogenic callus. At the same time, the key factors affecting the differentiation and maturation of somatic embryos were also comprehensively investigated. The results showed that the optimal medium for induction and proliferation of embryogenic callus was 1/2 LM + 2.2 mg·L^-1 2,4-D + 1.1 mg·L^-16-BA + 10 g·L^-1 sucrose + 2 g·L^-1 gellan gum + 1g·L^-1 casein hydrolysate. The best medium for differentiation of somatic embryos was 1/2 LM + 24 mg·L^-1 ABA + 50 g·L^-1 PEG4000 + 30 g·L^-1 sucrose + 1 g·L^-1activated charcoal+ 4 g·L^-1 gellan gum+ 1 g·L^-1 casein hydrolysate. The suitable medium of germination was 1/4 LM + 20 g·L^-1 sucrose + 1 g·L^-1 AC+ 4 g·L^-1 gellan gum + 1 g·L^-1 casein hydrolysate. All medium was supplemented with 0.5 g·L^-1 L-glutamine, and adjusted to pH 5.8.

Key words: Picea asperata, somatic embryogenesis, embryogenic callus, mature somatic embryos

中图分类号:

S718.46

张建伟, 王军辉, 李青粉, 马建伟. 云杉未成熟合子胚诱导体细胞胚胎发生[J]. 林业科学, 2014, 50(4): 39-46.

Zhang Jianwei, Wang Junhui, Li Qingfen, Ma Jianwei. Somatic Embryogenesis of Picea asperata Induced from Immature Embryos[J]. Scientia Silvae Sinicae, 2014, 50(4): 39-46.

参考文献

陈芳, 陈少瑜, 吴涛, 等. 2010. 丽江云杉体细胞胚胎发生. 林业科学, 46 (8): 162-167.
(Chen F, Chen S Y, Wu T, et al. 2010. Somatic embryogenesis of Picea likiangensis. Scientia Silvae Sinicae, 46 (8): 162-167. [in Chinese] )
郭小兵, 娄迎春, 叶东梅, 等. 2009. 沙地云杉胚性细胞悬浮培养研究. 内蒙古农业大学学报, 30 (2): 274-277.
(Guo X B, Lou Y C, Ye D M, et al. 2009. Research of embryogenic cells suspensi on cultrue on Picea mongolica. Journal of Inner Mongolia Agricultural University(Natural Science Edition), 30 (2): 274-277. [in Chinese] )
李奇, 张可, 罗建勋. 2007. 粗枝云杉无性系种子园生长性状变异和无性系选择初步研究. 四川林业科技, 28 (5): 21-27.
(Li Q, Zhang K, Luo J X. 2007. A prelimiary research on the variation of growth traits and clonal selection in clonal seed orchard of Picea asperata. Journal of Sichuan Forestry Science and Technology, 28 (5): 21-27. [in Chinese] )
李映红, 郭仲琛. 1990. 青杄在不同条件下的体细胞胚胎发生及苗的形成. 植物学报, 32 (7): 568-570.
(Li Y H, Guo Z S. 1990. Somatic embryogenesis and plantlet formation of Picea Wilsonii mast. in different conditions. Acta Botanica Sinica, 32 (7): 568-570. [in Chinese] )
刘宝光, 李成浩, 王伟达, 等. 2009. 红皮云杉胚性愈伤组织诱导技术研究. 植物研究, 29 (1): 69-73.
(Liu B G, Li C H, Wang W D, et al. 2009. Induction technique of Picea koraiensis nakai embryogenic callus. Bulletin Of Botanical Research, 29 (1): 69-73. [in Chinese] )
罗建勋, 孙鹏, 王乐辉, 等. 2006. 云杉种源苗期性状变异及种源选择初步研究. 西南林学院学报, 26 (4): 14-18.
(Luo J X, Sun P, Wang L H, et al. 2006. Growth trait variation of Picea asperata at seedling stage and provenance selection. Journal of Southwest Forestry College, 26 (4): 14-18. [in Chinese] )
吕守芳, 张守攻, 齐力旺, 等. 2005. 日本落叶松体细胞胚胎发生的研究. 林业科学, 41 (2): 48-53.
(Lü S F, Zhang S G, Qi L W, et al. 2005. Somatic Embryogenesis from Immature Embryos of Larix kaempferi. Scientia Silvae Sinicae, 41 (2): 48-53. [in Chinese] )
四川省粗枝云杉纸浆材协作组. 2001. 粗枝云杉纸浆材种源林分区划研究. 四川农业大学学报, 19 (1): 34-36.
(Sichuan Picea Asperata Woodpulp Cooperation Group. 2001. A study on division of provenances as woodpulp stands of Picea asperata. Journal of Sichuan Agricultural University, 19 (1): 34-36. [in Chinese] )
孙敬爽, 贾桂霞. 2010. 北美蓝云杉体细胞胚发生技术研究. 北京林业大学学报, 32 (1): 1-8.
(Sun J S, Jia G X. 2010. Somatic embryogenesis of Picea pungens Engelmann. Journal of Beijing Forestry University, 32 (1): 1-8. [in Chinese] )
吴丽君, 叶建仁. 2008. 火炬松体胚发生与几种专利培养基的应用. 南京林业大学学报: 自然科学版, 32 (1): 1-5.
(Wu L J, Ye J R. 2008. A study on somatic embryogenesis in loblolly pine and application of several patent media. Journal of Nanjing Forestry University(Natural Sciences Edition), 32 (1): 1-5. [in Chinese] )
席梦利, 施季森. 2006. 杉木成熟合子胚器官发生和体胚发生. 林业科学, 42 (9): 29-34.
(Xi M L, Shi J S. 2006. Organogenesis and somatic embryogenesis from mature zygotic embryos of Cunninghamia lanceolata. Scientia Silvae Sinicae, 42 (9): 29-34. [in Chinese] )
杨金玲, 桂耀林, 杨映根, 等. 1997. 白杄体细胞胚胎发生及其植株再生. 植物学报, 39 (4): 315-321.
(Yang J L, Gui Y L, Yang Y G, et al. 1997. Somatic embryogenesis and plantlet regeneration in mature zygotic embryos of Picea meyeri. Acta Botanica Sinica, 39 (4): 315-321. [in Chinese] )
赵晓敏, 沈海龙, 杨玲, 等. 2007. 兴安落叶松胚性愈伤组织诱导影响因子的研究. 植物研究, 27 (5): 538-543.
(Zhao X M, Shen H L, Yang L, et al. 2007. Factors Affecting Induction of embryogenic callus of Larix gmelinii. Bulletin of Botanical Research, 27 (5): 538-543. [in Chinese] )
中国植物志编辑委员会. 1978. 中国植物志: 第七卷. 北京: 科学出版社, 123-133.
(Editorialcommittee of flora of china. 1978. Flora reipublicae popularis sinicae(Vol.7). Beijing: Science Press, 123-133. [in Chinese] )
Birol I, Raymond A, Jackman S D, et al. 2013. Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data. Bioinformatics, 29 (12): 1492-1497.
Budimir S, Vujicic R. 1992. Benzyladenine induction of buds and somatic embryogenesis in Picea omorika Purk. Plant Cell, Tissue and Organ Culture, 31 (2): 89-94.
Stasolla C, Yeung E C. 2003. Recent advances in conifer somatic embryogenesis improving somatic embryo quality. Plant Cell, Tissue and Organ Culture, 74 (1): 15-35.
Gale S, John A, Harding K, et al. 2008. Developing cryopreservation for Picea sitchensis (Sitka spruce) somatic embryos: a comparison of vitrification protocols. Cryoletters, 29 (2): 135-144.
Hakman I, Arnold S V. 1985. Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce). Journal of Plant Physiology, 121 (2): 149-158.
Harry I S, Thorpe T A. 1991. Somatic embryogenesis and plant regeneration from mature zygotic embryos of red spruce. Botanical Gazette, 152 (4): 446-452.
Ishii. 1995. Somatic embryogenesis in Picea glehnii and P. jezoensis//Mohan Jain S, Gupta P K, Newton R J. Somatic embryogenesis in woody plants. vol 3. Dordrecht, The Netherlands: Kluwer Academic Publishers, 55-65.
Kim Y W, Newton R, Frampton J, et al. 2009. Embryogenic tissue initiation and somatic embryogenesis in Fraser fir (Abies fraseri[Pursh]Poir.). In Vitro Cellular & Developmental Biology-Plant, 45 (4): 400-406.
Klimaszewska K, Park Y S, Overton C, et al. 2001. Optimized somatic embryogenesis in Pinus strobus L. In Vitro Cellular & Developmental Biology-Plant, 37 (3): 392-399.
Krajňáková J, Häggman H, Gömöry D. 2009. Effect of sucrose concentration, polyethylene glycol and activated charcoal on maturation and regeneration of Abies cephalonica somatic embryos. Plant Cell, Tissue and Organ Culture, 96 (3): 251-262.
Krajňáková J, Sutela S, Aronen T, et al. 2011. Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology, 63 (1): 17-25.
Krogstrup P, Eriksen E N, Möller J D, et al. 1988. Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.). Plant Cell Rep, 7 (7): 115-123.
Lelu-Walter M A, Bernier-Cardou M, Klimaszewska K. 2006. Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.). Plant Cell Reports, 25 (8): 767-776.
Litvay J D, Verma D C, Johnson M A. 1985. Influence of a loblolly pine (pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Reports, 4 (6): 325-328.
Lu C Y, Thorpe T A. 1987. Somatic embryogenesis and plantlet regeneration in cultured immature embryos of Picea glauca. Journal of Plant Physiology, 128 (3): 297-302.
Nørgaard J V, Duran V, Johnsen Ø, et al. 1993. Variations in cryotolerance of embryogenic Picea abies cell lines and the association to genetic, morphological, and physiological factors. Canadian Journal of Forest Research, 23 (12): 2560-2567.
Nørgaard J V. 1997. Somatic embryo maturation and plant regeneration in Abies nordmanniana Lk. Plant Science, 124 (2): 211-221.
Nystedt B, Street N R, Wetterbom A, et al. 2013. The Norway spruce genome sequence and conifer genome evolution. Nature, 497 (7451): 579-584.
Pullman G S, Johnson S, Peter G, et al. 2003. Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination, and gene expression. Plant Cell Reports, 21 (8): 747-758.
Rai M K, Shekhawat N S, Gupta A K, et al. 2011. The role of abscisic acid in plant tissue culture: a review of recent progress. Plant Cell, Tissue and Organ Culture, 106 (2): 179-190.
Ramarosandratana A V, Harvengt L, Bouvet A, et al. 2001. Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal-suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos. In Vitro Cellular & Developmental Biology-Plant, 37 (1): 29-34.
Ramarosandratana A V, van Staden J. 2003. Tissue position, explant orientation and naphthaleneacetic acid (NAA) affect initiation of somatic embryos and callus proliferation in Norway spruce (Picea abies). Plant Cell, Tissue and Organ Culture, 74 (3): 249-255.
Roberts D R, Flinn B S, Webb D T, et al. 1989. Characterization of immature embryos of interior spruce by SDS-Page and microscopy in relation to their competence for somatic embryogenesis. Plant Cell Reports, 8 (5): 285-288.
Silveira V, Floh E I S, Handro W, et al. 2004. Effect of plant growth regulators on the cellular growth and levels of intracellular protein, starch and polyamines in embryogenic suspension cultures of Pinus taeda. Plant Cell, Tissue and Organ Culture, 76 (1): 53-60.
Stasolla C, Kong L, Yeung E C, et al. 2002. Maturation of somatic embryos in conifers: Morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cellular & Developmental Biology-Plant, 38 (2): 93-105.
Tautorus T E, Attree S M, Fowke L C, et al. 1990. Somatic embryogenesis from immature and mature zygotic embryos and embryo regegeration from protoplasts in Black spruce (Picea mariana Mill). Plant Science, 67 (1): 115-124.
Touchell D H, Chiang V L, Tsai C J. 2002. Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Reports, 21 (2): 118-124.
von Aderkas P, Label P, Lelu M A. 2002. Charcoal affects early development and hormonal concentrations of somatic embryos of hybrid larch. Tree Physiology, 22 (6): 431-434.
Webster F B, Roberts D R, McInnis S M,et al.1990. Propagation of interior spruce by somatic embryogenesis. Canadian Journal of Forest Research, 20 (11): 1759-1765.

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