红皮云杉体胚发生体系优化和超低温保存技术研究

高芳; 陈士刚; 秦彩云; 才巨锋; 王聪慧; 董环宇; 陶晶

doi:10.12302/j.issn.1000-2006.202007029

高芳, 陈士刚, 秦彩云, 才巨锋, 王聪慧, 董环宇, 陶晶

南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3) : 100-108.

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3) : 100-108. DOI: 10.12302/j.issn.1000-2006.202007029

研究论文

红皮云杉体胚发生体系优化和超低温保存技术研究

高芳 ¹^,² ,
陈士刚 ¹ ,
秦彩云 ¹ ,
才巨锋 ¹ ,
王聪慧 ³ ,
董环宇 ¹ ,
陶晶 ¹^,^*

作者信息 +

Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis

GAO Fang ¹^,² ,
CHEN Shigang ¹ ,
QIN Caiyun ¹ ,
CAI Jufeng ¹ ,
WANG Conghui ³ ,
DONG Huanyu ¹ ,
TAO Jing ¹^,^*

Author information +

文章历史 +

摘要

【目的】通过对红皮云杉(Picea koraiensis)体细胞胚胎发生条件的筛选和超低温保存技术研究, 建立其完整的体胚发生体系及超低温保存条件,为红皮云杉优良种质资源的大量繁殖及保存提供基础。【方法】以红皮云杉合子胚为外植体,以两种基本培养基、6种植物生长调节剂组合为培养条件,筛选胚性愈伤组织诱导的适宜条件。并将获得的胚性愈伤组织进行超低温保存和体胚发育与成熟萌发试验,以3种脱落酸(ABA)和Gelrite浓度以及4种基本培养基为培养条件,筛选出体胚成熟和萌发的适宜培养条件。【结果】①两种基本培养基相比较,改良RJW(诱导率为30.00%)优于1/2 LV(诱导率为22.50%)基本培养基。当萘乙酸(NAA)质量浓度为3.0 mg/L、6-苄氨基腺嘌呤(6-BA)质量浓度为0.5 mg/L时胚性愈伤组织诱导率最高为50.00%;② 胚性愈伤组织增殖培养3个月后进行超低温保存试验,经恢复培养10 d 左右可以清晰地观察到成活的胚性愈伤组织,3个细胞系的胚性愈伤组织均可成活;③ 体胚发育阶段,改良RJW培养基添加Gelrite 6.0 g/L和ABA 20 mg/L时,3个细胞系体胚数量均较高,但是畸形胚比例也偏高,特别是HY-1细胞系畸形胚比例高达77.14%;当Gelrite质量浓度增加到8.0 g/L时,3个细胞系畸形胚比例均降低, 其中HY-2细胞系体胚数量为396.00个/g,畸形胚比例为10.33%;④ 红皮云杉体胚成熟培养两个月进行体胚萌发,在1/2 LM上体胚萌发能力最弱,其次是LM基本培养基,改良RJW体胚萌发能力最佳。不同细胞系体胚萌发能力也不同,体胚萌发能力最高为65.00%。【结论】① 胚性愈伤组织诱导的适宜培养基为改良RJW基本培养基,适宜的激素组合为NAA 3.0 mg/L + 6-BA 0.5 mg/L;② 本试验进行超低温保存的细胞系均可成活;③ 体胚发育与成熟适宜的ABA质量浓度为20 mg/L,适宜Gelrite质量浓度为8.0 g/L;④ 体胚萌发的适宜基本培养基为改良RJW基本培养基。本研究建立了红皮云杉体胚发生体系并成功进行胚性愈伤组织的超低温保存条件,该技术体系可用于红皮云杉优良种质的快速繁殖。

Abstract

【Objective】This study aimed to establish a complete somatic embryogenesis system and optimal cryopreservation conditions to provide a basis for the breeding and preservation of Picea koraiensis (Korean spruce) planting resources. We screened somatic embryogenesis conditions and the cryopreservation of Korean spruce. 【Method】We explanted zygote embryos in two basal culture media with six plant growth regulators to define the optimal culture conditions in which the embryogenic mass was introduced. The embryogenic mass was cryopreserved, and the somatic embryo maturation and germination were assessed. We screened three abscisic acid (ABA) and Gelrite concentrations and four basic culture media to assess the optimal conditions for the somatic embryo maturation and germination. 【Result】①The two basal culture media were compared, and the modified induction ratio in RJW medium was 30.00% which was greater than that of 1/2 LV (induction ratio, 22.50%) basal culture medium. The highest induction ratio was 50.00% in the proliferation medium containing 3.0 mg/L of 1-naphthalacetic acid (NAA) and 0.5 mg/L of N6-benzyladenine (6-BA). ②The embryogenic mass was cultured for 3 months, then cryopreservation was assessed. The cryopreserved embryogenic mass from three cell lines survived after 10 days of restoration. ③The numbers of somatic embryos in the three cell lines were higher, but the ratio of abnormal HY-1 somatic embryos was 77.14% in the maturation medium containing Gelrite (6.0 g/L) and ABA (20 mg/L). Increasing the Gelrite concentration to 8.0 g/L decreased the ratio of abnormal somatic embryos in all three cell lines; the number of HY-2 somatic embryos was 396.00 per gram, and the ratio of abnormal somatic embryos was 10.33%. ④The somatic embryos of Korean spruce matured for two months in the germination medium. The amount of somatic embryo germination was the lowest on 1/2 LM, followed by on LM medium and was the highest on RJW medium. The germination ability of the cell lines differed, with a somatic germination ratio of 65.00%.【Conclusion】①The optimal basal culture medium for the embryogenic mass induction was modified RJW, and the appropriate plant growth regulator concentration was NAA 3.0 mg/L + 6-BA 0.5 mg/L. ②The embryogenic mass obtained survived cryopreservation. ③The optimal ABA and Gelrite concentrations for somatic embryo maturation were 20 mg/L and 8.0 g/L, respectively. ④Also, the optimal basal culture medium for somatic embryo germination was modified RJW. We established the ideal conditions for the regeneration, cryopreservation, and somatic embryogenesis of Korean spruce. This technique can be applied to propagate the best planting resources and to establish multi-varietal forestry by using Korean spruce.

导出引用

高芳, 陈士刚, 秦彩云, 等. 红皮云杉体胚发生体系优化和超低温保存技术研究[J]. 南京林业大学学报（自然科学版）. 2021, 45(3): 100-108 https://doi.org/10.12302/j.issn.1000-2006.202007029

GAO Fang, CHEN Shigang, QIN Caiyun, et al. Optimization of somatic embryogenesis system and cryopreservation of Picea koraiensis[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2021, 45(3): 100-108 https://doi.org/10.12302/j.issn.1000-2006.202007029

中图分类号： S722.3

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	HUMÁNEZ A, BLASCO M, BRISA C, et al. Somatic embryogenesis from different tissues of Spanish populations of maritime pine[J]. Plant Cell Tissue Organ Cult (PCTOC), 2012,111(3):373-383.DOI: 10.1007/s11240-012-0203-0. https://doi.org/10.1007/s11240-012-0203-0 10.1007/s11240-012-0203-0 本文引用 [1]

[2]	甄艳, 陈金慧, 施季森. 植物体细胞胚发生胚性潜势恢复的研究进展[J]. 南京林业大学学报(自然科学版), 2013,37(6):147-152. ZHEN Y, CHEN J H, SHI J S. Research progress on the reacquisition of embryogenic potentiality in plant somatic embryogenesis[J]. J Nanjing For Univ (Nat Sci Ed), 2013,37(6):147-152. 本文引用 [1]

[3]	GÓNGRA-CASTILLOE, NIC-CAN G I, GALAZ-AVALOS R M, et al. Elaboration of: transcriptome during the induction of somatic embryogenesis[J]. Plant Cell Culture Protoc, 2018,7(7) 411-427. DOI: 10.1007/978-1-4939-8594-4_29. 10.1007/978-1-4939-8594-4_29 本文引用 [1]

[4]	ELHITI M, STASOLLA C. The use of zygotic embryos as explants for in vitro propagation: an overview[M]//TREVOR A T, EDWARD C Y. Plant embryo culture methods and protocols. New York, Humana Press, 2011: 229-255.DOI: 10.1007/978-1-61737-988-8_17. 10.1007/978-1-61737-988-8_17 本文引用 [1]

[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(1):69-76. DOI: 10.12657/denbio.074.007. https://doi.org/10.12657/denbio.074.007 10.12657/denbio.074.007 本文引用 [2]

[6]	HERINGER A S, SANTA-CATARINA C, SILVEIRA V. Insights from proteomic studies into plant somatic embryogenesis[J]. Proteomics, 2018,18(5/6):e1700265.DOI: 10.1002/pmic.201700265. 10.1002/pmic.201700265 本文引用 [1]

[7]

MOREL

, TRONTIN J

, CORBINEAU

, et al. Cotyledonary somatic embryos of Pinus pinaster Ait.most closely resemble fresh,maturing cotyledonary zygotic embryos:biological,carbohydrate and proteomic analyses[J]. Planta, 2014,240(5):1075-1095.DOI: 10.1007/s00425-014-2125-z.

https://doi.org/10.1007/s00425-014-2125-z

10.1007/s00425-014-2125-z

本文引用 [1]

[8]

AHN C

, CHOI Y

. Erratum to:in vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis[J]. Plant Cell Tissue Organ Cult (PCTOC), 2017,131(3):525.DOI: 10.1007/s11240-017-1307-3.

https://doi.org/10.1007/s11240-017-1307-3

10.1007/s11240-017-1307-3

本文引用 [1]

[9]

刘宝光, 李成浩, 张含国. 红皮云杉球状胚诱导影响因子分析[J]. 北华大学学报(自然科学版), 2010,11(3):247-250.

LIU B

, LI C

, ZHANG H

. Analysis of the factors affecting globular embryos induction of Picea koraiensis[J]. J Beihua Univ (Nat Sci), 2010,11(3):247-250.DOI: 10.3969/j.issn.1009-4822.2010.03.015.

10.3969/j.issn.1009-4822.2010.03.015

本文引用 [3]

[10]

周磊, 吴慧, 王树力. 不同林分红皮云杉针叶养分含量及生态化学计量特征研究[J]. 植物资源与环境学报, 2020,29(3):19-25,33.

ZHOU

, WU

, WANG S

. Study on nutrient contents and ecological stoichiometric characteristics in needles of Picea koraiensis in different stands[J]. J Plant Resour Environ, 2020,29(3):19-25,33.DOI: 10.3969/j.issn.1674-7895.2020.03.03.

10.3969/j.issn.1674-7895.2020.03.03

本文引用 [1]

[11]

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.

LIU B

, LI C

, ZHANG H

. Screening of influence factors on maintenance and proliferation of embryogenic callus of Picea koraiensis[J]. J Northeast For Univ, 2010,38(7):56-60.DOI: 10.13759/j.cnki.dlxb.2010.07.028.

10.13759/j.cnki.dlxb.2010.07.028

本文引用 [2]

[12]	陈少瑜. 丽江云杉体胚发生体系优化及体胚发育过程蛋白质组分析[D]. 昆明:云南大学, 2010. CHEN S Y. Optimization of somatic embryogenesis in Picea likiangensis (Franch.) Pritz and proteomic analysis on somatic embryos at developmental stages[D]. Kunming:Yunnan University, 2010. 本文引用 [1]

[13]	孙敬爽, 贾桂霞. 北美蓝云杉体细胞胚发生技术研究[J]. 北京林业大学学报, 2010,32(1):44-51. SUN J S, JIA G X. Somatic embryogenesis of Picea pungens Engelmann[J]. J Beijing For Univ, 2010,32(1):44-51. DOI: 10.13332/j.1000-1522.2010.01.018. 10.13332/j.1000-1522.2010.01.018 本文引用 [1]

[14]	LI C H, LIU B G, KIM T D, et al. Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis[J]. Plant Biotechnol Rep, 2008,2(4):259-265.DOI: 10.1007/s11816-008-0073-4. https://doi.org/10.1007/s11816-008-0073-4 10.1007/s11816-008-0073-4 本文引用 [2]

[15]	刘宝光, 李成浩, 王伟达, 等. 红皮云杉胚性愈伤组织诱导技术研究[J]. 植物研究, 2009,29(1):69-73. LIU B G, LI C H, WANG W D, et al. Induction technique of Picea koraiensis nakai embryogenic callus[J]. Bull Bot Res, 2009,29(1):69-73. 本文引用 [1]

[16]	孙婷玉, 王艳丽, 沈李元, 等. 培养基成分对黑松体胚发育成熟的影响[J]. 林业科学, 2019,55(4):178-186. SUN T Y, WANG Y L, SHEN L Y, et al. Impact of medium components on somatic embryo maturation in Pinus thunbergii[J]. Sci Silvae Sin, 2019,55(4):178-186. DOI: 10.11707/j.1001-7488.20190419. 10.11707/j.1001-7488.20190419 本文引用 [2]

[17]

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.

GAO

, SHEN H

, LIU C

, et al. Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis[J]. J Nanjing For Univ (Nat Sci Ed), 2017,41(3):43-50.DOI: 10.3969/j.issn.1000-2006.201605034.

10.3969/j.issn.1000-2006.201605034

本文引用 [1]

[18]	PULLMAN G S, BUCALO K. Pine somatic embryogenesis using zygotic embryos as explants[C]//TREVOR A T, EDWARD C Y. Method in molecular biology. New York: Humana Press, 2011: 267-291. DOI: 10.1007/978-1-61737-988-8_19. 10.1007/978-1-61737-988-8_19 本文引用 [2]

[19]

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.

JI K

, WANG P

, WANG J

, et al. Review on in vitro culture of tree species in Pinaceae[J]. J Nanjing For Univ (Nat Sci Ed), 2015,39(1):142-148. DOI: 10.3969/J.ISSN.1000-2006.2015.01.026.

10.3969/J.ISSN.1000-2006.2015.01.026

本文引用 [1]

[20]	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):2118-2121,2180. WANG X X, YANG Y G. Study on the somatic embryogenesis of Larix leptolepis[J]. J Anhui Agric Sci, 2010,38(4):2118-2121,2180.DOI: 10.13989/j.cnki.0517-6611.2010.04.138. 10.13989/j.cnki.0517-6611.2010.04.138 本文引用 [1]

[21]

RUDUS

, WEILER E

, KEPCZYNSKA

. Do stress-related phytohormones,abscisic acid and jasmonic acid play a role in the regulation of Medicago sativa L.somatic embryogenesis?[J]. Plant Growth Regul, 2009,59(2):159-169.DOI: 10.1007/s10725-009-9399-3.

https://doi.org/10.1007/s10725-009-9399-3

10.1007/s10725-009-9399-3

本文引用 [1]

[22]	PULLMAN G S, BUCALO K. Pine somatic embryogenesis:analyses of seed tissue and medium to improve protocol development[J]. New For, 2014,45(3):353-377.DOI: 10.1007/s11056-014-9407-y. https://doi.org/10.1007/s11056-014-9407-y 10.1007/s11056-014-9407-y 本文引用 [1]

[23]	崔凯荣, 戴若兰. 植物体细胞胚发生的分子生物学:生命科学专论[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. 本文引用 [1]

[24]	KONG L S, ADERKAS P. A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer[J]. Plant Cell Tissue Organ Cult (PCTOC), 2011,106(1):115-125.DOI: 10.1007/s11240-010-9899-x. https://doi.org/10.1007/s11240-010-9899-x 10.1007/s11240-010-9899-x 本文引用 [1]

[25]

BRETON

, HARVENGT

, TRONTIN J

, et al. Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine[J]. Plant Cell Tissue Organ Cult, 2006,87(1):95-108.DOI: 10.1007/s11240-006-9144-9.

https://doi.org/10.1007/s11240-006-9144-9

10.1007/s11240-006-9144-9

本文引用 [1]

[26]	LI Q F, DENG C, ZHU T Q, et al. Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana[J]. Trees, 2019,33(2):469-480.DOI: 10.1007/s00468-018-1793-x. https://doi.org/10.1007/s00468-018-1793-x 10.1007/s00468-018-1793-x 本文引用 [1]

[27]

LINEROS

, BALOCCHI

, MUÑOZ

, et al. Cryopreservation of Pinus radiata embryogenic tissue:effects of cryoprotective pretreatments on maturation ability[J]. Plant Cell Tissue Organ Cult (PCTOC), 2018,135(2):357-366.DOI: 10.1007/s11240-018-1469-7.

https://doi.org/10.1007/s11240-018-1469-7

10.1007/s11240-018-1469-7

本文引用 [1]

[28]	GALE S, JOHN A, BENSON E E. Cryopreservation of Picea sitchensis (sitka spruce) embryogenic suspensor masses[J]. Cryo Letters, 2007,28(4) : 225-239. 本文引用 [1]

[29]	KLIMASZEWSKA K, HARGREAVES C, LELU-WALTER M A, et al. Advances in conifer somatic embryogenesis since year 2000[M]//GERMENA M A, LAMBARDI M. In vitro embryogenesis in higher plants. New York: Springer, 2016: 131-166. DOI: 10.1007/978-1-4939-3061-6_7. 10.1007/978-1-4939-3061-6_7 本文引用 [2]

[30]	VON ARNOLD S, SABALA I, BOZHKOV P, et al. Developmental pathways of somatic embryogenesis[J]. Plant Cell Tissue Organ Cult, 2002,69(3):233-249.DOI: 10.1023/A:1015673200621. https://doi.org/10.1023/A:1015673200621 10.1023/A:1015673200621 本文引用 [1]

[31]

KONG L

, ADERKAS

, OWEN S

, et al. Comparison of endogenous cytokinins,ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine[J]. Trees, 2011,25(6):1103-1110.DOI: 10.1007/s00468-011-0585-3.

https://doi.org/10.1007/s00468-011-0585-3

10.1007/s00468-011-0585-3

本文引用 [1]

[32]

BOZHKOV P

, FILONOVA L

, VON ARNOLD

. A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification[J]. Biotechnol Bioeng, 2002,77(6):658-667.DOI: 10.1002/bit.10228.

https://doi.org/10.1002/(ISSN)1097-0290

10.1002/bit.10228

本文引用 [1]

[33]

ZHANG

, HAN

, DE SMET

, et al. ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem[J]. Plant J, 2010,64(5):764-774.DOI: 10.1111/j.1365-313X.2010.04367.x.

https://doi.org/10.1111/tpj.2010.64.issue-5

10.1111/j.1365-313X.2010.04367.x

本文引用 [1]

[34]

ZHANG J

, ZHANG S

, LI S

, et al. Regulation of synchronism by abscisic-acid-responsive small noncoding RNAs during somatic embryogenesis in larch (Larix leptolepis)[J]. Plant Cell Tissue Organ Cult (PCTOC), 2014,116(3):361-370.DOI: 10.1007/s11240-013-0412-1.

https://doi.org/10.1007/s11240-013-0412-1

10.1007/s11240-013-0412-1

本文引用 [1]

[35]

LITVAY J

, VERMA D

, JOHNSON M

, et al. 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.)[J]. Plant Cell Rep, 1985,4(6):325-328. DOI: 10.1007/BF00269890.

https://doi.org/10.1007/BF00269890

10.1007/BF00269890

本文引用 [1]

[36]

GARIN

, BERNIER-CARDOU

, ISABEL

, et al. Effect of sugars,amino acids,and culture technique on maturation of somatic embryos of Pinus strobus on medium with two gellan gum concentrations[J]. Plant Cell Tissue Organ Cult, 2000,62(1):27-37.DOI: 10.1023/A:1006402215457.

https://doi.org/10.1023/A:1006402215457

10.1023/A:1006402215457

本文引用 [1]

[37]

MOREL

, TEYSSIER

, TRONTIN J

, et al. Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses[J]. Physiol Plant, 2014,152(1):184-201. DOI: 10.1111/ppl.12158.

https://doi.org/10.1111/ppl.12158

10.1111/ppl.12158

本文引用 [1]