Effects of Hormone Combinations, Carbon Source Type and Proliferation Cycle on Embryogenic Callus Proliferation of Pinus koraiensis

doi:10.7525/j.issn.1673-5102.2022.04.020

Abstract

Abstract:

In order to solve the poor growth and low somatic embryo production in the embryogenic callus proliferation of Pinus koraiensis， P. koraiensis embryogenic callus 1-100 cell line was used as material to explore its effect on the somatic embryogenesis and the changes of physiological and biochemical characteristics by adjusting hormone， carbon source and proliferation culture cycle in proliferation medium. The results were as follows： ①the optimum proliferation condition was 1 mg·L^-1 NAA+0.25 mg·L^-1 6-BA， the best carbon source was sucrose， and the best culture cycle was 14 days. Under the conditions， the largest number of somatic embryos were obtained（166·g^-1）； ②in the process of optimizing proliferation culture， the stored substances provided energy for somatic embryogenesis； There was no significant difference in soluble protein mass fraction between hormone types and their concentrations， carbon source types and proliferation culture cycles； ③high CAT activity in callus was harmful to somatic embryogenesis， which might be due to excessive accumulation of toxic metabolites such as H₂O₂.

Key words: Pinus koraiensis, embryogenic callus, somatic embryogenesis, proliferation medium

CLC Number:

S722.8

Yuhui REN, Shuai NIE, Chunxue PENG, Ling YANG, Hailong SHEN. Effects of Hormone Combinations, Carbon Source Type and Proliferation Cycle on Embryogenic Callus Proliferation of Pinus koraiensis[J]. Bulletin of Botanical Research, 2022, 42(4): 704-712.

References

1	周雪燕，高海燕，李召珉，等.基于生长与结实评价红松种子园亲本［J］.植物研究，2020，40（3）：376-385.
1	ZHOU X Y， GAO H Y， LI Z M，et al.Evaluating parents of Pinus koraiensis seeds orchard with growth and fruiting［J］.Bulletin of Botanical Research，2020，40（3）：376-385.
2	隋立龙，王芳，赵泉湖，等.不同林分红松生长与结实性状比较研究［J］.植物研究，2018，38（6）：886-893.
2	SUI L L， WANG F， ZHAO Q H，et al.Growth and fruiting characters of different Pinus koraiensis stands［J］.Bulletin of Botanical Research，2018，38（6）：886-893.
3	GAO F， PENG C X， WANG H，et al.Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis ［J］.Journal of Forestry Research，2021，32（2）：483-491.
4	PENG C X， GAO F， WANG H，et al.Optimization of maturation process for somatic embryo production and cryopreservation of embryogenic tissue in Pinus koraiensis ［J］.Plant Cell，Tissue and Organ Culture，2021，144（1）：185-194.
5	GAO F， PENG C X， WANG H，et al.Key techniques for somatic embryogenesis and plant regeneration of Pinus koraiensis ［J］.Forests，2020，11（9）：912.
6	PENG C X， GAO F， WANG H，et al.Physiological and biochemical traits in Pinus koraiensis somatic embryogenesis［J］.Forests，2020，11（5）：577.
7	姜国云，蒋路平，宋双林，等.红松半同胞家系遗传变异分析及果材兼用优良家系选择［J］.植物研究，2018，38（5）：775-784.
7	JIANG G Y， JIANG L P， SONG S L，et al.Genetic variance analysis and excellent fruit-timber families selection of half-sib Pinus koraiensis ［J］.Bulletin of Botanical Research，2018，38（5）：775-784.
8	莫迟，张海啸，张磊，等.红松不同杂交组合种实形态与营养成分变异研究［J］.植物研究，2017，37（5）：700-708.
8	MO C， ZHANG H X， ZHANG L，et al.Variations in nutrition compositions and morphology characteristics in different hybrid combination of Korean pine（Pinus koraiensis）［J］.Bulletin of Botanical Research，2017，37（5）：700-708.
9	陈士刚，秦彩云，王聪慧，等.红松5个家系未成熟种子的胚性愈伤组织诱导研究［J］.森林工程，2021，37（1）：13-17.
9	CHEN S G， QIN C Y， WANG C H，et al.Embryogenic callus induction of Pinus koraiensis immature seeds from five families［J］.Forest Engineering，2021，37（1）：13-17.
10	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.
10	GAO F， SHEN H L， LIU C P，et al.Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis ［J］.Journal of Nanjing Forestry University（Natural Sciences Edition），2017，41（3）：43-50.
11	LEE C L， CHANG H 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］.Journal of Integrative Plant Biology，1962，4（3）：179-186.
12	KIM O R， PARK Y G. In vitro studies on Pinus koraiensis.Ⅱ.Chromosomal variation in the callus［J］.Journal of Korean Society of Forest Science，1986（74）：56-60.
13	刘玉喜，唐翠，詹亚光.红松成熟胚离体培养器官发生组织学研究［J］.植物研究，1992，12（2）：189-194.
13	LIU Y X， TANG C， ZHAN Y G.Study on histogenesis and organogensis of the in vitro development from Pinus koraiensis embryos［J］.Bulletin of Botanical Research，1992，12（2）：189-194.
14	申晓辉，蒋湘宁，Yill Sung Park，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.
14	SHEN X H， JIANG X N， PARK Y S，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.
15	KLIMASZEWSKA K， PARK Y S， OVERTON C，et al.Optimized somatic embryogenesis in Pinus strobus L.［J］.in vitro Cellular & Developmental Biology-Plant，2001，37（3）：392-399.
16	张志良，瞿伟菁.植物生理学实验指导［M］.3版.北京：高等教育出版社，2003.
16	ZHANG Z L， QU W J.The experimental guide for plant physiology［M］.3ed.Beijing：Higher Education Press，2003.
17	张蕾.日本落叶松×华北落叶松体细胞胚胎发生的生化机制和分子机理研究［D］.北京：中国林业科学研究院，2008.
17	ZHANG L.Study on biochemical and molecular mechanisms of Larix leptolepis × L . principis-rupprechtii somatic embryogenesis［D］.Beijing：Chinese Academy of Forestry，2008.
18	ADIL M， REN X X， KANG D I，et al.Effect of explant type and plant growth regulators on callus induction，growth and secondary metabolites production in Cnidium officinale Makino［J］.Molecular Biology Reports，2018，45（6）：1919-1927.
19	CHAI M L， JIA Y F， CHEN S，et al.Callus induction，plant regeneration，and long-term maintenance of embryogenic cultures in Zoysia matrella［L.］Merr［J］.Plant Cell，Tissue and Organ Culture：PCTOC，2011，104（2）：187-192.
20	DHINDSA R S， PLUMB-DHINDSA P， THORPE T A.Leaf senescence：correlated with increased levels of membrane permeability and lipid peroxidation，and decreased levels of superoxide dismutase and catalase［J］.Journal of Experimental Botany，1981，32（1）：93-101.
21	李茜.白皮松体细胞胚胎发生的研究［D］.杨凌：西北农林科技大学，2006.
21	LI Q.Somatic embryogenesis in Pinus bungeana ［D］.Yangling：Northwest A&F University，2006.
22	宋跃，甄成，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.
22	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.
23	齐力旺，韩一凡，李玲，等.应用311-A最优回归设计研究ABA、PEG4000及AgNO₃对落叶松体细胞胚发生数量的影响［J］.生物工程学报，2001，17（1）：84-89.
23	QI L W， HAN Y F， LI L，et al.Study on effect of ABA，PEG4000 and AgNO₃ on number of somatic embryos of Larix principis-rupprechtii by 311-A regression design［J］.Chinese Journal of Biotechnology，2001，17（1）：84-89.
24	PASSAMANI L Z， REIS R S， VALE E M，et al.Long-term culture with 2，4-dichlorophenoxyacetic acid affects embryogenic competence in sugarcane callus via changes in starch，polyamine and protein profiles［J］.Plant Cell，Tissue and Organ Culture，2020，140（2）：415-429.
25	詹园凤，吴震，金潇潇，等.大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征［J］.西北植物学报，2006，26（9）：1799-1802.
25	ZHAN Y F， WU Z， JIN X X，et al.Anti-oxidative enzymes and some physiological features in somatic embryogenesis of garlic［J］.Acta Botanica Boreali-Occidentalia Sinica，2006，26（9）：1799-1802.
26	LISZKAY A， KENK B， SCHOPFER P.Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth［J］.Planta，2003，217（4）：658-667.
27	DE MARCO A， ROUBELAKIS-ANGELAKIS K A.Hydrogen peroxide plays a bivalent role in the regeneration of protoplasts［J］.Journal of Plant Physiology，1996，149（1/2）：109-114.
28	RAJESWARI V， PALIWAL K.Peroxidase and catalase changes during in vitro adventitious shoot organogenesis from hypocotyls of Albizia odoratissima L.f.（Benth）［J］.Acta Physiologiae Plantarum，2008，30（6）：825-832.
29	刘冬云，史宝胜，李银华，等.不同碳源及PP₃₃₃、GA₃对山丹组培苗鳞茎增大的影响［J］.河北农业大学学报，2005，28（2）：32-35.
29	LIU D Y， SHI B S， LI Y H，et al.The bulbs augmentation influenced by the carbon resources PP₃₃₃ and GA₃ during the low lily tissue culture［J］.Journal of Agricultural University of Hebei，2005，28（2）：32-35.
30	杨玲，刘虹男，张冬严，等.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.
30	YANG L， LIU H N， ZHANG D Y，et al.Effect of plant growth regulators and osmoticums on somatic embryogenesis of Fraxinus mandshurica Rupr.［J］.Bulletin of Botanical Research，2017，37（5）：682-689.
31	张建伟.粗枝云杉体胚发生和萌发前的干化调控机制［D］.北京：中国林业科学研究院，2014.
31	ZHANG J W.Somatic embryogenesis and partial desiccation mechanism of somatic embryos before regeneration in Picea asperata Mast.［D］.Beijing：Chinese Academy of Forestry，2014.
32	胡家恕，朱诚，曾广文，等.超干红花种子抗老化作用及其机理［J］.植物生理学报，1999，25（2）：171-177.
32	HU J S， ZHU C， ZENG G W，et al.Effect of aging resistance of ultradried safflower seeds and its machanism［J］.Acta Photophysiologica Sinica，1999，25（2）：171-177.
33	魏明，杨超英，姜绍通，等.继代周期对霍山石斛类原球茎悬浮培养动力学的影响［J］.中国农学通报，2008，24（9）：43-47.
33	WEI M， YANG C Y， JING S T，et al.Effect of subculture cycle on kinetics of suspension cultures of protocorm-like bodies from Dendrobium huoshanense［J］.Chinese Agricultural Science Bulletin，2008，24（9）：43-47.
34	秦彩云，李青梅，王聪慧，等.青海云杉愈伤组织增殖与体胚成熟阶段影响因子研究［J］.森林工程，2018，34（2）：21-25.
34	QIN C Y， LI Q M， WANG C H，et al.Study on the influence factors of the proliferation and the mature stage of somatic embryo of Picea crassifolia ［J］.Forest Engineering，2018，34（2）：21-25.
35	BRETON D， HARVENGT L， TRONTIN J F，et al.High subculture frequency，maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine［J］.in vitro Cellular & Developmental Biology-Plant，2005，41（4）：494-504.
36	CUI K R， LI J， XING G M，et al.Effect of hydrogen peroxide on synthesis of proteins during somatic embryogenesis in Lycium barbarum ［J］.Plant Cell Tissue and Organ Culture，2002，68（2）：187-193.

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