Somatic Embryo Induction and Histocytological Observation of Isodon rubescens

doi:10.7525/j.issn.1673-5102.2024.05.006

Abstract

Abstract:

Establishing the somatic embryogenesis system of Isodon rubescens to give full play to its value in traditional Chinese medicine， and promote the stability and utilization of its resources. The leaves of I. rubescens were used as explants to dedifferentiate into embryonic callus and further proliferate to induce somatic embryo. The histological and cytological observation of embryonic callus and somatic embryo at each stage was made by paraffin section， and the effect of hormones on plant regeneration was tested. The results showed that in B5+1.0 mg·L^-1 2，4-D medium， leaves could dedifferentiate into light yellow and granular embryonic callus. In MS+1.0 mg·L^-1 2，4-D+0.2 mg·L^-1 6-BA medium， the embryogenic callus had the best proliferation effect， and the maximum net proliferation was 2.81 g. The optimal medium for somatic embryo induction was MS+1.5 mg·L^-1 TDZ+0.2 mg·L^-1 NAA， and the highest induction rate was 91.33%. In MS+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 IBA medium， embryogenic callus containing somatic embryos could differentiate into small plants. In histological observation， the embryonic callus was yellowish or yellowish white granules with large and obvious nuclei. Two kinds of somatic embryogenesis were observed in the inner and outer origin of the callus， and the later developed into spherical， heart-shaped， torpedo-shaped， cotyledon-shaped embryo or cluster embryo structures containing starch granules. This study successfully established the indirect somatic embryogenesis system of I. rubescens， which laid a foundation for optimizing the “quality” and “quantity” of embryogenic callus， and also provided the theoretical basis for the research of molecular breeding and somatic embryo technology.

Key words: Isodon rubescens, embryonic callus, somatic embryo, histocytology

CLC Number:

S722.8

Wange WU, Baoyu JI, Lixin PEI, Conglong LIAN, Chengming DONG, Suiqing CHEN. Somatic Embryo Induction and Histocytological Observation of Isodon rubescens[J]. Bulletin of Botanical Research, 2024, 44(5): 692-701.

Figures/Tables 9

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References 31

1	高世勇，唐博琪，魏新瑞，等.冬凌草的本草考证、化学成分、药理作用及其临床应用［J］.中草药，2023，54（19）：6543-6554.
	GAO S Y， TANG B Q， WEI X R，et al.Textual research， chemical composition， pharmacological effects and clinical applications of Rabdosia rubescens ［J］.Chinese Traditional and Herbal Drugs，2023，54（19）：6543-6554.
2	张婉晴，夏小婧.冬凌草化学成分及药理作用研究进展［J］.云南化工，2020，47（10）：17-19.
	ZHANG W Q， XIA X J.Advances in studies on chemical components and pharmacological effects of oridonium［J］.Yunnan Chemical Technology，2020，47（10）：17-19.
3	孔睿，潘鹏宇，梁国标.冬凌草素药理作用及机制的研究进展［J］.中华中医药学刊，2021，39（11）：70-72.
	KONG R， PAN P Y， LIANG G B.Research progress on pharmacological role and mechanism of oridonin［J］.Chinese Archives of Traditional Chinese Medicine，2021，39（11）：70-72.
4	陈川川，刘越，郭凤仙，等.冬凌草生物学研究进展［J］.湖北农业科学，2016，55（19）：4901-4906.
	CHEN C C， LIU Y， GUO F X，et al.Progress of biological research for Rabdosia rubescens ［J］.Hubei Agricultural Sciences，2016，55（19）：4901-4906.
5	李美莹，李前，金子涵，等.蒙古栎体细胞胚胎发生技术研究［J］.辽宁林业科技，2021（1）：1-6.
	LI M Y， LI Q， JIN Z H，et al.Study on somatic embryogenesis of Quercus mongolica ［J］.Liaoning Forestry Science and Technology，2021（1）：1-6.
6	李志芬，杨扬，由香玲，等.通过体细胞胚发生快繁优良刺五加植株的技术［J］.东北林业大学学报，2012，40（10）：19-21.
	LI Z F， YANG Y， YOU X L，et al.Rapid propagation of Eleutherococcus senticosus via somatic embryogenesis［J］.Journal of Northeast Forestry University，2012，40（10）：19-21.
7	魏冬霞，张滕，郑严仪，等.芍药愈伤组织中体细胞胚发育过程的组织细胞学观察［J］.植物研究，2018，38（1）：56-63.
	WEI D X， ZHANG T， ZHENG Y Y，et al.Histocytology observation on the somatic embryogenesis in herbaceous peony callus［J］.Bulletin of Botanical Research，2018，38（1）：56-63.
8	VERGNE P， MAENE M， GABANT G，et al.Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush［J］.Plant Cell，Tissue and Organ Culture，2010，100（1）：73-81.
9	梁慧敏，杜雪玲，夏阳，等.沿阶草体细胞胚诱导及快繁技术研究［J］.安徽农业科学，2021，49（24）：136-140.
	LIANG H M， DU X L， XIA Y，et al.A rapid propagation potocol of Ophiopogon japonicus via somatic embryo culture in vitro ［J］.Journal of Anhui Agricultural Sciences，2021，49（24）：136-140.
10	张苏苏，孙丹，艾军，等.黑暗处理对五味子体细胞胚再生及其生理特性的影响［J］.植物生理学报，2022，58（5）：864-872.
	ZHANG S S， SUN D， AI J，et al.Effects of dark treatment on somatic embryo regeneration and physiological characteristics of Schisandra chinensis ［J］.Plant Physiology Journal，2022，58（5）：864-872.
11	邢建宏，彭漳清，刘希华，等.无患子优树叶片体细胞胚胎发生及植株再生的研究［J］.三明学院学报，2014，31（6）：93-100.
	XING J H， PENG Z Q， LIU X H，et al.Somatic embryogenesis and plant regeneration from the elite Tree’s leaves of Sapindus mukorossi ［J］.Journal of Sanming University，2014，31（6）：93-100.
12	徐莉莉，聂世现，黄文静，等.冬凌草组织培养及其愈伤组织诱导研究［J］.生物学杂志，2010，27（3）：24-26.
	XU L L， NIE S X， HUANG W J，et al.Research on tissue culture and callus induction of Rabdosia rubescens（Hcnsi） Hara［J］.Journal of Biology，2010，27（3）：24-26.
13	苏秀红，董诚明，王春雷，等.冬凌草离体培养体系的建立及主要次生代谢产物的测定［J］.西北植物学报，2008，28（2）：310-316.
	SU X H， DONG C M， WANG C L，et al.Establishment of culture system of Rabdosia rubescens（Hemsl.） Hara and content of the main secondary metabolites in its regenerated plantlets［J］.Acta Botanica Boreali-Occidentalia Sinica，2008，28（2）：310-316.
14	刘宝骏，刘国锋.安祖花胚性愈伤组织诱导及植株再生研究［J］.热带亚热带植物学报，2018，26（4）：407-414.
	LIU B J， LIU G F.Studies on embryogenic callus induction and plant regeneration of Anthurium andreanum ［J］.Journal of Tropical and Subtropical Botany，2018，26（4）：407-414.
15	梁忠泉，袁昌隆，张宝贺.微小组织石蜡切片制作体会［J］.中国组织化学与细胞化学杂志，2018，27（4）：368-373.
	LIANG Z Q， YUAN C L， ZHANG B H.Experience in making microtissue paraffin section［J］.Chinese Journal of Histochemistry and Cytochemistry，2018，27（4）：368-373.
16	任毓辉，聂帅，彭春雪，等.红松胚性愈伤组织增殖的激素配比、糖源类型和增殖周期效应研究［J］.植物研究，2022，42（4）：704-712.
	REN Y H， NIE S， PENG C X，et al.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.
17	许丁帆，刘艳军，陶则满，等.番茄胚性愈伤组织诱导与增殖研究［J］.天津农学院学报，2020，27（3）：22-25.
	XU D F， LIU Y J， TAO Z M，et al.Study on embryogenic callus induction and proliferation of tomato［J］.Journal of Tianjin Agricultural University，2020，27（3）：22-25.
18	薛敏，高艳芳，朱磊磊，等.油用亚麻胚性愈伤组织的诱导及组织细胞学分析［J/OL］.分子植物育种.（2022-09-28）［2024-01-24］..
	XUE M， GAO Y F， ZHU L L，et al.Induction and histocytological observation of embryogenic callus from oil flax［J/OL］.Molecular Plant Breeding.（2022-09-28）［2024-01-24］..
19	王晓春，高婷，杨炜迪，等.紫花苜蓿胚性愈伤组织诱导及胚状体分化研究［J］.作物杂志，2023（4）：98-103.
	WANG X C， GAO T， YANG W D，et al.Study on embryogenic callus induction and embryoid differentiation of alfalfa［J］.Crops，2023（4）：98-103.
20	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 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.
21	张媛媛，朱智慧，张苗苗，等.培养基类型和植物生长物质浓度对王枣子愈伤组织诱导和继代的影响［J］.世界科学技术-中医药现代化，2020，22（8）：2851-2856.
	ZHANG Y Y， ZHU Z H， ZHANG M M，et al.Effect of medium type and plant growth substance concentration on callus induction and subculture of Isodon amethystoides ［J］.World Science and Technology-Modernization of Traditional Chinese Medicine and Materia Medica，2020，22（8）：2851-2856.
22	姜治国，马鑫，胡胜，等.三叶木通体细胞胚胎发生及植株再生研究［J］.西南林业大学学报（自然科学），2022，42（3）：34-41.
	JIANG Z G， MA X， HU S，et al.Somatic embryogenesis and plantlet regeneration of Akebia trifoliate ［J］.Journal of Southwest Forestry University（Natural Sciences Edition），2022，42（3）：34-41.
23	NATARAJAN N， SUNDARARAJAN S， RAMALINGAM S，et al.Efficient and rapid in-vitro plantlet regeneration via somatic embryogenesis in ornamental bananas（Musa spp.）［J］.Biologia，2020，75（2）：317-326.
24	ASLAM J， KHAN S A， CHERUTH A J，et al.Somatic embryogenesis，scanning electron microscopy，histology and biochemical analysis at different developing stages of embryogenesis in six date palm（Phoenix dactylifera L.） cultivars［J］.Saudi Journal of Biological Sciences，2011，18（4）：369-380.
25	安文杰，王银杰，刘清泉，等.马蔺幼胚高效再生体系的建立［J］.植物研究，2023，43（3）：333-339.
	AN W J， WANG Y J， LIU Q Q，et al.Establishment of an efficient regeneration system in Iris lacteal var. chinensis with immature embryo［J］.Bulletin of Botanical Research，2023，43（3）：333-339.
26	吴丽芳，魏晓梅，陆伟东.白刺花胚性愈伤组织诱导和植株再生体系建立［J］.植物生理学报，2019，55（2）：218-224.
	WU L F， WEI X M， LU W D.Embryogenic callus induction and establishment of plant regeneration system of Sophora davidii ［J］.Plant Physiology Journal，2019，55（2）：218-224.
27	杨先泉，姚丽君，倪苏，等.TDZ对非洲紫罗兰叶外植体体细胞胚发生的效应［J］.西南农业学报，2010，23（6）：2030-2033.
	YANG X Q， YAO L J， NI S，et al.Effect of thidiazuron on somatic embryogenesis of African violets leaf explants［J］.Southwest China Journal of Agricultural Sciences，2010，23（6）：2030-2033.
28	荆茹月，霍坤，李志辉.香樟胚性与非胚性愈伤组织间的差异研究［J］.中南林业科技大学学报，2020， 40（10）：70-78.
	JING R Y， HUO K， LI Z H.Difference between embryogenic and non-embryogenic callus of Cinnamomum camphora L.［J］.Journal of Central South University of Forestry and Technology，2020，40（10）：70-78.
29	项伟波，赵金凯，吴家胜，等.香榧体细胞胚发生、发育的形态与细胞学观察［J］.园艺学报，2015，42（8）：1477-1486.
	XIANG W B， ZHAO J K， WU J S，et al.Morphological and cytological observation of somatic embryogenesis and development in Torreya grandis ‘Merrillii’［J］.Acta Horticulturae Sinica，2015，42（8）：1477-1486.
30	马镇荣，刘卫，王昌虎，等.香根草体细胞胚胎发生的细胞学特点与形成条件［J］.生态学报，2003，23（7）：1290-1296.
	MA Z R， LIU W， WANG C H，et al.Cytology observation and formation conditions of somatic embryogenesis in Vetiveria zizaniodes ［J］.Acta Ecologica Sinica，2003，23（7）：1290-1296.
31	孔冬梅，沈海龙，冯丹丹，等.水曲柳体细胞胚与合子胚发生的细胞学研究［J］.林业科学，2006，42（12）：130-133.
	KONG D M， SHEN H L， FENG D D，et al.Cytological investigation of somatic and zygotic embryogenesis of Fraxinus mandshurica ［J］.Scientia Silvae Sinicae，2006，42（12）：130-133.

基本培养基类型 Basic medium type	外植体数量 Number of explants	启动时间 Start-up time/d	生长状态 Growth situation
MS	25	3~4	白色，质地偏硬，表面干燥 White，hard texture，dry surface
B5	25	10~11	淡黄色，质地疏松，颗粒状 Light yellow，loose texture，granular
1/2MS	25	5~6	白色，黄褐色，部分水渍状，表面产生绒毛状根 White，yellowish brown，partially water-stained，with fluff-like roots on the surface
WPM	25	6~7	黄褐色，质地偏硬，易褐化 Yellowish brown，hard texture，easy browning

处理 Treatment	基本培养基 Basic medium	2，4-D质量浓度 2，4-D mass concentration/（mg·L^-1）	胚性愈伤组织诱导率 Embryogenic callus induction rate/%
1	B5	0.1	40.67±2.31^d
2		0.5	42.67±2.52^d
3		1.0	93.33±0.58^a
4		1.5	85.00±1.00^b
5		2.0	76.00±8.67^c

处理 Treatments	接种数量/块 Inoculation number	TDZ质量浓度 Mass concentration of TDZ/ （mg·L^-1）	NAA质量浓度 Mass concentration of NAA/ （mg·L^-1）	体胚诱导率 Somatic embryo induction rate/%
1	30	1.0	0	40.00±10.00^de
2	30	1.5	0	63.33±5.77^bc
3	30	2.0	0	43.33±15.28^de
4	30	2.5	0	33.33±5.77^ef
5	30	3.0	0	20.00±10.00^f
6	30	1.5	0.5	73.33±3.51^b
7	30	1.5	0.2	91.33±5.13^a
8	30	2.0	0.5	53.33±3.51^cd
9	30	2.0	0.2	60.00±3.00^bc

处理 Treatments	激素质量浓度 Hormone mass concentration/（mg·L^-1）			接种数量 Inoculation number	成苗率 Seedling percent/%	30 d生长状态 30 d growth state
处理 Treatments	6-BA	IBA	NAA	接种数量 Inoculation number	成苗率 Seedling percent/%	30 d生长状态 30 d growth state
1	0.5	0.5	0.0	15	26.67	表面体胚进一步发育成熟，少部分形成小植株 The embryo of the surface body further developed and matured， and a few formed small plants
2	0.5	0.0	0.1	15	0	表面形成芽点，少部分分化为丛生芽 The surface formed bud points，and a few differentiated into clumped buds
3	0.5	0.0	0.2	15	0	表面形成芽点，全部有丛生芽出现 The surface formed bud points，all have clumped buds

[1]	Yue REN, Cheng WEI, Tiantian XU, Hailong SHEN, Ling YANG. Effects of Cryopreservation on Physiological and Biochemical Characteristics of Fraxinus mandshurica Embryos [J]. Bulletin of Botanical Research, 2023, 43(3): 396-403.
[2]	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.
[3]	Chun-Xue PENG, Xue-Mei CUI, Hai-Long SHEN. Callus Induction and Somatic Embryogenesis and Physiological State Analysis from Mature Zygotic Embryo Explant of Syringa reticulata var. mandshurica [J]. Bulletin of Botanical Research, 2021, 41(4): 557-563.
[4]	ZHANG Lu, PAN Yuan-Zhi, LIU Shi-Liang, CHEN Yuan, SHEN Li-Jia. Embryonic Callus Induction and Plant Regeneration of Lilium leucanthum [J]. Bulletin of Botanical Research, 2019, 39(3): 338-346.
[5]	PENG Chun-Xue, CUI Xue-Mei, LIU Chun-Ping, YANG Ling, WANG Qiu-Shui, SHEN Hai-Long. Physiological Analysis of Three Anti-Browning Agents Affecting Somatic Embryogenesis in Fraxinus mandshurica [J]. Bulletin of Botanical Research, 2019, 39(2): 252-258.
[6]	WEI Dong-Xia, ZHANG Teng, ZHENG Yan-Yi, YU Xiao-Nan. Histocytology Observation on the Somatic Embryogenesis in Herbaceous Peony Callus [J]. Bulletin of Botanical Research, 2018, 38(1): 56-63.
[7]	YANG Ling, LIU Hong-Nan, ZHANG Dong-Yan, WEI Cheng, SHEN Hai-Long. Effect of Plant Growth Regulators and Osmoticums on Somatic Embryogenesis of Fraxinus mandshurica Rupr. [J]. Bulletin of Botanical Research, 2017, 37(5): 682-689.
[8]	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.
[9]	GAO Jie1；WANG Yuan-Zhong2；HUANG Heng-Yu1. Embryonic Callus Induction and Plant Regeneration of Lilium fargesii* [J]. Bulletin of Botanical Research, 2016, 36(1): 52-57.
[10]	HUANG Zhen1,2；LI Yun1,2；LI Yuan1,2；WANG Pei-Qi1,2；KANG Xiang-Yang1,2. Establishment and Regeneration of Suspension Cell Lines in Hybirds of （Populus pseudo-simonii×P.nigra* ‘Zhenyin3#’）×P.×beijingensis [J]. Bulletin of Botanical Research, 2015, 35(5): 696-703.
[11]	LIAO Jing;FANG Yan-Ming;YU Mu-Kui. Preliminary Report on Somatic Embryogenesis from Stem of Quercus acutissima* Carr. and Histological Observation [J]. Bulletin of Botanical Research, 2011, 31(5): 575-578.
[12]	YANG Ling;SHEN Hai-Long;LIU Chun-Ping;ZHAI Xiao-Jie. Somatic Embryogenesis from Mature Zygotic Embryo Explants of Sorbus pohuashanensis* Hedl [J]. Bulletin of Botanical Research, 2010, 30(2): 174-179.
[13]	ZHANG Jian-Ying;JIANG Si-Jia;XING Ya-Juan;GUO Shu-Ping;GUO Xi-Jun;WANG Fei;. Induction of Embryogenic callus and Somatic Embryogenesis of Juglans mandshurica* Maxim. [J]. Bulletin of Botanical Research, 2010, 30(1): 22-26.
[14]	LIU Bao-Guang;LI Cheng-Hao;WANG Wei-Da;ZHANG Han-Guo. Induction Technique of Picea koraiensis* Nakai Embryogenic Callus [J]. Bulletin of Botanical Research, 2009, 29(1): 69-73.
[15]	ZHAO Xiao-Min;SHEN Hai-Long;YANG Ling;LIU Chun-Ping. Factors Affecting Induction of embryogenic callus of Larix gmelinii* [J]. Bulletin of Botanical Research, 2007, 27(5): 538-543.