Rapid Micropropagation of Fraxinus mandshurica Rupr. Based on Liquid and Solid Alternate Culture

doi:10.7525/j.issn.1673-5102.2022.02.003

Abstract

Abstract:

A rapid and efficient regeneration system of liquid-solid alternate culture was developed for the tissue culture seedlings of Fraxinus mandshurica with lignificated stalk， aged leaves and dormant apical buds， and the axillary buds were germinated in a short time by liquid suspension culture， and the axillary buds were regenerated in vitro in solid culture to obtain the new tissue culture seedlings. On liquid and solid media supplemented of WPM with different plant growth regulators， axillary buds of F. mandshurica were induced to germinate and grow into seedlings. It was found that the axillary bud germination rate of F. mandshurica could reach 100% within 7-15 d after dark culture in WPM liquid medium supplemented with 0.6 mg·L^-1 TDZ. After cutting the sprouted buds and inoculating them into WPM solid medium of 0.05 mg·L^-1 TDZ and 0.6 mg·L^-1 BA， axillary buds could grow into shoots after 1-2 subcultures with an average height of 2.64 cm and a multiplication coefficient of 4.04. After transplanting， the survival rate of rooting seedlings was 90% after 50 d. The establishment of the method could be conducive to the large-scale propagation of F. mandshurica， and the liquid-solid alternate circulation culture， simple， controllable， easy to operate， suitable for different production conditions and reduces the cost.

Key words: Fraxinus mandshurica Rupr., dormant bud, tissue culture, rapid reproduction

CLC Number:

Q949.776.2

Fenghui Qi, Wenxuan Wang, Lin Liu, Mingshuo Tang, Feixiang Song, Yaguang Zhan. Rapid Micropropagation of Fraxinus mandshurica Rupr. Based on Liquid and Solid Alternate Culture[J]. Bulletin of Botanical Research, 2022, 42(2): 184-190.

Figures/Tables 10

Table 1

Fig.1

Table 2

Table 3

Fig.2

Table 4

Table 5

Fig.3

Fig.4

Fig.5

References 22

1	张丽杰，张丽玮，冯丹丹，等.水曲柳腋芽离体快繁研究初报［J］.植物研究，2007，27（3）：319-324.
	Zhang L J，Zhang L W，Feng D D，et al.In vitro culture of axillary bud sprouting of Fraxinus mandshurica［J］.Bulletin of Botanical Research，2007，27（3）：319-324.
2	李莉.我国水曲柳遗传育种研究进展［J］.辽宁林业科技，2014（2）：52-54.
	Li L.Research Progress on genetics and breeding of Fraxinus mandshurica in China［J］.Liaoning Forestry Science And Technology，2014（2）：52-54.
3	Kong D M，Shen H L，Li N.Influence of AgNO₃ on somatic embryo induction and development in Manchurian ash（Fraxinus mandshurica Rupr.）［J］.African Journal of Biotechnology，2012，11（1）：120-125.
4	Zhu Z，Qi F H，Yan C F，et al.Sexually different morphological，physiological and molecular responses of Fraxinus mandshurica flowers to floral development and chilling stress［J］.Plant Physiology and Biochemistry，2016，99：97-107.
5	沈海龙，赵霞，邢朝斌，等.水曲柳扦插繁殖影响因子的分析［J］.东北林业大学学报，2005，33（3）：5-6.
	Shen H L，Zhao X，Xing Z B，et al.Influencing factors of vegetative propagation of Fraxinus mandshurica by rooted cuttings［J］.Journal of Northeast Forestry University，2005，33（3）：5-6.
6	Bhardwaj D R，Mishra V K.Vegetative propagation of Ulmus villosa：effects of plant growth regulators，collection time，type of donor and position of shoot on adventitious root formation in stem cuttings［J］.New Forests，2005，29（2）：105-116.
7	Snedden J，Landhäusser S M，Lieffers V J，et al.Propagating trembling aspen from root cuttings：impact of storage length and phenological period of root donor plants［J］.New Forests，2010，39（2）：169-182.
8	Yang L，Bian L，Shen H L，et al.Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of manchurian ash（Fraxinus mandshurica Rupr.）［J］.Plant Cell，Tissue and Organ Culture，2013，115（2）：115-125.
9	Wickson M，Thimann K V.The antagonism of auxin and kinetin in apical dominance［J］.Physiologia Plantarum，1958，11（1）：62-74.
10	Beveridge C A.Axillary bud outgrowth：sending a message［J］.Current Opinion in Plant Biology，2006，9（1）：35-40.
11	Ferguson B J，Beveridge C A.Roles for auxin，cytokinin，and strigolactone in regulating shoot branching［J］.Plant Physiology，2009，149（4）：1929-1944.
12	Braun N，De Saint Germain A，Pillot J P，et al.The pea TCP transcription factor PsBRC1 acts downstream of strigolactones to control shoot branching［J］.Plant Physiology，2012，158（1）：225-238.
13	Tan M，Li G F，Chen X L，et al.Role of cytokinin，strigolactone，and auxin export on outgrowth of axillary buds in apple［J］.Frontuers in Plant Science，2019，10：616.
14	Barbier F，Péron T，Lecerf M，et al.Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida［J］.Journal of Experimental Botany，2015，66（9）：2569-2582.
15	Waldie T，Leyser O.Cytokinin targets auxin transport to promote shoot branching［J］.Plant Physiology，2018，177（2）：803-818.
16	Müller D，AuxinLeyser O.，cytokinin and the control of shoot branching［J］.Annals of Botany，2011，107（7）：1203-1212.
17	Guo B，Abbasi B H，Zeb A，et al.Thidiazuron：a multi-dimensional plant growth regulator［J］.African Journal of Biotechnology，2011，10（45）：8984-9000.
18	Tan M，Li G F，Qi S Y，et al.Identification and expression analysis of the IPT and CKX gene families during axillary bud outgrowth in apple（Malus domestica Borkh.）［J］.Gene，2018，651：106-117.
19	Balla J，Kalousek P，Reinöhl V，et al.Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth［J］.The Plant Journal，2011，65（4）：571-577.
20	Rathore M S，Mastan S G，Yadav P，et al.Shoot regeneration from leaf explants of Withania coagulans（Stocks） Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers［J］.South African Journal of Botany，2016，102：12-17.
21	Beasley R R，Pijut P M.Regeneration of plants from Fraxinus nigra Marsh．Hypocotyls［J］.HortScience，2013，48（7）：887-890.
22	Lee J H，Pijut P M.Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra［J］.Plant Cell，Tissue and Organ Culture，2017，130（2）：335-343.

培养基编号 Culture medium number	TDZ /（mg·L^-1）	芽总数量 The total number of shoots	7 d芽数量 7 d number of buds	7 d芽萌发率 7 d bud germination rate /%	24 d芽数量 24 d number of buds	24 d芽萌发率 24 d bud germination rate /%
WY1	0.4	129	30	23.25±3.72	41	31.78±4.10
WY2	0.6	106	41	38.68±4.73	51	48.11±4.85
WY3	0.8	96	41	45.83±5.09	51	53.13±5.09
CK	0	0	0	0	0	0

TDZ /（mg·L^-1）	光培养Light train			平均芽长度 Average length of bud /cm	暗培养Dark culture
TDZ /（mg·L^-1）	芽点总数量 Total number of shoots	萌发芽数量 Number of buds	萌芽率 Bud germination rate /%	平均芽长度 Average length of bud /cm	芽点总数量 Total number of shoots	萌发芽数量 Number of buds	萌发率 Bud germination rate /%	平均芽长度 Average length of bud /cm
0.4	63	20	31.75±5.86	0.68	66	21	31.82±5.73	0.56
0.6	54	26	48.15±6.80	0.63	52	25	48.08±6.93	0.67
0.8	47	20	42.55±7.21	0.98	49	28	57.14±7.70	0.50

培养基编号 Medium numbe	初始平均芽长度 Initial mean bud length /cm	平均幼苗高度 Mean seedling height /cm	平均伸长倍数 Average elongation	死亡率 Mortality rate /%	生长状态 The growth state
Z1	0.84	1.56	0.72	0	叶绿，茎较为健壮，少许褐化现象，几乎无玻璃化 The leaves are green，the stems are strong，a little browning，almost no vitrification
Z2	0.97	1.58	0.61	20	褐化明显，部分死亡，叶呈黄绿色，有玻璃化现象 The plants are significantly browning with vitrification and partially dead with yellowish green leaves
Z3	1.15	1.26	0.11	10	整体玻璃化和褐化现象明显，叶大部分呈现黄绿色，有愈伤化现象 The plants show obvious overall vitrification and browning，and most of the leaves appeare yellowish green with callus formation
Z4	1.10	1.89	0.79	30	褐化严重，玻璃化严重，未褐化的叶呈绿色 The plants are severely browning，vitrified，and nonbrowning leaves appear green
Z5	1.05	0.81	-0.24	60	过半数褐化死亡，还有一些褐化严重，未褐化的植株完全玻璃化 Over half of the plants died by browning，and still others are severely browning and the non browning plants are completely vitrified

[1]	Xu HE, Yuan GAO, Qunye ZHANG, Chenguang ZHOU, Wei LI, Shuang LI. Establishment and Application of Genetic Transformation System for Populus simonii×P. nigra ‘Baicheng’ [J]. Bulletin of Botanical Research, 2023, 43(5): 667-678.
[2]	Shi-Jun ZHONG, Bi-Dong ZHANG, Shi-Ting LAI, Hua-Min LIU. Tissue Culture and Rapid Propagation System of Gentiana rhodantha [J]. Bulletin of Botanical Research, 2021, 41(5): 753-759.
[3]	Sen SHI, Na MIAO, Yu-Tong SHI, Hui-Mei WANG. Effects of Sucrose on Growth and Physiological Characteristics in vitro Plantlets of 84K Poplar [J]. Bulletin of Botanical Research, 2021, 41(1): 74-78.
[4]	YUAN Yun-Xiang. Callus induction and Plant Regeneration of Euonymus microcarpus(Oliv.) Spraggue [J]. Bulletin of Botanical Research, 2020, 40(5): 673-678.
[5]	XIAO Zu-Fei, WANG Ling-Ling, CAO Lu-Yao, LIAO Wen-Xuan, JIN Zhi-Nong, LI Feng, Lü Xiong-Wei, ZHANG Bei-Hong, ZHAO Jiao. Tissue Culture Technology of Stem Segment of Cinnamomum bodinieri var. citralifera [J]. Bulletin of Botanical Research, 2020, 40(2): 196-201.
[6]	ZHANG Li, ZHANG Lei, HOU Dan, ZHANG Su-Fang, ZHANG Han-Guo. Establishment and Optimization of plant regeneration of Hybrid larch(L.gmelinii 10×L.kaempferi 13) [J]. Bulletin of Botanical Research, 2016, 36(3): 434-443.
[7]	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.
[8]	DU Zhao-Wei；ZHENG Tang-Chun；LI Shuang；ZANG Li-Na；QU Guan-Zheng；YOU Xiang-Ling. Rapid Propagation and Regeneration System of Populus simonii×Populus nigra* [J]. Bulletin of Botanical Research, 2015, 35(6): 904-907.
[9]	MA Yang-Yang1；LUO Yan-Jun1；LI Ya-Juan1；YUE Ding-Yi1；FENG Jia-Yue1,2. Tissue Culture System of Muscari botryoides* Mill Leaf and Flower Organs [J]. Bulletin of Botanical Research, 2015, 35(5): 679-683.
[10]	HU Feng1,2；SHI Qiong1；HUANG Lie-Jian1. Tissue Culture System of Axillary Proliferation of Acacia crassicarpa* [J]. Bulletin of Botanical Research, 2015, 35(2): 179-184.
[11]	ZHANG Jian-Ying；QI Yong-Hui；Lü Yue-Dong；LIU Jian-Ming；XING Ya-Juan. Regeneration System for Axillary Bud of Juglans mandshurica* Maxim. [J]. Bulletin of Botanical Research, 2015, 35(1): 22-26.
[12]	QU Wen-Jing;LI Qing*;LIU Yan. Contamination Control of Underground Buds in Peony Tissue Culture [J]. Bulletin of Botanical Research, 2014, 34(4): 524-528.
[13]	CHEN Suo-Liang;LV Xiu-Li;CUI Xin-Hong;WANG Cheng;ZHANG Qun;ZHU Yi;LU Liang. Tissue Culture of Gunnera manlcata* L. [J]. Bulletin of Botanical Research, 2014, 34(3): 328-332.
[14]	WANG Na;DOU Kai;WANG Zhi-Ying;ZHANG Rong-Shu;WANG Yu-Cheng;LIU Zhi-Hua. Rooting and Transplanting Method of Populus davidiana×P.bolleana* Tissue Culture Seedlings [J]. Bulletin of Botanical Research, 2014, 34(3): 380-385.
[15]	WU Bai-Yan;XIE Yan-Mei;WANG Zhen-Yue. Tissue Culture and Plant Regeneration of Rumex gmelini* Turcz. [J]. Bulletin of Botanical Research, 2012, 32(6): 685-688.