茶树茎段不定芽高效发生体系的建立

doi:10.11983/CBB22184

植物学报 ›› 2023, Vol. 58 ›› Issue (2): 308-315.DOI: 10.11983/CBB22184

茶树茎段不定芽高效发生体系的建立

任露露¹, 张有泽¹, 黄克林¹, 宛晓春¹, 张照亮¹^,^*(), 朱木兰²^,³^,^*(), 韦朝领¹^,^*()

¹安徽农业大学茶树生物学与资源利用国家重点实验室/农业农村部茶树生物学与茶叶加工重点实验室, 合肥 230036
²中国科学院分子植物科学卓越创新中心, 上海 200032
³上海辰山植物园, 上海市资源植物功能基因组学重点实验室, 上海 201602

收稿日期:2022-08-03 接受日期:2022-11-15 出版日期:2023-03-01 发布日期:2023-03-15
通讯作者: ^*E-mail: zhlzhang@ahau.edu.cn;mlzhu@cemps.ac.cn;weichl@ahau.edu.cn
基金资助:
“十三五”国家重点研发项目(2019YFD1001601);安徽省重大科技专项(202003a06020021)

An Efficient System for Regenerating Adventitious Buds in Stem Segments of Tea Plants

Lulu Ren¹, Youze Zhang¹, Kelin Huang¹, Xiaochun Wan¹, Zhaoliang Zhang¹^,^*(), Mulan Zhu²^,³^,^*(), Chaoling Wei¹^,^*()

¹Key Laboratory of Tea Biology and Tea Processing, Ministry of Agriculture and Rural Areas, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China
²Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences, Shanghai 200032, China
³Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai 201602, China

Received:2022-08-03 Accepted:2022-11-15 Online:2023-03-01 Published:2023-03-15
Contact: ^*E-mail: zhlzhang@ahau.edu.cn;mlzhu@cemps.ac.cn;weichl@ahau.edu.cn

摘要/Abstract

摘要： 茶树(Camellia sinensis)是重要的经济作物, 杂合度高且变异度大, 其高效离体再生体系鲜见报道。以舒茶早茎段为起始外植体, 进行不定芽高效发生影响因子研究。结果表明, MS+2 mg∙L^-1 6-BA为定芽诱导的最适配方, 定芽诱导率为84.44%, 吸收底盘膨大率为80%, 利于后续不定芽诱导; MS+2 mg∙L^-1 6-BA+0.2 mg∙L^-1 NAA+0.1 mg∙L^-1 KT+1 mg∙L^-1脯氨酸为不定芽增殖诱导的适宜配方, 不定芽诱导率为88.89%, 平均芽数为7.8。不定根诱导的适宜配方为1/2MS+3 mg∙L^-1 IBA, 生根率为85.56%。采用RAPD和ISSR技术对再生植株进行分子检测, 在连续2代离体再生植株中未发现明显变异。

关键词: 茶树, 离体再生, 不定芽诱导, 遗传稳定性

Abstract: Tea plant is an important economic crop with high heterozygosity and variation. There are few reports on the in vitro regeneration system with high efficiency. In this study, the stem segment of a tea cultivar Shuchazao was used as the initial explants to identify the factors which could efficiently influence the regeneration of adventitious buds. The results showed that MS+2 mg∙L^-1 6-BA was the best formula for normal buds induction, and the induction rate was 84.44%. The expansion rate of the absorption chassis was 80%, which was beneficial to the subsequent induction of adventitious buds. MS+2 mg∙L^-1 6-BA+0.2 mg∙L^-1 NAA+0.1 mg∙L^-1 KT+1 mg∙L^-1 proline was the suitable formula for the induction of adventitious buds, and the induction rate was 88.89%. The average number of shoots was 7.8. 1/2MS+3 mg∙L^-1 IBA was the suitable formula for adventitious root induction, and the rooting rate was 85.56%. The regenerated plants were subjected to molecular verification by RAPD and ISSR techniques. The results showed that no significant variation was found in the two consecutive generations of regenerated plants in vitro.

Key words: Camellia sinensis, in vitro regeneration, adventitious bud induction, genetic stability

任露露, 张有泽, 黄克林, 宛晓春, 张照亮, 朱木兰, 韦朝领. 茶树茎段不定芽高效发生体系的建立. 植物学报, 2023, 58(2): 308-315.

Lulu Ren, Youze Zhang, Kelin Huang, Xiaochun Wan, Zhaoliang Zhang, Mulan Zhu, Chaoling Wei. An Efficient System for Regenerating Adventitious Buds in Stem Segments of Tea Plants. Chinese Bulletin of Botany, 2023, 58(2): 308-315.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22184

https://www.chinbullbotany.com/CN/Y2023/V58/I2/308

图/表 10

图1 茎段不定芽发生体系 (A) 外植体(bar=1 cm); (B) 定芽诱导(bar=1 cm); (C) 不定芽诱导(bar=1 cm); (D) 不定芽伸长(bar=1 cm); (E) 不定根诱导(bar=1.5 cm); (F) 移栽存活的植株(bar=2 cm)

Figure 1 Adventitious budding system of stem segment (A) Explant (bar=1 cm); (B) Normal bud induction (bar=1 cm); (C) Adventitious bud induction (bar=1 cm); (D) Adventitious bud elongation (bar=1 cm); (E) Adventitious root induction (bar=1.5 cm); (F) Transplanted plants (bar=2 cm)

表1 不同基本培养基对茎段诱导的影响

Table 1 Effects of different basic media on stem induction

Basic medium	State of growth
WPM	The bottom callus starts quickly and grows well, the petiole turns red, a small number of normal buds
MS	The bottom callus is thickened, the fixed buds are robust, a small number of normal buds, the foliage is emerald green, and the whole culture is viable
N6	The bottom callus starts quickly, the petiole is reddish, and the leaves are thin
DCR	There is less callus at the bottom and the leaves are thin and yellowish

表2 不同浓度6-BA对茎段定芽诱导的影响

Table 2 Effect of different concentrations of 6-BA on the normal bud induction of stem segments

6-BA (mg?L^-1)	Bud induction rate (%)	Bottom expansion rate (%)	State of growth
0	5.56±1.92 d	1.11±1.92 d	The growth is weak and the base becomes brown
1	51.11±5.09 c	61.11±3.84 c	The growth is better, with fewer buds and calli form at the base
2	77.78±5.09 a	80.00±3.33 b	The growth is strong, with fixed buds at the axillary buds and callus at the base
3	84.44±1.92 a	78.89±6.93 b	The growth is better, with fixed buds at the axillary buds and more callus at the base
4	58.89±3.84 b	88.89±5.09 a	The growth is weak, the axillary buds are yellow, and the chassis is expanded and partially lobed

表4 植物生长调节剂对不定芽伸长的影响

Table 4 Effects of plant growth regulators on adventitious bud elongation

Number	6-BA (mg?L^-1)	NAA (mg?L^-1)	Adventitious bud elongation rate (%)
1	0.2	0.02	24.44±6.94 c
2	0.4	0.04	30.00±3.33 c
3	0.6	0.06	58.89±1.92 b
4	0.8	0.08	70.00±8.82 a
5	1	0.1	51.11±5.09 b

表5 不同浓度IBA和基本培养基对不定根诱导的影响

Table 5 Effects of different concentration of IBA and basal medium on the induction of adventitious root

Number	Medium	IBA (mg?L^-1)	Rooting rate (%)
1	1/2MS	1	38.89±5.88 b
2		2	42.22±4.01 b
3		3	85.56±2.94 a
4		4	46.67±5.77 b
5	MS	1	5.56±2.22 c
6		2	18.89±2.94 c
7		3	8.89±4.01 c
8		4	11.11±4.01 c

表6 随机扩增多态性DNA (RAPD)引物及其扩增谱

Table 6 The random amplification polymorphic DNA (RAPD) primers used in the study and their amplification profile

Primer	Nucleotide sequence (5°-3°)	Number of amplification band	Size range of band (bp)
S29	GGGTAACGCC	8	750-3000
S30	CTGCTGGGAC	12	300-5000
S31	TGTCATCCCC	9	400-3000
S49	CTCACCGTCC	7	500-3000
S132	CAGCTCACGA	8	250-2000
S133	CTCTCCGCCA	9	300-2000
S144	GGAAGTCGCC	6	500-2000
S169	TGGAGAGCAG	4	750-3000
S172	AGAGGGCACA	5	500-2000
S175	TCATCCGAGG	6	500-3000
S181	CTACTGCGCT	9	250-3000
S208	AACGGCGACA	8	300-3000
S221	TGACGCATGG	5	600-2000
S226	ACGCCCAGGT	10	500-3000
S230	GGACCTGCTG	7	500-2000
Total		113

表7 简单重复序列区间(ISSR)引物及其扩增谱

Table 7 The inter-simple sequence repeat (ISSR) primers used in the study and their amplification profile

Primer	Nucleotide sequence (5°-3°)	Number of amplification band	Size range of band (bp)
UBC824	TCTCTCTCTCTCTCTCG	4	500-2000
UBC835	CTCTCTCTCTCTCTCTYC	6	300-1500
UBC840	GAGAGAGAGAGAGAGAYT	2	500-1500
UBC844	CTCTCTCTCTCTCTCTRC	5	500-1500
UBC853	TCTCTCTCTCTCTCTCRT	5	600-1500
UBC854	TCTCTCTCTCTCTCTCRG	6	500-2000
UBC856	ACACACACACACACACYA	5	500-1500
UBC859	TGTGTGTGTGTGTGTGRC	4	500-3000
UBC873	GACAGACAGACAGACA	5	750-3000
UBC880	GGAGAGGAGAGGAGA	6	500-2000
Total		48

图2 舒茶早再生植株的随机扩增多态性DNA (RAPD)扩增图谱 (A) 引物S132; (B) 引物S133。1-18: 同一茶树茎段的离体再生植株群; 1-6: 舒茶早一代再生植株; 7-12: 舒茶早二代再生植株; 13-18: 舒茶早三代再生植株; CK: 母本材料

Figure 2 Random amplification polymorphic DNA (RAPD) banding profiles of regenerated plants of Shuchazao (A) Primer S132; (B) Primer S133. 1-18: In vitro regenerated plant population of the same tea plant stem segment; 1-6: First generation regenerated plants of Shuchazao; 7-12: Second generation regenerated plants of Shuchazao; 13-18: Third generation regenerated plants of Shuchazao; CK: Mother plant

图3 舒茶早再生植株的简单重复序列区间(ISSR)扩增图谱 (A) 引物UBC854; (B) 引物UBC880。1-18: 同一茶树茎段的离体再生植株群; 1-6: 舒茶早一代再生植株; 7-12: 舒茶早二代再生植株; 13-18: 舒茶早三代再生植株; CK: 母本材料

Figure 3 Inter-simple sequence repeat (ISSR) banding pro- files of regenerated plants of Shuchazao (A) Primer UBC854; (B) Primer UBC880. 1-18: In vitro regenerated plant population of the same tea plant stem segment; 1-6: First generation regenerated plants of Shuchazao; 7-12: Second generation regenerated plants of Shuchazao; 13-18: Third generation regenerated plants of Shuchazao; CK: Mother plant

表3 植物生长调节剂对不定芽增殖诱导的影响

Table 3 Effects of plant growth regulators on the induction of adventitious bud proliferation

Number	6-BA (mg?L^-1)	NAA (mg?L^-1)	Adventitious bud induction rate (%)	Average number of shoots
1	0	0	16.67±5.78 d	0.33±0.15 d
2	1	0.1	54.44±5.09 c	1.33±0.08 c
3	2	0.2	88.89±6.93 a	7.88±0.23 a
4	3	0.3	71.11±6.93 b	3.48±0.25 b

参考文献 21

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茶树茎段不定芽高效发生体系的建立

An Efficient System for Regenerating Adventitious Buds in Stem Segments of Tea Plants

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