手指柠檬茎段离体再生体系建立

doi:10.11983/CBB23060

摘要/Abstract

摘要： 以手指柠檬(Citrus australasica)茎段为外植体, 通过离体器官发生途径诱导形成不定芽, 探讨不同植物生长调节剂组合、培养基类型以及暗培养时间对其愈伤组织诱导和植株再生的影响, 建立手指柠檬离体再生体系。结果表明, 1/2MS+4.0 mg∙L^-1 ZT+30.0 g∙L^-1蔗糖为手指柠檬茎段不定芽诱导的最佳配方, 14天暗培养后转移到光下培养效果最好, 愈伤组织及不定芽诱导率均为100%, 每外植体平均再生不定芽数达4.83。诱导不定根的适宜培养基配方为1/2MS+0.5 mg∙L^-1NAA, 生根率达94.43%, 平均再生根数为3.9; 在草炭:珍珠岩:蛭石=2:1:1 (v/v/v)的混合基质中组培苗长势最好, 成活率在90%以上。该研究建立了手指柠檬茎段离体再生体系, 为手指柠檬的遗传改良和优良品种快繁奠定了基础。

关键词: 手指柠檬, 茎段, 组织培养, 离体再生

Abstract: In order to establish the regeneration system of Citrus australasica, the effects of different plant growth regulators combinations, medium types and dark culture time on callus induction and plant regeneration of C. australasica were studied using stem segments as explants. The results showed that the best medium for adventitious bud induction was 1/2MS+4.0 mg∙L^-1 ZT+30.0 g∙L^-1 sucrose. Dark culture for 14 days and then light culture had the best promoting effect. The induction rate of callus and adventitious bud was 100%, and the average number of adventitious bud regeneration per explants was 4.83. The optimal rooting medium was 1/2MS+0.5 mg∙L^-1 NAA, and the regenerated plants with the 94.43% rooting rate were obtained, and the average number of roots was 3.9. In the mixture of grass carbon:perlite: vermiculite=2:1:1 (v/v/v), tissue culture seedlings had the best growth, and the survival rate was more than 90%. This study established in vitro regeneration system of C. australasica, which laid the foundation for the genetic improvement and rapid propagation of C. australasica fine varieties.

Key words: Citrus australasica, stem segment, tissue culture, in vitro regeneration

谢纯刚, 刘哲, 章书声, 胡海涛. 手指柠檬茎段离体再生体系建立. 植物学报, 2023, 58(6): 926-934.

Chungang Xie, Zhe Liu, Shusheng Zhang, Haitao Hu. Establishment of In Vitro Regeneration System of Citrus australasica. Chinese Bulletin of Botany, 2023, 58(6): 926-934.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB23060

https://www.chinbullbotany.com/CN/Y2023/V58/I6/926

图/表 8

表1 不同浓度NAA与6-BA配比对手指柠檬茎段愈伤组织诱导及不定芽增殖的影响

Table 1 Effects of NAA and 6-BA ratio at different concentrations on Citrus australasica callus induction and adventitious bud proliferation

No.	NAA (mg?L^-1)	6-BA (mg?L^-1)	3 weeks	5 weeks		Growth condition
No.	NAA (mg?L^-1)	6-BA (mg?L^-1)	Callus induction rate (%)	Rate of adventitious bud differentiation (%)	Frequency of bud differentiation	Growth condition
1	0	0	61.11	55.57±5.57 cde	1.57±0.29 bc	++
2	0	0.5	100.00	72.23±5.53 efg	2.08±0.08 de	++++
3	0	1.0	100.00	94.43±5.57 g	2.53±0.03 e	++++
4	0	2.0	91.67	55.57±5.57 cde	1.19±0.10 ab	++
5	0	4.0	58.33	27.73±5.57 abc	1.17±0.17 ab	+
6	0.1	0.5	58.33	61.13±5.57 def	2.36±0.07 de	+++
7	0.1	1.0	66.67	44.43±5.57 bcde	1.89±0.11 bce	++
8	0.1	2.0	75.00	44.47±11.17 bcde	2.00±0.29 bce	++
9	0.1	4.0	41.67	16.70±0.00 a	1.17±0.29 ab	+
10	0.5	0.5	66.67	55.57±5.57 cde	2.33±0.19 de	+++
11	0.5	1.0	91.67	66.67±9.61 ef	2.57±0.23 e	+++
12	0.5	2.0	33.33	33.33±9.61 abcd	1.61±0.20 bc	++
13	0.5	4.0	33.33	22.23±5.53 ab	1.17±0.17 ab	+

图1 手指柠檬愈伤组织诱导及不定芽分化 (A)-(D) 手指柠檬茎段在添加6-BA的1/2MS培养基上的离体再生过程 (A) 手指柠檬茎段外植体; (B) 培养3周后, 茎段诱导出愈伤组织; (C) 培养4周后, 愈伤组织分化出不定芽; (D) 培养5周后, 诱导出大量不定芽; (E)-(H) 手指柠檬茎段在添加ZT的1/2MS培养基上的离体再生过程 (E) 手指柠檬茎段外植体; (F) 培养3周后, 茎段分化出愈伤组织; (G) 培养4周后, 愈伤组织分化出不定芽; (H) 培养5周后, 诱导出大量不定芽。Bars=0.5 cm

Figure 1 Callus induction and adventitious bud differentiation of Citrus australasica (A)-(D) In vitro regeneration process of C. australasica on 1/2MS medium with 6-BA (A) Stem explant of C. australasica; (B) The stem segments differentiated into callus after 3 weeks; (C) Adventitious buds were induced from the callus after 4 weeks; (D) Adventitious buds were induced after 5 weeks; (E)-(H) In vitro regeneration process of C. australasica on 1/2MS medium with ZT (E) Stem explant of C. australasica; (F) The stem segments differentiated into callus after 3 weeks; (G) Adventitious buds were induced from the callus after 4 weeks; (H) Adventitious buds were induced after 5 weeks. Bars=0.5 cm

表2 不同浓度NAA与ZT配比对手指柠檬茎段愈伤组织诱导及不定芽增殖的影响

Table 2 Effects of NAA and ZT ratio at different concentrations on Citrus australasica stem callus induction and adventitious bud proliferation

No.	NAA (mg?L^-1)	ZT (mg?L^-1)	3 weeks	5 weeks		Growth condition
No.	NAA (mg?L^-1)	ZT (mg?L^-1)	Callus induction rate (%)	Rate of adventitious bud differentiation (%)	Frequency of bud differentiation	Growth condition
1	0	0	61.11	55.57±5.57 b	1.56±0.29 a	++
2	0	0.5	77.80	77.76±5.53 c	3.53±0.42 cdef	+++
3	0	1.0	83.30	88.87±5.57 c	3.63±0.75 cdef	++++
4	0	2.0	100.00	100.00±0.00 c	3.94±0.92 def	++++
5	0	3.0	100.00	100.00±0.00 c	3.94±0.53 defg	+++
6	0	4.0	100.00	100.00±0.00 c	4.43±0.50 fg	++++
7	0	5.0	100.00	100.00±0.00 c	4.24±0.20 fg	+++
8	0	6.0	83.33	77.77±5.53 c	3.19±0.19 bcdef	+++
9	0.1	0.5	88.90	77.76±5.53 c	2.50±0.30 abcde	++
10	0.1	1.0	100.00	100.00±0.00 c	3.55±0.48 cdef	++++
11	0.1	2.0	100.00	100.00±0.00 c	3.28±0.79 cdef	+++
12	0.1	3.0	100.00	100.00±0.00 c	3.28±0.46 bcdef	+++
13	0.1	4.0	94.44	94.43±5.57 c	4.21±0.45 efg	++++
14	0.1	5.0	94.44	88.87±5.57 c	3.42±0.14 cdef	+++
15	0.1	6.0	66.67	61.10±5.55 b	2.69±0.26 abd	+++
16	0.5	0.5	100.00	72.20±11.10 bc	1.49±0.16 ab	++
17	0.5	1.0	85.70	33.33±8.33 a	1.50±0.50 ab	++
18	0.5	2.0	88.90	50.00±9.64 ab	1.83±0.29 abc	++
19	0.5	3.0	88.89	50.00±9.64 ab	1.83±0.17 ab	++
20	0.5	4.0	83.33	88.87±5.57 c	2.86±0.17 abcde	+++
21	0.5	5.0	50.00	44.43±5.57 ab	2.27±0.37 abc	++
22	0.5	6.0	33.33	27.77±5.53 a	2.11±0.11 abc	+

表3 不同基本培养基对手指柠檬茎段愈伤组织诱导及不定芽增殖的影响

Table 3 Effects of different basic media on callus induction and adventitious bud proliferation of Citrus australasica

No.	Different media	3 weeks	5 weeks		Growth condition
No.	Different media	Callus induction rate (%)	Rate of adventitious bud differentiation (%)	Frequency of bud differentiation	Growth condition
1	1/2MS	100.00	100.00±0.00 a	4.57±0.33 a	++++
2	MT	83.30	61.13±5.57 b	3.57±0.24 b	+++
3	MS	100.00	88.87±5.57 ab	3.64±0.38 b	+++
4	WPM	83.30	72.23±5.53 bc	2.83±0.17 bc	+++
5	White	66.70	33.33±0.00 e	1.56±0.29 c	+

表4 暗培养处理对手指柠檬茎段愈伤组织诱导及不定芽增殖的影响

Table 4 Effects of dark treatment on callus induction and adventitious bud proliferation of Citrus australasica

No.	Dark treatment (day)	3 weeks	5 weeks		Growth condition
No.	Dark treatment (day)	Callus induction rate (%)	Rate of adventitious bud differentiation (%)	Frequency of bud differentiation	Growth condition
1	0	55.57±5.57 a	44.43±5.57 a	2.28±0.15 a	++
2	7	88.87±5.57 b	83.33±9.61 bc	3.50±0.21 b	+++
3	14	100.00±0.00 b	100.00±0.00 c	4.83±0.38 c	++++
4	21	94.43±5.57 b	66.70±0.00 b	2.75±0.38 a	++
5	28	100.00±0.00 b	38.87±5.57 a	2.33±0.33 a	++

图2 手指柠檬茎段在优化后的培养基上不定芽再生状况手指柠檬茎段在配方为1/2MS+5 mg?L-1 ZT+30 g?L-1蔗糖的优化培养基上暗处理14天, 光下培养5周(A)和8周(B)离体不定芽的再生情况。ZT: 玉米素。Bars=1 cm

Figure 2 Adventitious buds from stem explants of Citrus australasica on optimized medium After 14 days dark treatment, adventitious buds from stem explants of C. australasica on 1/2MS+5 mg?L-1 ZT+30 g?L-1 sucrose, 5 weeks (A) and 8 weeks (B) in the light. ZT: Zeatin. Bars=1 cm

表5 不同浓度NAA对手指柠檬不定芽生根的影响

Table 5 Effects of NAA concentration on rooting induction of Citrus australasica adventitious buds

No.	NAA concentration (mg?L^-1)	Rooting rate (%)	Average rooting number	Growth condition
1	0	16.70±0.00 d	0.97±0.21 d	+
2	0.05	38.87±9.64 c	2.02±0.24 c	++
3	0.1	44.43±9.64 c	2.07±0.15 c	++
4	0.3	77.77±9.58 b	3.03±0.13 b	++
5	0.5	94.43±9.64 a	3.90±0.20 a	++++
6	1	61.13±9.64 c	2.35±0.40 c	+++

图3 手指柠檬再生植株的生根与移栽 (A), (B) 不定根诱导; (C) 再生植株; (D) 移栽成活的植株。Bars=1 cm

Figure 3 Rooting and transplanting of Citrus australasica regenerated plants (A), (B) Adventitious root induction; (C) Regenerated plants; (D) Transplant survived plants. Bars=1 cm

参考文献 23

[1]	常贝贝, 屈宜宝, 王智宇, 程晓帆, 杜晓云, 于晓丽, 赵玲玲, 张硕 (2023). 2个苹果新品种高效再生体系建立. 分子植物育种. https://kns.cnki.net/kcms/detail/46.1068.S.20220722.1647.008.html.
[2]	范永梅, 甘霖, 邓秀新 (2003). 冰糖橙胚性愈伤组织的诱导与植株再生. 华中农业大学学报 22, 399-402.
[3]	林颖, 龙自立, 张璐, 叶庆富, 刘永立 (2012). 猕猴桃胚乳再生植株体系的优化. 核农学报 26, 257-261, 310. DOI
[4]	逯锦春, 曹丽娜, 佟冠杰, 王鑫颖, 张利英, 喻锌, 李荟芳, 李彦慧 (2022). 大花银莲花愈伤组织诱导及再生体系的建立. 植物学报 57, 217-226. DOI
[5]	任露露, 张有泽, 黄克林, 宛晓春, 张照亮, 朱木兰, 韦朝领 (2023). 茶树茎段不定芽高效发生体系的建立. 植物学报 58, 308-315. DOI
[6]	魏佳, 路天宇, 王朝胜, 张红叶, 张晶, 王顺利, 李润芝 (2022). 不同粒级园艺基质原料物理性质差异分析. 北京农学院学报 37(4), 13-18.
[7]	杨圣涛, 吕岩, 贺元源, 刘婷婷, 马晓祯 (2021). 基于CT扫描的草炭土孔隙结构分析及渗流模拟. 工程地质学报 29, 1354-1365.
[8]	张郎郎, 张洁, 吕虹霖, 谭彬, 王伟, 程钧, 冯建灿 (2022). 欧洲李叶片再生体系的建立. 果树学报 39, 1945-1953.
[9]	张秀英, 鲁兴凯, 程安富, 胡志芳, 马勉娣, 张丹, 黄国嫣, 陈晨, 全勇, 汪琼 (2022). 基质对苹果砧木M26脱毒组培苗移栽成活率和生长的影响. 中国南方果树 51(5), 150-153.
[10]	张旭红, 王頔, 梁振旭, 孙美玉, 张金政, 石雷 (2018). 欧洲百合愈伤组织诱导及植株再生体系的建立. 植物学报 53, 840-847. DOI
[11]	Bernula D, Benkő P, Kaszler N, Domonkos I, Valkai I, Szőllősi R, Ferenc G, Ayaydin F, Fehér A, Gémes K (2020). Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots. Plant Cell Tissue Organ Cult 140, 327-339. DOI
[12]	Cioni E, Migone C, Ascrizzi R, Muscatello B, De Leo M, Piras AM, Zambito Y, Flamini G, Pistelli L (2022). Comparing metabolomic and essential oil fingerprints of Citrus australasica F. Muell (Finger Lime) varieties and their in vitro antioxidant activity. Antioxidants (Basel) 11, 2047. DOI URL
[13]	Conti G, Xoconostle-Cázares B, Marcelino-Pérez G, Hopp HE, Reyes CA (2021). Citrus genetic transformation: an overview of the current strategies and insights on the new emerging technologies. Front Plant Sci 12, 768197. DOI URL
[14]	Dai WH, Castillo C (2007). Factors affecting plant regeneration from leaf tissues of buddleia species. HortScience 42, 1670-1673. DOI URL
[15]	Huang T, Peng SL, Dong GF, Zhang LY, Li GG (2002). Plant regeneration from leaf-derived callus in Citrus grandis (pummelo): effects of auxins in callus induction medium. Plant Cell Tissue Organ Cult 69, 141-146. DOI URL
[16]	Jardak R, Boubakri H, Zemni H, Gandoura S, Mejri S, Mliki A, Ghorbel A (2020). Establishment of an in vitro regeneration system and genetic transformation of the Tunisian ‘Maltese half-blood’ (Citrus sinensis): an agro- economically important variety. 3 Biotech 10, 99. DOI PMID
[17]	Long Y, Yang Y, Pan GT, Shen YO (2022). New insights into tissue culture plant-regeneration mechanisms. Front Plant Sci 13, 926752. DOI URL
[18]	Loyola-Vargas VM, Ochoa-Alejo N (2018). An introduction to plant tissue culture:advances and perspectives. In: Loyola-Vargas VM, Ochoa-Alejo N, eds. Plant Cell Culture Protocols. New York: Humana Press. pp. 3-13.
[19]	Peña L, Pérez RM, Cervera M, Juárez JA, Navarro L (2004). Early events in Agrobacterium-mediated genetic transformation of citrus explants. Ann Bot 94, 67-74. DOI URL
[20]	Poles L, Licciardello C, Distefano G, Nicolosi E, Gentile A, La Malfa S (2020). Recent advances of in vitro culture for the application of new breeding techniques in citrus. Plants (Basel) 9, 938. DOI URL
[21]	Raspor M, Motyka V, Kaleri AR, Ninković S, Tubić L, Cingel A, Ćosić T (2021). Integrating the roles for cytokinin and auxin in de novo shoot organogenesis: from hormone uptake to signaling outputs. Int J Mol Sci 22, 8554. DOI URL
[22]	Skoog F, Miller CO (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11, 118-130.
[23]	Zenser N, Ellsmore A, Leasure C, Callis J (2001). Auxin modulates the degradation rate of Aux/IAA proteins. Proc Natl Acad Sci USA 98, 11795-11800. DOI PMID