苹果砧木B9再生体系的建立与优化

doi:10.16420/j.issn.0513-353x.2024-0707

摘要/Abstract

摘要：

为了建立高效的苹果砧木再生体系，以B9组培苗离体叶片为外植体，对不同浓度的TDZ/NAA植物激素组合、暗培养时间、苗龄以及潮霉素浓度等影响再生的重要因素进行筛选。结果表明：不定芽诱导培养基为MS + 3.0 mg · L^-1 TDZ + 0.3 mg · L^-1 NAA + 蔗糖30 g · L^-1 + 琼脂7 g · L^-1，苗龄45 d，暗培养14 d时不定芽再生效率最高；增殖培养基为MS + 0.8 mg · L^-1 6-BA + 0.2 mg · L^-1 IAA + 蔗糖30 g · L^-1 + 琼脂7 g · L^-1时，不定芽增殖效果最好；生根培养基为1/2MS + 0.6 mg · L^-1 IBA + 蔗糖30 g · L^-1 + 琼脂7 g · L^-1时，组培苗生根率和平均生根数最高。叶片再生时潮霉素筛选浓度为2 ~ 4 mg · L^-1，生根时潮霉素筛选浓度为2 ~ 3 mg · L^-1。

关键词: 苹果, 砧木, 再生, 植物激素, 不定芽

Abstract:

To establish an efficient regeneration system for apple rootstock，this study utilized detached in vitro leaves from tissue-cultured seedlings of apple rootstock B9 as explants. This study screened key factors influencing regeneration，including various concentrations of phytohormone combinations，durations of dark culture，seedling ages，and hygromycin concentration. The findings revealed that the optimal medium for inducing indefinite buds was MS + 3.0 mg · L^-1 TDZ + 0.3 mg · L^-1 NAA + 30 g · L^-1 sucrose + 7 g · L^-1 agar，with a seedling age of 45 days and a dark culture period of 14 days，leading to the highest regeneration efficiency. The proliferation medium consisted of MS + 0.8 mg · L^-1 6-BA + 0.2 mg · L^-1 IAA + 30 g·L^-1 sucrose + 7 g · L^-1 agar；The rooting medium was 1/2 MS + 0.6 mg · L^-1 IBA + 30 g · L^-1 sucrose + 7 g · L^-1 agar. The appropriate hygromycin concentration for leaf regeneration was 2-4 mg · L^-1，while for rooting，it was 2-3 mg · L^-1.

Key words: apple, rootstock, regeneration, phytohormone, indefinite buds

田玉凤, 马松亚, 杨安, 韩晓蕾, 张彩霞. 苹果砧木B9再生体系的建立与优化[J]. 园艺学报, 2025, 52(4): 947-958.

TIAN Yufeng, MA Songya, YANG An, HAN Xiaolei, ZHANG Caixia. Establishment and Optimization of Regeneration System for Apple Rootstock B9[J]. Acta Horticulturae Sinica, 2025, 52(4): 947-958.

图/表 12

图1 TDZ和NAA与浓度对叶片再生芽的影响以JM为基础培养基。下同

Fig. 1 The effect of TDZ and NAAand concentrations on leaf regeneration buds JM was used as the basic medium. The same below

表1 植物生长调节剂种类与浓度对叶片再生芽的影响

Table 1 Effect of type and concentration of plant growth regulators on leaf regeneration buds

TDZ/（mg · L^-1）		再生效率/% Regeneration efficiency	平均再生芽数/个 Average number of regenerated buds
1	0.1	0.83 ± 0.42 e	1.67 ± 0.88 b
1	0.3	22.08 ± 1.10 d	1.61 ± 0.07 b
1	0.5	16.67 ± 2.20 d	1.30 ± 0.03 b
2	0.1	26.25 ± 1.44 d	1.84 ± 0.02 b
2	0.3	44.58 ± 4.41 c	2.10 ± 0.10 ab
2	0.5	52.08 ± 4.70 bc	2.20 ± 0.04 ab
3	0.1	63.33 ± 6.86 bc	2.20 ± 0.11 ab
3	0.3	80.42 ± 7.95 a	2.93 ± 0.13 a
3	0.5	45.83 ± 4.23 c	1.94 ± 0.05 b

图2 暗培养时间对叶片再生芽的影响

Fig. 2 The effect of dark cultivation time on leaf regeneration buds

表2 暗培养时间对叶片再生芽的影响

Table 2 Effects of dark cultivation time on leaf regeneration buds

暗培养时间/d Dark incubation time	调查个数 Number of investigations	再生效率/% Regeneration efficiency	平均再生芽数/个 Average number of regenerated buds
7	80	37.50 ± 4.73 b	1.91 ± 0.16 a
14	80	66.25 ± 5.77 a	2.38 ± 0.07 a
21	80	48.33 ± 6.05 ab	2.36 ± 0.11 a

图3 苗龄对叶片再生芽的影响

Fig. 3 The effect of seedling age on leaf regeneration buds

表3 苗龄对叶片再生芽的影响

Table 3 Effect of seedling age on leaf regeneration buds

苗龄/d Seedling age	数量 Number	再生效率/% Regeneration efficiency	平均再生芽数/个 Average number of regenerated buds
35	80	35.00 ± 3.31 b	1.80 ± 0.58 a
45	80	69.58 ± 3.97 a	2.58 ± 0.17 a
55	80	40.42 ± 4.10 b	1.92 ± 0.22 a

图4 不同激素浓度对不定芽增殖的影响

Fig. 4 The effect of different hormone concentrations on the proliferation of adventitious buds

表4 不同植物生长调节剂浓度组合对不定芽增殖的影响

Table 4 Effects of various plant growth regulators concentration combinations on the proliferation of adventitious buds

6-BA/（mg · L^-1）	IAA/（mg · L^-1）	GA₃/（mg · L^-1）	增殖倍数 Multiplication multiple
0.4	0.1	0.2	1.13 ± 0.14 d
0.8	0.1	0.2	2.11 ± 0.11 bc
1.2	0.1	0.2	4.45 ± 0.16 a
0.4	0.2	0.2	2.89 ± 0.16 b
0.8	0.2	0.2	3.75 ± 0.21 a
1.2	0.2	0.2	1.55 ± 0.16 cd

图5 不同IBA浓度对B9组培苗生根的影响以Sg为基础培养基

Fig. 5 The impact of varying concentrations of IBA on the rooting of B9 tissue culture seedlings Sg based medium

表5 IBA浓度对B9生根的影响

Table 5 Effect of IBA concentration on B9 rooting

IBA /（mg · L^-1）	生根率/% Rooting rate	平均生根数 Average rooting number
0.2	21.11 ± 4.01 d	2.51 ± 0.24 b
0.4	40.00 ± 5.77 cd	2.66 ± 0.12 b
0.6	81.11 ± 4.84 a	5.99 ± 0.30 a
0.8	66.67 ± 5.09 ab	5.18 ± 0.72 a
1.0	52.22 ± 2.94 bc	2.77 ± 0.09 b
1.2	23.33 ± 5.09 d	3.17 ± 0.28 b

图6 不同潮霉素浓度对叶片不定芽诱导的影响不同小写字母表示经Duncan’s法多重比较后各处理间差异显著（P < 0.05）。下同

Fig. 6 The effect of different hygromycin on the induction of adventitious buds in leaves The different lowercase letters in the table indicate significant differences（P < 0.05）between the treatments after multiple comparisons by Duncan’s method. The same below

图7 不同潮霉素浓度对不定根诱导的影响以Sg为基础培养基

Fig. 7 The effect of different concentrations of hygromycin on adventitious root induction Sg based medium

参考文献 41

[1]	Cao Xiaomin, Chi Xin, Di Doudou, Hu Hui, Song Laiqing, Jiang Zhongwu, Zhao Lingling. 2020. Preliminary identification of cold resistance of six apple rootstocks. China Fruits,(4):12-17. (in Chinese)
	曹晓敏, 迟馨, 弟豆豆, 胡慧, 宋来庆, 姜中武, 赵玲玲. 2020. 6种苹果砧木的抗寒性比较研究. 中国果树,(4):12-17.
[2]	Dubranszki J, Teixeira d Silva J A. 2010. Micropropagation of apple - a review. Biotechnology Advances, 28 (4):462-488.
[3]	Famiani F, Ferradini N, Staffolani P, Standardi A. 1994. Effect of leaf excision time and age,BA concentration and dark treatments on in vitro shoot regeneration of M.26 apple rootstocks. Journal of Horticultural Science, 69 (4):679-685.
[4]	Fan Xiufang, Liu Xufeng, Zhang Jiantang, Shi Lianrang. 2004. Experiment on suitability of apple dwarf root-stocks grown in Weibei Area. Journal of Northwest A & F University(Natural Science Edition),(9):77-80. (in Chinese)
	樊秀芳, 刘旭峰, 张建堂, 史联让. 2004. B9等苹果矮化砧木在渭北地区适应性比较试验. 西北农林科技大学学报(自然科学版),(9):77-80.
[5]	Gao Bing, Sun Jun. 2017. ‘Gala’ apple leaf callus induction. Northern Horticulture,(9):88-92. (in Chinese)
	高兵, 孙俊. 2017. “嘎拉”苹果叶片愈伤组织的诱导. 北方园艺,(9):88-92.
[6]	Hao Hongmei, Tian Yi, Cong Peihua, Yang Zhenying. 2011. Research progress on plant regeneration of apple leaves in vitro. China Fruits,(1):55-57. (in Chinese)
	郝红梅, 田义, 丛佩华, 杨振英. 2011. 苹果离体叶片植株再生研究进展. 中国果树,(1):55-57.
[7]	He Huiyi, Chen Yongsheng, Zhang Nannan, Wu Jiayun, Wang Qinnan, Gao Xiaoning, Huang Yonghong, Chen Xinglong. 2023. Study on the tolerance of different sugarcane varieties to hygromycin B. Sugarcane and Canesugar, 52 (3):15-21. (in Chinese)
	何慧怡, 陈勇生, 张南南, 吴嘉云, 王勤南, 高小宁, 黄咏虹, 陈兴龙. 2023. 不同甘蔗品种对潮霉素B耐受性的研究. 甘蔗糖业, 52 (3):15-21.
[8]	James D J, Passey A J, Barbara D J, Bevan M. 1989. Genetic transformation of apple(Malus pumila Mill.) using a disarmed Ti-binary vector. Plant Cell Reports, 7 (8):658-661.
[9]	Jia Dongjie, Fan Lianmei, Shen Junling, Qin Song, Li Fuchao, Liu Chenglian, Yuan Yongbing. 2013. Genetic transformation and expressing of astaxanthin biosynthesis genes bkt into‘brookfield gala’apple. Acta Horticulturae Sinica, 40 (1):21-31. (in Chinese)
	贾东杰, 樊连梅, 沈俊岭, 秦松, 李富超, 刘成连, 原永兵. 2013. 虾青素合成关键酶基因bkt在‘Brookfield Gala’苹果中的遗传转化及表达. 园艺学报, 40 (1):21-31.
[10]	Li Haiwei, Ai Zhiguo, Gu Hongfei, Bai Baozhen, Ma Chunhong, Jia Yinsuo. 2010. Experimental study on cutting of B 9 young branches of apple dwarfing rootstock. Hebei Fruits,(2):4. (in Chinese)
	李海伟, 艾治国, 谷鸿飞, 白宝珍, 马春红, 贾银锁. 2010. 苹果矮化砧木B9嫩枝扦插试验. 河北果树,(2):4.
[11]	Li Kunkun, Xu Changjie. 2017. Advances in regeneration and genetic transformation of rosaceae fruit tree. Acta Horticulturae Sinica, 44 (9):1633-1644. (in Chinese)
	李坤坤, 徐昌杰. 2017. 蔷薇科果树离体再生与遗传转化研究进展. 园艺学报, 44 (9):1633-1644.
[12]	Li Qin. 2022. The establishment of rapid propagation and regeneration system of canna in vitro[Ph. D. Dissertation]. Guiyang: Guizhou University. (in Chinese)
	李琴. 2022. 美人蕉离体快繁及再生体系建立[博士论文]. 贵阳: 贵州大学.
[13]	Liao X, Guo X, Wang Q, Wang Y, Zhao D, Yao L, Wang S, Liu G, Li T. 2017. Overexpression of MsDREB6.2 results in cytokinin-deficient developmental phenotypes and enhances drought tolerance in transgenic apple plants. Plant Journal, 89 (3):510-526.
[14]	Liu Mingxiao, Jing Junli, Li Xiaohan, Sun Ye, Xu Jizhong, Zhou Shasha. 2023. Effects of apple MdPYL9 gene on salt tolerance of apple tissue culture seedlings. Jiangsu Agricultural Sciences, 51 (10):149-154. (in Chinese)
	刘铭潇, 井俊丽, 李晓涵, 孙晔, 徐继忠, 周莎莎. 2023. 苹果MdPYL9基因对苹果组培苗耐盐性的影响. 江苏农业科学, 51 (10):149-154.
[15]	Liu Xinglu, Wang Hongping, Sun Wentai, Dong Tie, Niu Junqiang, Ma Ming. 2021. Cold resistance evaluation of the shoots of 5 apple rootstocks. Journal of Fruit Science, 38 (8):1264-1274. (in Chinese)
	刘兴禄, 王红平, 孙文泰, 董铁, 牛军强, 马明. 2021. 5个砧木苹果枝条的抗寒性评价. 果树学报, 38 (8):1264-1274.
[16]	Liu Ying, Xu Jizhong, Shao Jianzhu, Gao Yi. 2006. The establishment of a highly efficient regeneration system from leaves in vitro of apple dwarf stock P59. Journal of Hebei Agricultural University,(3):10-12. (in Chinese)
	刘莹, 徐继忠, 邵建柱, 高仪. 2006. 苹果矮化砧木P_(59)离体叶片高效再生体系的建立. 河北农业大学学报,(3):10-12.
[17]	Olhoft P M, Flagel L E, Donovan C M, Somers D A. 2003. Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta, 216 (5):723-735.
[18]	Predieri S, Malavasi F F F. 1989. High-frequency shoot regeneration from leaves of the apple rootstock M26(Malus pumila Mill.). Plant Cell,Tissue and Organ Culture, 17 (2):133-142.
[19]	Sedira M, Holefors A, Welander M. 2001. Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting. Plant Cell Reports, 20 (6):517-524.
[20]	Siboza X I, Kotze W P, Cook N C, Steyn W J. 2020. Evaluation of apple rootstocks planted in different locations in South Africa. Acta Horticulturae,(1281):145-151.
[21]	Song Xin,Zhao Shuqing. 2017. Identification of 35S:MS606/myb26 transgenic plants in Arabidopsis thaliana. Journal of Shanxi Agricultural Sciences, 45 (5):680-683. (in Chinese)
	宋欣, 赵淑清. 2017. 拟南芥35S:MS606/myb26转基因植物的鉴定. 山西农业科学, 45 (5):680-683.
[22]	Sun Jinglang. 2023. Research on efficient in vitro regeneration and gene editing application of Pineapple[Ph. D. Dissertation]. Shanghai: Shanghai Normal University. (in Chinese)
	孙婧朗. 2023. 菠萝高效离体再生及基因编辑应用研究[博士论文]. 上海: 上海师范大学.
[23]	Sun Kecong. 2020. Establishment of the shoot regeneration system of apple dwarfing self-rootstock[Ph. D. Dissertation]. Tai’an: Shandong Agricultural University. (in Chinese)
	孙克聪. 2020. 苹果矮化自根砧再生芽体系的建立与优化[博士论文]. 泰安: 山东农业大学.
[24]	Sun Qingrong, Guan Qiuzhu, Li Linguang, He Ping, Wang Haibo, Sun Hongyan. 2021. Establishment of high-efficiency adventitious bud regeneration from leaf explants of Malus domestica var.‘Luli’. Northern Horticulture,(19):42-47. (in Chinese)
	孙清荣, 关秋竹, 李林光, 何平, 王海波, 孙洪雁. 2021. 苹果新品种‘鲁丽’离体叶片高效不定芽再生体系的建立. 北方园艺,(19):42-47.
[25]	Sun Qingrong, Guan Qiuzhu, Sun Hongyan, Li Linguang, Tao Jihan, Wang Haibo, He Ping. 2017. Tissue culture and shoot regeneration from leaf explants of cold-hardy and semi-dwarf apple rootstock‘54-118’. Plant Physiology Journal, 53 (11):2007-2012. (in Chinese)
	孙清荣, 关秋竹, 孙洪雁, 李林光, 陶吉寒, 王海波, 何平. 2017. 苹果抗寒半矮化砧木‘54-118’的组织培养及其离体叶片不定梢再生. 植物生理学报, 53 (11):2007-2012.
[26]	Sun Qingrong, Sun Hongyan, Liu Qingzhong, Shi Yinping. 2000. Regeneration of adventitious plants from leaves of different-ploidy apple trees. Deciduous Fruits,(2):12-14. (in Chinese)
	孙清荣, 孙洪雁, 刘庆忠, 石荫坪. 2000. 不同倍性苹果叶片不定植株再生研究. 落叶果树,(2):12-14.
[27]	Tian Q, Xu M, Wu D, Wang C, Wang X, Che Q, Li Z, Xu X. 2023. Integrated transgene and transcriptome reveal the molecular basis of MdWRKY87 positively regulate adventitious rooting in apple rootstock. Frontiers in Plant Science,14.
[28]	Wada M, Nishitani C, Komori S. 2020. Stable and efficient transformation of apple. Plant Biotechnology, 37 (2):163-170.
[29]	Wang Dan, Zou Li, Wang Yi,Yang Minbao. 2010. Study on the sensitivity of poplar to hygromycn. Journal of Jilin Agricultural University, 32 (1):47-50. (in Chinese)
	王丹, 邹莉, 王义, 杨民宝. 2010. 杨树对潮霉素的敏感性研究. 吉林农业大学学报, 32 (1):47-50.
[30]	Wang Guiying, Liu Xiaojie, Li Shanshan, Sun Chao, Yang Jinku, Yang Minsheng, Liu Meiling. 2015. Establishment of populus deltoides cv.JuBa regeneration system and its resistance to hygromycin. Northern Horticulture,(13):98-102. (in Chinese)
	王桂英, 刘晓杰, 李珊珊, 孙超, 杨金库, 杨敏生, 刘美玲. 2015. 巨霸杨组培再生体系的建立及潮霉素抗性试验. 北方园艺,(13):98-102.
[31]	Wang Jiameng, Hou Mengjuan, Kong Hui, Xie Zhaosong, Wang Jinyuan, Zhu Jianbo, Li Jin, Peng Guowei. 2024. Established of Agrobacterium-mediated ‘Yeda No. 1’efficient genetic transformation system. Soybean Science, 43 (6):708-716. (in Chinese)
	王嘉萌, 侯梦娟, 孔辉, 谢照松, 王晋元, 祝建波, 李锦, 彭国威. 2024. 农杆菌介导的“野大1号”高效遗传转化体系建立. 大豆科学, 43 (6):708-716.
[32]	Wang Jian, Li Yongling, Liu Wei. 2017. Research progress on application of hygromycin B in genetic transformation. Journal of Ningxia Agriculture and Forestry Science and Technology, 58 (12):36-43. (in Chinese)
	王坚, 李永玲, 刘炜. 2017. 潮霉素B在遗传转化中应用的研究进展. 宁夏农林科技, 58 (12):36-43.
[33]	Wang Yan,Sun Zhongxu,Xia Yang. 2004. Study of regeneration form leaves of apple in viteo. Journal of Shandong Agricultural University(Natural Science Edition),(2):196-200. (in Chinese)
	王艳, 孙仲序, 夏阳. 2004. 苹果砧木品种试管苗叶片再生体系的建立. 山东农业大学学报(自然科学版),(2):196-200.
[34]	Wang Zhe, Sun Jingke, Wang Shixiang, Xiao Jingjing. 2009. Hygromycin resistance to the concentration of tohacco selection and reserch, Journal of Henan University of Urban Construction, 18 (4):65-67. (in Chinese)
	王哲, 孙敬克, 王世翔, 肖婧婧. 2009. 烟草潮霉素抗性浓度的筛选与研究. 河南城建学院学报, 18 (4):65-67.
[35]	Welander M, Zhu L H, Li X Y. 2004. Transformation of dwarfing apple and pear rootstocks with the rolB gene and its influence on rooting and growth. 11th Eucarpia Symposium on Fruit Breeding and GeneticsProceedings of the XIth Eucarpia Symposium on Fruit Breeding and Genetics,Vols 1 and 2,663:437-442.
[36]	Yu Fushun, Jiao Gongqiang, Jiang Zhixun. 2002. Three excellent rootstocks for apples. Deciduous Fruits,(2):16. (in Chinese)
	于福顺, 焦功强, 江志训. 2002. 苹果的3种优良砧木. 落叶果树,(2):16.
[37]	Zimmerman J L. 1993. Somatic embryogenesis:a model for early development in higher plants. The Plant Cell, 5 (10):1411-1423.
[38]	Zhang Guangdi, Yu Xiaoyan, Xu Qinglin, Feng Xiaorong. 2006. Analysis on introducing culture and application in landscape scheme of B9,N29. Journal of Agricultural Sciences,(1):30-33. (in Chinese)
	张光弟, 俞晓艳, 徐庆林, 冯小蓉. 2006. B9、N29的引种栽培及在园林景观配置中的应用分析. 农业科学研究,(1):30-33.
[39]	Zhang Jian, Wang Lu, Yuan Jiawei, Tian Shimin, Zhang Zhanbei, Liang Zhejun. 2022. Regeneration system from leaf explants of Luli Apple. Seed, 41 (9):127-133. (in Chinese)
	张健, 王璐, 袁嘉玮, 田时敏, 张战备, 梁哲军. 2022. 鲁丽苹果植株再生体系的建立. 种子, 41 (9):127-133.
[40]	Zhang Ling, Peng Rihe, Fu Xiaoyan, Gao Jianjie, Wang Mingqing, Yao Quanhong. 2020. Establishment of efficient regeneration system and study of hygromycin resistance test of Malus robusta Rehd. Northern Horticulture,(15):82-88. (in Chinese)
	张苓, 彭日荷, 付晓燕, 高建杰, 王明清, 姚泉洪. 2020. 八棱海棠高效再生体系的建立及潮霉素的抗性试验. 北方园艺,(15):82-88.
[41]	Zhou Aiqin, Zhu Jun, Yang Qingshan, Wang Zhenmeng, Li Yongtao, Wei Haixia. 2017. Optimization of embryoid regeneration system from‘Lujia No.5’columnar apple. Northern Horticulture,(10):95-99. (in Chinese)
	周爱琴, 祝军, 杨庆山, 王振猛, 李永涛, 魏海霞. 2017. “鲁加5号”柱型苹果胚状体再生体系优化. 北方园艺,(10):95-99.