Plantlet Regeneration from Leaves of Tissue Culture Seedlings of Hydrangea macrophylla ‘Endless Summer’

doi:10.11924/j.issn.1000-6850.casb20190400010

Abstract

Abstract:

The paper aims to establish a large-scale asexual reproduction system and efficient genetic transformation system of Hydrangea macrophylla ‘Endless Summer’. The effects of leaf position, dark culture time and different hormone combinations on callus induction of leaves from ‘Endless Summer’ tissue culture seedlings and adventitious bud differentiation were studied. The results showed that the callus induction effect of the upper and middle leaves of a plantlet was good, and the 3 ^rd to 6^th leaves were suitable for explant sampling. Initial dark culture was beneficial to leaf callus formation and the best effect of dark culture was 15-25 days. The optimum medium for inducing callus formation in leaves of ‘Endless Summer’ tissue-culture seedlings was MS+CPPU 3.0 mg/L+IBA 0.1 mg/L, the callus induction rate was over 98%. The medium for callus proliferation and adventitious bud differentiation was MS+6-BA 1.0-2.25 mg/L+IBA 0.1 mg/L.

Key words: Hydrangea macrophylla, leaves of tissue culture plantlets, callus, plantlet regeneration

CLC Number:

S685.21

Sun Xiaobo, Su Jiale, Chen Shuangshuang, Liang Lijian, Deng Yanming. Plantlet Regeneration from Leaves of Tissue Culture Seedlings of Hydrangea macrophylla ‘Endless Summer’[J]. Chinese Agricultural Science Bulletin, 2020, 36(16): 67-72.

Figures/Tables 5

References 27

[1]	彭亮, 彭尽晖, 赵盈盈 , 等. 八仙花组织培养研究进展[J]. 安徽农业科学, 2014,10:2863-2864,2892.
[2]	姜波, 沈宗根, 黄四娣 , 等. 草绣球与绣球的光合作用和叶绿素荧光特性比较[J]. 浙江农业学报, 2012,24(4):582-588.
[3]	黄林, 黄小云, 何平 , 等. 四川省及重庆市绣球属(Hydrangea Linn.)的分类研究(I):研究历史及地理分布[J]. 西南师范大学学报:自然科学版, 2001,26(3):317-322.
[4]	彭尽晖, 周朴华, 周红灿 , 等. 湖南省绣球属植物资源调查[J]. 湖南农业大学学报:自然科学版, 2008,34(5):563-567.
[5]	Zhang H, Matsuda H, Kumahara A , et al. New type of anti-diabetic compounds from the processed leaves of Hydrangea macrophylla var. thunbergii (Hydrangeae Dulcis Folium)[J]. Bioorg Med Chem Lett, 2007,17:4972-4976. doi: 10.1016/j.bmcl.2007.06.027 URL
[6]	贾新平, 孙晓波, 梁丽建 , 等. 绣球SSR-PCR反应体系的建立与优化[J]. 华北农学报, 2016,31(4):68-73. doi: 10.7668/hbnxb.2016.04.012 URL
[7]	谭巍 . 八仙花栽培技术[J]. 北方园艺, 2006(1):96-97.
[8]	闫海霞, 蒋月喜, 邓杰玲 , 等. 绣球花组织培养的研究进展[J]. 广西农学报, 2017,32(3):39-43.
[9]	王忠武, 建德锋 . 八仙花茎尖离体培养技术研究[J]. 北方园艺, 2012,9:129-130.
[10]	雷亚灵, 李周岐 . 八仙花茎段组织培养技术研究[J]. 西北林学院学报, 2008,23(4):101-103.
[11]	Donna I L, John E P . Thidiazuron stimulates adventitious shoot production from Hydrangea quercifolia Bartr. leaf explants[J]. Scientia Hort, 2004,101:121-126. doi: 10.1016/j.scienta.2003.09.014 URL
[12]	Liu F, Huang L L, Li Y L , et al. Shoot organogenesis in leaf explants of Hydrangea macrophylla ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers[J]. Plant Cell Tiss Organ Cult, 2011,104:111-117. doi: 10.1007/s11240-010-9797-2 URL
[13]	Latifa H, Linda V, Didier P , et al. Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs[J]. Scientia Horticulturae, 2011,127:378-387. doi: 10.1016/j.scienta.2010.09.021 URL
[14]	范晓峰, 杨建霞, 杨颖丽 . 八仙花愈伤组织诱导与快速繁殖[J]. 经济林研究, 2009,27(1):41-44.
[15]	Ruffoni B, Sacco E, Savona M . In Vitro Propagation of Hydrangea spp[J]. Methods Mol Biol, 2013,110(13):231-244.
[16]	白芝兰, 李果果, 吴晓冉 , 等. 野生毛桃叶片愈伤组织诱导及不定芽再生[J]. 果树学报, 2012,29(5):814-819.
[17]	吕经娟, 李淑斌, 周宁宁 , 等. ‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究[J]. 植物科学学报, 2014,32(2):139-147. doi: 10.3724/SP.J.1142.2014.20139 URL
[18]	张文泉, 邓洁 , 青钱柳叶片诱导愈伤组织研究[J]. 内蒙古农业大学学报:自然科学版, 2016,37(2):28-33.
[19]	莫竹承, 庞万伟, 刘珏 , 等. 膝柄木叶片诱导愈伤组织研究[J]. 中南林业科技大学学报, 2015,35(10):13-17.
[20]	杨秀, 莲胡蝶, 宋佳妮 , 等. 海州常山叶片愈伤组织诱导及不定芽再生[J]. 东北林业大学学报, 2018,46(7):28-32.
[21]	杨春 . 生长素和细胞分裂素在马铃薯愈伤组织分化中的作用[J]. 山西农业科学, 2008,36(7):40-42.
[22]	Kakahashi S, Shudo K, Okamoto T , et al. Cytokinin activity of N-phenyl-N-(4-pyridyl)urea derivatives. Phytochemistry, 1978,17:1201-1207.
[23]	张桂芬, 常中开 . CPPU应用研究概况[J]. 企业科技与发展, 2010,23:22-24.
[24]	Reynolds A G, Wardle D A, Zurowski C , et al. Phenylureas CPPU and thidiazuron affect yield components, fruit composition, and storage potential of four seedless grape selections[J]. J Amer Soc Hort Sci, 1992,117(1):85-89. doi: 10.21273/JASHS.117.1.85 URL
[25]	王关林, 方宏筠, 那杰 . 高活性细胞激动素TDZ在植物组织培养中的应用[J]. 植物学通报, 1997,14(3):47-53.
[26]	吴晓冉, 王秀, 白芝兰 , 等. 氯吡苯脲对野生毛桃叶片植株再生和愈伤组织诱导的影响[J]. 园艺与种苗, 2013,10:24-28,54.
[27]	余宏傲, 叶朝军, 康华靖 , 等. 高丛蓝莓‘薄雾’愈伤组织高效诱导及胚性转化[J]. 分子植物育种, 2018.

叶位	出愈率/%	愈伤长势	愈伤状态
3~4	100a	+++	切面处逐渐形成绿色颗粒状愈伤组织,致密
5~6	99.8±2.3a	+++	切面处逐渐形成绿色颗粒状愈伤组织,致密
7~8	82.5±6.2b	++	切面处逐渐形成绿色颗粒状愈伤组织,偶有黄褐色颗粒,致密
9~10	55.3±4.9c	+	切面处逐渐形成浅绿色颗粒状愈伤组织,偶有红褐色颗粒,致密

叶位	出愈率/%	愈伤长势	愈伤状态
3~4	100a	+++	切面处逐渐形成绿色颗粒状愈伤组织,致密
5~6	99.8±2.3a	+++	切面处逐渐形成绿色颗粒状愈伤组织,致密
7~8	82.5±6.2b	++	切面处逐渐形成绿色颗粒状愈伤组织,偶有黄褐色颗粒,致密
9~10	55.3±4.9c	+	切面处逐渐形成浅绿色颗粒状愈伤组织,偶有红褐色颗粒,致密

暗培养时间/d	出愈率/%	愈伤组织长势	愈伤组织形态
0	70.1±13.8c	+	深绿颗粒状,致密,偶见丛生芽
5	98.7±4.6a	++	深绿颗粒状,致密,部分丛生芽
10	97.3±8.2a	++	绿色颗粒状,致密,部分丛生芽
15	98.1±5.1a	+++	绿色颗粒状,致密,少见丛生芽
20	99.5±2.9a	+++	绿色颗粒状,致密,少见丛生芽
25	100a	+++	绿色颗粒状,偶有黄色颗粒,致密,偶见丛生芽
30	91.1±3.5b	++	浅绿颗粒状,偶有黄色颗粒,致密,偶见丛生芽

暗培养时间/d	出愈率/%	愈伤组织长势	愈伤组织形态
0	70.1±13.8c	+	深绿颗粒状,致密,偶见丛生芽
5	98.7±4.6a	++	深绿颗粒状,致密,部分丛生芽
10	97.3±8.2a	++	绿色颗粒状,致密,部分丛生芽
15	98.1±5.1a	+++	绿色颗粒状,致密,少见丛生芽
20	99.5±2.9a	+++	绿色颗粒状,致密,少见丛生芽
25	100a	+++	绿色颗粒状,偶有黄色颗粒,致密,偶见丛生芽
30	91.1±3.5b	++	浅绿颗粒状,偶有黄色颗粒,致密,偶见丛生芽

序号	ZT/(mg/L)	KT/(mg/L)	6-BA/(mg/L)	CPPU/(mg/L)	IBA/(mg/L)	不定芽诱导率/%	愈伤诱导率/%
1	2.25	0	0	0	0.1	0	0
2	0	2.25	0	0	0.1	0	0
3	0	0	2.25	0	0.1	15.6±6.9	0
4	0	0	0	3.0	0.1	0	99.5±2.3