‘阳光玫瑰’葡萄试管苗热处理结合茎尖和腋芽培养脱毒技术研究

doi:10.16420/j.issn.0513-353x.2023-0204

摘要/Abstract

摘要：

为进一步优化葡萄试管苗脱毒体系，以携带葡萄蚕豆萎蔫病毒（GFabV）、沙地葡萄茎痘相关病毒（GRSPaV）、葡萄灰比诺病毒（GPGV）、葡萄卷叶相关病毒-3（GLRaV-3）和葡萄病毒E（GVE）共5种病毒的‘阳光玫瑰’葡萄试管苗为材料，研究了热处理方式和时间以及热处理后茎尖和腋芽培养对脱毒的影响。结果表明，热处理方式影响试管苗的存活率和脱毒效率，以38 ℃/光照8 h + 32 ℃/黑暗8 h变温处理效果最佳，其次为38 ℃/光照16 h + 32 ℃/黑暗8 h变温处理以及38℃恒温/（光照16 h + 黑暗8 h）处理。取38 ℃/光照8 h + 32 ℃/黑暗8 h热处理10、15、20、25、30和40 d的试管苗1.5 mm左右的茎尖和茎尖下第1、2腋芽接种培养，茎尖存活率高于腋芽，繁殖成苗后检测，均脱除了上述5种病毒，其中热处理10 d的平均脱毒率为52.38%，25和30 d的脱毒率均达100%；随热处理时间延长，接种茎尖和腋芽的存活率下降，故热处理时间以25 d为宜。

关键词: 葡萄, 试管苗, 热处理, 茎尖, 腋芽, 脱毒

Abstract:

In order to optimize the virus elimination system of grape plantlets in vitro，research on virus elimination was carried out using the‘Shine Muscat’grape plantlets in vitro，which have been detected carrying five viruses including grapevine fabavirus（GFabV），grapevine rupestris stem pitting associated virus（GRSPaV），grapevine Pinot gris virus（GPGV），grapevine leafroll-associated virus-3 （GLRaV-3），and grapevine virus E（GVE）. The effects on virus elimination regarding heat treatment ways and duration，as well as plant materials（shoot tip or axillary bud）used for inoculation after heat treatment were studied. The results showed that the heat treatment of 38 ℃/light 8 h and 32 ℃/ dark 8 h in turn generated the highest survival rate and virus elimination efficiency，followed by the treatment of 38 ℃/light 16 h and 32 ℃/ dark 8 h in turn and constant 38 ℃（light 16 h + dark 8 h）. Then，about 1.5 mm long shoot tips or the first or the second axillary buds under the shoot tips were cut from the plantlets cultured under the heat treatment of 38 ℃/light 8 h and 32 ℃/ dark 8 h in turn for 10，15，20，25，30，and 40 days，respectively，for inoculation. The survival rate of the shoot tips was higher than that of the axillary buds. The new plantlets were detected for the viruses，and virus-free plantlets were obtained from all kinds of the materials. The virus elimination rate was 52.38% when the heat treatment duration was 10 d，and reached 100% when that was 25 d or 30 d. With the further increased duration，the survival rate of the inoculated shoot tips and the axillary buds decreased. The duration of 25 d was recommended.

Key words: grape, plantlet in vitro, heat treatment, shoot tip, axillary bud, virus elimination

杜易静, 刘文林, 乔月莲, 王莉, 安德志, 杜国强, 师校欣. ‘阳光玫瑰’葡萄试管苗热处理结合茎尖和腋芽培养脱毒技术研究[J]. 园艺学报, 2024, 51(4): 893-902.

DU Yijing, LIU Wenlin, QIAO Yuelian, WANG Li, AN Dezhi, DU Guoqiang, SHI Xiaoxin. Virus Elimination from‘Shine Muscat’Grape Plantlets in vitro via Heat Treatment Combined with Shoot Tip and Axillary Bud Culture[J]. Acta Horticulturae Sinica, 2024, 51(4): 893-902.

图/表 8

参考文献 29

[1]	Aljuaid B S, Attia A O, Ismail I A, Dessoky E S, Sadik A S, Khalifa M E. 2022. Production of virus-free rose plants using meristem-tip culture and in vitro thermotherapy. Pakistan Journal of Biological Sciences, 25 (2):160-167.
[2]	Bai Xianjin, Lou Binghai, Han Jiayu, Wang Bo, Song Yaqin, Bai Yang, Cao Xiongjun, Cheng Guo. 2022. Graft transmission of leaf crinkle and mosaic symptoms on‘Shine Muscat’grapevines. Sino-Overseas Grapevine & Wine, 46 (2):48-51. (in Chinese)
	白先进, 娄兵海, 韩佳宇, 王博, 宋雅琴, 白扬, 曹雄军, 成果. 2022. ‘阳光玫瑰’葡萄皱叶花叶症状嫁接传染研究. 中外葡萄与葡萄酒, 46 (2):48-51.
[3]	Cao Dong-xu, Zhou Hai-feng, Yu Sheng-cheng, Nian Yu-xin. 2012. Study on heat-treated stem tip culture of Kyoho grape. Northern Horticulture, 34 (9):116-118. (in Chinese)
	曹冬煦, 周海峰, 于生成, 年玉欣. 2012. “巨峰”葡萄热处理茎尖培养技术研究. 北方园艺, 34 (9):116-118.
[4]	Cao Xiongjun, Han Jiayu, Cheng Guo, Wang Bo, Ma Guangren, Lin Ling, Tan Zongkun, Huang Qiumi, Chen Xiao, Chen Fuyi, Shi Xiaofang, Pan Fengping, Bai Xianjin. 2023. Effects of light duration and intensity on yield formation of‘Shine-Muscat’grape. Acta Horticulturae Sinica, 50 (8):1739-1746. (in Chinese)
	曹雄军, 韩佳宇, 成果, 王博, 马广仁, 林玲, 谭宗琨, 黄秋秘, 陈潇, 陈孚仪, 时晓芳, 盘丰平, 白先进. 2023. 光照时数及强度对‘阳光玫瑰’葡萄产量形成的影响. 园艺学报, 50 (8):1739-1746.
[5]	Chellappan P, Vanitharani R, Ogbe F, Fauquet C M. 2005. Effect of temperature on geminivirus-induced RNA silencing in plants. Plant Physiology, 138 (4):1828-1841. doi: 10.1104/pp.105.066563 pmid: 16040661
[6]	Cheng Xiaohua, Xu Zhiwei, Wang Li, Shi Xiaoxin, Du Guoqiang. 2021. Efficacy of low temperature and mineral oil covering in prolonging subculture interval during grape germplasm preservation in vitro. Acta Horticulturae Sinica, 48 (3):477-486. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0304
	成晓华, 徐志微, 王莉, 师校欣, 杜国强. 2021. 葡萄种质离体保存中低温和矿物油覆盖延长继代间隔的效应. 园艺学报, 48 (3):477-486. doi: 10.16420/j.issn.0513-353x.2020-0304
[7]	Fan Xu-dong, Dong Ya-feng, Zhang Zun-ping, Zhang Meng-yan, Ren Fang, Hu Guo-jun. 2019. Small RNA sequencing and RT-PCR detection of viruses infecting‘Shine Muscat’grapevines. Acta Phytopathologica Sinica, 49 (6):749-755. (in Chinese)
	范旭东, 董雅凤, 张尊平, 张梦妍, 任芳, 胡国君. 2019. ‘阳光玫瑰’葡萄病毒小RNA测序鉴定及RT-PCR检测. 植物病理学报, 49 (6):749-755.
[8]	Fan Xu-dong, Zhang Zun-ping, Ren Fang, Hu Guo-jun, Li Zheng-nan, Dong Ya-feng. 2018. Detection and sequence analyses of Grapevine Pinot gris virus isolates from China. Acta Phytopathologica Sinica, 48 (4):466-473. (in Chinese)
	范旭东, 张尊平, 任芳, 胡国君, 李正男, 董雅凤. 2018. 我国灰比诺葡萄病毒分离物检测及基因序列分析. 植物病理学报, 48 (4):466-473.
[9]	Feng C H, Wang R R, Li J W, Wang B, Yin Z F, Cui Z H, Li B Q, Bi W L, Zhang Z B, Li M F, Wang Q C. 2013. Production of pathogen-free horticultural crops by cryotherapy of in vitro-grown shoot tips. Methods in Molecular Biology, 11013:463-482.
[10]	Gambino G, Bondaz J, Gribaudo I. 2006. Detection and elimination of viruses in callus,somatic embryos and regenerated plantlets of grapevine. European Journal of Plant Pathology, 114 (4):397-404.
[11]	Gao Hui-qing, Fan Lan-ying, Wang Xiu-hong, Gao Hong-zhang. 2010. Study of merited tip culture combined with heat treatment on virus elimination in Lilium longiflorum. Journal of Shanxi Agricultural University(Natural Science Edition), 30 (6):528-532. (in Chinese)
	高慧卿, 樊兰瑛, 王秀红, 高宏樟. 2010. 茎尖培养及热处理技术在百合脱毒中的应用研究. 山西农业大学学报(自然科学版), 30 (6):528-532.
[12]	Habili N, Krake L R, Barlass M, Rezaian M A. 1992. Evaluation of biological indexing and dsRNA analysis in grapevine virus elimination. Annals of Applied Biology, 121 (2):227-283.
[13]	Hu G J, Dong Y F, Zhang Z P, Fan X D, Ren F, Li Z N, Zhang S N. 2018. Elimination of Grapevine rupestris stem pitting-associated virus from Vitis vinifera‘Kyoho’by an antiviral agent combined with shoot tip culture. Scientia Horticulturae, 229:99-106.
[14]	Jin Xiaofei, Chen Zhe. 2019. Positioning cultivation techniques of‘Shine Muscat’grapevine. Sino-Overseas Grapevine & Wine, 43 (4):55-57. (in Chinese)
	金晓飞, 陈哲. 2019. ‘阳光玫瑰’葡萄定位栽培技术. 中外葡萄与葡萄酒, 43 (4):55-57.
[15]	Li Zhi-qiang, Wang Jing, Ding Guo-liang, Cheng Jian-jun, Fan Hong, Zuo Li-juan. 2012. Study on heat treatment and shoot tip virus elimination technique of strawberry. Northern Horticulture, 34 (5):125-127. (in Chinese)
	李志强, 王晶, 丁国亮, 程建军, 范红, 左立娟. 2012. 草莓热处理结合茎尖脱毒技术研究. 北方园艺, 34 (5):125-127.
[16]	Lin Qian, Gao Yingying, Qin Huanling, Huang Tiankun, Zhao Yu, Wang Zhongxia, Chen Shuyuan. 2021. Study on rapid propagation technology of virus-free seedlings by tissue culture in‘Shine Muscat’grape. Journal of Fruit Science, 38 (3):435-443. (in Chinese)
	林茜, 高营营, 覃换玲, 黄天琨, 赵宇, 王钟霞, 陈淑媛. 2021. ‘阳光玫瑰’葡萄组培脱毒快繁技术研究. 果树学报, 38 (3):435-443.
[17]	Liu Dan, Wang Rong, Chen Tian-lang, Wu Hai-feng, Zhang Bin-bin, Fang Ping, Chen Wei, He Wen-tao. 2022. Study on virus-free technique of cut chrysanthemum“Hongchang”. Journal of Anhui Agricultural Sciences, 50 (19):114-118. (in Chinese)
	刘丹, 王容, 陈天烺, 吴海峰, 张宾宾, 方萍, 陈炜, 赫文韬. 2022. 切花小菊“红昌”脱毒技术研究. 安徽农业科学, 50 (19):114-118.
[18]	Liu Ke-hong, Zhou Yan, Li Zhong-an. 2016. Technical progress on shoot-tip grafting of citrus. Acta Horticulturae Sinica, 43 (9):1665-1674. (in Chinese) doi: 10.16420/j.issn.0513-353x.2016-0079
	刘科宏, 周彦, 李中安. 2016. 柑橘茎尖嫁接脱毒技术研究进展. 园艺学报, 43 (9):1665-1674. doi: 10.16420/j.issn.0513-353x.2016-0079
[19]	Liu Xingyue, Zhu Qianglong, Li Huiying, Sun Jingyu, Li Bicong, Zhang Hongyu, Huang Yingjin, Fang Jiajun, Zhou Qinghong. 2020. Induction and plant regeneration of virus-free microtuber in red bud taro. Acta Horticulturae Sinica, 47 (12):2427-2438. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0470
	刘星月, 朱强龙, 李慧英, 孙静宇, 李必聪, 张宏玉, 黄英金, 方加军, 周庆红. 2020. 红芽芋脱毒试管芋诱导及植株再生. 园艺学报, 47 (12):2427-2438.
[20]	Lou Binghai, Bai Xianjin, Song Yaqin, Bai Yang, Wang Bo, Chen Aijun. 2017. The detection of main viruses in‘Shine Muscat’grape. Journal of Plant Protection, 44 (2):345-346. (in Chinese)
	娄兵海, 白先进, 宋雅琴, 白扬, 王博, 陈爱军. 2017. ‘阳光玫瑰’葡萄中主要葡萄病毒病原的检测. 植物保护学报, 44 (2):345-346.
[21]	Maliogka V I, Skiada F G, Eleftheriou E P, Katis N I. 2009. Elimination of a new ampelovirus(GLRaV-Pr)and Grapevine rupestris stem pitting associated virus(GRSPaV)from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture. Scientia Horticulturae, 123 (2):280-282.
[22]	Ren Fang, Dong Ya-feng, Zhang Zun-ping, Fan Xu-dong, Hu Guo-jun, Zhou Jun. 2014. Recent advances in viruses infecting grapevine. Acta Horticulturae Sinica, 41 (9):1777-1792. (in Chinese)
	任芳, 董雅凤, 张尊平, 范旭东, 胡国君, 周俊. 2014. 葡萄病毒研究最新进展. 园艺学报, 41 (9):1777-1792.
[23]	Umer M, Liu J W, You H F, Xu C, Dong K L, Luo N, Kong H, Li X P, Hong Ni, Wang G P, Fan X D, Kotta-Loizou I, Xu W X. 2019. Genomic,morphological and biological traits of the viruses infecting major fruit trees. Viruses, 11 (6):515.
[24]	Wei Wei, Shan Shouming. 2022. Research progress on virus-free grape and rapid propagation technology. Molecular Plant Breeding, 20 (1):259-265. (in Chinese)
	韦伟, 单守明. 2022. 葡萄脱毒与快繁技术研究进展. 分子植物育种, 20 (1):259-265.
[25]	Yang Guang, Jin Gui-hua, Dong Jun, Zhang Qing, Gong Na. 2014. Study on virus-free and rapid propagation techniques of Zhuangyuanhong grape. Acta Agriculturae Zhejiangensis, 26 (1):72-76. (in Chinese)
	杨光, 金桂花, 董俊, 张青, 龚娜. 2014. 状元红葡萄的脱毒与快繁技术研究. 浙江农业学报, 26 (1):72-76.
[26]	Zhang Feixue, Zhang Shuhong, Ma Yandong, Chen Feng, Song Xianjie, Liu Ruijie. 2023. Effects of TDZ,CPPU and GA₃ on fruit quality of‘Shine Muscat’grape. Acta Horticulturae Sinica, 50 (12):2633-2640. (in Chinese) doi: 10.16420/j.issn.0513-353x.2023-0083
	张飞雪, 张书红, 马延东, 陈峰, 宋贤杰, 刘锐杰. 2023. TDZ、CPPU和GA₃对‘阳光玫瑰’葡萄果实品质的影响. 园艺学报, 50 (12):2633-2640. doi: 10.16420/j.issn.0513-353x.2023-0083
[27]	Zhang Zun-ping, Fan Xu-dong, Hu Guo-jun, Ren Fang, Zhu Hong-juan, Dong Ya-feng. 2013. Studied on technics of virus elimination via heat treatment in grape in vitro. China Fruits, 37(1):39-41. (in Chinese)
	张尊平, 范旭东, 胡国君, 任芳, 朱红娟, 董雅凤. 2013. 葡萄试管苗热处理脱毒技术研究. 中国果树, 37 (1):39-41.
[28]	Zhao L, Wang M R, Cui Z H, Chen L, Volk G M, Wang Q C. 2018. Combining thermotherapy with cryotherapy for efficient eradication of Apple stem grooving virus from infected in vitro-cultured apple shoots. Plant Disease, 102 (8):1574-1580. doi: 10.1094/PDIS-11-17-1753-RE pmid: 30673422
[29]	Zhou Mei-ling, Zeng Jun, Zhang Zhi-yong, Cai Jian-rong, Jiang Wei, Zhang Wen-bin. 2022. Establishment of virus-free callus culture and rapid propagation system of virus-free seedlings from stem segments of yellow passion fruit. Acta Agriculturae Jiangxi, 34 (9):97-101. (in Chinese)
	周美玲, 曾军, 张志勇, 蔡建荣, 江巍, 张文斌. 2022. 黄果百香果茎段愈伤组织培养脱毒及脱毒苗快繁体系建立. 江西农业学报, 34 (9):97-101.

病毒名称 Virus	引物序列（5′-3′） Primer sequence	片段大小/bp Product length	退火温度/℃ Annealing temperature	延伸时间/s Extension time
GLRaV-1	F：TCTTTACCAACCCCGAGATGAA；R：GTGTCTGGTGACGTGCTAAACG	232	56.0	30
GLRaV-2	F：GGTGATAACCGACGCCTCTA；R：CCTAGCTGACGCAGATTGCT	597	57.8	60
GLRaV-3	F：CGCTAGGGCTGTGGAAGTATT；R：GTTGTCCCGGGTACCAGATAT	546	56.0	60
GLRaV-4	F：ACATTCTCCACCTTGTGCTTTT；R：CATACAAGCGAGTGCAATTAC	319	57.8	60
GRSPaV	F：TGAAGGCTTTAGGGGTTAG；R：CTTAACCCAGCCTTGAAAT	905	57.8	60
GVA	F：CCAGAGGAGTTTGAGACAATA；R：GTCCCGACCAAGGCGATGTACCC	196	56.0	60
GVB	F：ATCAGCAAACACGCTTGAACCG；R：GTGCTAAGAACGTCTTCACAGC	460	56.0	40
GVE	F：TGTGGGGTGCATAGTCATAGGTTT；R：GCTTTTGACTCCATTGGCTTTCTC	478	56.0	40
GFLV	F：CCAAAGTTGGTTTCCCAAGA；R：ACCGGATTGACGTGGGTGAT	321	56.0	40
GFabV	F：GGAAATAACATTCCCAGGAAG；R：TCCTGCTGTCAAATTTCATTC	325	50.0	40
GINV	F：WMGKGTTTCTGGGAAGATTGC；R：KACSAGATCCCTCAATTCCTG	968	55.0	70
GPGV	F：GGTTTCTGGGAGGATTGCAAC；R：RCTTYGCGATCAGCTCTCTCC	1 041	55.0	70

病毒名称 Virus	引物序列（5′-3′） Primer sequence	片段大小/bp Product length	退火温度/℃ Annealing temperature	延伸时间/s Extension time
GLRaV-1	F：TCTTTACCAACCCCGAGATGAA；R：GTGTCTGGTGACGTGCTAAACG	232	56.0	30
GLRaV-2	F：GGTGATAACCGACGCCTCTA；R：CCTAGCTGACGCAGATTGCT	597	57.8	60
GLRaV-3	F：CGCTAGGGCTGTGGAAGTATT；R：GTTGTCCCGGGTACCAGATAT	546	56.0	60
GLRaV-4	F：ACATTCTCCACCTTGTGCTTTT；R：CATACAAGCGAGTGCAATTAC	319	57.8	60
GRSPaV	F：TGAAGGCTTTAGGGGTTAG；R：CTTAACCCAGCCTTGAAAT	905	57.8	60
GVA	F：CCAGAGGAGTTTGAGACAATA；R：GTCCCGACCAAGGCGATGTACCC	196	56.0	60
GVB	F：ATCAGCAAACACGCTTGAACCG；R：GTGCTAAGAACGTCTTCACAGC	460	56.0	40
GVE	F：TGTGGGGTGCATAGTCATAGGTTT；R：GCTTTTGACTCCATTGGCTTTCTC	478	56.0	40
GFLV	F：CCAAAGTTGGTTTCCCAAGA；R：ACCGGATTGACGTGGGTGAT	321	56.0	40
GFabV	F：GGAAATAACATTCCCAGGAAG；R：TCCTGCTGTCAAATTTCATTC	325	50.0	40
GINV	F：WMGKGTTTCTGGGAAGATTGC；R：KACSAGATCCCTCAATTCCTG	968	55.0	70
GPGV	F：GGTTTCTGGGAGGATTGCAAC；R：RCTTYGCGATCAGCTCTCTCC	1 041	55.0	70

温度/光照 + 温度/黑暗 Temperature/light + Temperature/dark	调查株数 Investigation number	存活株数 Survival number	存活率/% Survival rate
38 ℃/16 h + 38 ℃/8 h	30	10	33.33 ± 5.77 c
38 ℃/8 h + 32 ℃/8 h	30	28	93.33 ± 5.77 a
38 ℃/16 h + 32 ℃/8 h	30	23	76.67 ± 5.77 b

温度/光照 + 温度/黑暗 Temperature/light + Temperature/dark	调查株数 Investigation number	存活株数 Survival number	存活率/% Survival rate
38 ℃/16 h + 38 ℃/8 h	30	10	33.33 ± 5.77 c
38 ℃/8 h + 32 ℃/8 h	30	28	93.33 ± 5.77 a
38 ℃/16 h + 32 ℃/8 h	30	23	76.67 ± 5.77 b

温度/光照 + 温度/黑暗 Temperature/light + Temperature/dark	接种部位 Inoculation site	接种数 Inoculation number	存活率/% Survival rate
38 ℃/16 h + 38 ℃/8 h	茎尖 Shoot tip	28	50.00 ± 10.00 bc
	第1腋芽 First axillary bud	27	33.33 ± 2.87 c
	第2腋芽 Second axillary bud	27	0 d
38 ℃/8 h + 32 ℃/8 h	茎尖 Shoot tip	30	88.89 ± 9.62 a
	第1腋芽 First axillary bud	30	94.44 ± 0.96 a
	第2腋芽 Second axillary bud	30	94.44 ± 0.96 a
38 ℃/16 h + 32 ℃/8 h	茎尖 Shoot tip	29	91.67 ± 7.63 a
	第1腋芽 First axillary bud	28	100.00 a
	第2腋芽 Second axillary bud	28	55.56 ± 0.98 b