Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (10): 48-54    DOI: 10.13523/j.cb.20181006
技术与方法     
‘贵长’猕猴桃叶片高效直接再生体系的建立 *
赵许朋,赵晓朋,施豪,陈学梅,姜婷,刘燕()
贵阳学院 贵阳 550005
Establishment of High Frequency Regeneration via Leaf Explants of ‘Guichang’ Kiwifruit (Actinidia chinensis)
Xu-peng ZHAO,Xiao-peng ZHAO,Hao SHI,Xue-mei CHEN,Ting JIANG,Yan LIU()
Guiyang University, Guiyang 550005, China
 全文: PDF(813 KB)   HTML
摘要：

以‘贵长’猕猴桃叶片为外植体,直接脱分化产生不定芽,并对不定芽增殖以及生根体系进行优化,建立了其高效直接再生体系。结果表明,叶片在MS+4.0mg/L 6-BA+0.4mg/L NAA培养基中,不定芽诱导率达95.8%,平均出芽数达15.7个/叶片;不定芽在MS+3.0mg/L 6-BA+0.3mg/L NAA+0.2mg/L GA3培养基中,增殖率达100%,且1~6代平均繁殖系数达8.15;不定芽先在添加1.0mg/L IBA的1/2 MS固体培养基中诱导7d,然后再先后在1/2 MS固体培养基和充分吸附1/2 MS培养液的珍珠岩中各培养14d,生根率达98.61%,且根系发育良好;50株试管苗移栽到以珍珠岩和田间土壤(其体积比为1∶4)为基质的营养钵中,2周后成活49株,成活率达98%。该研究成功建立了‘贵长’猕猴桃叶片高效再生体系,该方法不定芽诱导周期短,出芽率高且数目多,不定芽增殖系数大,生根率高且试管苗根系发达,为‘贵长’猕猴桃离体快速繁殖和遗传转化奠定了基础。
关键词： ‘贵长’猕猴桃;;  叶片;  不定芽;  植株再生    
Abstract:

A high efficient in vitro regeneration system was developed from leaf explants of ‘Guichang’ kiwifruit (Actinidia chinensis) and the multiplication coefficient and rooting rate of adventitious buds were also optimized. Results revealed that the adventitious buds developing directly from leaf explants were noticed after 30d of culture. The maximum regeneration frequency of adventitious buds is 95.8% and 15.7 shoots was observed in each leaf explants when MS medium was supplemented with 4.0mg/L 6-BA+0.4mg/L NAA. The optimal culture medium for bud proliferation is MS+3.0mg/L 6-BA+0.3mg/L NAA+0.2mg/L GA3 and the proliferation coefficient reached 8.15 in every subculture during 1 to 6 subcultures. On the rooting medium with 1/2 MS+1.0mg/L IBA for 15d, the adventitious plantlets were successively transferred into agar power and matrix perlite supplied with 1/2 MS liquid medium for 15d and 95.83% of them rooted and the roots were also heavy and strong very much. 49 out of 50 plantlets (99%) survived acclimatization in the greenhouse after transplanting them to the Mixed matrix of pearlite and soil(1∶4)for two weeks. In conclusion, a highly efficient regeneration protocol via leaf explants of ‘Guichang’ kiwifruit was successfully established and it may provide theoretical and technical guidance for micropropagation and genetic transformation studies of ‘Guichang’ kiwifruit.
Key words: ‘Guichang’ kiwifruit (Actinidia chinensis);    Leaves    Adventitious buds    Plant regeneration
收稿日期: 2018-07-01 出版日期: 2018-11-09
ZTFLH:  Q813.1+2  
基金资助: * 贵州省教育厅重点(黔教合KY字[2015]382号);贵州省科技厅联合基金(黔科合LH字[2015]7310号);贵州省一流大学 一流师资团队建设(培育)(2017158322);贵州省大学生创新创业训练计划(201610976005)
通讯作者: 刘燕     E-mail: gyly68@sina.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
赵许朋
赵晓朋
施豪
陈学梅
姜婷
刘燕

引用本文:

赵许朋,赵晓朋,施豪,陈学梅,姜婷,刘燕. ‘贵长’猕猴桃叶片高效直接再生体系的建立 *[J]. 中国生物工程杂志, 2018, 38(10): 48-54.

Xu-peng ZHAO,Xiao-peng ZHAO,Hao SHI,Xue-mei CHEN,Ting JIANG,Yan LIU. Establishment of High Frequency Regeneration via Leaf Explants of ‘Guichang’ Kiwifruit (Actinidia chinensis). China Biotechnology, 2018, 38(10): 48-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181006    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I10/48

编号
No.		 植物生长调节剂/mg/L
Plant growth regulators		 接种外植体数
No. of leaves		 出芽叶片数/个
Explants No.of
buds formation		 出芽率/%
Induction rate of
adventitious buds		 平均芽长/cm
Average No.of buds
A1 6-BA 3.0+NAA 0.2 24 9.3±0.88d 37.5±4.17f 3.7±0.33d
A2 6-BA 4.0+NAA 0.2 24 10.7±1.20d 45.8±6.36ef 7.3±0.67c
A3 6-BA 5.0+NAA 0.2 24 15.0±2.08c 62.5±8.67de 4.3±0.67d
A4 6-BA 3.0+NAA 0.4 24 16.0±1.73bc 66.7±7.22cd 10.7±1.45b
A5 6-BA 4.0+NAA 0.4 24 23.0±1.00a 95.8±4.17a 15.7±0.88a
A6 6-BA 5.0+NAA 0.4 24 19.7±1.20ab 83.3±4.17abc 10.3±0.88b
A7 6-BA 3.0+NAA 0.6 24 16.3±1.76bc 70.8±4.81bcd 11.0±1.25b
A8 6-BA 4.0+NAA 0.6 24 23.0±0.58a 95.8±2.41a 11.3±1.20b
A9 6-BA 5.0+NAA 0.6 24 21.0±1.53a 87.5±6.36ab 10.0±0.58bc
表1  植物生长调节剂对‘贵长’猕猴桃叶片分化不定芽的影响
编号
No.		 植物生长调节剂/mg/L
Plant growth regulators		 增殖率/%
Multiplied
rate/%		 各代繁殖系数/倍 Propagation coefficients
in clonal generations /Times		 平均增殖倍数Average
multiplied times
1 2 3 4 5 6
B1 6-BA 2.0+NAA 0.3+GA3 0.2 73.6±3.67b 2.11 2.07 2.00 1.89 1.88 1.97 1.99±0.03e
B2 6-BA 3.0+NAA 0.3+GA3 0.2 100±0.00a 8.14 8.22 8.17 8.29 8.03 8.03 8.15±0.04a
B3 6-BA 4.0+NAA 0.3+GA3 0.2 100±0.00a 5.36 5.40 5.38 5.44 5.25 5.23 5.34±0.03d
B4 6-BA 2.0+NAA 0.6+GA3 0.2 61.1±3.67b 1.76 1.79 1.72 1.86 1.78 1.76 1.78±0.02f
B5 6-BA 3.0+NAA 0.6+GA3 0.2 95.8±2.41a 5.68 5.67 5.53 5.75 5.60 5.60 5.64±0.03c
B6 6-BA 4.0+NAA 0.6+GA3 0.2 100±0.00a 6.21 6.22 6.06 6.19 5.97 6.14 6.13±0.04b
表2  植物生长调节剂浓度‘贵长’猕猴桃不定芽增殖的影响
培养基
编号
Media No.		 吲哚丁酸
IBA/mg-1		 接种不定芽数
No. of adventitious
bud inoculated		 生根不定芽数
No. of adventitious
bud rooted		 生根率/%
Rate of rooting		 平均根数/条
Average No.
of root		 平均根长/cm
Average length
of root
C1 0 24 10.33±0.88e 43.06±3.67e 0.78±0.12d 0.44±0.08b
C2 0.4 24 12.00±2.31de 50.00±9.62de 1.22±0.22d 0.51±0.05b
C3 0.6 24 15.67±1.45cd 65.28±6.05cd 2.01±0.14c 0.85±0.01a
C4 0.8 24 20.33±1.20ab 84.72±5.01ab 3.47±0.26b 0.86±0.01a
C5 1.0 24 23.67±0.33a 98.61±1.39a 5.31±0.15a 0.81±0.01a
C6 1.2 24 19.33±0.67bc 80.56±2.78bc 3.53±0.10b 0.76±0.02a
表3  IBA浓度对‘贵长’猕猴桃不定芽生根的影响
图1  ‘贵长’猕猴桃叶片植株再生
[1] 赵许朋, 周月, 杨立 , 等. ‘红阳’猕猴桃茎段高效再生体系的建立. 西南大学学报(自然科学版), 2013,35(2):6-10.
Zhao X P, Zhou Y, Yang L , et al. Establishment of a high frequently regeneration system from stems segments of ‘Gui chang’ kiwifruit(Actinidia chinensis). Journal of Southwest University(Natural Science Edition), 2013,35(2):6-10.
[2] 蒲文江, 满玉萍, 雷瑞 , 等. 猕猴桃观花品‘江山娇’与中华猕猴桃回交后代的性别分离与开花性状变异. 植物科学学报, 2017,35(5):723-734.
doi: 10.11913/PSJ.2095-0837.2017.50723
Pu W J, Man Y P, Lei R , et al. Variation in sex ratio and flowering traits in backcross hybrid populations between ornamental Jiangshanjiao and male Actinidia chinensis planch. Plant Science Journal, 2017,35(5):723-734.
doi: 10.11913/PSJ.2095-0837.2017.50723
[3] 金方伦, 黎明, 韩成敏 . ‘贵长’猕猴桃在黔北地区的生物学特性及丰产优质栽培技术. 贵州农业科学, 2009,37(10):175-177.
Jin F L, Li M, Han C M . Biological characteristic of Guichang Chinese gooseberry and its cultivation technique with high yield and quality in Qianbei areas. Guizhou Agricultural Sciences, 2009,37(10):175-177.
[4] 王金华, 杜超, 梁晨 , 等. ‘贵长’猕猴桃多糖提取工艺及体外抗氧化功能. 食品科学, 2016,37(20):19-23.
doi: 10.7506/spkx1002-6630-201620004
Wang J H, Du C, Liang C , et al. Extraction and antioxidant activity of polysaccharides from Guichang kiwifruit. Food Science, 2016,37(20):19-23.
doi: 10.7506/spkx1002-6630-201620004
[5] 阳小成, 王伯初, 叶志义 , 等. 中华猕猴桃的组织培养及其实用快速繁殖. 重庆大学学报(自然科学版), 2002,25(6):75-77.
doi: 10.3969/j.issn.1000-582X.2002.06.021
Yang X C, Wang B C, Ye Z Y , et al. Tissue culture and rapid propagation of Actinidia chinensis. Journal of Chongqing University(Natural Science Edition), 2002,25(6):75-77.
doi: 10.3969/j.issn.1000-582X.2002.06.021
[6] 刘长春, 陈泽雄, 龚雪芹 , 等. 金富猕猴桃离体培养与植株再生的优化研究. 西南师范大学学报(自然科学版), 2007,32(5):124-128.
doi: 10.3969/j.issn.1000-5471.2007.05.028
Liu C C, Chen Z X, Gong X Q , et al. Studies on optimization of the conditions for in vitro culture and plantlet regeneration of Actinidia chinensis cv. JinFu. Journal of Southwest China Normal University(Natural Science Edition), 2007,32(5):124-128.
doi: 10.3969/j.issn.1000-5471.2007.05.028
[7] 汪克强, 周建峰, 王晓兵 . 红阳猕猴桃的栽培与管理. 陕西林业科技, 2010,2010(5):68-71.
doi: 10.3969/j.issn.1001-2117.2010.05.020
Wang K Q, Zhou J F, Wang X B . Cultivation and management of red sun kiwifruit. Shanxi Forest Science and Technology, 2010,2010(5):68-71.
doi: 10.3969/j.issn.1001-2117.2010.05.020
[8] 刘占德, 吕岩 . 猕猴桃生产中存在的几个问题. 西北园艺, 2011,2011(4):5-6.
Liu Z D, Lv Y . Some problems in the production of kiwifruit. Northwest Horticulture, 2011,2011(4):5-6.
[9] 安婷, 季静, 王昱蓉 等. 百合鳞片的诱导分化及遗传转化效率分析. 中国生物工程杂志, 2018,38(1):25-31
An T, Ji J, Wang Y R , et al. Analysis of the transformation efficiency and induced differentiation Lilium brownii of scales. China Biotechnology, 2018,38(1):25-31.
[10] 郑云凤, 张晓曼, 刘晓 . 欧报春上下胚轴再生体系的建立. 分子植物育种, 2018,16(4):1250-1256.
Zheng Y F, Zhang X M, Liu X . Establishment of regeneration system of epicotyls and hypocotyls of primrose (Primula vulgaris). Molecular Plant Breeding, 2018,16(4):1250-1256.
[11] Sablok G, Budak H, Ralph P. Brachypodium genomics: methods and protocols. State of New Jersey: Humana Press, 2017: 37-72.
[12] Yan Q, Karau M J, Greenwood-Quaintance K E , et al. Comparison of diagnostic accuracy of periprosthettic tissue culture in blood culture bottles to that of prosthesis sonication fluid culture for diagnosis of prosthetic joint infection (PJI) by use of Bayesian latent class modeling and IDSA PJI criteria for classification. Journal of Clinical Microbiology, 2018,56(6):1-11.
[13] Bi J H, Liu Y L, Asghar S . In vitro organogenesis and plant regeneration from leaf explants of Actinidia latifolia. Journal of Fruit Science, 2005,22(4):405-408.
[14] Abdin M Z, Kiran U, Kamaluddin , et al. Plant biotechnology: principles and applications. Berlin: Springer, 2017: 289-310.
doi: 10.1007/978-981-10-2961-5
[15] 胡选萍, 秦公伟, 曹小勇 . 蓝莓组织培养技术的研究进展. 分子植物育种, 2018,16(3):960-965.
Hu X P, Qin G W, Cao X Y . Research progress on tissue culture technology of blueberry. Molecular Plant Breeding, 2018,16(3):960-965.
[16] Alhasnawi A N . Tissue culture technician and in vitro screening of rice (Oryza sativa L.) callus for salt tolerance. Journal of Global Pharma Technology, 2018,11(9):67-74.
[17] 赵许朋, 罗克明, 周月 , 等. ‘红阳’猕猴桃叶盘高频直接再生体系的建立. 生物工程学报, 2013,29(11):1599-1606.
Zhao X P, Luo K M, Zhou Y , et al. Establishment of high frequency regeneration via leaf explants of ‘Red Sun’ kiwifruit (Actinidia chinensis). Chinese Journal of Biotechnology, 2013,29(11):1599-1606.
[18] Ibrahim R, Debergh P C . Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.). Scientia Horticulturae, 2001,88(1):41-57.
doi: 10.1016/S0304-4238(00)00189-8
[19] Akba? F, I?ikalan C, Namli S . Callus induction and plant regeneration from different explants of Actinidia deliciosa. Applied Biochemistry and Biotechnology, 2009,158(2):470-475.
doi: 10.1007/s12010-008-8401-2 pmid: 18975140
[20] Kim M, Kim S C, Moon D Y , et al. Rapid shoot propagation from micro-cross sections of kiwifruit (Actinidia deliciosa cv. ‘Hayward’). Journal of Plant Biology, 2007,50(6):681-686.
doi: 10.1007/BF03030613
[21] 尚霄丽, 马春华, 冯建灿 . 中华猕猴桃叶片再生体系的建立. 江西农业学报, 2010,22(4):50-52.
doi: 10.3969/j.issn.1001-8581.2010.04.016
Shang X L, Ma C H, Feng J C . Establishment of regeneration system from leaves of Actinidia chinensis. Acta Agriculturae Jiangxi, 2010,22(4):50-52.
doi: 10.3969/j.issn.1001-8581.2010.04.016
[22] Takahashi W, Sugawara F, Yamamoto N , et al. Plant regeneration in Actinidia polygama Miq. by leaf, stem, and petiole culture with zeatin, and from stem-derived calli on low-sucrose medium. Journal of Forestry Research, 2004,9(1):85-88.
doi: 10.1007/s10310-003-0053-z
[23] Prado M J, Gonzalez M V, Romo S , et al. Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation. Plant Cell Tissue and Organ Culture, 2007,88(1):1-10.
doi: 10.1007/s11240-006-9116-0
[24] Lan D W, Liu Y L, Yuan T L . Organogenesis, somatic embryogenesis and plantlet regeneration from leaf explants of Actinidia kolomikta cultured in vitro. Journal of Fruit Science, 2007,24(2):218-222.
[25] 严姜黎, 张翼, 邢梅 , 等. 红肉猕猴桃离体快速繁殖技术研究. 华中农业大学学报, 2008,27(1):101-104.
doi: 10.3321/j.issn:1000-2421.2008.01.022
Yan J L, Zhang Y, Xing M , et al. Studies on rapid micro-propagation technique of Actinidia chinensis var. rufopulpa. Journal of Huazhong Agricultural University, 2008,27(1):101-104.
doi: 10.3321/j.issn:1000-2421.2008.01.022
[26] Sotiropoulos T E, Dimassi K N . Response to increasing rates of boron and NaCl on shoot proliferation and chemical composition of in vitro kiwifruit shoot cultures. Plant Cell Tissue & Organ Culture, 2004,79(3):285-289.
doi: 10.1007/s11240-004-4609-1
[1] 任美艳,唐宽刚,薛敏,郭慧琴,王茅雁. 沙冬青AmNAC3转录因子基因在抗旱性和抗寒性中的功能鉴定 *[J]. 中国生物工程杂志, 2019, 39(8): 32-39.
[2] 岳文冉,岳俊燕,张秀娟,杨杞,韩晓东,王瑞刚,李国婧. 中间锦鸡儿CiNAC1基因促进转基因拟南芥叶片的衰老[J]. 中国生物工程杂志, 2018, 38(4): 24-29.
[3] 杨天瑞,李娜,张秀娟,杨杞,王瑞刚,李国婧. 中间锦鸡儿CibHLH027基因的克隆和功能研究*[J]. 中国生物工程杂志, 2018, 38(3): 33-40.
[4] 何官榕,何碧珠,吴沙沙,石京山,陈集双,兰思仁. 多叶斑叶兰繁殖体系建立及基于转录组的发育调控途径功能基因研究[J]. 中国生物工程杂志, 2018, 38(12): 57-64.
[5] 欧巧明, 厚毅清, 包梅年, 胡达, 陈玉梁, 岳春玲. 中药半夏单细胞悬浮培养、胚胎发生及植株再生[J]. 中国生物工程杂志, 2012, 32(10): 39-49.
[6] 卢松冲, 朱金启, 李杰, 张洪霞. 采用俄罗斯橄榄叶片和茎段诱导产生不定芽的高效再生方法[J]. 中国生物工程杂志, 2011, 31(04): 113-118.
[7] 侯春喜 赵寿经 梁彦龙 王建华. 人参遗传转化研究进展[J]. 中国生物工程杂志, 2009, 29(09): 0-0.
[8] 姚庆荣,郭运玲,孔华,郭安平. 木薯体细胞胚胎发生及植株再生研究[J]. 中国生物工程杂志, 2008, 28(12): 52-56.
[9] 王维飞,韩烈保,曾会明. 高羊茅转基因研究进展[J]. 中国生物工程杂志, 2006, 26(08): 15-21.
[10] 张永彦, 徐子勤, 高丽美, 黄萱, 王健. 多年生黑麦草成熟胚再生体系的建立及基因枪转化[J]. 中国生物工程杂志, 2005, 25(3): 53-57,59.
[11] 贾彩凤, 李悦, 瞿超. 木本植物体细胞胚胎发生技术[J]. 中国生物工程杂志, 2004, 24(3): 26-29.
[12] 李集临, 徐香玲, 陈金山. 发根农杆菌Ri质粒及其应用[J]. 中国生物工程杂志, 1993, 13(2): 8-14.
[13] 江洪. 植物细胞生物反应器的新成就[J]. 中国生物工程杂志, 1992, 12(2): 61-62.
[14] 高山真策, 何文竹. 植物的工厂化生产[J]. 中国生物工程杂志, 1987, 7(5): 68-73.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会