草莓β-1，4-葡聚糖酶基因的克隆与遗传转化
摘要
草莓(Fragaria ananassa Duch)属于蔷薇科(Rosaceae)多年生草本植物。其果实营养丰富，鲜美多汁，具有很高的营养价值和经济价值。但草莓不耐贮运，草莓果实采摘后的贮运保鲜仍是目前生产实践中亟待解决的一大难题。另外，在草莓生产中多采用无性繁殖，经多年栽培后，很容易感染多种病毒，致使种性严重退化、产量降低、品质变劣和抗性降低等。因此，如何改善草莓品质和提高其抗病性已越来越受到人们的关注。近年来飞速发展的现代生物技术为草莓品种遗传改良提供了新途径。
本研究以目前国内主栽品种丰香和章姬为试材，较为系统地研究了影响草莓离体再生的诸多因素，如外植体类型、外源激素种类、外植体生理状态等，建立了草莓叶片高效离体再生体系，从而为草莓的遗传转化研究与应用奠定了基础。试验结果表明：不同激素组合及配比对草莓叶盘不定芽再生有不同的影响。TDZ对芽再生的效果明显比6-BA强，丰香叶盘在MS+TDZ2.0mg／L+2，4-D0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达83.2％，平均每叶盘再生芽数达1.4个；章姬则在MS+TDZ4.0mg／L+NAA0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达65.7％，平均每叶盘再生芽数为1.3个。同一基因型草莓叶盘再生效果比叶柄好，而其中又以10d左右叶龄的叶片再生能力最高。8.0mg／L AgNO_3能较好地控制外植体的褐化，促进芽的分化。再生芽在1／2MS+IBA0.1mg／L生根培养基中，生根率达100％。本文还对草莓花药离体培养进行了研究，筛选出适合草莓赵屯一号品种花药培养最佳诱导愈伤组织培养基，即F+6-BA1.0mg／L+NAA0.5mg／L+CH300mg／L，并成功诱导出再生芽获得再生植株。这为草莓花药脱毒培养和将来利用花药进行目的基因遗传转化研究提供了理论依据。
为了研究β-1,4-葡聚糖酶基因在草莓果实成熟过程中的作用及探讨利用β-1，4-葡聚糖酶反义基因培育耐贮草莓新品种，从草莓果实中提取总RNA，根据Gen Bank中β-1，4-葡聚糖酶基因FaEG1和FaEG3序列设计合成特异性引物，利
用RT一PcR技术分别扩增了两个基因1 491bp和1863bp的cDNA，反向插入到植
物表达载体PROKZ中，将该载体转入农杆菌AGL一I中，构建了石hEGI和卢h百G了
的反义基因的表达载体。通过农杆菌介导法转化草墓品种丰香和章姬后，从104
块抗卡那霉素转化愈伤中随机抽取24块经PCR检测，其中17块扩增出目的带，
PCR阳性率为70.8%，初步证实这两个基因整合到草葛染色体基因组中，抗Kan
愈伤现已分化形成12个转化芽。试验通过对农杆菌转化前外植体预培养、农杆
菌侵染时间及转化后外植体共培养等方面的探索发现，农杆菌介导的草荀基因遗
传转化的最佳条件为:预培养1d一共培养3d，农杆菌侵染时间为巧一ZOmin效
果最好。
Strawberry (Fragaria ananassa Duch) , which is a kind of herbage and belongs to the rose family in the plant kingdom. The fruit of Strawberry contains plentiful nutrient materials and richer sweet juice, and therefore has maximum nutritive value and significant market prospect. However, the present problem which severely affects strawberry production is how to keep the fruit green or to retard softening for longer time after harvesting. In addition, the vegetative propagation may result in many negative effects such as virus attacks, strain degeneration, and both yield and quality reductions. Recently, more and more technics of molecular and cell biology including high efficient plant regeneration, plant genetic transformation, have been used in the studies of improvement of the quality and disease resistance in Strawberry.
The present primary cultivars of strawberry 'Fengxiang' and 'Zhangji' were used as experimental materials. To establish a reliable, rapid and effective in vitro regeneration system and to carry the strawberry genetic transformation out successfully, the factors affecting adventitious shoots regeneration were studied. The results indicated that the frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. Compared with 6-BA, TDZ had a better effect on inducing shoot regeneration. The regeneration frequency of cultivar 'Fengxiang' reached the highest level of 83.2% in MS+TDZ2.0mg/L+2,4-D 0.1mg/L+IBA0.2mg/L, while cultivar 'Zhangji' obtained the highest level of 65.7% in MS+TDZ4.0mg/L + NAA0.1mg/L+IBA0.2mg/L medium. The corresponding numbers of shoots regeneration were 1.4 and 1.3 per leaf disc, respectively. The regeneration of the leaf disc was better than that of petiole in the same genotype and the 10-day-old leaf had higher regeneration a
bility. Our data also showed that AgNO3 played an important role in controlling the browning of explants, and the concentration of 8.0mg/L was recommended. While using the rooting medium of l/2MS+IBA0.1mg/L, the rooting rate achieved nearly 100%. In addition, the anther culture of 'Zhaotunl' was studied and the regeneration plants were obtained when cultured with F+6-BA1.0mg/L+NAA0.5mg/L+CH300mg/L medium.
To investigate the role of endo-l,4-glucanase gene in strawberry fruit ripening, total RNAs were isolated from the pink strawberry fruits. By using the specific primers designed from the FaEGl and FaEGS cDNAs, 1491bp and 1863bp fragments were amplified by reverse transcriptional polymerase chain reaction (RT-PCR). The FaEGl and FaEG3 cDNAs were inserted into expressional vector pROK2 in reverse orientations.The antisense clonings pROK2-FaEGl and pROK2-FaEG3 were therefore mobilized into Agrobacterium tumefaciens AGL-1 by electroporation. The strawberry transgenic plants were developed by Agrobacterium-mediated plant transformation with kanamycin resistance selection. Among the twenty-four Kan-resistant calli, seventeen of them showed PCR positive. At the same time, we have further studied the factors such as the pre-culture of explants, the time of infection with Agrobacterium and co-cultivation to demonstrate the appropriate conditions for genetic transformation in Strawberry. The results have clearly shown
that the conditions of pre-cultured for 1 day, co-cultured for 3 day and infected for 15-20min were favourable for the transformation!.
本研究以目前国内主栽品种丰香和章姬为试材，较为系统地研究了影响草莓离体再生的诸多因素，如外植体类型、外源激素种类、外植体生理状态等，建立了草莓叶片高效离体再生体系，从而为草莓的遗传转化研究与应用奠定了基础。试验结果表明：不同激素组合及配比对草莓叶盘不定芽再生有不同的影响。TDZ对芽再生的效果明显比6-BA强，丰香叶盘在MS+TDZ2.0mg／L+2，4-D0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达83.2％，平均每叶盘再生芽数达1.4个；章姬则在MS+TDZ4.0mg／L+NAA0.1mg／L+IBA0.2mg／L分化培养基上芽再生频率可达65.7％，平均每叶盘再生芽数为1.3个。同一基因型草莓叶盘再生效果比叶柄好，而其中又以10d左右叶龄的叶片再生能力最高。8.0mg／L AgNO_3能较好地控制外植体的褐化，促进芽的分化。再生芽在1／2MS+IBA0.1mg／L生根培养基中，生根率达100％。本文还对草莓花药离体培养进行了研究，筛选出适合草莓赵屯一号品种花药培养最佳诱导愈伤组织培养基，即F+6-BA1.0mg／L+NAA0.5mg／L+CH300mg／L，并成功诱导出再生芽获得再生植株。这为草莓花药脱毒培养和将来利用花药进行目的基因遗传转化研究提供了理论依据。
为了研究β-1,4-葡聚糖酶基因在草莓果实成熟过程中的作用及探讨利用β-1，4-葡聚糖酶反义基因培育耐贮草莓新品种，从草莓果实中提取总RNA，根据Gen Bank中β-1，4-葡聚糖酶基因FaEG1和FaEG3序列设计合成特异性引物，利
用RT一PcR技术分别扩增了两个基因1 491bp和1863bp的cDNA，反向插入到植
物表达载体PROKZ中，将该载体转入农杆菌AGL一I中，构建了石hEGI和卢h百G了
的反义基因的表达载体。通过农杆菌介导法转化草墓品种丰香和章姬后，从104
块抗卡那霉素转化愈伤中随机抽取24块经PCR检测，其中17块扩增出目的带，
PCR阳性率为70.8%，初步证实这两个基因整合到草葛染色体基因组中，抗Kan
愈伤现已分化形成12个转化芽。试验通过对农杆菌转化前外植体预培养、农杆
菌侵染时间及转化后外植体共培养等方面的探索发现，农杆菌介导的草荀基因遗
传转化的最佳条件为:预培养1d一共培养3d，农杆菌侵染时间为巧一ZOmin效
果最好。
Strawberry (Fragaria ananassa Duch) , which is a kind of herbage and belongs to the rose family in the plant kingdom. The fruit of Strawberry contains plentiful nutrient materials and richer sweet juice, and therefore has maximum nutritive value and significant market prospect. However, the present problem which severely affects strawberry production is how to keep the fruit green or to retard softening for longer time after harvesting. In addition, the vegetative propagation may result in many negative effects such as virus attacks, strain degeneration, and both yield and quality reductions. Recently, more and more technics of molecular and cell biology including high efficient plant regeneration, plant genetic transformation, have been used in the studies of improvement of the quality and disease resistance in Strawberry.
The present primary cultivars of strawberry 'Fengxiang' and 'Zhangji' were used as experimental materials. To establish a reliable, rapid and effective in vitro regeneration system and to carry the strawberry genetic transformation out successfully, the factors affecting adventitious shoots regeneration were studied. The results indicated that the frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. Compared with 6-BA, TDZ had a better effect on inducing shoot regeneration. The regeneration frequency of cultivar 'Fengxiang' reached the highest level of 83.2% in MS+TDZ2.0mg/L+2,4-D 0.1mg/L+IBA0.2mg/L, while cultivar 'Zhangji' obtained the highest level of 65.7% in MS+TDZ4.0mg/L + NAA0.1mg/L+IBA0.2mg/L medium. The corresponding numbers of shoots regeneration were 1.4 and 1.3 per leaf disc, respectively. The regeneration of the leaf disc was better than that of petiole in the same genotype and the 10-day-old leaf had higher regeneration a
bility. Our data also showed that AgNO3 played an important role in controlling the browning of explants, and the concentration of 8.0mg/L was recommended. While using the rooting medium of l/2MS+IBA0.1mg/L, the rooting rate achieved nearly 100%. In addition, the anther culture of 'Zhaotunl' was studied and the regeneration plants were obtained when cultured with F+6-BA1.0mg/L+NAA0.5mg/L+CH300mg/L medium.
To investigate the role of endo-l,4-glucanase gene in strawberry fruit ripening, total RNAs were isolated from the pink strawberry fruits. By using the specific primers designed from the FaEGl and FaEGS cDNAs, 1491bp and 1863bp fragments were amplified by reverse transcriptional polymerase chain reaction (RT-PCR). The FaEGl and FaEG3 cDNAs were inserted into expressional vector pROK2 in reverse orientations.The antisense clonings pROK2-FaEGl and pROK2-FaEG3 were therefore mobilized into Agrobacterium tumefaciens AGL-1 by electroporation. The strawberry transgenic plants were developed by Agrobacterium-mediated plant transformation with kanamycin resistance selection. Among the twenty-four Kan-resistant calli, seventeen of them showed PCR positive. At the same time, we have further studied the factors such as the pre-culture of explants, the time of infection with Agrobacterium and co-cultivation to demonstrate the appropriate conditions for genetic transformation in Strawberry. The results have clearly shown
that the conditions of pre-cultured for 1 day, co-cultured for 3 day and infected for 15-20min were favourable for the transformation!.
引文
1．邓明琴．草莓科研文选．沈阳：辽宁科技出版社．1990
2．薛光荣，扬振英，朱秋英．草莓花药培养获得无病毒植株的技术研究．植物学通报，1990，7(1)：22-26
3．薛光荣，费开伟，胡军．草莓花药离体培养获得单倍体植株．园艺学报，1981，8(1)：9-12
4. Owea H R, Miller A R. Haploid plant regeneration from anther culture of three North American cultivars of strawberry (Fragaria Ananassa Duch). Plant Cell Roports, 1996, 15(1):905-909
5. Foley I, Hennerty M J, Mass J L. et al. Field performance of androgenic strawberry plants. Acta Horticulturae, 1993,348:188-195
6. Faedi W, Quarta R, Persano S, et al. Somaclonal variations in strawberry plants regenerated by anther culture of cv. pajaro. Acta Horticulturae, 1993,348:427-431
7. Fowler C. W, Huhges HG. Janick J callus formation from strawberry anther. Hort Res, 1971, (2):116-117
8．大泽胜次，西贞夫(日)．用花药培养草莓脱毒苗的研究．农业与园艺，1974，(4)：537-540
9. Roati PM, Devrcus, laneri U. Anther culture from strawberry. Hort Science, 1975(10):119-120
10．侯喜林．不同品种和激素条件对草莓花药培养的影响．南京农业大学学报，1992，15(2)：21-28
11. Simon I, Racz E, Zatyko J M. Preliminary notes on somaclonal variations of strawberry. Fruit Science Reports, 1987,14(4):151-154
12．Niizekl H．马换普译．用聚氟苯基丙氨酸处理排除草莓体细胞的染色体．国外果树科学文摘，1984，2：161
13. Toyoda H, Horikshi K. Selection for Fusariuim wilt desease resistance from regenerates derived from leaf callus of strawberry. Plant Cell Reports, 1991,10:167-170
14. Oosawa K et at. Studies on the anther culture of vegetable crops. Bull. Veg. Ornam. Res. Station Japan, 1974, AI:41-57
15．马洪民，高公泓．草莓花药离体条件下形态发牛的研究．西北植物学报 1992，12(4)：287-294
16．杭玲，苏国秀，陈丽娟等．草莓花药培养再生植株研究．广西农业学，1999，5：227-229
17．吴伟民，余桂红，马鸿翔等．草莓花药愈伤组织继代培育和不定芽发生研究．江苏农业研究，1999，20(4)：19-23
18．韩雪梅．草莓试管苗分化培养基优化的研究．生物技术，1997，7(2)：27—29
19．尹淑萍，金万梅，王萍等．Thidiazuron对草莓外植体再生不定芽的影响．农业生物技术学报，2003，11(4)：379—382
20．柯善强，洪树荣．植物激素对草莓叶片不定芽形成的影响．武汉植物学研究，1988，6(2)：133—138
21．牛建新，鲁晓燕，于艳华．外植体和培养因子对草莓不定芽诱导的影响．北方园艺，1999，3：30—31
22. Deng X, Hu W Y. Shoot regeneration and transformation from strawberry lesf disks. Chinese Bull Bot, 2000,17:174-178
23. Badaui M A, Alphonse M and Bondok A Z et al. Propagation of some strawberry cultivars by means of tissue culture technique. Egyption Journal of Horticulture, 1990, 17(1): 9-16
24. Liu Z R and Sanford J G. Plant regeneration by organogenesis from strawberry leaf and runner tissue. Hort Science, 1998,23(6):1057-1069
25. Nehra N S, Stushnoff C and Kartha K K. Direct shoot regeneration from strawberry leaf disk. J. Amer. Soc. Hort. Sci. 1989,114(6):1014-1018
26. Nehra N S and Chebbar R N Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant cell reports, 1990(9):293-298
27．孙惊波，谢鸣，蒋桂华等．草莓主栽品种丰香高效离体再生体系的研究．浙江农业学报，2003，15(2)：69—72
28. Greene A E and davis T M. Regeneration of Fragaria vesca plants from leaf tissue. Edited by Dale; Lubby, J J, Portland, Oregon, USA. Timber press Inc. 1991, 124-126
29. Sorvari S, Ulvinen S and Hietarnta T et al. Preculture medium promotes direct shoot regeneration from micropropagated strawberry leaf disk. Hortscience, 1993,28(1):55-57
30. Marta Barcelo, Iman Mansourit, Miguel A et al. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandle. Plant Cell Tissue and Orga Culture, 1998,54:29-36
31. Murashige T. Plant propagation through tissue culture. Ann. Rev. Plant Physiol, 1974,25:135-166
32. Zhang Z H, Wu L P, Dai H Y et al. Regeneration and transformation in vitro of the strawberry varieties. Acta Hort Sinica, 2001, 28:189-193
33. Li H, Murch S J, Saxena P K. Thidiazuron-induced develop shoot organogenesis on seedings etiolated hypocotyls and stem segments of huang-qin. Plant Cell Tissue Org Cult, 2000, 62:169-173
34．覃兰英，徐光霞，邓世秀．草莓茎尖离体培养大量繁殖研究初报．中国果树，1981，3：38-41
35．邓明琴，任秀云．草莓茎尖组织培养瓶外生根试验．中国果树，1981，3：46-49
36．李青，邓世秀，张瑞清．降低草莓生根培养基大量元素及糖浓度的试验．北京农业科学，1992，10(3)：18-21
37. Nyman M, Wallin A. Plant regeneration from strawberry (Fragaria ananassa Duch.)mesophyll protoplasts. Journal Plant Physiol, 1988,133:375-377
38. Nyman M, Wallin A. Improved culture technique for strawberry (Fragaria ananassa Duch.)protoplasts and the determination of DNA contained in protoplast derived plants. Plant Cell, Tissue and Organ Culture, 1992,30:127-133
39. Infante R, Rosoti P. Fragaria vesca L. Alpine protoplast culture and regeneration. Acta Horticulturae, 1993, 348:432-434
40．史永忠，万蜀渊，薛光荣．草莓花药愈伤组织原生质体培养再生多细胞团．华中农业大学学报，1995，14(2)：187-190
41．李卫东．草莓细胞悬浮系的建立及叶肉原生质体培养技术研究．保定：河北农业大学，1997
42．周春江．草莓悬浮细胞培养及原生质体技术研究．保定：河北农业大学，1999．
43. Blando F, Niglio A, Frattarelli a. Cell suspension cultures in strawberry: growth characterization and variability. Acta Horticulturae. 1993, 336:257-262
44. McGranahan G H. Lislic C A, Uratsu S L. Agrobacterium-mediated transformatiom of walnut somatic embryos and regeneration of transgenic plants. Bio—Technology, 1998,6:800-804
45. Jamea D J, Passey A J, et al. Genetic transformation of apple using a disarmed binary vector. Plant Cell Reports, 1989,7:658-661
46. James D J, Passey A J, et al. Agribacterium-mediated transformation of the cultivated strawberry using disarmed binary vectors. Plant Science, 1990,69:79-94
47. Nehra N S, chibbar R N, kartha K Ket al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants. Plant Cell Reports, 1990,9:10-13
48. James D J, Passey A J, Easterbrook M A et al. Progress in the introduction of transgenes for pest resistance in apple and strawberry. Phytoparasitica, 1992,20(suppi):83-87
49. Finstad K, Martin R R. Transformation of strawberry for virus resistance. Acta Horticulturae, 1995,385:86-90
50. Martinelli L, rugini E, Saccardo F. Genetic transformation for biotic stress resistance in horticultral plants. In vitro, 1996,32(3):69-70
51. Hennie J, Plessis D U, Brand R J. Efficient genetic transformation of strswberry cultivar Selekta. Acta Horticulturae, 1997,447:289-293
52. Bachelier C, Graham J, Machray G et al. Integration of an invertase gene to control sucrose metabolism in strawberry cultivars. Acta Horticulture, 1997,436:161-163
53. Dolfov S V, Lebedev V G, Firsov A P, et al. Expression of thaumatin gene in horticultural crops. Sacrasicia M, Porceddu E. Genetics and breeding for crop quality and resistance. Dordrecht, Gemany:Kluwer academic publishers, 1999,165-172
54. Mathews H, Wagoner W, Kellogg J, et al. Genetic transformation of strawberry: stable integration of a gene to control biofythesis of enthylene. In vitro Cellular and Developmental Biology Plant, 1995,31(1):36-43
55. Firsov A P, Dolgov S V. Agrobacterial transformation and transfer of the antifreeze protein gene of winter flounder to the strawberry. Acta Horticulturae, 1999,484:581-586
56．金万梅，巩振辉，李桂荣等．植物遗传转化方法和转基因植株的鉴定．陕西农业科学，2000，(1)：24-29
57. Nyman M, Wallin A. Transient gene expression in strawberry protoplasts and the recovery of transgenic plant. Plant Cell Reports, 1992,11:105-108
58. Boulter D, Edwards G A, Gatehouse A M R et al. Additive effects of different plant derived insect resistance genes in transgenic tobacco plants. Crop Protection, 1989,9:351-354
59. Hilder V A, Powell K S, Gatehouse A M R et al. Expression of snoedrop lectin in transgenic tobacco plants results in added protection against aphids. Trangenic Research, 1995,4:18-24
60. Collinge D B, Slusarenko A J. Plant gene expression in response to pathogens. Plant Molecular Biology, 1987,9:389-410
61. Bennett A, Rode J. Fruit-specific and rippening-regulation expansion genes to control fruit texture and softening. US Patent:5929303, 1997-07-27
62. Nehra N S, Stshnoof C, Kartha K K. Regeneration of plants from immature leaf derived callus of strawberry. Hort. Science, 1998,23:56
63. Jones O P, Waller B J, Beech M G. The production of strawberry plants from callus culture. Plant Cell, Tissue and Organ Culture, 1998,12:235-241
64. Mansouri E, Mercado J A, Valpuesta et al Shoot regeneration and Agrobacterium-mediated transformation of Fragaria Vesca L Plant Cell, Tissue and Organ Culture, 1996, 15(8): 642—646
65. Barcelo M, Mansouri E, Mercado J A et al. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar chandler. Plant Cell, Tissue and Organ Culture, 1998, 54(1): 29—36
66．于冬梅，胡文玉，王关林．基因型和培养因子对诱导草莓叶片再生芽的影响．沈阳农业大学学报，1998，29(2)：138—143
67．张志宏，吴禄平．草莓主栽品种Tudla遗传转化体系的建立．农业生物技术学报，1998，6(2)：200—204
68. Nyman M, Wallin A. Plant regeneration from sttawberry mesophyll protoplasts. Plant Physiol, 1988,133:375-377
69. Nyman M, Wallin A. Improved culture technique for strawberry protoplasts and determination of DNA content in protoplasts derived plants. Plant Cell, Tissue and Organ Culture, 1992,30:127-133
70. Kathen A, Coboson H J, Agrobacterium tumefaciens mediated transformation of pium sativum L. using binary and contegrate vectors. Plant Cell Reports, 1990,9:276-279
71．李卫，郭光沁，郑昌．根癌农杆菌介导遗传转化研究的若干新进展．科学通报，2000，45(8)：798-807
72. Brummel, D A., Lashbrook, E. C. and Bennett, A. B. Plant endo-1, 4-β-D-glucanasea:structure, properties and physiological function. American Chemical Society, pp. 1994:100-129
73．兰海燕，陈正华．葡聚糖酶及其在植物中的发育调节和防卫反应．生物技术通报，1998(4)：10—15
74．生吉萍，申琳，罗云波．果实成熟衰老相关酶的研究进展．食品与机械，2000，3：7—9
75. Redgwell RJ, MacRace E, Hallett I et al. In vivo and in vitro swelling of cell walls during fruit ripening. Planta, 1997,203:162-173.
76. Huber, D.J. The role of cell wall hydrolases in fruit softening. Horticultural Reviews, 1983, pp:169-219
77. Brady, C.J. Fruit ripening. Annu, Rev. Plant Physiol,1987,38:155-178
78. Rhodes, M.J.C. The maturation and ripening of fruit. In Senescence in Plants, 1980, pp:157-205
79. Harpster, M.H. Brummell, D.A. and Dunsmuir, P. Expression analysis of a ripening-specific, auxin-repressed endo-1, 4-β-D-glucanasea gene in strawberry. Plant Physiol, 1998,118:1037-1316
80. Trainotti L., Spolaore, S., Pavanello, A., et al. A novel E-type endo-1, 4-β-D-glucanasea with a putative cellulose-binding domain is highly espressed in ripening strawberry fruits. Plant Mpl. Biol. 1999b, 40:323—332
81. Llop-Tous, L., Donminguez-Puigianer, E., Palomer, X. et al. Characterization of two divergent endo-1, 4-β-D-glucanasea cDNA clones highly express in non-climacteric strawberry fruit. Plant Physiol, 1999, 119:1415-1421
82. Trainotti, L., Ferrarese, L., Dalla Vecchia, F., er al. Two different endo-1, 4-β-D-glucanasea contribute to the softening of strawberry fruit. J. Plant Physiol, 1999a, 154:355-362
83. Brummell, D.A. and Harpster, M.H. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology, 2001,47:311-340
84. Abeles F.B., Takeda F. Cellulase activity and ethylene in ripening strawberry and apple fruits. Sci Hortic, 1990,42:269-275
Lashbrook, C.C., Gonzalez-Bosch and Bennette A.B., Two divergent endo-β-1, 4-glucanase genes exhibit overbit overlapping expression in ripening fruit and abscising flowers. Plant Cell, 1994,6:1485-1493
85. Lashbrook C.C., Giovannoni J.J., Hall B.D. et al. Transgenic analysis of tomato endo-1, 4-β-glucanase gene function. Role of cell in floral abscission. Plant J, 1998,13:303-310
86. Silvia B, Jose R N, Juan M Bet al. Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiology, 2002,128,751-759
87．潘景丽，高公泓．小麦花粉胚的产生及不同激素水平等因子对诱导频率的影响．花药培养讨论会文集．北京：科学出版社，1977：99—106
88. Nitsch C., Kasha K J. Haploids in higher plant-advance and potentials. Univ. Gueiphx, 1974:123-135
89. Sunderland N. Plant Tissue Culture, Beijing:Sci. Press, 1978:65-71
90. Rashid A. Advance in anther culture. Physiol. Plant, 1983, 58:544-551
91. Saini N S et al. Requirement for ethylene synthesis and action during relief of thermoinhibition of seed germination by combinations of GA, KT and Carbon dioxide. Plant Physiol. 1986, 81:950-953
92. Guye M G et al. Chilling-induced ethylene production in relation to chill-sensitivity in phaseolus spp. J. Exp. Bot. 1987, 38:680-688
93. Songstad D D, Armstrong C L, Retersan W L. AgNO_3 increase type Ⅱ callus induction from immature embryos of maize inbred B_(73) and its derivatives. Plant Cell Reports, 1991, 9: 699-702
94．卫志明，黄健秋，徐淑平等．甘蓝下胚轴的高效再生和农杆菌介导B．t基因转化甘蓝．上海农业学报，1998，4(2)：11-18
95．黎定军．辣椒高效离体再生与遗传转化体系建立及抗病基因的研究．湖南农业大学博士学位论文，2001
2．薛光荣，扬振英，朱秋英．草莓花药培养获得无病毒植株的技术研究．植物学通报，1990，7(1)：22-26
3．薛光荣，费开伟，胡军．草莓花药离体培养获得单倍体植株．园艺学报，1981，8(1)：9-12
4. Owea H R, Miller A R. Haploid plant regeneration from anther culture of three North American cultivars of strawberry (Fragaria Ananassa Duch). Plant Cell Roports, 1996, 15(1):905-909
5. Foley I, Hennerty M J, Mass J L. et al. Field performance of androgenic strawberry plants. Acta Horticulturae, 1993,348:188-195
6. Faedi W, Quarta R, Persano S, et al. Somaclonal variations in strawberry plants regenerated by anther culture of cv. pajaro. Acta Horticulturae, 1993,348:427-431
7. Fowler C. W, Huhges HG. Janick J callus formation from strawberry anther. Hort Res, 1971, (2):116-117
8．大泽胜次，西贞夫(日)．用花药培养草莓脱毒苗的研究．农业与园艺，1974，(4)：537-540
9. Roati PM, Devrcus, laneri U. Anther culture from strawberry. Hort Science, 1975(10):119-120
10．侯喜林．不同品种和激素条件对草莓花药培养的影响．南京农业大学学报，1992，15(2)：21-28
11. Simon I, Racz E, Zatyko J M. Preliminary notes on somaclonal variations of strawberry. Fruit Science Reports, 1987,14(4):151-154
12．Niizekl H．马换普译．用聚氟苯基丙氨酸处理排除草莓体细胞的染色体．国外果树科学文摘，1984，2：161
13. Toyoda H, Horikshi K. Selection for Fusariuim wilt desease resistance from regenerates derived from leaf callus of strawberry. Plant Cell Reports, 1991,10:167-170
14. Oosawa K et at. Studies on the anther culture of vegetable crops. Bull. Veg. Ornam. Res. Station Japan, 1974, AI:41-57
15．马洪民，高公泓．草莓花药离体条件下形态发牛的研究．西北植物学报 1992，12(4)：287-294
16．杭玲，苏国秀，陈丽娟等．草莓花药培养再生植株研究．广西农业学，1999，5：227-229
17．吴伟民，余桂红，马鸿翔等．草莓花药愈伤组织继代培育和不定芽发生研究．江苏农业研究，1999，20(4)：19-23
18．韩雪梅．草莓试管苗分化培养基优化的研究．生物技术，1997，7(2)：27—29
19．尹淑萍，金万梅，王萍等．Thidiazuron对草莓外植体再生不定芽的影响．农业生物技术学报，2003，11(4)：379—382
20．柯善强，洪树荣．植物激素对草莓叶片不定芽形成的影响．武汉植物学研究，1988，6(2)：133—138
21．牛建新，鲁晓燕，于艳华．外植体和培养因子对草莓不定芽诱导的影响．北方园艺，1999，3：30—31
22. Deng X, Hu W Y. Shoot regeneration and transformation from strawberry lesf disks. Chinese Bull Bot, 2000,17:174-178
23. Badaui M A, Alphonse M and Bondok A Z et al. Propagation of some strawberry cultivars by means of tissue culture technique. Egyption Journal of Horticulture, 1990, 17(1): 9-16
24. Liu Z R and Sanford J G. Plant regeneration by organogenesis from strawberry leaf and runner tissue. Hort Science, 1998,23(6):1057-1069
25. Nehra N S, Stushnoff C and Kartha K K. Direct shoot regeneration from strawberry leaf disk. J. Amer. Soc. Hort. Sci. 1989,114(6):1014-1018
26. Nehra N S and Chebbar R N Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant cell reports, 1990(9):293-298
27．孙惊波，谢鸣，蒋桂华等．草莓主栽品种丰香高效离体再生体系的研究．浙江农业学报，2003，15(2)：69—72
28. Greene A E and davis T M. Regeneration of Fragaria vesca plants from leaf tissue. Edited by Dale; Lubby, J J, Portland, Oregon, USA. Timber press Inc. 1991, 124-126
29. Sorvari S, Ulvinen S and Hietarnta T et al. Preculture medium promotes direct shoot regeneration from micropropagated strawberry leaf disk. Hortscience, 1993,28(1):55-57
30. Marta Barcelo, Iman Mansourit, Miguel A et al. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandle. Plant Cell Tissue and Orga Culture, 1998,54:29-36
31. Murashige T. Plant propagation through tissue culture. Ann. Rev. Plant Physiol, 1974,25:135-166
32. Zhang Z H, Wu L P, Dai H Y et al. Regeneration and transformation in vitro of the strawberry varieties. Acta Hort Sinica, 2001, 28:189-193
33. Li H, Murch S J, Saxena P K. Thidiazuron-induced develop shoot organogenesis on seedings etiolated hypocotyls and stem segments of huang-qin. Plant Cell Tissue Org Cult, 2000, 62:169-173
34．覃兰英，徐光霞，邓世秀．草莓茎尖离体培养大量繁殖研究初报．中国果树，1981，3：38-41
35．邓明琴，任秀云．草莓茎尖组织培养瓶外生根试验．中国果树，1981，3：46-49
36．李青，邓世秀，张瑞清．降低草莓生根培养基大量元素及糖浓度的试验．北京农业科学，1992，10(3)：18-21
37. Nyman M, Wallin A. Plant regeneration from strawberry (Fragaria ananassa Duch.)mesophyll protoplasts. Journal Plant Physiol, 1988,133:375-377
38. Nyman M, Wallin A. Improved culture technique for strawberry (Fragaria ananassa Duch.)protoplasts and the determination of DNA contained in protoplast derived plants. Plant Cell, Tissue and Organ Culture, 1992,30:127-133
39. Infante R, Rosoti P. Fragaria vesca L. Alpine protoplast culture and regeneration. Acta Horticulturae, 1993, 348:432-434
40．史永忠，万蜀渊，薛光荣．草莓花药愈伤组织原生质体培养再生多细胞团．华中农业大学学报，1995，14(2)：187-190
41．李卫东．草莓细胞悬浮系的建立及叶肉原生质体培养技术研究．保定：河北农业大学，1997
42．周春江．草莓悬浮细胞培养及原生质体技术研究．保定：河北农业大学，1999．
43. Blando F, Niglio A, Frattarelli a. Cell suspension cultures in strawberry: growth characterization and variability. Acta Horticulturae. 1993, 336:257-262
44. McGranahan G H. Lislic C A, Uratsu S L. Agrobacterium-mediated transformatiom of walnut somatic embryos and regeneration of transgenic plants. Bio—Technology, 1998,6:800-804
45. Jamea D J, Passey A J, et al. Genetic transformation of apple using a disarmed binary vector. Plant Cell Reports, 1989,7:658-661
46. James D J, Passey A J, et al. Agribacterium-mediated transformation of the cultivated strawberry using disarmed binary vectors. Plant Science, 1990,69:79-94
47. Nehra N S, chibbar R N, kartha K Ket al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants. Plant Cell Reports, 1990,9:10-13
48. James D J, Passey A J, Easterbrook M A et al. Progress in the introduction of transgenes for pest resistance in apple and strawberry. Phytoparasitica, 1992,20(suppi):83-87
49. Finstad K, Martin R R. Transformation of strawberry for virus resistance. Acta Horticulturae, 1995,385:86-90
50. Martinelli L, rugini E, Saccardo F. Genetic transformation for biotic stress resistance in horticultral plants. In vitro, 1996,32(3):69-70
51. Hennie J, Plessis D U, Brand R J. Efficient genetic transformation of strswberry cultivar Selekta. Acta Horticulturae, 1997,447:289-293
52. Bachelier C, Graham J, Machray G et al. Integration of an invertase gene to control sucrose metabolism in strawberry cultivars. Acta Horticulture, 1997,436:161-163
53. Dolfov S V, Lebedev V G, Firsov A P, et al. Expression of thaumatin gene in horticultural crops. Sacrasicia M, Porceddu E. Genetics and breeding for crop quality and resistance. Dordrecht, Gemany:Kluwer academic publishers, 1999,165-172
54. Mathews H, Wagoner W, Kellogg J, et al. Genetic transformation of strawberry: stable integration of a gene to control biofythesis of enthylene. In vitro Cellular and Developmental Biology Plant, 1995,31(1):36-43
55. Firsov A P, Dolgov S V. Agrobacterial transformation and transfer of the antifreeze protein gene of winter flounder to the strawberry. Acta Horticulturae, 1999,484:581-586
56．金万梅，巩振辉，李桂荣等．植物遗传转化方法和转基因植株的鉴定．陕西农业科学，2000，(1)：24-29
57. Nyman M, Wallin A. Transient gene expression in strawberry protoplasts and the recovery of transgenic plant. Plant Cell Reports, 1992,11:105-108
58. Boulter D, Edwards G A, Gatehouse A M R et al. Additive effects of different plant derived insect resistance genes in transgenic tobacco plants. Crop Protection, 1989,9:351-354
59. Hilder V A, Powell K S, Gatehouse A M R et al. Expression of snoedrop lectin in transgenic tobacco plants results in added protection against aphids. Trangenic Research, 1995,4:18-24
60. Collinge D B, Slusarenko A J. Plant gene expression in response to pathogens. Plant Molecular Biology, 1987,9:389-410
61. Bennett A, Rode J. Fruit-specific and rippening-regulation expansion genes to control fruit texture and softening. US Patent:5929303, 1997-07-27
62. Nehra N S, Stshnoof C, Kartha K K. Regeneration of plants from immature leaf derived callus of strawberry. Hort. Science, 1998,23:56
63. Jones O P, Waller B J, Beech M G. The production of strawberry plants from callus culture. Plant Cell, Tissue and Organ Culture, 1998,12:235-241
64. Mansouri E, Mercado J A, Valpuesta et al Shoot regeneration and Agrobacterium-mediated transformation of Fragaria Vesca L Plant Cell, Tissue and Organ Culture, 1996, 15(8): 642—646
65. Barcelo M, Mansouri E, Mercado J A et al. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar chandler. Plant Cell, Tissue and Organ Culture, 1998, 54(1): 29—36
66．于冬梅，胡文玉，王关林．基因型和培养因子对诱导草莓叶片再生芽的影响．沈阳农业大学学报，1998，29(2)：138—143
67．张志宏，吴禄平．草莓主栽品种Tudla遗传转化体系的建立．农业生物技术学报，1998，6(2)：200—204
68. Nyman M, Wallin A. Plant regeneration from sttawberry mesophyll protoplasts. Plant Physiol, 1988,133:375-377
69. Nyman M, Wallin A. Improved culture technique for strawberry protoplasts and determination of DNA content in protoplasts derived plants. Plant Cell, Tissue and Organ Culture, 1992,30:127-133
70. Kathen A, Coboson H J, Agrobacterium tumefaciens mediated transformation of pium sativum L. using binary and contegrate vectors. Plant Cell Reports, 1990,9:276-279
71．李卫，郭光沁，郑昌．根癌农杆菌介导遗传转化研究的若干新进展．科学通报，2000，45(8)：798-807
72. Brummel, D A., Lashbrook, E. C. and Bennett, A. B. Plant endo-1, 4-β-D-glucanasea:structure, properties and physiological function. American Chemical Society, pp. 1994:100-129
73．兰海燕，陈正华．葡聚糖酶及其在植物中的发育调节和防卫反应．生物技术通报，1998(4)：10—15
74．生吉萍，申琳，罗云波．果实成熟衰老相关酶的研究进展．食品与机械，2000，3：7—9
75. Redgwell RJ, MacRace E, Hallett I et al. In vivo and in vitro swelling of cell walls during fruit ripening. Planta, 1997,203:162-173.
76. Huber, D.J. The role of cell wall hydrolases in fruit softening. Horticultural Reviews, 1983, pp:169-219
77. Brady, C.J. Fruit ripening. Annu, Rev. Plant Physiol,1987,38:155-178
78. Rhodes, M.J.C. The maturation and ripening of fruit. In Senescence in Plants, 1980, pp:157-205
79. Harpster, M.H. Brummell, D.A. and Dunsmuir, P. Expression analysis of a ripening-specific, auxin-repressed endo-1, 4-β-D-glucanasea gene in strawberry. Plant Physiol, 1998,118:1037-1316
80. Trainotti L., Spolaore, S., Pavanello, A., et al. A novel E-type endo-1, 4-β-D-glucanasea with a putative cellulose-binding domain is highly espressed in ripening strawberry fruits. Plant Mpl. Biol. 1999b, 40:323—332
81. Llop-Tous, L., Donminguez-Puigianer, E., Palomer, X. et al. Characterization of two divergent endo-1, 4-β-D-glucanasea cDNA clones highly express in non-climacteric strawberry fruit. Plant Physiol, 1999, 119:1415-1421
82. Trainotti, L., Ferrarese, L., Dalla Vecchia, F., er al. Two different endo-1, 4-β-D-glucanasea contribute to the softening of strawberry fruit. J. Plant Physiol, 1999a, 154:355-362
83. Brummell, D.A. and Harpster, M.H. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology, 2001,47:311-340
84. Abeles F.B., Takeda F. Cellulase activity and ethylene in ripening strawberry and apple fruits. Sci Hortic, 1990,42:269-275
Lashbrook, C.C., Gonzalez-Bosch and Bennette A.B., Two divergent endo-β-1, 4-glucanase genes exhibit overbit overlapping expression in ripening fruit and abscising flowers. Plant Cell, 1994,6:1485-1493
85. Lashbrook C.C., Giovannoni J.J., Hall B.D. et al. Transgenic analysis of tomato endo-1, 4-β-glucanase gene function. Role of cell in floral abscission. Plant J, 1998,13:303-310
86. Silvia B, Jose R N, Juan M Bet al. Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiology, 2002,128,751-759
87．潘景丽，高公泓．小麦花粉胚的产生及不同激素水平等因子对诱导频率的影响．花药培养讨论会文集．北京：科学出版社，1977：99—106
88. Nitsch C., Kasha K J. Haploids in higher plant-advance and potentials. Univ. Gueiphx, 1974:123-135
89. Sunderland N. Plant Tissue Culture, Beijing:Sci. Press, 1978:65-71
90. Rashid A. Advance in anther culture. Physiol. Plant, 1983, 58:544-551
91. Saini N S et al. Requirement for ethylene synthesis and action during relief of thermoinhibition of seed germination by combinations of GA, KT and Carbon dioxide. Plant Physiol. 1986, 81:950-953
92. Guye M G et al. Chilling-induced ethylene production in relation to chill-sensitivity in phaseolus spp. J. Exp. Bot. 1987, 38:680-688
93. Songstad D D, Armstrong C L, Retersan W L. AgNO_3 increase type Ⅱ callus induction from immature embryos of maize inbred B_(73) and its derivatives. Plant Cell Reports, 1991, 9: 699-702
94．卫志明，黄健秋，徐淑平等．甘蓝下胚轴的高效再生和农杆菌介导B．t基因转化甘蓝．上海农业学报，1998，4(2)：11-18
95．黎定军．辣椒高效离体再生与遗传转化体系建立及抗病基因的研究．湖南农业大学博士学位论文，2001