草莓离体再生及根癌农杆菌介导CBF1基因遗传转化的研究
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摘要
草莓（Fragaria×ananassa Duch）是世界上最重要的经济浆果之一。多年来草莓新品种选育的主要途径是杂交育种，但由于草莓本身遗传性状的高度杂合和多倍性，使得常规育种费时费力、周期长，见效慢，而且目标性状与不良性状连锁，造成育种效率低，不能适应草莓生产发展的需要。耐寒耐旱、抗虫、抗病等优良品种的缺乏仍是目前草莓大田生产上一个严重的问题。植物基因工程的发展和应用则为草莓育种开辟了一条新途径。由于农杆菌介导的遗传转化主要依赖于受体材料的再生能力及农杆菌的转化效率，因此，首先良好而高效的受体再生体系建立是进行成功的基因转化关键所在。本试验以草莓品种达斯莱克特、童子1号、枥乙女、意大利1号为试材，研究了草莓离体再生过程中基因型、基本培养基、激素配比、蔗糖及琼脂浓度、苗龄和外植体等对草莓离体再生不定芽的影响，获得了草莓高效的离体再生体系。在此基础上，对影响农杆菌转化效率的主要因素进行筛选，得到一个最佳的转化体系。通过根癌农杆菌介导将转录激活因子CBF1基因导入草莓基因组，获得转化植株。Kan筛选、PCR检测证明目的基因已经整合到草莓基因组中。叶片抗寒电导率测定和植株抗寒生长恢复实验均证实了CBF1基因已在草莓转化植株中得到表达。
主要研究结果如下：
（一）建立了草莓离体再生体系
1 草莓不同基因型再生能力差异很大，再生频率94%~0%。在四个供试草莓品种中达斯莱克特再生能力最强，童子1号和意大利1号次之，枥乙女再生能力最弱，几乎不能诱导出芽。
2 在不定芽诱导中以3.0mg/L6-BA+0.1mg/L 2,4-D对达斯莱克特叶片诱导效果最佳，低浓度的2,4-D有利于不定芽再生。IAA 、IBA、NAA分别以浓度1.0、0.5、0.1 mg/L诱导较好，但它们对不定芽的总体诱导效果都不及2,4-D。高浓度2,4-D、IBA、NAA不利叶片再生。TDZ对达斯莱克特无明显促进效果，且不及6-BA。
3 基本培养基MS+B5大大促进了叶片不定芽的分化。蔗糖、琼脂浓度分别以3%、0.7%诱导效果最好。
4 叶片再生能力远大于叶柄和根。叶片采取近轴面接触培养基效果最佳。以继代35～42天的试管苗幼嫩、平展叶片作为接种外植体为宜。叶柄最高再生频率只达40%，根未诱导出不定芽。
（二）建立了草莓遗传转化体系并获得一批转化植株。
1 在农杆菌介导的基因转化中，确定了有效的Kan选择压为20 mg/L；通过抑菌实验，确定了合适的抗菌素及其浓度(即500mg/L Cef)。
2 对影响农杆菌转化效率的各种因素进行筛选，得到了一条最佳的转化途径 ：叶片在分化培养基上预培养时间为2～4天；农杆菌培养至对数生长期，用MS液体培养基重新稀释悬浮培养，调整菌液浓度为OD6000.3-0.5；侵染叶盘组织约3～5分钟；共培养2天；然后，延迟2周筛选，即将外植体接到含有500 mg/L Cef的诱导培养基上进行培养，2周后再转接到含有选择压20 mg/LKan和500 mg/LCef的诱导培养基上进行选择培养。
（三） 转基因植株PCR检测及抗寒性状鉴定
1 经20 mg/L Kan选择压初步筛选出28棵转化植株。以未转化植株为阴性对照，质粒DNA为阳性对照，CBF1基因的两端序列作为特异引物进行PCR扩增检测。检测结果表明：在这些植株中有10棵植株经PCR扩增出的特异条带和阳性对照一致，阴性对照未能扩增出任何条带。这表明CBF1基因已整合进这些植株的基因组DNA中。
2 离体叶片抗寒性鉴定。对植株离体叶片进行抗寒电导率测定，结果表明在一定的低温范围内，转基因植株电导率明显低于未转化植株，说明转基因植株的抗寒能力较未转化植株有明显提高，且在不同转基因株系之间抗寒性提高程度有着差异。
3 植株抗寒性鉴定。对植株进行抗寒生长恢复实验，转基因植株和未转化植株冻害程度及生长恢复状况表现出较大差异，证实转基因植株抗寒性明显强于未转化植株。
Strawberry is one of the most important commercial fruit crops in the world. Because of the genetic limitations associated with high heterozygosity and polyploidy, it is laborious, costly, and time-consuming to improve strawberry quality and yield by the conventional methods of breeding, which usually results in a low frequency. A major challenge remains in commercial strawberry cultivars for the lack of good varieties with resistance to coldness, insects and disease. Recent advances and applications in genetic engineering technology provide a new path for strawberry breeding and selection by way of incorporating foreign genes into plant genome for desired agronomic traits. However, an efficient tissue culture system is thought to be crucial to the success of plant genetic engineering, since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors, one being the regeneration ability of the infected tissue, and the other, the infection efficiency of Agrobacterium.
In this research, we have studied the effects of a series of cultural factors (genotypes, basic media, hormone concentrations, sucrose and agar concentrations, leaf ages and explants) in the inducing adventitious shoot of strawberry, respectively. Then on the basic of high efficient regeneration system obtained above, we established an efficient and quick agrobacterium-mediated transgenic system of strawberry, with which the transcription factor CBF1 gene was successfully transformed into the strawberry genome. The major results can be summarized as below:
1. The strawberry regeneration system has been established successfully.
Regeneration frequency varied dramatically with genotype of strawberry, ranging from 94% to 0%. Of four strawberry cultivars, the regeneration frequency of Dasrlect was the most highest, whereas the strawberry Liyinv was observed to be very difficult to regenerate.
The combination of 3mg/L 6-BA and 0.1mg/L 2,4-D were found to be the most efficient of Dasrlect. And 2, 4-D was better than IAA, IBA and NAA, all of 2, 4-D, IBA and NAA with high concentration were not beneficial to induce shoot of Dasrlect. In addition, it had no obvious effects on the shoot formation of Dasrlect with TDZ at the levels tested in this study.
It was suitable to induce shoot with 0.7% agar and 3% sucrose. And the regeneration
frequency was significantly enhanced by the MS+B5 basic medium.
The regeneration capacity of leaf was higher far than petiole and root. Leaf age can affect also the regeneration frequency and 28～35 days was the best. The leaf disks were cultured with the adaxial surface in contact with the regeneration media. The maximum regeneration frequency of petiole only got 40%, whereas the roots did not induced any advenvious bud.
2. An efficient strawberry transgenic system has been established.
In the agrobacterium-mediated gene transformation, the selecting pressure is 20mg/L Kan. And the optical bacteriophage is 500mg/L Cef.
At first, the leaf disks should be precultured for 2～4 days in the inducing medium. While bacterium concentration for inoculation is OD6000.3-0.5, the inoculation time within 3～5 minutes is very suitable, then cocultured for 2 days. The cocultured leaf disks were transferred to the inducing medium containing 500mg/L Cef. After two weeks, the disks were transferred to the inducing medium supplementing with 20mg/L Kan and 500mg/L Cef .
3. PCR assaying and chilling-resistance test
Twenty eight regenerated Kanamycin resistant plants were obtained from the transformation above, and they were assayed by PCR reaction with the untransformed plants as negative control, plasmid DNA as positive control and the sequence of CBF1gene as specific primers. Ten plants showed specific positive band same as the plasmid control, while untransformed plants have not any band. The result indicated that the CBF1gene has been integrated into the strawberry genome.
Electrolyte leakage test of leaf demonstrated that the freezing tolerance of the transgenic plants, whic
主要研究结果如下：
（一）建立了草莓离体再生体系
1 草莓不同基因型再生能力差异很大，再生频率94%~0%。在四个供试草莓品种中达斯莱克特再生能力最强，童子1号和意大利1号次之，枥乙女再生能力最弱，几乎不能诱导出芽。
2 在不定芽诱导中以3.0mg/L6-BA+0.1mg/L 2,4-D对达斯莱克特叶片诱导效果最佳，低浓度的2,4-D有利于不定芽再生。IAA 、IBA、NAA分别以浓度1.0、0.5、0.1 mg/L诱导较好，但它们对不定芽的总体诱导效果都不及2,4-D。高浓度2,4-D、IBA、NAA不利叶片再生。TDZ对达斯莱克特无明显促进效果，且不及6-BA。
3 基本培养基MS+B5大大促进了叶片不定芽的分化。蔗糖、琼脂浓度分别以3%、0.7%诱导效果最好。
4 叶片再生能力远大于叶柄和根。叶片采取近轴面接触培养基效果最佳。以继代35～42天的试管苗幼嫩、平展叶片作为接种外植体为宜。叶柄最高再生频率只达40%，根未诱导出不定芽。
（二）建立了草莓遗传转化体系并获得一批转化植株。
1 在农杆菌介导的基因转化中，确定了有效的Kan选择压为20 mg/L；通过抑菌实验，确定了合适的抗菌素及其浓度(即500mg/L Cef)。
2 对影响农杆菌转化效率的各种因素进行筛选，得到了一条最佳的转化途径 ：叶片在分化培养基上预培养时间为2～4天；农杆菌培养至对数生长期，用MS液体培养基重新稀释悬浮培养，调整菌液浓度为OD6000.3-0.5；侵染叶盘组织约3～5分钟；共培养2天；然后，延迟2周筛选，即将外植体接到含有500 mg/L Cef的诱导培养基上进行培养，2周后再转接到含有选择压20 mg/LKan和500 mg/LCef的诱导培养基上进行选择培养。
（三） 转基因植株PCR检测及抗寒性状鉴定
1 经20 mg/L Kan选择压初步筛选出28棵转化植株。以未转化植株为阴性对照，质粒DNA为阳性对照，CBF1基因的两端序列作为特异引物进行PCR扩增检测。检测结果表明：在这些植株中有10棵植株经PCR扩增出的特异条带和阳性对照一致，阴性对照未能扩增出任何条带。这表明CBF1基因已整合进这些植株的基因组DNA中。
2 离体叶片抗寒性鉴定。对植株离体叶片进行抗寒电导率测定，结果表明在一定的低温范围内，转基因植株电导率明显低于未转化植株，说明转基因植株的抗寒能力较未转化植株有明显提高，且在不同转基因株系之间抗寒性提高程度有着差异。
3 植株抗寒性鉴定。对植株进行抗寒生长恢复实验，转基因植株和未转化植株冻害程度及生长恢复状况表现出较大差异，证实转基因植株抗寒性明显强于未转化植株。
Strawberry is one of the most important commercial fruit crops in the world. Because of the genetic limitations associated with high heterozygosity and polyploidy, it is laborious, costly, and time-consuming to improve strawberry quality and yield by the conventional methods of breeding, which usually results in a low frequency. A major challenge remains in commercial strawberry cultivars for the lack of good varieties with resistance to coldness, insects and disease. Recent advances and applications in genetic engineering technology provide a new path for strawberry breeding and selection by way of incorporating foreign genes into plant genome for desired agronomic traits. However, an efficient tissue culture system is thought to be crucial to the success of plant genetic engineering, since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors, one being the regeneration ability of the infected tissue, and the other, the infection efficiency of Agrobacterium.
In this research, we have studied the effects of a series of cultural factors (genotypes, basic media, hormone concentrations, sucrose and agar concentrations, leaf ages and explants) in the inducing adventitious shoot of strawberry, respectively. Then on the basic of high efficient regeneration system obtained above, we established an efficient and quick agrobacterium-mediated transgenic system of strawberry, with which the transcription factor CBF1 gene was successfully transformed into the strawberry genome. The major results can be summarized as below:
1. The strawberry regeneration system has been established successfully.
Regeneration frequency varied dramatically with genotype of strawberry, ranging from 94% to 0%. Of four strawberry cultivars, the regeneration frequency of Dasrlect was the most highest, whereas the strawberry Liyinv was observed to be very difficult to regenerate.
The combination of 3mg/L 6-BA and 0.1mg/L 2,4-D were found to be the most efficient of Dasrlect. And 2, 4-D was better than IAA, IBA and NAA, all of 2, 4-D, IBA and NAA with high concentration were not beneficial to induce shoot of Dasrlect. In addition, it had no obvious effects on the shoot formation of Dasrlect with TDZ at the levels tested in this study.
It was suitable to induce shoot with 0.7% agar and 3% sucrose. And the regeneration
frequency was significantly enhanced by the MS+B5 basic medium.
The regeneration capacity of leaf was higher far than petiole and root. Leaf age can affect also the regeneration frequency and 28～35 days was the best. The leaf disks were cultured with the adaxial surface in contact with the regeneration media. The maximum regeneration frequency of petiole only got 40%, whereas the roots did not induced any advenvious bud.
2. An efficient strawberry transgenic system has been established.
In the agrobacterium-mediated gene transformation, the selecting pressure is 20mg/L Kan. And the optical bacteriophage is 500mg/L Cef.
At first, the leaf disks should be precultured for 2～4 days in the inducing medium. While bacterium concentration for inoculation is OD6000.3-0.5, the inoculation time within 3～5 minutes is very suitable, then cocultured for 2 days. The cocultured leaf disks were transferred to the inducing medium containing 500mg/L Cef. After two weeks, the disks were transferred to the inducing medium supplementing with 20mg/L Kan and 500mg/L Cef .
3. PCR assaying and chilling-resistance test
Twenty eight regenerated Kanamycin resistant plants were obtained from the transformation above, and they were assayed by PCR reaction with the untransformed plants as negative control, plasmid DNA as positive control and the sequence of CBF1gene as specific primers. Ten plants showed specific positive band same as the plasmid control, while untransformed plants have not any band. The result indicated that the CBF1gene has been integrated into the strawberry genome.
Electrolyte leakage test of leaf demonstrated that the freezing tolerance of the transgenic plants, whic
引文
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40 Grahan B J, Mcnicol R J, Greig K. Towards genetic based insect resistance in strawberry using the Cowpea trypsin inhabitor gene. Ann. Appl. Biol, 1995, 127: 163~173
41 Georges F, Saleem M, et al. Design and cloning of a synthetic gene of the flounder antifreeze protein and its expression in plant cells.1990,91:159~165
42 Hennie J, Plessis D U, Brand R J. Efficient genetic transformation of strawberry(Fragaria×Anannassa Duch) cultivar Selecta. Acta Horticulturae, 1997 ,447:
289~ 293
43 Highttower R and Baden C. Expression of antifreeze proteins in transgenic plant.Plant Mol. Biol., 1991,17:1013～1021.
44 Hutteman C A, Preece J E. Thidiazuron,a potent cytokinin for woody plant culture. Plant Cell Tissue Organ Culture,1993,33:105
45 James D J, Passey A. Factors affecting high frequency plant regeneration from apple leaf tissues cultured in vitro.Journal of Plant Physiology, 1988,132,:148~154
46 James D J, Pessey A J, Barbara D J. Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria×Anannassa Duch) using disarmed binary vectors. Plant Sci., 1990，69:79~94
47 Jaglo-Ottosen k R, Gilmour S J, Zarka D G et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 1998, 280:140~144
48 Janssen B J, Gardner R C. The use of transient GUS expression to develop an Agrobacterium-mediated gene transfer system for kiwifruit. Plant Cell Rep, 1993,3:28~31
49 Kasuga M, Liu Q, Miura S et al. Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotech,1999,
17:287~291
50 Liu Q, Kasuga M, Sakuina Y et al. Two transcription flactors, DREB1 and DREB2,with an EREB/Ap2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression respectively, in Arabidopsis. Plant Cell,1998, 10: 1391～1406.
51 Lyons J M and RaisonJ K. Oxidative activity mitochondria isolated from plant tissued sentive and resistant to chilling injury. Plant Physiol. 1970, 45: 386～389
52 Martinelli L, Rugini E, Saccardo F. Genetic transformation for biotic stress resistance in horticultural plants. In Vitro, 1996, 32(3):69~70
53 Murata N, Yamaki T. Temperature-dependent phase behavior of phosphatidylgylcerols from chilling-sentive and chilling-resistant plant. Plant Physiol., 1984,74:1016～1024.
54 Murata N. Molecular species composition of phosphatidylgycerols from chilling-sentive plant. 1992, Nature, 365:710.
55 Murata N, Lshizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y and Nishida I. Genetically engineered alteration in the chilling sensitivity of plants. Nature, 1992,356(23):710～713
56 Murata N, Yamaki T. Temperature-dependent phase behavior of phosphatidylgylcerols from chilling-sentive and chilling-resistant plant[J]. Plant Physiol, 1984,74:1016～1024.
57 Metter I J. A simple and rapid method for minipreparation of DNA from tissue-cultured plant cells. Plant Mol Biol Rep, 1987, 5:346~349
58 Nehra N S, Stushnoff C. Direct shoot regeneration from strawberry leaf disks. J Amer Soc Hort Sci, 1989, 114(6):1014~1018
59 Nehra N S, Chibbar R N, Kartha K K et al. Genatic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Reports, 1990, 9:293~298
60 Nehra N S, Chibbar R N,Kartha K K et al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants. Plant Cell Reports, 1990,1:10~13
61 Nyman M, Wallin A. Plant regeneration from strawberry mesophy II protoplasts. J Plant Physiol, 1988, 133:375~377
62 Nyman M, Wallin A. Improved cultured technique for strawberry protoplasts and the determination of DNA contend in protoplasts derived plants. Plant Cell, Tiss ue and Organ Culture,1992,30:127~133
63 Owens L D, Smigocki A C. Transformation of soybean cells using mixed strains of
Agrobacterium turrefaciens and phenolic compounds. Plant Physiol,1988, 88:570~573
64 Passey A J, Barrent K J, James D J. Adrentitrous shoot regeneration from seven commercial strawberry cultivars using a range of explants types. Plant Cell Rep, 2003,21:397~401
65 Palmer C E. Enhanced shoot regeneration from Brassica campestris by silver nitrate. Plant Cell Rep, 1992, 11:541~545
66 Sukumaran N P, Weiser C J. An excised leaflet test for evaluating potato frost tolerance. Hortiscience, 1972,7:467
67 Rugini E, Orlando R. High-efficiency shoot regeneration from calluses of strawberry stipules of in vitro shoot cultures. J Hortic Sci,1992,67:577~582
68 Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DR E, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997,94:1035～1 040
69 Gilmour S J, Zarka D G, Stockinger E J et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step incold-induced COR gene expression. Plant J, 1998,16(4):433~442
70 Thomashow M F. Role of cold responsive gene in plant freezing tolerance. Plant Physiol, 1998, 118:1~7
71 Thomshow M F. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol, 1999,50:571~599
72 Teng W L. Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap .Plant Cell Rep, 1999, 18:363~368
73 Zhu B. Expression of an ABA-responsive osmotion-like gene during the induction of freezing tolerance in solanum commersonii. Plant Mol Physiol.1990, 93:81～84.
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27张志宏, 方宏筠, 景士西 等. 苹果主载品种高效遗传转化系统的建立及其影响因子的研究. 遗传学报,1998,25(2):160~165
28张松, 温孚江, 魏毓棠. 不同基因型大白菜芽分化及激素浓度的优化模型. 山东农业大学学报, 2002,33（1）:127-135
29张小红, 张红燕, 武军 等. TDZ对香椿愈伤组织诱导及芽增殖生长等的影响. 西北农林科技大学学报, 2002,30(5):35~38
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33赵政阳, 曹晓玲, 黄英 等. 叶片成熟度对苹果试管苗叶片再生植株的影响. 陕西农业科学，1997，（1）：20~21
34 Ashby A M, Watsan M D, Loake G J. Ti-plasmid specified chemofaxis of Agrobacterium tumefaciens toward vir-induction phenolic compounds and soluble factors from monocotyledonous plants. J. Bacteriol, 1998,170:4181~4187
35 Atkinson R G. Plant Cell Reports, 1993,12:341~351
36 Caboni E, Tonelli M G. Effect of 1.2-benzisoxcizole-3-aotic acid on adventitious shoot regeneration and in vitro rooting in apple. Plant Cell Reports, 1999,18:985~ 988
37 Chi G L. Effect of AgNo3 and amincethokyvinyl glycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes. PlantCell Rep,1990, 9:195~198
38 F.-L.zhang, Y.Takahata, J.-B.Xu. Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage L Brassia campestrisL.ssp.Pekinensis). Plant Cell Report,1998,17:780~78
39 Finstad K, Martin R R. Transformation of strawberry for virus resistance. Acta Horticulturace,1995, 385:86~90
40 Grahan B J, Mcnicol R J, Greig K. Towards genetic based insect resistance in strawberry using the Cowpea trypsin inhabitor gene. Ann. Appl. Biol, 1995, 127: 163~173
41 Georges F, Saleem M, et al. Design and cloning of a synthetic gene of the flounder antifreeze protein and its expression in plant cells.1990,91:159~165
42 Hennie J, Plessis D U, Brand R J. Efficient genetic transformation of strawberry(Fragaria×Anannassa Duch) cultivar Selecta. Acta Horticulturae, 1997 ,447:
289~ 293
43 Highttower R and Baden C. Expression of antifreeze proteins in transgenic plant.Plant Mol. Biol., 1991,17:1013～1021.
44 Hutteman C A, Preece J E. Thidiazuron,a potent cytokinin for woody plant culture. Plant Cell Tissue Organ Culture,1993,33:105
45 James D J, Passey A. Factors affecting high frequency plant regeneration from apple leaf tissues cultured in vitro.Journal of Plant Physiology, 1988,132,:148~154
46 James D J, Pessey A J, Barbara D J. Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria×Anannassa Duch) using disarmed binary vectors. Plant Sci., 1990，69:79~94
47 Jaglo-Ottosen k R, Gilmour S J, Zarka D G et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 1998, 280:140~144
48 Janssen B J, Gardner R C. The use of transient GUS expression to develop an Agrobacterium-mediated gene transfer system for kiwifruit. Plant Cell Rep, 1993,3:28~31
49 Kasuga M, Liu Q, Miura S et al. Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotech,1999,
17:287~291
50 Liu Q, Kasuga M, Sakuina Y et al. Two transcription flactors, DREB1 and DREB2,with an EREB/Ap2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression respectively, in Arabidopsis. Plant Cell,1998, 10: 1391～1406.
51 Lyons J M and RaisonJ K. Oxidative activity mitochondria isolated from plant tissued sentive and resistant to chilling injury. Plant Physiol. 1970, 45: 386～389
52 Martinelli L, Rugini E, Saccardo F. Genetic transformation for biotic stress resistance in horticultural plants. In Vitro, 1996, 32(3):69~70
53 Murata N, Yamaki T. Temperature-dependent phase behavior of phosphatidylgylcerols from chilling-sentive and chilling-resistant plant. Plant Physiol., 1984,74:1016～1024.
54 Murata N. Molecular species composition of phosphatidylgycerols from chilling-sentive plant. 1992, Nature, 365:710.
55 Murata N, Lshizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y and Nishida I. Genetically engineered alteration in the chilling sensitivity of plants. Nature, 1992,356(23):710～713
56 Murata N, Yamaki T. Temperature-dependent phase behavior of phosphatidylgylcerols from chilling-sentive and chilling-resistant plant[J]. Plant Physiol, 1984,74:1016～1024.
57 Metter I J. A simple and rapid method for minipreparation of DNA from tissue-cultured plant cells. Plant Mol Biol Rep, 1987, 5:346~349
58 Nehra N S, Stushnoff C. Direct shoot regeneration from strawberry leaf disks. J Amer Soc Hort Sci, 1989, 114(6):1014~1018
59 Nehra N S, Chibbar R N, Kartha K K et al. Genatic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Reports, 1990, 9:293~298
60 Nehra N S, Chibbar R N,Kartha K K et al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants. Plant Cell Reports, 1990,1:10~13
61 Nyman M, Wallin A. Plant regeneration from strawberry mesophy II protoplasts. J Plant Physiol, 1988, 133:375~377
62 Nyman M, Wallin A. Improved cultured technique for strawberry protoplasts and the determination of DNA contend in protoplasts derived plants. Plant Cell, Tiss ue and Organ Culture,1992,30:127~133
63 Owens L D, Smigocki A C. Transformation of soybean cells using mixed strains of
Agrobacterium turrefaciens and phenolic compounds. Plant Physiol,1988, 88:570~573
64 Passey A J, Barrent K J, James D J. Adrentitrous shoot regeneration from seven commercial strawberry cultivars using a range of explants types. Plant Cell Rep, 2003,21:397~401
65 Palmer C E. Enhanced shoot regeneration from Brassica campestris by silver nitrate. Plant Cell Rep, 1992, 11:541~545
66 Sukumaran N P, Weiser C J. An excised leaflet test for evaluating potato frost tolerance. Hortiscience, 1972,7:467
67 Rugini E, Orlando R. High-efficiency shoot regeneration from calluses of strawberry stipules of in vitro shoot cultures. J Hortic Sci,1992,67:577~582
68 Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DR E, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997,94:1035～1 040
69 Gilmour S J, Zarka D G, Stockinger E J et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step incold-induced COR gene expression. Plant J, 1998,16(4):433~442
70 Thomashow M F. Role of cold responsive gene in plant freezing tolerance. Plant Physiol, 1998, 118:1~7
71 Thomshow M F. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol, 1999,50:571~599
72 Teng W L. Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap .Plant Cell Rep, 1999, 18:363~368
73 Zhu B. Expression of an ABA-responsive osmotion-like gene during the induction of freezing tolerance in solanum commersonii. Plant Mol Physiol.1990, 93:81～84.