‘岱玉杏’抗寒基因AmEBP1转化试验研究
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摘要
杏(Armeniaca vulgaris Lam.)系蔷薇科(Rosaceac)杏属植物。杏原产于我国,种质资源极为丰富,是中国栽培历史最久的果树之一。但由于春季低温和晚霜,常使花芽、花,甚至幼果受冻,产量和经济效益受到极大的损失,因此选育抗寒性强的品种有重要意义。近十几年来,国内外迅速发展的转基因技术已经在育种中得到了广泛的应用,并且已经取得了长足发展,转基因技术已经成为改良品种最有前途的技术方法之一。花粉管导入法在农作物上取得了令人鼓舞的成绩,但是在果树上应用的研究却极少,由于果树育种周期长,不断地摸索试验快速有效的育种方法是很有意义的。本研究对杏进行了抗寒基因转化试验研究,并成功将沙冬青抗寒基因AmEBP1通过花粉管成功转化入岱玉杏T0代中,并对组培苗进行生理指标测定,其主要研究内容如下:
1.岱玉杏×凯特杏杂交亲和,坐果率达到57%,果实横径、纵经、侧径可达4.081cm,4.403cm,4.412cm,均高于自然授粉的岱玉杏,表现出现杂种优势。不同处理导入DNA对转化率有一定影响,切除1/2柱头滴注外源DNA能提高转化率,但对坐果率有一定影响;花粉基因处理的坐果率较正常花粉坐果率下降9.5%;
2.采用不同浓度DNA导入对转化率有一定的影响,当DNA浓度分别为200μg/mL、500μg/mL、800μg/mL时,坐果率分别为21.5%、26.4%、12.7%,坐果率没有随着外源DNA浓度高低而逐渐升高或降低,而是以500μg/mL的浓度相对比较高,3个DNA浓度处理的坐果率远低于对照;导入DNA浓度在500μg/mL时,切除花柱1/2时转基因成功率最高,在第7天时能达到75%,60天后为30%,但基因导入后随时间的延长,阳性比率仍然出现下降的趋势,说明基因丢失仍然存在。
3.采用改良的WPM培养基,对岱玉杏不同胚发育时期培养结果得出,在改良WPM培养基上,PF值高于0.3时才能形成愈伤,附加激素BA在0.6 mg/mL-0.8 mg/mL时,愈伤诱导率最高,在PF=0.7时成苗率最高;成熟胚在MS和改良WPM上均能萌动,但有叶小、卷曲、畸形、或玻璃化,根芽生长不良的情况出现。
4.在对岱玉杏,大果杏,凯特杏,金太阳4个品种成熟胚培养结果表明,低温处理的时间和4个品种杏的萌发呈现正相关,随着低温处理时间的延长,4个品种杏的萌动迹象越发明显,成苗率都有提升。大果杏在处理60天后,大田出苗率基本可以达到100﹪,是最易成苗的品种。岱玉杏无论种胚腐烂率很高,在60﹪以上。
5.本研究得到岱玉杏转基因苗木。16株经过PCR检测阳性,Southern杂交检测有11个分别出现1-3条杂交带,另一个转基因植株中只出现了微弱的杂交带,证明AmEBP1基因已经整合到T0代组培苗中。
6.通过对转基因株系与未转基因株系组培苗抗寒生理对比,转基因株系(组培苗)LT50为-4.37℃<对照-4.12℃;SOD、POD活性与可溶性蛋白含量在-5℃到-10℃之前达到最大值,总体均有先升后降的趋势,并有转基因高于对照组的;游离脯氨酸含量总体上均呈现“上升”的趋势;随着温度的降低MDA含量增加,-5℃之后转基因植株增幅速率明显低于对照。
7.试验饲喂转基因杏果后,鸡群与羊群没有明显病症,测量血常规得出对照组与转基因组血液中红细胞数、白细胞数、血红蛋白量、红细胞压积、淋巴细胞比率,其指标值均无显著差异(P>0.05),并未对动物造成病害。
Apricot department rosaceae apricot genus, Apricot is one own fruit trees of China, it distributing east-north of our country. Because of poignant chance of the temperature in early spring, the bud, flower and even young fruit will be hurt by the low temperature, and the production of the apricot fruit will be lower.So it is very important to breeding frost-resistant strong varieties .Last ten years it has made rapid development in transgenic technology.It has been widely used in fruit breeding at home and abroad. Transgenic has become one of the most promising methods to improve fruit varieties.Now to import the DNA by pollen tube it has been successfully used in ccrops, but rarely in fruit, which is reason to the long breeding cycle in fruit.So it is very meaningful to find a effective methods in breeding. My study use Daiyu apricot transfer the cold hardiness gene (AmEBP1) from Sand Holly by pollen tube and then determin the physiological indexes of the seedling cultivation. We have gain results followed.
1.After Haidai jade apricot♀cross Kate♂and transferred with outer DNA, fruit-bearing rate reached 57%, horizontal diameter,vertical diameter and side diameter got 4.081 cm, can reach 4.403 cm, 4.412 cm,which are higher than Tarzan haidai jade apricot in the natural pollination., There heterosis performance appears.We gained the highest rate of conversion of DNA by cutting the 1/2 tube of flower,But there were certain influence to fruit-bearing rate.So take the method of cutting the 1/2 instead. Fruit-bearing rate by pollen gene treatment were less down 9.5% than normal pollen fruit-bearing rate;
2.Using different concentrations of DNA conversion rate have certain effect, when DNA concentration for 200μg/mL respectively/mL, 500μg/mL, 800μg/mL, fruit-bearing rate for targeting were 21.5%,26.4%, 12.7%. Fruit-bearing rate was not as exogenous DNA concentration levels and gradually raise or lower, but the most with 500μg/mL and cutting the 1/2 tube of flower.which can achieve 75% in the first seven days, 30% 60 days later. But there were still that gene lost trend still exists after gene transgenic.
3.Take the modified WPM media for the different embryos period, The results show that it can form callus only when PF>0.3,and the callus induction get the most by PF=0.7 and 6-BA=0.6 mg/ml. Mature embryos can budding on the the modified WPM and MS medias.But there appears little leaves, curly, deformity, or vitrification, stressing the situation. poor growth situations.
4. The results show that low processing time and the germination of 4 species apricot was positively correlated. With the extension of treatment time, signs of germination about the 4 varieties of apricot are more obvious, seedling rate has increase. In dealing with Daguo apricot 60 days, the field emergence almost can reach 100%,which is the the most vulnerable to seedling specie.Taishan Daiyu has a high rate of embryo rot either under 4℃or 0℃,and it has the low rate of seedling. That shows embryo of Taishan Daiyu is dysplasia and perishable.
5. We get some transgenic seedlings of Taishan Daiyu apricot in this study ,16 tested positive for PCR.And Southern hybridization with digoingenin labeled DNA probe results showed 11 tissue culture had 1-3 respectively.It showed that exogenous AmEBPI gene were successfully transformed into T0 tissue culture of Taishan Daiyu.
6. By the cold resistance contrast detection between transgenic plants and non-transgenic plants,it showed that LT50 of the transgenic plants was -4.37℃ 7. Test feeding genetically modified apricots, chickens and flocks have no obvious symptoms, measuring blood tests that control group and transgenic group, the number of the the indexes such as RBC, WBC, HGB, HCT, LYM, were no significant difference (P > 0.05), which did not cause disease to animals.
1.岱玉杏×凯特杏杂交亲和,坐果率达到57%,果实横径、纵经、侧径可达4.081cm,4.403cm,4.412cm,均高于自然授粉的岱玉杏,表现出现杂种优势。不同处理导入DNA对转化率有一定影响,切除1/2柱头滴注外源DNA能提高转化率,但对坐果率有一定影响;花粉基因处理的坐果率较正常花粉坐果率下降9.5%;
2.采用不同浓度DNA导入对转化率有一定的影响,当DNA浓度分别为200μg/mL、500μg/mL、800μg/mL时,坐果率分别为21.5%、26.4%、12.7%,坐果率没有随着外源DNA浓度高低而逐渐升高或降低,而是以500μg/mL的浓度相对比较高,3个DNA浓度处理的坐果率远低于对照;导入DNA浓度在500μg/mL时,切除花柱1/2时转基因成功率最高,在第7天时能达到75%,60天后为30%,但基因导入后随时间的延长,阳性比率仍然出现下降的趋势,说明基因丢失仍然存在。
3.采用改良的WPM培养基,对岱玉杏不同胚发育时期培养结果得出,在改良WPM培养基上,PF值高于0.3时才能形成愈伤,附加激素BA在0.6 mg/mL-0.8 mg/mL时,愈伤诱导率最高,在PF=0.7时成苗率最高;成熟胚在MS和改良WPM上均能萌动,但有叶小、卷曲、畸形、或玻璃化,根芽生长不良的情况出现。
4.在对岱玉杏,大果杏,凯特杏,金太阳4个品种成熟胚培养结果表明,低温处理的时间和4个品种杏的萌发呈现正相关,随着低温处理时间的延长,4个品种杏的萌动迹象越发明显,成苗率都有提升。大果杏在处理60天后,大田出苗率基本可以达到100﹪,是最易成苗的品种。岱玉杏无论种胚腐烂率很高,在60﹪以上。
5.本研究得到岱玉杏转基因苗木。16株经过PCR检测阳性,Southern杂交检测有11个分别出现1-3条杂交带,另一个转基因植株中只出现了微弱的杂交带,证明AmEBP1基因已经整合到T0代组培苗中。
6.通过对转基因株系与未转基因株系组培苗抗寒生理对比,转基因株系(组培苗)LT50为-4.37℃<对照-4.12℃;SOD、POD活性与可溶性蛋白含量在-5℃到-10℃之前达到最大值,总体均有先升后降的趋势,并有转基因高于对照组的;游离脯氨酸含量总体上均呈现“上升”的趋势;随着温度的降低MDA含量增加,-5℃之后转基因植株增幅速率明显低于对照。
7.试验饲喂转基因杏果后,鸡群与羊群没有明显病症,测量血常规得出对照组与转基因组血液中红细胞数、白细胞数、血红蛋白量、红细胞压积、淋巴细胞比率,其指标值均无显著差异(P>0.05),并未对动物造成病害。
Apricot department rosaceae apricot genus, Apricot is one own fruit trees of China, it distributing east-north of our country. Because of poignant chance of the temperature in early spring, the bud, flower and even young fruit will be hurt by the low temperature, and the production of the apricot fruit will be lower.So it is very important to breeding frost-resistant strong varieties .Last ten years it has made rapid development in transgenic technology.It has been widely used in fruit breeding at home and abroad. Transgenic has become one of the most promising methods to improve fruit varieties.Now to import the DNA by pollen tube it has been successfully used in ccrops, but rarely in fruit, which is reason to the long breeding cycle in fruit.So it is very meaningful to find a effective methods in breeding. My study use Daiyu apricot transfer the cold hardiness gene (AmEBP1) from Sand Holly by pollen tube and then determin the physiological indexes of the seedling cultivation. We have gain results followed.
1.After Haidai jade apricot♀cross Kate♂and transferred with outer DNA, fruit-bearing rate reached 57%, horizontal diameter,vertical diameter and side diameter got 4.081 cm, can reach 4.403 cm, 4.412 cm,which are higher than Tarzan haidai jade apricot in the natural pollination., There heterosis performance appears.We gained the highest rate of conversion of DNA by cutting the 1/2 tube of flower,But there were certain influence to fruit-bearing rate.So take the method of cutting the 1/2 instead. Fruit-bearing rate by pollen gene treatment were less down 9.5% than normal pollen fruit-bearing rate;
2.Using different concentrations of DNA conversion rate have certain effect, when DNA concentration for 200μg/mL respectively/mL, 500μg/mL, 800μg/mL, fruit-bearing rate for targeting were 21.5%,26.4%, 12.7%. Fruit-bearing rate was not as exogenous DNA concentration levels and gradually raise or lower, but the most with 500μg/mL and cutting the 1/2 tube of flower.which can achieve 75% in the first seven days, 30% 60 days later. But there were still that gene lost trend still exists after gene transgenic.
3.Take the modified WPM media for the different embryos period, The results show that it can form callus only when PF>0.3,and the callus induction get the most by PF=0.7 and 6-BA=0.6 mg/ml. Mature embryos can budding on the the modified WPM and MS medias.But there appears little leaves, curly, deformity, or vitrification, stressing the situation. poor growth situations.
4. The results show that low processing time and the germination of 4 species apricot was positively correlated. With the extension of treatment time, signs of germination about the 4 varieties of apricot are more obvious, seedling rate has increase. In dealing with Daguo apricot 60 days, the field emergence almost can reach 100%,which is the the most vulnerable to seedling specie.Taishan Daiyu has a high rate of embryo rot either under 4℃or 0℃,and it has the low rate of seedling. That shows embryo of Taishan Daiyu is dysplasia and perishable.
5. We get some transgenic seedlings of Taishan Daiyu apricot in this study ,16 tested positive for PCR.And Southern hybridization with digoingenin labeled DNA probe results showed 11 tissue culture had 1-3 respectively.It showed that exogenous AmEBPI gene were successfully transformed into T0 tissue culture of Taishan Daiyu.
6. By the cold resistance contrast detection between transgenic plants and non-transgenic plants,it showed that LT50 of the transgenic plants was -4.37℃
引文
陈永芳,弓成林,罗安才.果树基因转移研究进展[J].四川农业大学学报,2000:186～141 卞坡,霍裕平,秦广雍,等.生物物理学报[J],1999,15(3):551～555
陈火英,张建华,钟建江,等.野生番茄耐盐性研究及其利用[J].华东理工大学学报,2001(1): 151～154
曹阳,赵东利,王仁军,等.高粱花粉管通道法导入抗虫基因的研究[J].大连大学学报,2001(6):37～42
崔叶龙,柳学余.离子注入稻胚的M1代效应[J].安徽农业大学学报,1994,21(3):306～309
常振田,赵习平,杨莉,等极早熟杏种子发芽方法的试验[J].中国果树,1998(2):27～28
陈学森,张艳敏,张连忠,等.特早熟杏育种[J].园艺学报,1996, (1): 23～27
陈永芳,弓成林,罗安才,等.果树基因转移研究进展[J].四川农业大学学报,2000,18(2):186～190
楚宗丽.核果类果树胚珠培养及子叶再生体系构建[D].农业科学院郑州果树研究所,2007
程家胜,DandekarAM,UratsaSL.苹果基因转移技术研究[J].园艺学报,1992,19(2):101～10
邓江明,简令成.植物抗冻机理研究新进展:抗冻基因表达及其功能[J].植物学报,2001,18(5):521～530
丁国华,徐仲,朱祥春,等.花粉管通道法导入抗赤星病烤烟总DNA D1代性状变异的研究[J].东北农业大学学报.2000,11(2)76～80
杜杰,兰州百合外源DNA花粉管通道法导入的初步研究[D].西北师范大学,2003
董晓颖,孟新法,李培环,等.极早熟油桃幼胚离体培养成苗相关因素探讨[J].园艺学报2007,34 (3): 739～742
冯殿齐,刘静,王玉山,等.利用氮离子注入技术转化抗寒基因(afp)初步研究[J].山东农业大学学报(自然科学版),2005,36(1): 86~92
范成民,核桃快速繁殖技术的研究[D].西北农林科技大学,2008
樊力行.北京师范大学离子注入出新花[J].中国花卉报,2004,4(20)112~115
方宏筠,王关林.黑核桃体细胞胚状体发生及其基因转化系统的建立[J].园艺学报,2000,27(6):406～412
冯莎莎.冷诱导基因转录因子CBFs导入苹果及转化株系抗寒性指标分析[D]河北农业大学,2007
郭生武,大扁杏组织培养的研究[D]西北农林科技大学,2004
何迎春,高必达.花粉管通道法转化水稻研究进展[J].福建稻麦科技,2001,120(1)123~130
李长缨,简元才.花粉管通道法在植物遗传转化中的应用[J].生物学杂志.2000,(1):105~110
侯立群,李秀芬,崔刚,等.几种遗传转化技术在核桃转基因育种中的应用[J].山东林业科技,2004,150(1):8~9
李雪君.花粉管通道法培育烟草新品系[D].河南农业大学,2007
李秀梅.梨幼胚培养与子叶不定器官再生体系的研究[D]四川农业大学,2004
李玉晖.核果类果树远缘杂交不亲和性调控研究[D].山东农业大学,2003
李梦玲,李瑞家,陶正平等.杏叶片离体繁殖研究[J].中国农学通报,2001,17(21):8～10
李永红.离子束的生物学效应及诱变机理研究进展[J].河北农业大学学报,2002(2):43~45
刘静,王长宪,王斌等.石楠抗寒基因AmGS高效转化体系的研究[J].山东农业大学学报(自然科学版),2009,40(2):191～194
刘海坤,卫志明.利用根癌农杆菌介导转化大都成熟种子胚尖获得转基因植株[J].植物生理与分子生物学学报,2004,30(6):631～636
刘庆忠,赵红军.培育苹果转基因植株的研究[J].落叶果树,2001(1):5～9
刘学春,潘春欣,宋云枝,等.一种单子叶植物总DNA提取方法的改进及应用[J].山东农业大学学报,1995,24(4):45~48
倪大虎,向太和,张盘娣,等.应用低能离子束将抗菌肽Shiva A基因导入恢复系明恢63的研究[J].安徽农业科学,2000,28(4):408～409,419
师校欣,王斌,杜国强,等.根癌农杆菌介导豇豆胰蛋白酶抑制剂基因转入苹果主栽品种[J].园艺学报,2000,27(4):282~284
孙清荣,孙洪雁,石荫坪.早熟杏幼胚子叶再生不定梢[J].植物生理学通讯,2001,37(1):38~39
桑庆亮,赖钟雄.荔枝.基枪转化研究初报[J].福建农业大学学报,2001,30(2):266～267
孙芳.沙冬青CBLI基因的功能研究[D].北京林业大学,2009
覃兰英,克热木·伊力,郑华云,等.杏砧木的组织培养[J].植物组织培养简报,1994,30(5):354～359
王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998.193～2323
王立新,孟荣华,郭仁俊,等.应用花粉管通道法进行小麦抗白粉病转基因的研究初报[J]. 农业生物技术学报,2001,(3):51~55
王玖瑞,吕增仁,刘孟军,等.杏离体根愈伤组织诱导及起源的研究[J].河北农业大学学报2000,23(2):48～51
王玖瑞,吕增仁,刘孟军,等.杏组织培养研究进展[J].河北果树,2000,(2):6～11
王玉柱,张大鹏,杨丽,等.未成熟杏离体胚培养技术的研究[J].农业生物技术学报,2000,8(4):389～391
吴爱忠.花粉管导入法培育抗除草剂的转基因水稻[J].上海农学院学报1999,17(4):237～241
吴秀红.大豆花粉管通道法导入外源DNA的适宜时期与方法探讨[J].黑龙江农业科学,2001,11(2):43~47
阎国华,周宇,张开春,等.核果类果树转基因研究进展[J].果树学报,2001,18(6):358～364
杨成民.基因枪轰击法获得转P5CS基因黑麦草植株[D]内蒙古农业大学,2004. 张玲.杏转化抗寒基因的初步研究[D].山东农业大学,2004
张茂银,刘庆昌,王子霞等.用花粉管通道法将新疆大赖草DNA导入普通小麦的研究[J].农业生物技术学报,2000(2):43～46
张志宏,方宏筠,景士西,等.苹果主栽品种高效转化系统的建立及其影响因子的研究[J].遗传学报,1998,25(2):160～165
张志宏,景士西,王关林.果树基因工程研究进展[J].果树科学,1995,12(3):188～1932
张志宏,景士西,王关林.苹果基因转移技术研究初报[J].园艺学报,1997,24(4):378～381
张志宏,吴禄平.草莓主栽品种Tudla遗传转化体系的建立[J].农业生物技术学报,1997,5(3):305～307
张志宏,王关林,方宏筠,等.果树基因工程研究进展[J].果树科学,1995,12(3):188～193
赵淑清,郭剑波,常留印.特早熟杏胚培养试验[J].中国果树,2001(1):17～18
周广芳,王成强.果树多倍体育种途径与成就[J].落叶果树,1997,22(3):21~25
A.M.Foti, F.MilanoandL. Torrisi: Amino Acid Decomposition Induced by KevIon Irradiation. Nucl.Instr. [J].Meth 1990, B46:361-363
Ahmet Esitken Embryo Culture of Hasanbey Apricot CV. Acta Hort. 488.ISHS[J].1999, 441~443
CaoJ, ZhangW, McEloryD. etal.Biotechnology in Agriculture (No.6) Walling ford UR:CAB [J].Intrna tional,1991,175～198
C.M.Miguel.M.M.Oliveira Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants [J].Plant cell Reports 18:387~393, 1999
DengMD, CornuD,Zay-AllemandC.Genetic transformation of walnutsomatic embryo stostudy in vitrorhizo genesis [J].PlantPhysiol, 1991, 96:suppl,1537
D.A.Thompson: High Density Cascade Effects [J].Rad.Effects.1981, 56:105-150
DandekarAM, UratsuSL, MatsutaN. Factors influencing virulencein Agrobacterium-mediated transformation of apple [J].Acta Horti culture, 1990 ,280 :483~494
FitchM, ManshardtRM, GonsalvesD.S Table transformation of papayavia microprojectile bombardmen [J].PlantCellRep.1990, 9:189~194
GrahamJ, Mc. NicolRJ, KumarA. Use of the GUS gene as aselec Table marker for Agrobacterium-mediated transformation of Rubus [J].PlantCellTissOrgCult,1990, 20:35~39
G.K.Wolf: Chemical and Catalytic Effects of Ion Implantation [J]. Radiation Effects, 1982, 65:107-116
Grazia Marino comparative effects of sorbitol and soucrose as main carbon energy sources in micropropagation of apricot [J].Plant cell, Tissue and Organ culture 34:235~244, 1993
Gella R, Errea P. Application of in vitro therapy for ilarvirus elimination in three prunus specied [J].Journal of Phytopathogy, 1998, 146(8/9):445
HebertD,Kikkert JR, Smith FD. Optimiz atio of bio-listic transformation of embryo genic grapccellsuspensio-ns [J].PlantCellRep.1993,12:585～589
HaseY, InoueM, KikuchiM, etal [J].Review, 1997,21～23
HebertD, KikkertJR, SmithFD. Optimization of bio-listic transformation of embryo genicgrapccell suspensio-ns[J].PlantCellRep.1993,12:585～589
I.Kamensky, P.Hakansson, B.Sundqvist,etal: Comparison of Biomolecule Desorption Yields for Low and High Energy Primary Ions[J].Nucl.Instr.Meth. 1982, 198:65-68
Janice J, JNMooreed edited.Advance in fruit [J].Breeding. Purdue Univ, 1975, 285~383
JamesDJ, PasseyAJ, BarbaraDJ. Gentic transformation of apple (MaluspumilaMill.) using adisarmed Ti-binary vector [J].PlantCellRep.1989, 7:658~66110
K.G. Standing, B.T.Chait, W.Ens, etal: Time-of-Flight Measurement of Secondary Organic Ions Produced by 1keV to16keV Ions [J].Nucl. Instr. Meth.1982,198 :33-38
KareivaP.Transgenic plants on trail [J].Nature,1993,363:580~5811
KobayashiS, UchimiyaH. Expression and integration of a foreign gene inorange (CitrussinensisOsb.) protop lasts by direct gene transfer [J].JapanJGenet, 1989, 64:91~97
LambertC,TepferD.Use of Agrobac teriumr hizogenesto creat transgenic apple trees having an altered organo genic response 'ohormones [J].TheorAppleGenet,1992,85:105～1091
Luo Z. WuR.PlantMol [J].Biol.Rep.1980, 6(3):6～10
Larisa A.Kramarenko Micropopagation of apricot and Fild Performance of In vitro [J].Propagated Plants Acta Hort.488.ISHS1999417~420
Lane W D, Cossilo F.Adventious shoots from cotyledon of immatured cherry and apricot embryos [J].Canadian Journal of Plant Science,1986,66
M.Antonelli The use of a brief 2,4-Dtreatment to induce leaf regeneration on prunus canescens bois [J]. Acta Hort 280:45~51,1990
MachadoMC, Machado AdaC. Regeneration of transge-nic plants of Prunusa rmeniacacontaining the coat protein gene of Plum PoxVirus [J].PlantCellRep.1992 11:25~29
M.laimer Regeneration of shoots from leaf discs and stem microcuttings of fruit trees as a tool for transformation [J]. Acta Hort 1988, 235:85~92
Marino G, Magnanini E, Battistini S.Effect of hormon and main carbon energy sources on in vitro propagation of apricot(Prunus armeniaca L.) cvs’San ,castrese’and‘portili’[J].Acta Horti 1991,293:355
Main.M.L, Marin J A. Excised rootstock roots cultured in vitro [J].Plant Cell Report.1998, 18(3/4):350
Miaja.M.L, Vallawia R Regeneration through organogenesis and somatic embryogenesis in some fruit species [J].Italus Hortus, 1997,4(6):8~12
McGranahan G H.LeslicCA, UratsuSL. Agroba cterium-mediated transformation of walnutsomatic embryo sander generation of transgenic plants [J].Biotechnology, 1998,6:800～804
Marino G. Isolation and culture of apricot (Prunus armeniaca) protoplast[J].Invital results. ActaHorti 1991, 293:391
Mullins. MA, TansFCA, FasciottiD. Agrobacterium-mediated transformation of grapevines: [J].transgenic plants of Vitisrupestris Schelle and shoots of Vitisvinifera L.Bio/Technology, 1990, 8: 1041~1045
Murai Y, Harada H In vitro propagation of apricot (prunus armeniaca L) cv.’[J].Bakuoh junkjou’Journal of the Japanese society for horticultural scienc 1997,66(3/4):475
N.Hammatt.N.j.Grant Shoot regeneration from leaves of Prunus serotina Ehrh.(black cherry) [J].Horticulture Research International, Springer-Verlag 1998,17:526~530
Nedelchera S, Tsonra E. Effect of some auxins and cytokinins on organogenensis in callus ultures from apricot rosses;I callusogenensis [J].Rasteniev dni nauki,1998,35(7):540
Nedelchera S, Tsonra E. Callus formation and the possibility of regenerating complete apricot plants I .induced primary and organogenic callus from isolated cotyledons [J]. Rasteniev dninauki , 1998,35(4):296
Nedelchera S. Effect of some auxins and cytokines on organogenensis [J].Rasteniev dni nauki, 1998,35(8):626~631
NymanM, WallinA. Transient gene expression in stra-wberry (FragariaananassaDuch) protop last sand there covery of trans genic plants [J].PlantCellRep.1992,11:105～108
NehraNS, ChibbarRN, KarthaKK. Gentic transform-ation of straw berry by Agrobac terium tume faciensusinga leaf diskre generation system [J].PlantCellRep.1990, 9:293~298
O.Perez-Tornero advent itious shoot gegeneration from in vitro cultured leaves of apricot [J].Acta Hort 2000, 538:659~662
O.Perez-Tornero Effect of basal media and growth regulatoes on the vitro propagation of apricot (Prunus armenica L.)cv .canino Journal of Horticultural Science&Biotechnology 2000,75(3):283~286
O.Perez-Tornero assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot [J].Plant Science 2000,15(8):61~70
O.Perez-Tornero Medium-term Storage of Apricot shoot Tips In Vitro by minimal Growth Method Hort Science 34(7):1277-1278.1999
O.Perez-Tornero Different media requirements for micropropagation of apricor cultivars [J].Plant cell,Tissue and Organ culture 2000,63:133~141.
R.H. Veltman. Internal browning in pear fruit (Pyrus communis L. cv Conference) may be a result of a limited availability of energy and antioxidants [J]. Postharvest Biology and Technology, 2003, 28(2): 295-302.
Shaolan Yang, Changjie Xu, Bo Zhang, et al. Involvement of Both Subgroups A and B of Expansin Genes in Kiwifruit Ripening. [J].Hort-Science, 2007, 42: 315-319.
Shin Y, Ryu Jung-A, Liu R H, eta1. Harvest maturity, storage temperature and relative humidity affect fruit quality, antioxidant contents and activity, and inhibition of cell proliferation of strawbery fruit [J]. Postharvest Biology and Technology, 2008, 49: 201-209.
Soderhall K. Isolation and partial purification of polyphenol oxidase from Daucus carota L. [J].Cell Cultures, 1985, (78): 730-733.
Thomas P, Oke MS. Improvement in quality and storage of` Alphonso'mangos by acclimation [J]. Sci Hort, I983, 19: 257-262.
Snir J.in vitro propagation of‘Ganino’apricot [J].Horti-science 1984,19(2):229
Skirvin R M Tissue culture of peach, sweet and sour cherry, and apricot shoot-tips 1979,Proc.124, [J].Annumtg I11,state Hort Culture Socal 113:30
TodaY, Nishimura H, Inoue M, etal.[J].Breeding science (JP), 1998, 48(2):139.
VardiA, BleichanS, AvivD.Genetic transformation of transgenic plants [J].Plant Sci, 1990, 169:190~206
Watanabe H, Tanaka A,Inoue M,etal. [J].Application,1997,No:09-331708(JP97331708).
YuZengliang,Yang Jianbo,Wuyuejin,etal. [J].Nuclear Instrumen tsand Methodsin Physics Research,1993,B80/81:1328～1331
陈火英,张建华,钟建江,等.野生番茄耐盐性研究及其利用[J].华东理工大学学报,2001(1): 151～154
曹阳,赵东利,王仁军,等.高粱花粉管通道法导入抗虫基因的研究[J].大连大学学报,2001(6):37～42
崔叶龙,柳学余.离子注入稻胚的M1代效应[J].安徽农业大学学报,1994,21(3):306～309
常振田,赵习平,杨莉,等极早熟杏种子发芽方法的试验[J].中国果树,1998(2):27～28
陈学森,张艳敏,张连忠,等.特早熟杏育种[J].园艺学报,1996, (1): 23～27
陈永芳,弓成林,罗安才,等.果树基因转移研究进展[J].四川农业大学学报,2000,18(2):186～190
楚宗丽.核果类果树胚珠培养及子叶再生体系构建[D].农业科学院郑州果树研究所,2007
程家胜,DandekarAM,UratsaSL.苹果基因转移技术研究[J].园艺学报,1992,19(2):101～10
邓江明,简令成.植物抗冻机理研究新进展:抗冻基因表达及其功能[J].植物学报,2001,18(5):521～530
丁国华,徐仲,朱祥春,等.花粉管通道法导入抗赤星病烤烟总DNA D1代性状变异的研究[J].东北农业大学学报.2000,11(2)76～80
杜杰,兰州百合外源DNA花粉管通道法导入的初步研究[D].西北师范大学,2003
董晓颖,孟新法,李培环,等.极早熟油桃幼胚离体培养成苗相关因素探讨[J].园艺学报2007,34 (3): 739～742
冯殿齐,刘静,王玉山,等.利用氮离子注入技术转化抗寒基因(afp)初步研究[J].山东农业大学学报(自然科学版),2005,36(1): 86~92
范成民,核桃快速繁殖技术的研究[D].西北农林科技大学,2008
樊力行.北京师范大学离子注入出新花[J].中国花卉报,2004,4(20)112~115
方宏筠,王关林.黑核桃体细胞胚状体发生及其基因转化系统的建立[J].园艺学报,2000,27(6):406～412
冯莎莎.冷诱导基因转录因子CBFs导入苹果及转化株系抗寒性指标分析[D]河北农业大学,2007
郭生武,大扁杏组织培养的研究[D]西北农林科技大学,2004
何迎春,高必达.花粉管通道法转化水稻研究进展[J].福建稻麦科技,2001,120(1)123~130
李长缨,简元才.花粉管通道法在植物遗传转化中的应用[J].生物学杂志.2000,(1):105~110
侯立群,李秀芬,崔刚,等.几种遗传转化技术在核桃转基因育种中的应用[J].山东林业科技,2004,150(1):8~9
李雪君.花粉管通道法培育烟草新品系[D].河南农业大学,2007
李秀梅.梨幼胚培养与子叶不定器官再生体系的研究[D]四川农业大学,2004
李玉晖.核果类果树远缘杂交不亲和性调控研究[D].山东农业大学,2003
李梦玲,李瑞家,陶正平等.杏叶片离体繁殖研究[J].中国农学通报,2001,17(21):8～10
李永红.离子束的生物学效应及诱变机理研究进展[J].河北农业大学学报,2002(2):43~45
刘静,王长宪,王斌等.石楠抗寒基因AmGS高效转化体系的研究[J].山东农业大学学报(自然科学版),2009,40(2):191～194
刘海坤,卫志明.利用根癌农杆菌介导转化大都成熟种子胚尖获得转基因植株[J].植物生理与分子生物学学报,2004,30(6):631～636
刘庆忠,赵红军.培育苹果转基因植株的研究[J].落叶果树,2001(1):5～9
刘学春,潘春欣,宋云枝,等.一种单子叶植物总DNA提取方法的改进及应用[J].山东农业大学学报,1995,24(4):45~48
倪大虎,向太和,张盘娣,等.应用低能离子束将抗菌肽Shiva A基因导入恢复系明恢63的研究[J].安徽农业科学,2000,28(4):408～409,419
师校欣,王斌,杜国强,等.根癌农杆菌介导豇豆胰蛋白酶抑制剂基因转入苹果主栽品种[J].园艺学报,2000,27(4):282~284
孙清荣,孙洪雁,石荫坪.早熟杏幼胚子叶再生不定梢[J].植物生理学通讯,2001,37(1):38~39
桑庆亮,赖钟雄.荔枝.基枪转化研究初报[J].福建农业大学学报,2001,30(2):266～267
孙芳.沙冬青CBLI基因的功能研究[D].北京林业大学,2009
覃兰英,克热木·伊力,郑华云,等.杏砧木的组织培养[J].植物组织培养简报,1994,30(5):354～359
王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998.193～2323
王立新,孟荣华,郭仁俊,等.应用花粉管通道法进行小麦抗白粉病转基因的研究初报[J]. 农业生物技术学报,2001,(3):51~55
王玖瑞,吕增仁,刘孟军,等.杏离体根愈伤组织诱导及起源的研究[J].河北农业大学学报2000,23(2):48～51
王玖瑞,吕增仁,刘孟军,等.杏组织培养研究进展[J].河北果树,2000,(2):6～11
王玉柱,张大鹏,杨丽,等.未成熟杏离体胚培养技术的研究[J].农业生物技术学报,2000,8(4):389～391
吴爱忠.花粉管导入法培育抗除草剂的转基因水稻[J].上海农学院学报1999,17(4):237～241
吴秀红.大豆花粉管通道法导入外源DNA的适宜时期与方法探讨[J].黑龙江农业科学,2001,11(2):43~47
阎国华,周宇,张开春,等.核果类果树转基因研究进展[J].果树学报,2001,18(6):358～364
杨成民.基因枪轰击法获得转P5CS基因黑麦草植株[D]内蒙古农业大学,2004. 张玲.杏转化抗寒基因的初步研究[D].山东农业大学,2004
张茂银,刘庆昌,王子霞等.用花粉管通道法将新疆大赖草DNA导入普通小麦的研究[J].农业生物技术学报,2000(2):43～46
张志宏,方宏筠,景士西,等.苹果主栽品种高效转化系统的建立及其影响因子的研究[J].遗传学报,1998,25(2):160～165
张志宏,景士西,王关林.果树基因工程研究进展[J].果树科学,1995,12(3):188～1932
张志宏,景士西,王关林.苹果基因转移技术研究初报[J].园艺学报,1997,24(4):378～381
张志宏,吴禄平.草莓主栽品种Tudla遗传转化体系的建立[J].农业生物技术学报,1997,5(3):305～307
张志宏,王关林,方宏筠,等.果树基因工程研究进展[J].果树科学,1995,12(3):188～193
赵淑清,郭剑波,常留印.特早熟杏胚培养试验[J].中国果树,2001(1):17～18
周广芳,王成强.果树多倍体育种途径与成就[J].落叶果树,1997,22(3):21~25
A.M.Foti, F.MilanoandL. Torrisi: Amino Acid Decomposition Induced by KevIon Irradiation. Nucl.Instr. [J].Meth 1990, B46:361-363
Ahmet Esitken Embryo Culture of Hasanbey Apricot CV. Acta Hort. 488.ISHS[J].1999, 441~443
CaoJ, ZhangW, McEloryD. etal.Biotechnology in Agriculture (No.6) Walling ford UR:CAB [J].Intrna tional,1991,175～198
C.M.Miguel.M.M.Oliveira Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants [J].Plant cell Reports 18:387~393, 1999
DengMD, CornuD,Zay-AllemandC.Genetic transformation of walnutsomatic embryo stostudy in vitrorhizo genesis [J].PlantPhysiol, 1991, 96:suppl,1537
D.A.Thompson: High Density Cascade Effects [J].Rad.Effects.1981, 56:105-150
DandekarAM, UratsuSL, MatsutaN. Factors influencing virulencein Agrobacterium-mediated transformation of apple [J].Acta Horti culture, 1990 ,280 :483~494
FitchM, ManshardtRM, GonsalvesD.S Table transformation of papayavia microprojectile bombardmen [J].PlantCellRep.1990, 9:189~194
GrahamJ, Mc. NicolRJ, KumarA. Use of the GUS gene as aselec Table marker for Agrobacterium-mediated transformation of Rubus [J].PlantCellTissOrgCult,1990, 20:35~39
G.K.Wolf: Chemical and Catalytic Effects of Ion Implantation [J]. Radiation Effects, 1982, 65:107-116
Grazia Marino comparative effects of sorbitol and soucrose as main carbon energy sources in micropropagation of apricot [J].Plant cell, Tissue and Organ culture 34:235~244, 1993
Gella R, Errea P. Application of in vitro therapy for ilarvirus elimination in three prunus specied [J].Journal of Phytopathogy, 1998, 146(8/9):445
HebertD,Kikkert JR, Smith FD. Optimiz atio of bio-listic transformation of embryo genic grapccellsuspensio-ns [J].PlantCellRep.1993,12:585～589
HaseY, InoueM, KikuchiM, etal [J].Review, 1997,21～23
HebertD, KikkertJR, SmithFD. Optimization of bio-listic transformation of embryo genicgrapccell suspensio-ns[J].PlantCellRep.1993,12:585～589
I.Kamensky, P.Hakansson, B.Sundqvist,etal: Comparison of Biomolecule Desorption Yields for Low and High Energy Primary Ions[J].Nucl.Instr.Meth. 1982, 198:65-68
Janice J, JNMooreed edited.Advance in fruit [J].Breeding. Purdue Univ, 1975, 285~383
JamesDJ, PasseyAJ, BarbaraDJ. Gentic transformation of apple (MaluspumilaMill.) using adisarmed Ti-binary vector [J].PlantCellRep.1989, 7:658~66110
K.G. Standing, B.T.Chait, W.Ens, etal: Time-of-Flight Measurement of Secondary Organic Ions Produced by 1keV to16keV Ions [J].Nucl. Instr. Meth.1982,198 :33-38
KareivaP.Transgenic plants on trail [J].Nature,1993,363:580~5811
KobayashiS, UchimiyaH. Expression and integration of a foreign gene inorange (CitrussinensisOsb.) protop lasts by direct gene transfer [J].JapanJGenet, 1989, 64:91~97
LambertC,TepferD.Use of Agrobac teriumr hizogenesto creat transgenic apple trees having an altered organo genic response 'ohormones [J].TheorAppleGenet,1992,85:105～1091
Luo Z. WuR.PlantMol [J].Biol.Rep.1980, 6(3):6～10
Larisa A.Kramarenko Micropopagation of apricot and Fild Performance of In vitro [J].Propagated Plants Acta Hort.488.ISHS1999417~420
Lane W D, Cossilo F.Adventious shoots from cotyledon of immatured cherry and apricot embryos [J].Canadian Journal of Plant Science,1986,66
M.Antonelli The use of a brief 2,4-Dtreatment to induce leaf regeneration on prunus canescens bois [J]. Acta Hort 280:45~51,1990
MachadoMC, Machado AdaC. Regeneration of transge-nic plants of Prunusa rmeniacacontaining the coat protein gene of Plum PoxVirus [J].PlantCellRep.1992 11:25~29
M.laimer Regeneration of shoots from leaf discs and stem microcuttings of fruit trees as a tool for transformation [J]. Acta Hort 1988, 235:85~92
Marino G, Magnanini E, Battistini S.Effect of hormon and main carbon energy sources on in vitro propagation of apricot(Prunus armeniaca L.) cvs’San ,castrese’and‘portili’[J].Acta Horti 1991,293:355
Main.M.L, Marin J A. Excised rootstock roots cultured in vitro [J].Plant Cell Report.1998, 18(3/4):350
Miaja.M.L, Vallawia R Regeneration through organogenesis and somatic embryogenesis in some fruit species [J].Italus Hortus, 1997,4(6):8~12
McGranahan G H.LeslicCA, UratsuSL. Agroba cterium-mediated transformation of walnutsomatic embryo sander generation of transgenic plants [J].Biotechnology, 1998,6:800～804
Marino G. Isolation and culture of apricot (Prunus armeniaca) protoplast[J].Invital results. ActaHorti 1991, 293:391
Mullins. MA, TansFCA, FasciottiD. Agrobacterium-mediated transformation of grapevines: [J].transgenic plants of Vitisrupestris Schelle and shoots of Vitisvinifera L.Bio/Technology, 1990, 8: 1041~1045
Murai Y, Harada H In vitro propagation of apricot (prunus armeniaca L) cv.’[J].Bakuoh junkjou’Journal of the Japanese society for horticultural scienc 1997,66(3/4):475
N.Hammatt.N.j.Grant Shoot regeneration from leaves of Prunus serotina Ehrh.(black cherry) [J].Horticulture Research International, Springer-Verlag 1998,17:526~530
Nedelchera S, Tsonra E. Effect of some auxins and cytokinins on organogenensis in callus ultures from apricot rosses;I callusogenensis [J].Rasteniev dni nauki,1998,35(7):540
Nedelchera S, Tsonra E. Callus formation and the possibility of regenerating complete apricot plants I .induced primary and organogenic callus from isolated cotyledons [J]. Rasteniev dninauki , 1998,35(4):296
Nedelchera S. Effect of some auxins and cytokines on organogenensis [J].Rasteniev dni nauki, 1998,35(8):626~631
NymanM, WallinA. Transient gene expression in stra-wberry (FragariaananassaDuch) protop last sand there covery of trans genic plants [J].PlantCellRep.1992,11:105～108
NehraNS, ChibbarRN, KarthaKK. Gentic transform-ation of straw berry by Agrobac terium tume faciensusinga leaf diskre generation system [J].PlantCellRep.1990, 9:293~298
O.Perez-Tornero advent itious shoot gegeneration from in vitro cultured leaves of apricot [J].Acta Hort 2000, 538:659~662
O.Perez-Tornero Effect of basal media and growth regulatoes on the vitro propagation of apricot (Prunus armenica L.)cv .canino Journal of Horticultural Science&Biotechnology 2000,75(3):283~286
O.Perez-Tornero assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot [J].Plant Science 2000,15(8):61~70
O.Perez-Tornero Medium-term Storage of Apricot shoot Tips In Vitro by minimal Growth Method Hort Science 34(7):1277-1278.1999
O.Perez-Tornero Different media requirements for micropropagation of apricor cultivars [J].Plant cell,Tissue and Organ culture 2000,63:133~141.
R.H. Veltman. Internal browning in pear fruit (Pyrus communis L. cv Conference) may be a result of a limited availability of energy and antioxidants [J]. Postharvest Biology and Technology, 2003, 28(2): 295-302.
Shaolan Yang, Changjie Xu, Bo Zhang, et al. Involvement of Both Subgroups A and B of Expansin Genes in Kiwifruit Ripening. [J].Hort-Science, 2007, 42: 315-319.
Shin Y, Ryu Jung-A, Liu R H, eta1. Harvest maturity, storage temperature and relative humidity affect fruit quality, antioxidant contents and activity, and inhibition of cell proliferation of strawbery fruit [J]. Postharvest Biology and Technology, 2008, 49: 201-209.
Soderhall K. Isolation and partial purification of polyphenol oxidase from Daucus carota L. [J].Cell Cultures, 1985, (78): 730-733.
Thomas P, Oke MS. Improvement in quality and storage of` Alphonso'mangos by acclimation [J]. Sci Hort, I983, 19: 257-262.
Snir J.in vitro propagation of‘Ganino’apricot [J].Horti-science 1984,19(2):229
Skirvin R M Tissue culture of peach, sweet and sour cherry, and apricot shoot-tips 1979,Proc.124, [J].Annumtg I11,state Hort Culture Socal 113:30
TodaY, Nishimura H, Inoue M, etal.[J].Breeding science (JP), 1998, 48(2):139.
VardiA, BleichanS, AvivD.Genetic transformation of transgenic plants [J].Plant Sci, 1990, 169:190~206
Watanabe H, Tanaka A,Inoue M,etal. [J].Application,1997,No:09-331708(JP97331708).
YuZengliang,Yang Jianbo,Wuyuejin,etal. [J].Nuclear Instrumen tsand Methodsin Physics Research,1993,B80/81:1328～1331
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