摘要
本试验以越橘四大种类(矮丛越橘、半高丛越橘、高丛越橘、兔眼越橘),14个越橘品种(‘园蓝’、‘日出’、‘塞埃罗’、‘都克’、‘伯克利’、‘埃利奥特’、‘蓝丰’、‘泽西’、‘乔治亚姆’、‘瑞维尔’、‘夏普蓝’、‘北陆’、‘美登’、‘芬蒂’)为材料,用叶片作为外植体进行离体再生培养,并对影响越橘离体再生的因素进行了初步的研究。
通过试验发现,影响越橘离体再生的因素可以从内因和外因两个方面进行探讨。内因主要是基因型,它是越橘离体再生很重要的限制因素;外因指激素种类及其浓度、培养基类型、蔗糖浓度、pH值、暗培养时间等因子。在从多外在因子中,培养基类型、激素种类及其浓度、暗培养时间是影响越橘离体再生的重要因素。
不同基因型的品种在再生能力上有很大的差别。供试的各品种中,‘日出’、‘塞埃罗’、‘乔治亚姆’、‘芬蒂’的再生能力很强,在适宜的培养基再生频率可达95%以上。‘埃利奥特’、‘北陆’、‘都克’、‘伯克利’再生能力次之,在适宜的培养基上再生频率可达85%以上,单叶片平均不定芽数达25个。‘泽西’、‘蓝丰’、‘美登’的再生能力很低,再生频率接近零。这三个品种的再生培养条件仍须继续摸索。对多种培养基进行研究得出结论:WPM培养基对越橘离体再生最为有利,各品种以其为基本培养基再生频率高于其他类型的培养基。试验中采用的细胞分裂素,包括ZT、TDZ、CPPU、2ip、BA,其中ZT、CPPU对越橘离体再生的效果最好。CPPU处理中品种再生频率最高可达100%,单叶片平均不定芽数最高可达41个。多数应试品种的再生对CPPU的反应良好,再生频率和平均不定芽数均高于ZT处理。如南高丛品种‘乔治亚姆’在ZT处理中的再生频率最高只有42.3%,单叶片平均不定芽数为6.8个;但在CPPU处理中再生率最高可达100%,但叶片平均不定芽数达最高可达20个。TDZ和2ip只对个别品种的再生有效果,如‘日出’、‘塞埃罗’、‘伯克利’、‘埃利奥特’,再生频率最高只有69%,不定芽数明显低于ZT、CPPU的处理。BA对越橘离体叶片的再生没有效果。此外暗培养对越橘叶片的再生也有很大的影响,虽然有些品种,如‘日出’和‘塞埃罗’,在不经暗处理的情况下再生率仍能达到85%以上,但不定芽的质量和数量远不如经过暗处理的。试验中多数品种都需要暗处理,才能获得较高的再生
频率和不定芽数。还有一些因子如蔗糖浓度、pH、外植体的摆放方向等,对越橘叶片
的再生影响不显著。
A procedure to regenerate shoots in vitro from leaf sections of fourteen blueberries genotypes, 'Gardenblue', 'Sunrise', 'Sierra', 'Duke', 'Berkeley', 'Elliot', 'Bluecrop', 'Jersey', 'Georgia Gem', 'Reveil', 'Sharpblue', 'Northland', 'Blomidon', 'Fundy', in Woody Plant Medium was established, and influencing factors such as genotype, medium, hormone, sugar concentration, pH, were also studies.
Cultivars with different genotype had different regeneration ability. Among the fourteen genotypes tested , 'Sunrise', 'Sierra', 'Georgia Gem' and 'Fundy' had the strongest ability in shoot regeneration with shoot induction frequency of 95% and mean number 20 shoots per leaf. Hormone types and concentration in medium were the most important factors influencing shoot regeneration from leaf segments of blueberry. The effect of Zeatin and CPPU on leaf segments regeneration was better than other hormones tested in experiment. Cytoinin TDZ or 2ip had effect on some cultivars. For example, regeneration percent of Sunrise obtained from 2ip medium was 59.4% or 69%, which was fewer than that from Zeatin or CPPU medium. Medium supplemented with the cytokinin CPPU was compared with medium supplemented with Zeatin. Use of 1.0 mg/L ~ 2.0 mg/L CPPU resulted in the higher regeneration rate and larger number of shoots per leaf section than 5.0 mg/L Zeatin did. Such as Georgia Gem , its highest regeneration percent on CPPU m
edium was 100%, while on the Zeatin medium was 42.3%. Consequently, we concluded that the cytokinin CPPU was more effective than either of the cytokinins, Zeatin, 2ip or TDZ, in promoting shoot regeneration from leaf sections of blueberry. Dark treatment was also important factor in blueberry leaf section regeneration. Ten days dark treatment after leaf sections inoculated on the medium promised the best adventitious buds and highest regeneration rate. And shorter or longer than ten days would decrees shoot regeneration rate. In addition, sugar concentration and pH had effect on blueberry leaf sections regeneration. Medium with 20mg/L sugar was optimum of leaf regeneration , produced
more shoots than any other concentrations of sugar. The optimal pH of medium was 5.4. The shoot regeneration rate of blueberry would be decreasing with pH of medium increasing. For example, the shoot regeneration rate of Georgia Gem on pH 5.4 medium was 97%, while on pH 6.5 medium the shoot regeneration was 10%. The temperature during dark treatment influenced shoot regeneration too. The optimal temperature of low bush blueberry was 20 ± 2 ℃ , for high bush blueberry was 25 ±2 ℃. The optimal temperature of rabbiteye was higher than 25 ± 2 ℃., but lower than 30℃.
引文
[1]郝瑞,越桔及其栽培.中国果树1987.1
[2]李亚东,吴林,张志东.《越橘(蓝莓)栽培与加工利用》.长春:吉林科学技术出版社,2000
[3]李亚东,吴林.越橘果实中营养成分分析.北方园艺,1996.3
[4]祖容主编,郝瑞编著.《浆果学》.北京:中国农业出版社,1996
[5]Hall I. V.. Callus formation in stem internode section of lowbush blueberry (Vaccinium angustifolium ait.) cultured on a medium containing plant growth regulation. Hort. Rev.,1976, 16:29-35
[6]Ahmed EI-shiekh. Long-term effects of propagation by tissue culture or soft-wood single-node cuttings on growth habit, yield and berry weight of "Northblue" blueberry. J.Amer. Soc. Hort. Sci. 1996,12 (2): 339-342
[7]Nickerson N. L.. In vitro shoot formation in lowbush blueberry seedling explants.Hortscience, 1978,13:698
[8]Lyrene P. M.. Juvenility and production of fast rooting cuttings from blueberry shoot cultures. J. Amer. Soc. Hort. Sci. 1981, 106: 396~398.
[9]Sharon G. Billings, Chee K. Chin, and G. Jelenkovic. Regeneration of blueberry plantlets from leaf segments. Hortscience, 1988, 23:763~766
[10]Callow P., Haghighi K., Giroux M. and Hancock J.. In vitro shoot regeneration on leaf tissue from microprogated highbush blueberry. Hortscience, 1989, 24:373~375
[11]Chandler C. K., Draper A. D.. Effect on zeatin and zip on shoot proliferation of three highbush blueberry clones in vitro. Hortscience, 1986, 21:1065~1066
[12]Wolfe D. E., Eck P., Chin C.. Evaluation of seven media for micropropagation of highbush blueberry. Hortscience, 1983, 18:703~705
[13]Read P. E, Hartley C. A., Sandahl J. G., Wilding D. K.. Field performance of in vitro propagated blueberries. Proc. Intl. Plant Prop. Soc, 1987, 37:450~452
[14]Rowland L. J., Elizabeth L. O.. Use of a cytokinin conjugate for efficient shoot
regeneration from leaf sections of Highbush blueberry. Hortscience, 1992, 27 (10):11127~1129
[15]Frett, J. J. and Smagula J. M.. In vitro shoot production of lowbush blueberry. Can. J.Plant Sci., 1982, 63: 467~472.
[16]Garley B., Stus hnoff C., Wildung D., Read P. E.. In vitro micropropagation of blueberry hybrids using single bud stem segments and stem tips. Hortscinece ,1979, 14:477 (Abst.)
[17]Brissette L., Tremblay L., Lord D.. Micropropagation of lowbush blueberry from mature field-grown plants. Hortscience, 1990,25 (3): 349~351
[18]Rida A., Shibli M. A., Smith L.. Direct shoot regeneration from vaccinium pahalae(Ohelo) and V. myrtills (Bilberry) leaf explant. Hortscience, 1996, 31 (7): 1225~1228
[19]Lyrene. P. M.. Micropropagation of rabbiteye blueberries. Hortscience, 1980, 15:80~81
[20]Scorza R., Welker W. V., Dunn L. J. The effects of glyphosate, auxin and cytokinin combinations on in vitro development of cranberry nodes. Hortscience, 1984, 19: 66~68.
[21]Serres R. A, Mc Cown B. H., Zeldin E., Mc cabe D. E., Stang E. J.. Application of biotechnology to cranberry: A model for fruit improvements ⅩⅩⅢ Intl. Hort. Congr.Florence, Italy, 1990, P. 127 (Abst.)
[22]Marcotrigiano M., P. McGlew A S.. Two-stage micropropagation system for cranberry J.Amer so Holt sci, 1991, 116 (5): 911-916
[23]Gerbhard T K., Friedrich M.. In vitro shoot regeneration of lingonberry clones. Garten bauwissen schwft, 1986,51:170-175
[24]Horier M. A, Flatebo G., Read P. E.. In vitro propagation of lirgonberry. Hortscience,1985, 20: 304-365.
[25]Riechers V., Bunemann G.. Mikrovev-mehvung vonpreise lbeeren (V. vitis-idaea). Erwerbsobstbau, 1989, 31:129-132
[26]Seeres R. A.,S.pan, Mccown B. H.. E. J.stang. micropropation of several lirgonberry clol tivars. Fruit var J. 1994,48:7-14
[27]Gustavsson B. A.. Field performanle of sanra linngonberry Derived by Microprogagation Vs stem Cuttings. Houtsience, 2000, 35 (4): 742-744
[28]陈慧都,郝瑞,关爱年,曲路平,等. 越桔组织培养与工厂化育苗技术研究.中国农业科学,1990,23(3):44-50
[29]金炜,陈品良,顾渊.植物激素对越桔组织培养中侧芽增殖的影响.植物学通报,1991,8 (2):53-54
[30]李文安,余叔文编.植物细胞分化及试管植物.植物生理与分子生物学,北京科学出版社,1992,37-52
[31]陈振光编著.果树组织培养.上海科技出版社,1987
[32]杨增海.园艺植物组织培养.北京农业出版社会,1989,60-90
[33]张宇和.果树繁殖.上海科技出版社,1989
[34]Hernik V. E. Plant regenwration from explant of sterile seelings in two introcluceel uarietids of Vaxxinium corymbosum. L.J vestsi Akakemi Navak rilarusi seryya Riywla gich nykh Navak no, 1992,34, 6-9 121.
[35]Shaorn G.B., Chee K, C.. Regeneretion of blue berry plant tets from leaf segmerrts. Hortscience, 1991, 23 (4): 763-766
[36]Hrlsko(1) D., Read P. E.. In vitro shoot regeraration from internode segments and internode-clerived callus off blueberry. Acta Horti Cwlturae, 1993, NO. 346:127-132
[37]Kvtas E.. Morphogenesk of introduced varieties of Vaccinium vitis-idaca in aseptic culture. Mets and slikud Lurimused, 1998, 30:87-93
[38]Chandler C. , Draper. Effect of zeatin and 2ip on shoot proliferation of three highbush blueberry clones in vitro. Hortscience, 1986,21 (4): 1065-1066
[39]Hen D.. Application of micro propagation methods for blueberry and tamayillos. The Intl.pleant prop. Soc., 1980,30:114-146
[40]Zimmerman R. H., Broome O. C.. Blueberry micropropagation. 1980, p: 4447
[41]Jin Wei, Cheng Pinliang, Gu Yuan. The influence of plant hormone on the multipletion of auxiliary bud in blueberry tissue culture. Chinese Bulletin of Botany, 1991 (2): 53-54
[42]James D. H., Read P. E.. In Vitro Shoot Regeneration From Internode Segments and Internode-Derived Callus of Blueberry (Vaccinium SPP.). Acta Horticulture, 1993, 346
[43]张庆松.育苗穴盘中的光照.中花园艺,2001,NO.119-25
[44]曹孜义,刘国民.主编《实用植物组织培养技术教程》.甘肃科学技术出版社,1998,50-51
[45]Penal, Cervera M., JuraeJ,et al. Genetic teansformation of lime:factor affecting transformation and regeneration. Plant Cell Rep, 1997, 16:731~737
[46]Penal L.,Cervera M., Juare J., et al.Agrobacterium-mediated trorsformation of sweet orange and regeneration of transgenic plants. Plant Cell Rep., 1995, 14:616~619
[47]Pena L., Cervera M., Juare J, et al. High efficiency of Agorbacterium-mediated transfortion and regeneration of citrus. Plant Sci., 1995, 104:183~191
[48]孙仲序,陈受宜,王建设,等.农杆菌介导BADH基因转化葡萄的研究.果树科学,2003,20(2):89~92
[49]石玮,李东栋,邓秀新,等.根癌农杆菌介导绿色荧光蛋白基因转化印度酸橘的研究,园艺学报,2002,29(2):109~112
[50]钟华鑫,杜勤,周利民,等.黄瓜子叶有丝分裂与离体培养反应关系的研究.科学通报,1993,38(16):1506~1509
[51]章美云,韩碧文.烟草薄层培养的花芽诱导和开花梯度研究.作物学报,1992,16(1):17~22
[52]杜胜利,魏惠军,魏爱民,等.苗龄基因型和外植体对黄瓜离体器官发生的影响.天津农业科学,2000,6(4):1~5
[53]王熊.细胞生物学杂志.1983,5(2):40~45
[54]刘涤.植物生理学报.1986,12(1):104~108
[55]泷宗英.植物生理学报.1985,11(1):17~24
[56]王关林,李春斌,方宏筠.提高银杏悬浮培养细胞内酯合成的研究.园艺学报,2002,29,(4):321~325
[57]刘庆忠,赵红军,Hammerschlag F.A.提高苹果基因转化效率的研究.果树科学,2000,(3):159~163
[58]Liu Q, Salihs, Hammerschlag F. A. Etiolation of 'Royal Gala' apple(Malus domestica) shoots promotes high frequency organagenesis and enhanced β-glucuronidase expression from stem internodes. Plant Cell Reports, 1998, 18:32~36
[59]刘庆中,赵红军,刘鹏,Mong Rengong,等.抗菌肽从B_(39)基因导入‘皇家嘎拉’苹果及四倍体植株的培育.园艺学报,2001,28(5):392~398
[60]张志宏,吴绿平,等.草莓主栽品种再生和转化的研究.园艺学报,2001,28(3):189~193
[61]张志宏,景士西,王关林,等.苹果品种新乔纳金离体再生体系的建立.农业生物技术学报,1997,5:305~306
[62]方金豹,田莉莉,等.CPPU对猕猴桃光和产物库源强度的影响.园艺学报,2000,27 (6):444~446
[63]Wooley D. J., Lawes G. S., Cruz-Castillo J. G.. The growth and competitive ability of Actinidia deliciosa 'Haywand' fruit: carbohydrate availability and response to the cutobinin-active compound CPPU. Acta Horticulturae, 1991, 297:467~473
[64]李宗霆.植物激素及其免疫检测技术.上海:江苏科学技术出版社,1996,128~129
[65]Lewis D.H., Burge G.K., Schmiere D.M., et al. Cytokinins and fruit development in kiwifruit (Actinidia deliciose). I. Changes during fruit development physiologia. Plantarum,1996, 98:179~186
[66]Maejima S., Kitahara H., Ohra H.. Effects of KT-3 on the growth and development of apples. In: Proceedings of Japanese society for chemical regulation of plants. [s.l.]; [s n.]1987
[67]Ohara H., kato M., Matsui H.. Comparison of the levels of endogenous plant growth substances in CPPU-treated and antreated kiwifruit. Joural of the Japanese society for horticultural science, 1997, 65 (4): 692~705
[68]闫国华,甘立军,孙瑞红,等.赤酶素和细胞分裂素调控苹果果实早期生长发育机理的研究.园艺学报,2000,27(1):11~16
[69]李三玉.CPPU对葡萄果粒生长过程中内源激素和细胞分裂素的影响.中国植物生理学会第七次全国议论文汇编.1996,78~83
[70]周俊彦,郭夫兴.苯基脲衍生物的细胞分裂素活性.植物生理学通讯.1990,4:7~13
[71]Murata M. and Orthoh T. J.. Callus initiation and regeneration capacities in Brassica. Plant Cell Tissue and Organ Culture, 1987, 11:111~123
[72]Narender S. N. and Stushnoff C.. Direct shoot regeneration from strawberry leaf disks.[J].Amer. Soc Hort. 1989, 23 (6): 1014~1018
[73]Zong R. L. and John C. S.. Plant regeneration by organogenesis from strawberry leaf and runner tissue Hortiscience. 1988, 23 (6): 1057~1059
[74]张志宏.苹果叶片离体再生及遗传转化的研究.沈阳农业大学博士学位论文,1996
[75]刘世强主编.细胞工程的理论与实践.辽宁大学出版社,1994
[76]夏秀英.樱桃砧木的离体再生及抗菌肽基因转化研究.沈阳农业大学博士学位论文,2000
[77]黄学林主编.高等植物组织离体培养的形态建成及其调控.科学出版社,1995
[78]汪开治.利用基因工程培育抗病葡萄.[J]生物技术通报,2000(1):45~46
[79]夏秀英.草莓叶片的离体再生及遗传转化的研究.沈阳农业大学硕士学位论文,1997
[80]刘庆昌,吴国良主编.植物细胞组织培养.中国农业大学出版社,2003