油橄榄胚和茎段离体培养研究
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摘要
本研究以油橄榄(Olea europaea L.)种胚和带芽茎段为试材,探索其组织培养体系,为油橄榄快速繁殖及相关研究奠定基础。主要研究结果如下：
1、在3月、4月、5月、6月分别采取‘Frantoio’品种的健壮枝条进行接种,结果表明4月和5月采取的外植体进行接种的效果最好,其褐化率最低,均为23.4%,而其它时间(3月和6月)采样进行接种的褐化率分别为45%,33.6%。
2、油橄榄的不同节段间,消毒效果存在一定的差异。表现为顶节、次顶节、第三节和第四节的污染率呈递减趋势,以第三节和第四节作为外植体更容易建立起无菌培养体系。而且以消毒处理18min的效果最适宜。
3、试验研究表明White培养基适宜诱导芽萌发,MS培养基适宜诱导愈伤组织。
4、胚培养对于启动培养基中各激素的要求不明显,9个处理的出芽率均较高,均在50%以上。而三个品种中‘Tanche’的胚培养小苗增殖效果最好,增殖系数为1.58。最佳增殖培养基为White+6-BA 2.0 mg.L-1+NAA 0.3 mg.L-1+GA32.0 mg.L-1。
5、茎段初代培养品种间差异很大,出芽率不高,最高的为16.7%。其中‘Frantoio’品种表现最好,出愈率和出芽率均较高。茎段培养小苗的增殖系数为1.72,最好的品种是‘Frantoio’,最佳增殖培养基为White+6-BA2.5 mg.L-1+NAA0.1 mg.L-1.
6、最佳生根培养基是：1/2 MS+NAA 2.0 mg.L-1+6-BA 0.3 mg.L-1,生根率可达86.7%。
7、瓶苗在室内放置7天,再将其置于室外进行炼苗,30天后开瓶,再炼苗7天后移栽,本次试验用于炼苗的植株为30株,成活了7株,成活率为23.33%。
In this study, olive (Olea europaea L.) embryo and stems were used as materials to establish an in vitro culture system. The main results were as follows.
1.'Frantoio' were inoculated in March、April、May and June, explants isolated in April and May resulted in the lowest browning rate,23.4%, the other time (in March and June) were inoculated with the browning sampling rate of 45%,33.6%.
2. Sterilization effects were different in different parts of the stems. Performance for the top section, second top section.ⅢandⅣof the contamination rate decreased, the section III and section IV were the best, And 18min to the effect of disinfection best.
3. White was the suitable basic medium with high sprouting rate, while MS was suitable for callus induction.
4. The embryo culture was less strict in the requirement for hormones in the medium in the primary culture. However, proliferation was cultivar dependent and'Tanche'showed the best proliferation performance in the medium White+6-BA2.0mg.L-1+NAA0.3 mg.L-1+GA32.0mg.L-1.
5. The stem culture was more cultivar dependent, the hightest was 16.7%. Of the cultivars used in the study,'Frantoio' was the most responsive whose callus induction rate and sprouting rate were 58.3% and 6.7%, respectively.
6. The best rooting medium was 1/2 MS+NAA2.0mg.L-1+6-BA0.3mg.L-1, in which rooting rate could reach 86.7%.
7. In vitro plantlets were put inside the room at the normal temperature for 7 days, then were put outside for hardening.30 days later, bottles were opened for 7 days. Then transplantation was followed, resulting in a survival rate of 11.9%.
1、在3月、4月、5月、6月分别采取‘Frantoio’品种的健壮枝条进行接种,结果表明4月和5月采取的外植体进行接种的效果最好,其褐化率最低,均为23.4%,而其它时间(3月和6月)采样进行接种的褐化率分别为45%,33.6%。
2、油橄榄的不同节段间,消毒效果存在一定的差异。表现为顶节、次顶节、第三节和第四节的污染率呈递减趋势,以第三节和第四节作为外植体更容易建立起无菌培养体系。而且以消毒处理18min的效果最适宜。
3、试验研究表明White培养基适宜诱导芽萌发,MS培养基适宜诱导愈伤组织。
4、胚培养对于启动培养基中各激素的要求不明显,9个处理的出芽率均较高,均在50%以上。而三个品种中‘Tanche’的胚培养小苗增殖效果最好,增殖系数为1.58。最佳增殖培养基为White+6-BA 2.0 mg.L-1+NAA 0.3 mg.L-1+GA32.0 mg.L-1。
5、茎段初代培养品种间差异很大,出芽率不高,最高的为16.7%。其中‘Frantoio’品种表现最好,出愈率和出芽率均较高。茎段培养小苗的增殖系数为1.72,最好的品种是‘Frantoio’,最佳增殖培养基为White+6-BA2.5 mg.L-1+NAA0.1 mg.L-1.
6、最佳生根培养基是：1/2 MS+NAA 2.0 mg.L-1+6-BA 0.3 mg.L-1,生根率可达86.7%。
7、瓶苗在室内放置7天,再将其置于室外进行炼苗,30天后开瓶,再炼苗7天后移栽,本次试验用于炼苗的植株为30株,成活了7株,成活率为23.33%。
In this study, olive (Olea europaea L.) embryo and stems were used as materials to establish an in vitro culture system. The main results were as follows.
1.'Frantoio' were inoculated in March、April、May and June, explants isolated in April and May resulted in the lowest browning rate,23.4%, the other time (in March and June) were inoculated with the browning sampling rate of 45%,33.6%.
2. Sterilization effects were different in different parts of the stems. Performance for the top section, second top section.ⅢandⅣof the contamination rate decreased, the section III and section IV were the best, And 18min to the effect of disinfection best.
3. White was the suitable basic medium with high sprouting rate, while MS was suitable for callus induction.
4. The embryo culture was less strict in the requirement for hormones in the medium in the primary culture. However, proliferation was cultivar dependent and'Tanche'showed the best proliferation performance in the medium White+6-BA2.0mg.L-1+NAA0.3 mg.L-1+GA32.0mg.L-1.
5. The stem culture was more cultivar dependent, the hightest was 16.7%. Of the cultivars used in the study,'Frantoio' was the most responsive whose callus induction rate and sprouting rate were 58.3% and 6.7%, respectively.
6. The best rooting medium was 1/2 MS+NAA2.0mg.L-1+6-BA0.3mg.L-1, in which rooting rate could reach 86.7%.
7. In vitro plantlets were put inside the room at the normal temperature for 7 days, then were put outside for hardening.30 days later, bottles were opened for 7 days. Then transplantation was followed, resulting in a survival rate of 11.9%.
引文
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[2]张宇和.果树繁殖[M].上海：上海科技出版社,1984
[3]包慈华,马经凤,王凯基,等.油橄榄茎尖培养成完整植株的初步研究[J].科学通报,1979,2(2)
[4]罗士伟.我国植物组织培养工作的进展[J].自然杂志年鉴.1979
[5]王怀智.经济植物组织培养[M].北京：科学出版社,1988
[6]张松,李纪蓉,李滨,等.葱组织培养体细胞胚胎发生的研究[J].园艺学报.1997,24(3)：264-268
[7]慈忠玲.磁场对防风体细胞胚发生发育和亚显微结构的影响[J].植物生理学报.1999,25(3)：309-312
[8]张东向,梅玲,张崇浩,等.Co2+对芹菜胚状体发生过程中乙稀释放量的影响[J].园艺学报.1999,26(3)：273-274
[9]Torrey.J.G. Cellular differentiation of cultured cells and tissucs[J]. Hort. Science,1977:12(2), 127-130
[10]Sangman.R.S.and H.Harada, Chemical regulation of callus growth, Organogenesis, plant regeneration and somatic embryogenesis in Autirrhinum majus tissue and cell cultures[J].1975
[11]王凯基,张丕方,倪德样,等,油橄榄组织培养的细胞组织学研究1.愈伤组织的建成[J].植物学报,1979：21(2)：124-130
[12]王凯基,张丕方,倪德样,等,油橄榄组织培养的细胞组织学研究Ⅱ.组织分化和器官发生[J].植物学报,1979：21(3)
[13]王丽,邹明谦,王晓光.香雪兰种子胚的组织培养和植株再生.园艺学报[J].1996,23(3)：281-284
[14]Canas, L.A, Carramolino and M. Vicente. Vegetative propagation of the olive tree from in vitro cultured embryos[J]. Plant Science,1987,50, (1):85-90
[15]伊华林,邓秀新,史永忠,等.三倍体柑桔幼胚离体培养研究[J],园艺学报.1797,24(3)：289-290.
[16]曾宋君,程式君,张京丽,等.五种石斛兰的胚培养及其快速繁殖研究[J].园艺学报.1998,25(1)：75-80.
[17]章宁,黄维南.马拉巴栗胚胎发育及离体胚再生植株的研究[J].园艺学报.2000,27(1)：71-73.
[18]漆燕玲,栗孟飞,孙萍等.桃儿七成熟胚的离体培养研究[J].生物学杂志,2008,25(4)：39-41
[19]吴翼,武耀廷,马子龙等.椰子胚的离体培养与植株再生(简报)[J].亚热带植物科学.2008,37(1)：63-64
[20]Amstrong C L, Green C E. Establishment and maintenance maintenance friable,embryogenic maize callus and involvement of L-praline[J].Plan ta,1985,164:207-214
[21]亚历山大洛夫,B.T.1954：植物解剖学上册[M].王凯基等译,高等教育出版社,1960：260-278
[22]崔澄,汤兆达.植物激素与组织分化和器官形成[M].罗士苇等编“植物激素”,上海科学技术出版社,1964
[23]Yeoman, M. M. and J.P.Mitchell, Changes accompanying the addition of 2,4-D to excised Jerusalem artichoke tuber tissres[J], Ann. Bot,1970:34:799-810
[24]Yeoman, M.M.G..G.. Naik,and A.I.Robertson, Growth and differentiation of plant tissue cultures, The initiation and pattern of cell division in developing callus cultures Ann. Bot,1968:32: 301-313
[25]Grant, M.E.and K.W.Fuller, Tissue culture of root cells of Vicia faba[J]. j.exp. Bot.1968:19: 667-680
[26]Blackly, L. M, and F.C.Steward, Growthinduction in crltures of Happlopappus gracilis.I. The behavuor of the cultured cells.Amer. J.Bot,1961:48:351-358
[27]Esau.K.1965:Vascular Differentiation in Plants[J]. Holt, Rinevart and Winston,New York.
[28]Reinert, J,1978:Aspects of organization-organogenesis and embryogenesis[M].In "Plant Tissue and Cell Cultures"(Street,H.E.ed.)Blackwell Scientific Publieations, Oxford, pp.338-355
[29]Torrey, J.G Cellular differentiation of cultured cells and tissues[J]. Hort. Science,1977:12(2), 127-130
[30]Murashiga. Tn, Plant pronagation through tissue cultures.Ann[J], Rev. Plant Physiol.1974:25: 135-166
[31]赵军良.植物茎尖培养与无毒种苗生产[J].北方园艺,1995(6)：10-11
[32]Fouad M M,et al. Factors influencing rooting ofpeach shoots culture in vitro[J].Acta Horticulture,1995,409:197-202.
[33]Hammerschlag F et al.Factors influencing in vitro multiplication and rooting ofpeach cultivars[J].Plant Cell Tissue and Organ Culture,1987,8:235-242.
[34]包慈华,马经凤,王凯基,等.油橄榄茎尖培养成完整植株的初步研究[J].科学通报；1979,2(2)
[35]陈正华.木本植物组织培养]M].北京：高等教育出版社.1986,34-36；408-419,456-465；466-480
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