湿地松抗病家系组培繁殖技术研究
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摘要
松针褐斑病(Lecanosticta acicola)的大面积流行严重制约了湿地松的进一步发展。在病害严重地区，抗病育种是经济有效地防治松针褐斑病的唯一途径。所建的抗病种子园已进入结实阶段，子代抗病性测定工作也已进一步展开之中。然而，种子园有限的产量远远不能满足生产的需要，快速扩大繁殖现有材质优良、抗病性强的湿地松材料已迫在眉睫。故此，本文以湿地松为材料，研究其快速繁育系统。其主要研究结果如下：
以湿地松成熟合子胚为外植体诱导胚性愈伤组织，其中培养基组合为LP+2,4-D12mg／L+6-BA4mg／L和LP+2,4-D4.5mg／L+6-BA0.1mg／L+KT0.1mg／L可产生少量类似胚性愈伤组织的培养物。
以幼嫩子叶为外植体诱导不定芽的发生，培养基组合为改良GD+NAA0.1mg／L+6-BA5mg／L，诱导频率仅有7.1％；直接诱导湿地松成熟合子胚的不定芽分化，较为适宜的培养基组合为改良GD+NAA0.01mg／L+6-BA5mg／L，其诱导率可达61％，但同时褐化率也高达56.5％。这两种途径获得的不定芽对培养条件要求较高，本实验中没有研究出适合其继续生长的培养基。
以带子叶顶芽为外植体比较容易建立起湿地松组织培养的再生体系。实验结果表明，改良GD是离体胚生长和丛生芽分化的较优基本培养基。改良GD培养基中附加6-BA(0.5～5mg／L)和NAA(0.01～0.1mg／L)可诱导丛生芽的发生。丛生芽诱导的较优培养基组合为改良GD+NAA0.05mg／L+6-BA3mg／L，诱导率73.3％，芽分化系数为4.7。丛生芽在培养基组合为改良GD+NAA0.07mg／L中可以继续生长。1／2改良GD培养基中添加0.05％的活性炭可促进丛生芽茎伸长，一个月内平均可伸长0.7cm。培养基组合为1／2改良GD+NAA0.05mg／L+6-BA1.5mg／L相对较适合丛生芽扩大繁殖，能使50％的丛生芽分化出第二代芽。在本实验中没有研究出适合第二代芽继续生长的培养基。
1／2MS培养基是湿地松试管苗生根的较优基本培养基。湿地松试管苗对NAA较为敏感，0.01mg／LNAA便有少量不定根的形成；而对IBA则不太敏感，1mg／L以下的IBA都不能诱导根的形成。湿地松试管苗的不定根诱导培养较为适宜的组合是1／2MS+NAA0.05mg／L，诱导率可达55.5％。无激素1／2改良GD不但能够使少量试管苗直接分化出不定根，而且还能使已分化出的不定根迅速伸长。
Brown spot needle blight has become a serious obstacle in the further expanding of Pinus elliottii. A great deal of studies indicate that breeding for the disease resistance is only efficient way to control the disease at hight disease hazard sites. The seed orchards of resistance to the disease have come into the stage of seeding. Furthermore, it is going that the work of the filial generation's resistance to the disease. Nevertheless, the seed supply of the orchards are far away from the needs of afforestation. Therefore, it is impendent to rapidly proliferate the material of Pinus elliottii which has good wood quality and can strongly resist to the brown spot needle blight. So, this article researched the system of the rapid proliferation in Pinus elliottii by the way of tissue culture. The results are as followed:
Embryogenic callus development was initiated in cultured mature embryo explant of Pinus elliottii. There have a few tissue that resemble the embryogenic callus in appearance, cultured in LP medium supplemented with 2,4-D(12mg/L) and 6-BA(4mg/L) or 2,4-D(4.5mg/UK 6-BA(0.1mg/L) and KT(0.1mg/L).
Adventitious bud formation was obtained from green cotyledons and mature embros of Pinus elliottii cultured in modified GD with hormones. Adventitious buds were achieved in the modified GD supplemented with 6-BA(5mg/L) and NAA(0.1mg/L), which differentiation frequency was 7.1%. In addition, it was suitable for the direct adventitious buds induction in the modified GD supplemented with 6-BA(5mg/L) and NAA(0.01mg/L). It was 61% for the differentiation frequency of direct adventitious buds in this medium. However, the rate of browning was very high, accounting for 56.5%. These adventitious buds induced by the two ways were very difficult to be cultured. In this study, no medium was found to meet the requirements of the adventitious buds to grow up.
It was showed by the whole experiment that selecting the tip sprout c-arrying cotyledons as explants was more easily to build the regeneration system for Pinus elliottii. The modified GD medium was the best elementary medium for axillary buds differentiation of Pinus elliottii. Axillary buds we-re induced from modified GD medium supplemented with different levels of6-BA(0.5~5mg/L) and NAA(0.01~0.1mg/L). The better medium for Pinus
elliottii to induce axillary buds was modified GD supplemented with 6-BA(3mg/L) and NAA(0.05mg/L). The differentiation frequency was very high, reaching to 73.3%. At the same time, the buds regeneration coefficient was 4.7.
The axillary buds could continue to develop in the modified GD medi-um supplemented
with NAA(0.07mg/L). The elongation of the shoot stem was in the half of modified GD supplemented with AC(0.05%), in which the average length of the stem reached at 0.07cm in a month. The combination of modified GD+NAA0.05mg/L+6-BA1.5mg/L was relatively suitable to the p-roliferation of the axillary buds in Pinus elliottii, in which the differentiation frequency of the second generation buds accounted for 50%. In this study, no medium was found to meet the requirements of the adventitious buds to grow up.
The half of MS medium was the better basic medium for the rooting of the regenerated plantlets. It was quite sensitive to NAA for the rooting of the regenerated plantlets. A few of root had come into being when the NAA concentration was 0.01 mg/L. However, it was not so sensitive to IB A. No root formed when the IB A concentration was below 1 mg/L. The half MS medium supplemented with NAA(0.05mg/L) was suitable to the root induction of the regenerated plantlets in Pinus elliottii, in which the rate of induction was 60%. In addition, not only could a few regenerated plantlets direct form root, but the root induced quickly elongated in the half of modified GD.
以湿地松成熟合子胚为外植体诱导胚性愈伤组织，其中培养基组合为LP+2,4-D12mg／L+6-BA4mg／L和LP+2,4-D4.5mg／L+6-BA0.1mg／L+KT0.1mg／L可产生少量类似胚性愈伤组织的培养物。
以幼嫩子叶为外植体诱导不定芽的发生，培养基组合为改良GD+NAA0.1mg／L+6-BA5mg／L，诱导频率仅有7.1％；直接诱导湿地松成熟合子胚的不定芽分化，较为适宜的培养基组合为改良GD+NAA0.01mg／L+6-BA5mg／L，其诱导率可达61％，但同时褐化率也高达56.5％。这两种途径获得的不定芽对培养条件要求较高，本实验中没有研究出适合其继续生长的培养基。
以带子叶顶芽为外植体比较容易建立起湿地松组织培养的再生体系。实验结果表明，改良GD是离体胚生长和丛生芽分化的较优基本培养基。改良GD培养基中附加6-BA(0.5～5mg／L)和NAA(0.01～0.1mg／L)可诱导丛生芽的发生。丛生芽诱导的较优培养基组合为改良GD+NAA0.05mg／L+6-BA3mg／L，诱导率73.3％，芽分化系数为4.7。丛生芽在培养基组合为改良GD+NAA0.07mg／L中可以继续生长。1／2改良GD培养基中添加0.05％的活性炭可促进丛生芽茎伸长，一个月内平均可伸长0.7cm。培养基组合为1／2改良GD+NAA0.05mg／L+6-BA1.5mg／L相对较适合丛生芽扩大繁殖，能使50％的丛生芽分化出第二代芽。在本实验中没有研究出适合第二代芽继续生长的培养基。
1／2MS培养基是湿地松试管苗生根的较优基本培养基。湿地松试管苗对NAA较为敏感，0.01mg／LNAA便有少量不定根的形成；而对IBA则不太敏感，1mg／L以下的IBA都不能诱导根的形成。湿地松试管苗的不定根诱导培养较为适宜的组合是1／2MS+NAA0.05mg／L，诱导率可达55.5％。无激素1／2改良GD不但能够使少量试管苗直接分化出不定根，而且还能使已分化出的不定根迅速伸长。
Brown spot needle blight has become a serious obstacle in the further expanding of Pinus elliottii. A great deal of studies indicate that breeding for the disease resistance is only efficient way to control the disease at hight disease hazard sites. The seed orchards of resistance to the disease have come into the stage of seeding. Furthermore, it is going that the work of the filial generation's resistance to the disease. Nevertheless, the seed supply of the orchards are far away from the needs of afforestation. Therefore, it is impendent to rapidly proliferate the material of Pinus elliottii which has good wood quality and can strongly resist to the brown spot needle blight. So, this article researched the system of the rapid proliferation in Pinus elliottii by the way of tissue culture. The results are as followed:
Embryogenic callus development was initiated in cultured mature embryo explant of Pinus elliottii. There have a few tissue that resemble the embryogenic callus in appearance, cultured in LP medium supplemented with 2,4-D(12mg/L) and 6-BA(4mg/L) or 2,4-D(4.5mg/UK 6-BA(0.1mg/L) and KT(0.1mg/L).
Adventitious bud formation was obtained from green cotyledons and mature embros of Pinus elliottii cultured in modified GD with hormones. Adventitious buds were achieved in the modified GD supplemented with 6-BA(5mg/L) and NAA(0.1mg/L), which differentiation frequency was 7.1%. In addition, it was suitable for the direct adventitious buds induction in the modified GD supplemented with 6-BA(5mg/L) and NAA(0.01mg/L). It was 61% for the differentiation frequency of direct adventitious buds in this medium. However, the rate of browning was very high, accounting for 56.5%. These adventitious buds induced by the two ways were very difficult to be cultured. In this study, no medium was found to meet the requirements of the adventitious buds to grow up.
It was showed by the whole experiment that selecting the tip sprout c-arrying cotyledons as explants was more easily to build the regeneration system for Pinus elliottii. The modified GD medium was the best elementary medium for axillary buds differentiation of Pinus elliottii. Axillary buds we-re induced from modified GD medium supplemented with different levels of6-BA(0.5~5mg/L) and NAA(0.01~0.1mg/L). The better medium for Pinus
elliottii to induce axillary buds was modified GD supplemented with 6-BA(3mg/L) and NAA(0.05mg/L). The differentiation frequency was very high, reaching to 73.3%. At the same time, the buds regeneration coefficient was 4.7.
The axillary buds could continue to develop in the modified GD medi-um supplemented
with NAA(0.07mg/L). The elongation of the shoot stem was in the half of modified GD supplemented with AC(0.05%), in which the average length of the stem reached at 0.07cm in a month. The combination of modified GD+NAA0.05mg/L+6-BA1.5mg/L was relatively suitable to the p-roliferation of the axillary buds in Pinus elliottii, in which the differentiation frequency of the second generation buds accounted for 50%. In this study, no medium was found to meet the requirements of the adventitious buds to grow up.
The half of MS medium was the better basic medium for the rooting of the regenerated plantlets. It was quite sensitive to NAA for the rooting of the regenerated plantlets. A few of root had come into being when the NAA concentration was 0.01 mg/L. However, it was not so sensitive to IB A. No root formed when the IB A concentration was below 1 mg/L. The half MS medium supplemented with NAA(0.05mg/L) was suitable to the root induction of the regenerated plantlets in Pinus elliottii, in which the rate of induction was 60%. In addition, not only could a few regenerated plantlets direct form root, but the root induced quickly elongated in the half of modified GD.
引文
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67. Jain SM, Dong N and Newton RJ. Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryos cultured in vitro. Plant science Limerck, 1989, 65(2): 233～242
68. Tautorus TE, Fowke LC. Dunstan DI. Somatic embryogenesis in conifers. Can J Bot. 1991, 69:1873
69．唐巍，杨映根，桂耀林．松柏类植物体细胞胚胎发生的研究和应用．植物学通报，1996，13(1)：25～31
70. Liao YK, Amerson HV. Slash pine (Pinus ellittii Engelm) somatic embryogenesis I. Initiation of embtyogenic cultures from immature zygotic embyos, New Forests, 1995, 10(2): 145～163
71. Jones XB. Van. Somatic plantlets production from somatic embryos of Pinus patula. Plant physiol,1995, 145:519～525
72．高国训．植物组织培养中的褐变问题．植物生理学通讯，1999，35(6)：501～506
73．姚洪军，罗晓芳，田硕亭．植物组织培养外植体褐变的研究进展．北京林业大学学报，1999，21(3)：78～83
74. Brown L and Robert H, Lawnrence. Culture of Pinus callus on a medium. Forest Science, 1968,
14 (1)
75. Browson MR, Dixon RK, et al. In-vitro plantlet formation from embryonic cotyledons of Pinus elliottii Engelm. Proceedings, 10th North American Forest Biology Workshop, 'physiology and genetics of reforedtation'. Vacouver, British Columbia, July 10-22, 1988, 1988, 261～267
76. Brown MR, Dixon RK. Culture factors influencing adventitious shoot and plantlet formation from slash pine cotyldons. New Forests, 1991, 5: 4, 277～288
77. Burns JA, Schwarz OJ, SchLarboum SE. Multiple shoot production from seedling explants of slash pine (Pine elliottii Engelm). Plant Cell Reports. 1991, 10: 9, 439～443
78. Meyer HJ. In vitro formation of adventitious buds on mature embryos of Pinus elliottii Engelm. XP. Caribaea Morelet Hybrids. South Afican Journal of Botany, 1998, 64: 3, 220～225
79．阕国宁等．火炬松、湿地松、晚松组培繁殖的研究．林业科学研究，1997，10(3)：227～232
80．雷泽勇，周凤艳，周莉．关于离体胚组织培养在植物育种中的应用进展．防护林科技，2001，48(3)：57～59
81. Attree. SM, et al. Plant Cell Report, 1987, 6:480～483
82. David T Webb, Olga Diaz Santiage . Cytokinin induced bud formation on Caribean Pine (Pinus caribaea ) embryos in vitro. Plant Science Lett, 1983, 32. 17
83. Becwar MR, Nagmani R, Warm SR. Initiation of embryogenni culture and somatic embryo development in loblolly pine . Can Jfor Res, 1990, 20:81～11
84．唐巍，欧阳藩，郭仲琛．火炬松成熟合子胚培养直接器官发生和植株再生．云南植物研究，1997，19(3)：285～288
85．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
86．高国训，李光晨，袁丽慧．柱型苹果茎尖培养中增殖与植物激素的关系．天津农业科学，5(2)：18～22
87．吕芝香等．碳源种类和浓度愈伤组织生长的影响．植物生理学报，1981，(6)：1-5
88．丁世萍，严菊强，季道潘．糖类在植物组织培养植物培养中的效应．植物学通报，1998，15(6)：42～46
89. Flowler MW, WastsonRlyons I. 1982, In proc. 5th Intl. Cong. Plant Tissue and Cell Culture, Tissue Culture pp 225
90．杜晓光，袁巧平，李玲．兴安落叶松组织培养中丛生芽的形成研究．林业科技通讯，1992(2)：15～17
91．金韵琴，朱鹿鸣．提高试管苗及其移栽成活技术的研究．江苏林业科技，1989，(3)：9～13
92．赖家业，杨振德．活性炭在土柠檬组织培养中的作用．广西热作科技，1991，73(4)：10～11
93．李传道，韩正敏，叶建仁等．松针褐斑病的发生与防治．森林病虫通讯，1986，1
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