湿地松、火炬松和黑松的组培繁殖技术研究
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摘要
湿地松和火炬松具有许多优良性状，然而，松针褐斑病的大面积流行制约了其在我国南方的发展；黑松作为优良的园林绿化和造林树种，在日本深受欢迎，但却受到松材线虫病的严重危害。目前，通过抗病选育的方式选出了一批优良的湿地松、火炬松和黑松家系并建立了种子园，但抗病种子产量有限，不能满足生产需要，因此，利用组织培养方式快速扩大繁殖现有的抗性材料具有重要的意义。
(1) 以成熟胚为外植体诱导不定芽发生建立了湿地松组织培养再生体系。用含高浓度6-BA的液体培养基处理湿地松成熟胚12～36小时，吸干后转移到无激素的培养基上，一个月后即可诱导产生不定芽。经60 mg／L6-BA的液体培养基处理12～24小时后，不定芽诱导频率及平均每外植体产芽数达到最高，但芽体较小，在继代培养基中伸长较慢。经30mg／L 6-BA的液体培养基处理12-36小时后，不定芽诱导频率较低，但不定芽较健壮，在继代培养基中伸长较快。6-BA浓度大于80mg／L时不利于外植体生长及不定芽的分化。伸长的不定芽在改良1／2GD+NAA0.05mg／L的培养基中培养4周后可形成不定根。
(2) 以带子叶顶芽为外植体诱导丛生芽建立了湿地松组织培养的再生体系。诱导培养基中单独添加细胞分裂素即可诱导湿地松丛生芽的发生，6-BA效果优于KT；培养基中配合使用生长素较单独使用细胞分裂素效果更理想。基本培养基、6-BA及NAA的浓度、处理时间等对丛生芽诱导有较大影响。在改良GD+6-BA4～5 mg／L+NAA0.05～0.1 mg／L的培养基上培养4～5周，其丛生芽诱导率最高达95％；已分化的丛生芽继代在无激素或添加活性炭或生长素(NAA或IBA)的GD培养基中伸长较快；丛生芽单个切下继代培养在改良GD+6-BA3.0 mg／L+NAA0.1 mg／L的培养基上增殖较快。将嫩梢切下置于附加NAA0.05mg／L或IBA2.0 mg／L或NAA0.05 mg／L+IBA2.0 mg／L的1／2GD培养基中培养30天后均可形成不定根，生根率分别为53.3％、51.6％、64.7％。再生植株移栽成活率达85％。
(3) 采用带子叶顶芽为外植体建立了黑松、火炬松组织培养的再生体系。较适合黑松丛生芽诱导的培养基为改良GD+6-BA4～5 mg／L+NAA0.1 mg／L，诱导率在90％左右。火炬松在改良GD+6-BA4 mg／L+NAA0.02 mg／L的培养基中诱导率较高(86.7％)。改良GD培养基中添加活性炭或微量生长素明显促进火炬松的伸长，对黑松丛生芽则无促进作用。较适合黑松丛生芽伸长的培养基为无激素的改良GD培养基，但其伸长速率比火炬松、湿地松低。将黑松、火炬松嫩梢切下置于附加NAA0.05 mg／L的1／2GD培养基中培养30天后均可形成不定根，但生根率很低，分别为5％、12.5％。
Brown spot needle blight caused by Lecannosticta acicola is one of the important diseases on P. elliottii and P. taeda which are major tree species in Southern China. Silvicultural characteristics and excellent wood quality of Japanese black pine make it a favoured reforestation species in Japan, but it is susceptible to Pine wood nematode. Resistant varieties have been developed through selective breeding. But seeds supply are limited. So, it is of great value to propagate rapidly excellent breeds which can strongly resist to diseases by the means of tissue culture.
Adventitious bud formation was obtained from mature embryos of P. elliottii by a short soaking of embryos in a liquid medium with high concentration of 6-BA and then transfered to 1/2 modified Gresshoff and Doy (GD) medium without hormone. Six weeks later , the highest differentiation frequency and the largest number of adventitious buds were obtained in the pretreatment with 60 mg/L 6-BA for 24h. Relatively high frequencies of large buds were obtained after a pretreatment with 30 mg/L 6-BA for 24h. On 1/2 GD medium supplemented with NAA 0.05 mg/L, adventitious shoots derived from a pretreatment with 60 mg/L 6-BA elongated more slowly and were smaller in size than those pretreated with 30 mg/L 6-BA. Roots were formed when excised shoots planted on 1/2 GD supplemented with NAA 0.05 mg/L for 4weeks, and the rooting rate was 30%.,
Axillary buds were induced when the explant sources were the aseptic seedlings without roots of P. elliottii. 6-BA and KT were both effective at inducing buds formation ,but 6-BA was more potent than KT. 6-BA in combination with NAA in GD medium increased bud production comparing with the medium with cytokinin only. Basal media composition, the concentration of 6-BA and NAA and length of exposure to the cytokinin significantly influenced bud induction. The highest inducing rate of axillary buds was up to 95% on the GD medium supplemented with 4.0-5.0 mg/L6-BA and 0.05-0.1 mg/L NAA for 4-5 weeks. The addition of NAA(0.05~0.1 mg/L) or IBA (1.0 mg/L) to basal medium could greatly promote slash pine shoots elongation and growth. Reduced concentration of minerals failed to enhance shoot elongation. The shoots were propagated most rapidly when subcultured on the medium of modified GD containing 3.0 mg/L 6-BA and 0.1 mg/L NAA. Roots were formed when excised shoots were planted on modified GD 1/2 medium supplemented
with 0.05 mg/LNAA or 2.0 mg/LIBA or 0.05 mg/L NAA plus 1.0 mg/L IBA and 20g/L sucrose for 30 days. The rooting rate was 53.3%, 51.6%, 64.7% respectively. The shoots elongation with different treatments influence root-induction significantly. The surviving rate was 85% when the rooting shoots
were moved to the greenhouse.
Axillary buds were also induced when the explant sources were the aseptic seedlings without roots of P. taeda and P. thunbergii. For bud induction, modified GD medium supplemented with 4.0-5.0 mg/L 6-BA and 0.1 mg/L NAA was best for P. thunbergii whereas a lower concentration of 6-BA(4 mg/L) and NAA (0.02 mg/L) was best for P. taeda. Addition of activated charcoal( 0.5 g/L )or NAA(0.02~0.05mg/L) to GD medium improved the growth of shoots of P. taeda but not of P. thunbergii. The most suitable medium for shoot elongation of P. thunbergii was GD without any growth regulators. Roots were formed when excised shoots were planted on modified GD 1/2 medium supplemented with 0.05 mg/L NAA , but the rooting rate of both plants were very low, only 5% ( P. thunbergii) and 12.5% ( P. taeda) respectively. Addition of activated charcoal inhibited root induction. So it would be necessary to optimize the rooting medium.
(1) 以成熟胚为外植体诱导不定芽发生建立了湿地松组织培养再生体系。用含高浓度6-BA的液体培养基处理湿地松成熟胚12～36小时，吸干后转移到无激素的培养基上，一个月后即可诱导产生不定芽。经60 mg／L6-BA的液体培养基处理12～24小时后，不定芽诱导频率及平均每外植体产芽数达到最高，但芽体较小，在继代培养基中伸长较慢。经30mg／L 6-BA的液体培养基处理12-36小时后，不定芽诱导频率较低，但不定芽较健壮，在继代培养基中伸长较快。6-BA浓度大于80mg／L时不利于外植体生长及不定芽的分化。伸长的不定芽在改良1／2GD+NAA0.05mg／L的培养基中培养4周后可形成不定根。
(2) 以带子叶顶芽为外植体诱导丛生芽建立了湿地松组织培养的再生体系。诱导培养基中单独添加细胞分裂素即可诱导湿地松丛生芽的发生，6-BA效果优于KT；培养基中配合使用生长素较单独使用细胞分裂素效果更理想。基本培养基、6-BA及NAA的浓度、处理时间等对丛生芽诱导有较大影响。在改良GD+6-BA4～5 mg／L+NAA0.05～0.1 mg／L的培养基上培养4～5周，其丛生芽诱导率最高达95％；已分化的丛生芽继代在无激素或添加活性炭或生长素(NAA或IBA)的GD培养基中伸长较快；丛生芽单个切下继代培养在改良GD+6-BA3.0 mg／L+NAA0.1 mg／L的培养基上增殖较快。将嫩梢切下置于附加NAA0.05mg／L或IBA2.0 mg／L或NAA0.05 mg／L+IBA2.0 mg／L的1／2GD培养基中培养30天后均可形成不定根，生根率分别为53.3％、51.6％、64.7％。再生植株移栽成活率达85％。
(3) 采用带子叶顶芽为外植体建立了黑松、火炬松组织培养的再生体系。较适合黑松丛生芽诱导的培养基为改良GD+6-BA4～5 mg／L+NAA0.1 mg／L，诱导率在90％左右。火炬松在改良GD+6-BA4 mg／L+NAA0.02 mg／L的培养基中诱导率较高(86.7％)。改良GD培养基中添加活性炭或微量生长素明显促进火炬松的伸长，对黑松丛生芽则无促进作用。较适合黑松丛生芽伸长的培养基为无激素的改良GD培养基，但其伸长速率比火炬松、湿地松低。将黑松、火炬松嫩梢切下置于附加NAA0.05 mg／L的1／2GD培养基中培养30天后均可形成不定根，但生根率很低，分别为5％、12.5％。
Brown spot needle blight caused by Lecannosticta acicola is one of the important diseases on P. elliottii and P. taeda which are major tree species in Southern China. Silvicultural characteristics and excellent wood quality of Japanese black pine make it a favoured reforestation species in Japan, but it is susceptible to Pine wood nematode. Resistant varieties have been developed through selective breeding. But seeds supply are limited. So, it is of great value to propagate rapidly excellent breeds which can strongly resist to diseases by the means of tissue culture.
Adventitious bud formation was obtained from mature embryos of P. elliottii by a short soaking of embryos in a liquid medium with high concentration of 6-BA and then transfered to 1/2 modified Gresshoff and Doy (GD) medium without hormone. Six weeks later , the highest differentiation frequency and the largest number of adventitious buds were obtained in the pretreatment with 60 mg/L 6-BA for 24h. Relatively high frequencies of large buds were obtained after a pretreatment with 30 mg/L 6-BA for 24h. On 1/2 GD medium supplemented with NAA 0.05 mg/L, adventitious shoots derived from a pretreatment with 60 mg/L 6-BA elongated more slowly and were smaller in size than those pretreated with 30 mg/L 6-BA. Roots were formed when excised shoots planted on 1/2 GD supplemented with NAA 0.05 mg/L for 4weeks, and the rooting rate was 30%.,
Axillary buds were induced when the explant sources were the aseptic seedlings without roots of P. elliottii. 6-BA and KT were both effective at inducing buds formation ,but 6-BA was more potent than KT. 6-BA in combination with NAA in GD medium increased bud production comparing with the medium with cytokinin only. Basal media composition, the concentration of 6-BA and NAA and length of exposure to the cytokinin significantly influenced bud induction. The highest inducing rate of axillary buds was up to 95% on the GD medium supplemented with 4.0-5.0 mg/L6-BA and 0.05-0.1 mg/L NAA for 4-5 weeks. The addition of NAA(0.05~0.1 mg/L) or IBA (1.0 mg/L) to basal medium could greatly promote slash pine shoots elongation and growth. Reduced concentration of minerals failed to enhance shoot elongation. The shoots were propagated most rapidly when subcultured on the medium of modified GD containing 3.0 mg/L 6-BA and 0.1 mg/L NAA. Roots were formed when excised shoots were planted on modified GD 1/2 medium supplemented
with 0.05 mg/LNAA or 2.0 mg/LIBA or 0.05 mg/L NAA plus 1.0 mg/L IBA and 20g/L sucrose for 30 days. The rooting rate was 53.3%, 51.6%, 64.7% respectively. The shoots elongation with different treatments influence root-induction significantly. The surviving rate was 85% when the rooting shoots
were moved to the greenhouse.
Axillary buds were also induced when the explant sources were the aseptic seedlings without roots of P. taeda and P. thunbergii. For bud induction, modified GD medium supplemented with 4.0-5.0 mg/L 6-BA and 0.1 mg/L NAA was best for P. thunbergii whereas a lower concentration of 6-BA(4 mg/L) and NAA (0.02 mg/L) was best for P. taeda. Addition of activated charcoal( 0.5 g/L )or NAA(0.02~0.05mg/L) to GD medium improved the growth of shoots of P. taeda but not of P. thunbergii. The most suitable medium for shoot elongation of P. thunbergii was GD without any growth regulators. Roots were formed when excised shoots were planted on modified GD 1/2 medium supplemented with 0.05 mg/L NAA , but the rooting rate of both plants were very low, only 5% ( P. thunbergii) and 12.5% ( P. taeda) respectively. Addition of activated charcoal inhibited root induction. So it would be necessary to optimize the rooting medium.
引文
1 Heybrock H M. et al. Resistance to diseases and pests in forest tree. Preceedings of the Third International Workshop on the Genetics of Host-Parasite Interactions in Forestry, 1982:14-21
2 周仲铭．森林病害防治理论和技术进展．世界林业研究，1992，2：36-42
3 Evans HC. The genus mycospharella and its anamorphs certoseptoria. Dothistrona and Lecanosticta pinus mycological papers, 1984, No.153, 102pp
4 Phleps WR, Kais AG, Nicholls TH. Brown-spot needle of Pines Forest Inset and disease. Forest service. U.S.A, 1978
5 Gibson IAS. Disease of forest trees widely planted as exotics in the tropics and southern hemisphere. Part 11. The genus Pinus. Commonwealth Forestry in stitate and Commonwealth mycological institute. Oxford an kew, UK, 1979
6 Skilling DD, Nicholls RH. Brown-spot needle disease biology and control in scotch pine plantations USA For. Ser.
7 李传道，朱熙樵等．松针褐斑病调查和病原鉴定。南京林学院学报，1986，10(2)：11-18
8 Boyer. W.D. Brown-spot resistance in natural stands of longleaf pine seedlings. USDA For. Service Res. Note.. 1972, 142p
9 Henru.B.W and Wills.O.O. Vstisyion in brown-spot infection of longleaf pine from several geographic sources. USDA For. Service Res. Note.. 1967, 52p
10 Kais. A. G. Influence of needle age and spore density on susceptibility of long leaf pine to scirrhia acicola. Phy(?)opathology. Vol67:686-688
11 Kais A. G. Variation between southern and northern isolates of Scirrhia acicola. Phytopathology. 1972: 62-78
12 Derr H.J and Melder T.W. Brown-spot resistance in longleaf pine Forest Science, 1970, 16:204-209
13 Longleaf x Slash hybrids at age 7: survival growth and disease susceptibility So: 1996 J. For 64(4): 236-239
14 Saydder. E Bayne and Derr H.J. Breeding longleaf pines for resistance to brown-spot needle blight. Phytopathology. 1972:325-329
15 叶建仁．湿地松、火炬松种源抗褐斑病试验和抗病优树选择．南京林学院学报，1986，10(2)：27-35
16 叶建仁，韩正敏，李传道等．湿地松抗松针褐斑病无性系种子园的营建技术的研究．南京林业大学学报，1991，15(2)：23-29
17 詹国辉．湿地松抗性子代抗病性遗传评价及其病原菌互作关系的研究．南京林业大学硕士论文．2001
18 杨宝君等编著．松材线虫病．中国林业出版社，2003
19 柴希民，蒋平主编．松材线虫病的发生和防治，中国农业出版社，2003
20 杨宝君，朱克恭，朱正昌，严敖金编．中国松材线虫病的流行与治理．中国林业出版社，1995
21 潘宏阳．当前我国松材线虫病的治理对策．森林病虫通讯．2000(6)：44-47
22 杨在琨．日本松材线虫抗性育种．周外林业科技，1984，(1)：11-12
23 马常耕．国际林木抗病育种的基本经验．世界林业研究，1995，4：13-21
24 Yoichi Kishi. The Pine Wood Nematode and the Japanese Pine Sawyew., Thomas Company Limited, Tokyo, Japan, 1995
25 片山 重俊，小林 慎一．Resistance experiment of a nematode onto seedlings from Japanese red pine passed primary or secondary inoculation test. TMJF 1987, 98: 259-260
26 Kurokawa, Y., Yamashita, M., Kasuya, K., T. and Kishi, Y.. Inoculation of Bursaphelenchus lignicolus to supposedly resistant pine trees. TMJF 1980, 91:365-366
27 Kishi, Y.. Selective breeding of Pinus densiflora and Pinus thunbergii trees having resistance to Bursaphelenchus lignicolus disease. Growth conditions of the native pine trees under direct inoculation of Bursaphelenchus lignicolus during 3 years. TMJF-KT 1982, 34:149-150
28 赵路．日本林木育种事业概观．世界林业研究，1988，3：34-40
29 Itoya, Y..The initial performances of interspecific hybrids, Pinus thunbergii x P. massoniana and P. tabulaeformis-cone setting, seed quantity, seedling growth and resistance for timber nematode. TMJF 1976, 87:183-184
30 林木育种协会．日本造林协会编．林木育种事业30年的进展．藤原印刷株式会社，昭和62年，东京
31 杨宝君，王秋丽，邹卫东．不同松树品种对松材线虫的抗性．植物病理学报，1987，17(4)：211-214
32 胡凯基，王秋丽，杨宝君．松材线虫和拟松材线虫不同株系致病性研究．林业科学研究，1994，7(4)：381-386
33 徐福元，葛明宏，朱正昌等．南京地区不同松种和马尾松种源对松材线虫病的抗性及病害流行规律．林业科学研究，1996，9(5)：521-524
34 徐福元，葛明宏，张培．不同马尾松种源对松材线虫病的抗性．南京林业大学学报，2000，24(4)：85-88
35 杨宝君，胡凯基，王秋丽等．松树对松材线虫的抗性的研究．林业科学研究，1993，6(3)：250-255
36 徐福元，席客，徐刚．不同龄级马尾松对松材线虫抗性的探讨．南京林业大学学报，1994，18(3)：27-33
37 周国梁，程瑚瑞．马尾松感染松材线虫的研究．植物病理学报，1993，23(1)：81-84
38 Well O O,Wakeley P C.Geographic variation in survival, groeth, and fusiforma-rust infection of planted loblolly pine. For. Sci., 1996, Monograph 11, 40pp.
2 周仲铭．森林病害防治理论和技术进展．世界林业研究，1992，2：36-42
3 Evans HC. The genus mycospharella and its anamorphs certoseptoria. Dothistrona and Lecanosticta pinus mycological papers, 1984, No.153, 102pp
4 Phleps WR, Kais AG, Nicholls TH. Brown-spot needle of Pines Forest Inset and disease. Forest service. U.S.A, 1978
5 Gibson IAS. Disease of forest trees widely planted as exotics in the tropics and southern hemisphere. Part 11. The genus Pinus. Commonwealth Forestry in stitate and Commonwealth mycological institute. Oxford an kew, UK, 1979
6 Skilling DD, Nicholls RH. Brown-spot needle disease biology and control in scotch pine plantations USA For. Ser.
7 李传道，朱熙樵等．松针褐斑病调查和病原鉴定。南京林学院学报，1986，10(2)：11-18
8 Boyer. W.D. Brown-spot resistance in natural stands of longleaf pine seedlings. USDA For. Service Res. Note.. 1972, 142p
9 Henru.B.W and Wills.O.O. Vstisyion in brown-spot infection of longleaf pine from several geographic sources. USDA For. Service Res. Note.. 1967, 52p
10 Kais. A. G. Influence of needle age and spore density on susceptibility of long leaf pine to scirrhia acicola. Phy(?)opathology. Vol67:686-688
11 Kais A. G. Variation between southern and northern isolates of Scirrhia acicola. Phytopathology. 1972: 62-78
12 Derr H.J and Melder T.W. Brown-spot resistance in longleaf pine Forest Science, 1970, 16:204-209
13 Longleaf x Slash hybrids at age 7: survival growth and disease susceptibility So: 1996 J. For 64(4): 236-239
14 Saydder. E Bayne and Derr H.J. Breeding longleaf pines for resistance to brown-spot needle blight. Phytopathology. 1972:325-329
15 叶建仁．湿地松、火炬松种源抗褐斑病试验和抗病优树选择．南京林学院学报，1986，10(2)：27-35
16 叶建仁，韩正敏，李传道等．湿地松抗松针褐斑病无性系种子园的营建技术的研究．南京林业大学学报，1991，15(2)：23-29
17 詹国辉．湿地松抗性子代抗病性遗传评价及其病原菌互作关系的研究．南京林业大学硕士论文．2001
18 杨宝君等编著．松材线虫病．中国林业出版社，2003
19 柴希民，蒋平主编．松材线虫病的发生和防治，中国农业出版社，2003
20 杨宝君，朱克恭，朱正昌，严敖金编．中国松材线虫病的流行与治理．中国林业出版社，1995
21 潘宏阳．当前我国松材线虫病的治理对策．森林病虫通讯．2000(6)：44-47
22 杨在琨．日本松材线虫抗性育种．周外林业科技，1984，(1)：11-12
23 马常耕．国际林木抗病育种的基本经验．世界林业研究，1995，4：13-21
24 Yoichi Kishi. The Pine Wood Nematode and the Japanese Pine Sawyew., Thomas Company Limited, Tokyo, Japan, 1995
25 片山 重俊，小林 慎一．Resistance experiment of a nematode onto seedlings from Japanese red pine passed primary or secondary inoculation test. TMJF 1987, 98: 259-260
26 Kurokawa, Y., Yamashita, M., Kasuya, K., T. and Kishi, Y.. Inoculation of Bursaphelenchus lignicolus to supposedly resistant pine trees. TMJF 1980, 91:365-366
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