濒危植物峨眉拟单性木兰群落特征及繁育技术研究
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摘要
峨眉拟单性木兰(Parakmeria omeiensis Cheng)为我国特有种,分布于四川峨眉山,野生植株仅存二十几株,是国家一级濒危保护植物。本文研究了峨眉拟单性木兰的群落特征、组培技术、嫁接技术、多酚氧化酶(PPO)活性和总酚含量。研究结果如下:
1.峨眉拟单性木兰群落特征峨眉拟单性木兰零星分布于峨眉山红椿坪扁担岩常绿阔叶林中,群落内植物50种,隶属于37科48属,其中双子叶植物占绝对优势,占整个群落的80%,把含有三个属以上的科看作优势科,分别有木兰科、壳斗科、樟科、蔷薇科,占总科数的10.8%。该群落植物区系具有热带亚热带性质,可分为9个类型,热带分布区的植物成份所占的比率高于温带分布区的植物成份,而热带分布区的植物成份中又以泛热带为主。群落中高位芽植物占物种数的74%,其中,中高位芽植物和矮高位芽植物约占64%;地上芽植物占4%,地面芽植物占6%,隐芽植物和一年生植物均占8%。乔木层郁闭度达70%,以重要值较高的山茶科、罗汉松科、壳斗科、木兰科植物为主,其次为槭树科和樟科植物。其中峨眉拟单性木兰在群落中呈零星分布,平均株高20m,平均枝下高13.8m左右,胸径达31cm,平均冠幅6m;更新层数量较多的有麦叶罗汉松、巫山新木姜子、南方红豆杉,以巫山新木姜子占绝对优势,在该层未见峨眉拟单性木兰幼苗;灌木层平均盖度达72%,鄂西玉山竹占绝对优势,其次是刺叶铁仔和西南菝葜;草本层物种单一,盖度5%～30%,主要为青绿苔草。分别计算各层次物种多样性指数,得出乔木层生物多样性最高,辛普森指数0.8610,Shannon-Weiner指数2.3062,均匀度最大0.8508,生态优势度最低,为0.1390,其次是更新层0.1823,灌木层生态优势度最高0.8521,均匀度最小0.1578。
2.峨眉拟单性木兰组织培养组培取材的最佳时间是4～5月,平均污染率在20%以下,升汞对茎段外植体的最佳处理时间是(7+8)min,此时污染率低,且腋芽诱导不会受到抑制,诱导率达74.3%;在培养基中添加10mg/L的VC和1g/L的PVP都能有效地控制褐化,其中10mg/L的VC效果最好,褐化率仅占对照的1/2,顶芽启动率达77.8%;有利于峨眉拟单性木兰带芽茎段腋芽诱导的最佳培养基配方组合为:B5+6-BA1.5 mg/L+IBA0.1 mg/L,诱导率达92.9%;适合腋芽茎段伸长生长的培养基是:MS+6-BA1.0+IBA0.1+NAA0.1或B5+6-BA1.5 mg/L+IBA0.1 mg/L;有利于顶芽启动培养的最佳培养基配方组合为:MS+BA 0.5 mg/L+IBA 0.05 mg/L;适合峨眉拟单性木兰茎段愈伤组织诱导的最佳培养基组合为:B5+6-BA1.5 mg/L+NAA0.5mg/L,培养条件为暗培养;4月取材为诱导茎段愈伤组织的最佳时间;MS(B5)+6-BA0.2mg/L+ZT 0.2mg/L+NAA0.1mg/L是适合芽增殖培养的最佳培养基组合。
3.峨眉拟单性木兰嫁接技术不同的砧木之间嫁接成活率的比较结果为:河南辛夷＞白玉兰＞江油辛夷,不同的嫁接季节成活率比较:春季＞秋季。
4.峨眉拟单性木兰PPO活性和总酚含量的测定对峨眉拟单性木兰新鲜茎段材料进行PPO活性的最佳试验条件及性质进行了研究,旨在为进一步探索峨眉拟单性木兰组织培养过程中的褐变机理提供可靠的试验方法,并为褐变的控制提供科学依据。试验结果发现,测定峨眉拟单性木兰PPO活性的最适PH值为7.0,最适温度为25℃,或高或低都会降低酶活性,邻苯二酚浓度为0.14 mol/L时PPO活性最大,VC、柠檬酸和Na_2SO_4三种抑制物中,VC的抑制效果最好;峨眉拟单性木兰PPO活性和总酚含量与物候期有较为密切的关系,生长季节的PPO活性高于休眠季节,而生长旺盛的季节和休眠季节的总酚含量高于抽稍季节;研究组培初代的茎段外植体PPO活性和总酚含量动态变化得出,接种后的一、二周PPO活性呈上升趋势,两周后就不断下降,而总酚含量呈上升一下降一上升的趋势变化。
Parakmeria omeiensis Cheng is the rare plant endemic to Emei mountain in China, which was listed as the first level protected paints of China.The total number of the Parakmeria omeiensis Cheng in wild is only more than 20.This article studied the community characteristics,tissue culture,grafting,PPO activity and total phenols content. The main results were as follows:
1.The community characteristics of Parakmeria omeiensis Cheng.There were some scattered Parakmeria omeiensis Cheng in evergreen broad-leaved forest of Biandanyan in Emei Mountain.The community contained 50 plant species,belonging to 37 families and 48 genera,among them the dicotyledon had comparative advantage, accounting for 80%of the total community.Magnoliaceae,Fagaceae,Lauraceae and Rosaceae accounted for 10.8%of the total families in community,which contained more than 3 genera be regarded as the superiority.The distributions of the plants in this community were mostly in the tropic and subtropic area,which could be divided into 9 types,the ratio of the plants which was to be tropic was higher than temperate zone,and the universal tropical plant was dominated.In this community,the phanerophytes accounted for 74%,mesophanerophyta and nanophanerophyta accounted for 64%, chamaephytes,hemicryptophytes,geophytes and therophytes accounted for 4%,6%,8% and 8%.The coverage of the arbor layer was 70%,Theaceae,Podocarpaceae,Fagaceae and Magnoliaceae was dominated with high importance value,then the Aceraceae and Lauraceae.Parakmeria omeiensis Cheng were scattered in this community,the average height was 20m,the height of clearance was about 13.8m,the diameter reached 31cm and the average crown width was about 6m.The number of the Podocarpus neriifolius D.don, Neolitsea wushanica(Chun)Merr and Taxus chinenwsis var.mairei Cheng et L.K was ralativly much more in regeneration layer,and the Neolitsea wushanica(Chun)Merr was dominated,but the seedling of Parakmeria omeiensis Cheng was not found in this layer. The average coverage of the shrub layer was 72%,Y.confusa(McClure)Z.P.Wang et G.H.Ye was dominated,then the Myrisne semiserrata and Smilax bockii Warb.The plant species in herb layer was single,the coverage was 5%～30%,the Carex leucochlora Gge.was dominated.The biodiversity of the arbor layer was the highest,Simpson index of which was 0.8610, Shannon-Weiner index was 2.3062,the evenness was the highest,0.8508,the ecological dominance was the lowest,0.1390,then the regeneration layer,0.1823,the ecological dominance of the shrub layer was the highest,0.8521,the evenness was the lowest,0.1578.
2.The tissue culture of Parakmeria omeiensis Cheng.The period from April to May was the optimal time for explants collection in cause of the pollution rate nearly 20%, Continuously sterilize twice(7+8) min was the suitable sterilizing condition for stem segment,the pollution rate was the lowest and the axillary buds induction was 74.3%.Medium supplemented with 10mg/L VC and 1g/L PVP could control browning efficiently,and the former was better,the browning rate was about 1/2 of the control,the induction of apical buds was 77.8%.The stems of Parakmeria omeiensis Cheng were taken as explants,the optional culture medium for the initiation was B5+6-BA1.5 mg/L+IBA0.1 mg/L,the startup rate was 92.9%.The optional culture medium for the elongation growth was MS+6-BA1.0+IBA0.1+NAA0.1或B5+6-BA1.5 mg/L+IBA0.1 mg/L.The optional culture medium for the induction of apical buds was MS+BA 0.5 mg/L +IBA 0.05 mg/L. B5+6-BA1.5 mg/L+NAA0.5mg/L was the optimal culture medium for callus induction of the stem segment of Parakmeria omeiensis Cheng,the explants were palced in darkness. April was the optimal time for explants collection to induct callus.MS(B5) +6-BA 0.2mg/L+ZT 0.2mg/L+NAA0.1mg/L was the optimal culture medium for bud multiplication.
3.The grafting of Parakmeria omeiensis Cheng.The comparison of survival among different rootstocks and in different season,Magnolia liliflora Desr(Henan)＞Magnolia denudate＞Magnolia liliflora Desr(Jiangyou),Spring＞Autumn.
4.The measuremen of the PPO activity and total phenols content of Parakmeria omeiensis Cheng.Studied on the best test condition and property of the PPO activity with fresh stem segment of Parakmeria omeiensis Cheng,provided the reliable testing method on browning mechanism in tissue culture for further exploring.The results showed that the best test condition on PPO activity of Parakmeria omeiensis Cheng was PH 7.0, 25℃,the temperature be too high or too below would inhibit the PPO activity,the optimal concentration of o-dihydroxybenzene was 0.14mol/L,among VC,Citric Acid and Na_2SO_4, VC was the best to control browning.PPO activity and total phenols content of Parakmeria omeiensis Cheng had close relationship with phenophase,PPO activity in growing season was higher than dormancy season,the total phenols content in growing season and dormancy season was higher than stooling stage.Studied on the dynamic change of PPO activity and total phenols content of stem segment explants in initial cultivation,the results showed that,in inoculation two weeks,PPO activity was rising trend,then decreased two weeks later,the dynamic change of total phenols content was rising,decreasing,and then rising.
1.峨眉拟单性木兰群落特征峨眉拟单性木兰零星分布于峨眉山红椿坪扁担岩常绿阔叶林中,群落内植物50种,隶属于37科48属,其中双子叶植物占绝对优势,占整个群落的80%,把含有三个属以上的科看作优势科,分别有木兰科、壳斗科、樟科、蔷薇科,占总科数的10.8%。该群落植物区系具有热带亚热带性质,可分为9个类型,热带分布区的植物成份所占的比率高于温带分布区的植物成份,而热带分布区的植物成份中又以泛热带为主。群落中高位芽植物占物种数的74%,其中,中高位芽植物和矮高位芽植物约占64%;地上芽植物占4%,地面芽植物占6%,隐芽植物和一年生植物均占8%。乔木层郁闭度达70%,以重要值较高的山茶科、罗汉松科、壳斗科、木兰科植物为主,其次为槭树科和樟科植物。其中峨眉拟单性木兰在群落中呈零星分布,平均株高20m,平均枝下高13.8m左右,胸径达31cm,平均冠幅6m;更新层数量较多的有麦叶罗汉松、巫山新木姜子、南方红豆杉,以巫山新木姜子占绝对优势,在该层未见峨眉拟单性木兰幼苗;灌木层平均盖度达72%,鄂西玉山竹占绝对优势,其次是刺叶铁仔和西南菝葜;草本层物种单一,盖度5%～30%,主要为青绿苔草。分别计算各层次物种多样性指数,得出乔木层生物多样性最高,辛普森指数0.8610,Shannon-Weiner指数2.3062,均匀度最大0.8508,生态优势度最低,为0.1390,其次是更新层0.1823,灌木层生态优势度最高0.8521,均匀度最小0.1578。
2.峨眉拟单性木兰组织培养组培取材的最佳时间是4～5月,平均污染率在20%以下,升汞对茎段外植体的最佳处理时间是(7+8)min,此时污染率低,且腋芽诱导不会受到抑制,诱导率达74.3%;在培养基中添加10mg/L的VC和1g/L的PVP都能有效地控制褐化,其中10mg/L的VC效果最好,褐化率仅占对照的1/2,顶芽启动率达77.8%;有利于峨眉拟单性木兰带芽茎段腋芽诱导的最佳培养基配方组合为:B5+6-BA1.5 mg/L+IBA0.1 mg/L,诱导率达92.9%;适合腋芽茎段伸长生长的培养基是:MS+6-BA1.0+IBA0.1+NAA0.1或B5+6-BA1.5 mg/L+IBA0.1 mg/L;有利于顶芽启动培养的最佳培养基配方组合为:MS+BA 0.5 mg/L+IBA 0.05 mg/L;适合峨眉拟单性木兰茎段愈伤组织诱导的最佳培养基组合为:B5+6-BA1.5 mg/L+NAA0.5mg/L,培养条件为暗培养;4月取材为诱导茎段愈伤组织的最佳时间;MS(B5)+6-BA0.2mg/L+ZT 0.2mg/L+NAA0.1mg/L是适合芽增殖培养的最佳培养基组合。
3.峨眉拟单性木兰嫁接技术不同的砧木之间嫁接成活率的比较结果为:河南辛夷＞白玉兰＞江油辛夷,不同的嫁接季节成活率比较:春季＞秋季。
4.峨眉拟单性木兰PPO活性和总酚含量的测定对峨眉拟单性木兰新鲜茎段材料进行PPO活性的最佳试验条件及性质进行了研究,旨在为进一步探索峨眉拟单性木兰组织培养过程中的褐变机理提供可靠的试验方法,并为褐变的控制提供科学依据。试验结果发现,测定峨眉拟单性木兰PPO活性的最适PH值为7.0,最适温度为25℃,或高或低都会降低酶活性,邻苯二酚浓度为0.14 mol/L时PPO活性最大,VC、柠檬酸和Na_2SO_4三种抑制物中,VC的抑制效果最好;峨眉拟单性木兰PPO活性和总酚含量与物候期有较为密切的关系,生长季节的PPO活性高于休眠季节,而生长旺盛的季节和休眠季节的总酚含量高于抽稍季节;研究组培初代的茎段外植体PPO活性和总酚含量动态变化得出,接种后的一、二周PPO活性呈上升趋势,两周后就不断下降,而总酚含量呈上升一下降一上升的趋势变化。
Parakmeria omeiensis Cheng is the rare plant endemic to Emei mountain in China, which was listed as the first level protected paints of China.The total number of the Parakmeria omeiensis Cheng in wild is only more than 20.This article studied the community characteristics,tissue culture,grafting,PPO activity and total phenols content. The main results were as follows:
1.The community characteristics of Parakmeria omeiensis Cheng.There were some scattered Parakmeria omeiensis Cheng in evergreen broad-leaved forest of Biandanyan in Emei Mountain.The community contained 50 plant species,belonging to 37 families and 48 genera,among them the dicotyledon had comparative advantage, accounting for 80%of the total community.Magnoliaceae,Fagaceae,Lauraceae and Rosaceae accounted for 10.8%of the total families in community,which contained more than 3 genera be regarded as the superiority.The distributions of the plants in this community were mostly in the tropic and subtropic area,which could be divided into 9 types,the ratio of the plants which was to be tropic was higher than temperate zone,and the universal tropical plant was dominated.In this community,the phanerophytes accounted for 74%,mesophanerophyta and nanophanerophyta accounted for 64%, chamaephytes,hemicryptophytes,geophytes and therophytes accounted for 4%,6%,8% and 8%.The coverage of the arbor layer was 70%,Theaceae,Podocarpaceae,Fagaceae and Magnoliaceae was dominated with high importance value,then the Aceraceae and Lauraceae.Parakmeria omeiensis Cheng were scattered in this community,the average height was 20m,the height of clearance was about 13.8m,the diameter reached 31cm and the average crown width was about 6m.The number of the Podocarpus neriifolius D.don, Neolitsea wushanica(Chun)Merr and Taxus chinenwsis var.mairei Cheng et L.K was ralativly much more in regeneration layer,and the Neolitsea wushanica(Chun)Merr was dominated,but the seedling of Parakmeria omeiensis Cheng was not found in this layer. The average coverage of the shrub layer was 72%,Y.confusa(McClure)Z.P.Wang et G.H.Ye was dominated,then the Myrisne semiserrata and Smilax bockii Warb.The plant species in herb layer was single,the coverage was 5%～30%,the Carex leucochlora Gge.was dominated.The biodiversity of the arbor layer was the highest,Simpson index of which was 0.8610, Shannon-Weiner index was 2.3062,the evenness was the highest,0.8508,the ecological dominance was the lowest,0.1390,then the regeneration layer,0.1823,the ecological dominance of the shrub layer was the highest,0.8521,the evenness was the lowest,0.1578.
2.The tissue culture of Parakmeria omeiensis Cheng.The period from April to May was the optimal time for explants collection in cause of the pollution rate nearly 20%, Continuously sterilize twice(7+8) min was the suitable sterilizing condition for stem segment,the pollution rate was the lowest and the axillary buds induction was 74.3%.Medium supplemented with 10mg/L VC and 1g/L PVP could control browning efficiently,and the former was better,the browning rate was about 1/2 of the control,the induction of apical buds was 77.8%.The stems of Parakmeria omeiensis Cheng were taken as explants,the optional culture medium for the initiation was B5+6-BA1.5 mg/L+IBA0.1 mg/L,the startup rate was 92.9%.The optional culture medium for the elongation growth was MS+6-BA1.0+IBA0.1+NAA0.1或B5+6-BA1.5 mg/L+IBA0.1 mg/L.The optional culture medium for the induction of apical buds was MS+BA 0.5 mg/L +IBA 0.05 mg/L. B5+6-BA1.5 mg/L+NAA0.5mg/L was the optimal culture medium for callus induction of the stem segment of Parakmeria omeiensis Cheng,the explants were palced in darkness. April was the optimal time for explants collection to induct callus.MS(B5) +6-BA 0.2mg/L+ZT 0.2mg/L+NAA0.1mg/L was the optimal culture medium for bud multiplication.
3.The grafting of Parakmeria omeiensis Cheng.The comparison of survival among different rootstocks and in different season,Magnolia liliflora Desr(Henan)＞Magnolia denudate＞Magnolia liliflora Desr(Jiangyou),Spring＞Autumn.
4.The measuremen of the PPO activity and total phenols content of Parakmeria omeiensis Cheng.Studied on the best test condition and property of the PPO activity with fresh stem segment of Parakmeria omeiensis Cheng,provided the reliable testing method on browning mechanism in tissue culture for further exploring.The results showed that the best test condition on PPO activity of Parakmeria omeiensis Cheng was PH 7.0, 25℃,the temperature be too high or too below would inhibit the PPO activity,the optimal concentration of o-dihydroxybenzene was 0.14mol/L,among VC,Citric Acid and Na_2SO_4, VC was the best to control browning.PPO activity and total phenols content of Parakmeria omeiensis Cheng had close relationship with phenophase,PPO activity in growing season was higher than dormancy season,the total phenols content in growing season and dormancy season was higher than stooling stage.Studied on the dynamic change of PPO activity and total phenols content of stem segment explants in initial cultivation,the results showed that,in inoculation two weeks,PPO activity was rising trend,then decreased two weeks later,the dynamic change of total phenols content was rising,decreasing,and then rising.
引文
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