磨盘柿生物技术的研究
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摘要
本论文以磨盘柿当年生休眠芽为外植体，研究了接种形态及基本培养基对初代培养的影响；以组培苗为材料，研究了影响其离体快繁的诸因素，确定了适于磨盘柿早期和中期继代增殖的培养基成分；利用组培苗叶片及无节茎段为外植体，较为系统地研究了影响其不定芽再生的各因素，建立了不定芽再生体系；从成熟期的磨盘柿果实中成功克隆了2个ACC合成酶基因片段。以期为通过生物技术改善柿果贮运性能、简化贮运操作奠定基础。主要结果如下：
以剥离的休眠芽为外植体，在含BA7.5mg/L的MS，MS(1/2N)和1/2 MS培养基中均能建立初代培养。其中在MS(1/2N)+BA7.5mg/L中的外植体，生长状态最好，转入MS(1/2N)+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中，继代培养2代后，增殖系数达2.37，平均苗高为11.8mm。
组培苗在MS(1/2N)+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中继代培养10～12代，转入MS+ZT1.0mg/L中IAA0.1mg/L+BA1.0mg/L培养基中后，增殖系数较前者提高了44.4％，平均苗高增加5mm。方差分析结果显示，基本培养基对磨盘柿继代增殖的影响极为显著，ZT的作用次之，IAA作用较弱，BA最弱。与MS(1/2N)和1/2 MS相比，MS基本培养基更有利于组培苗早期继代增殖。
在MS+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中继代10～12代的组培苗，转入DKW+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中，组培苗生长极为旺盛、健壮，增殖系数较MS基本培养基中提高了43.1％，平均苗高增加6mm。R值结果表明，DKW+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基为磨盘柿组培苗中期继代增殖的最佳培养基。
通过三因素三水平正交试验，对基本培养基、ZT及IAA对叶片不定芽再生的影响进行了研究。结果表明，ZT浓度对叶片不定芽再生的影响作用最大，基本培养基次之，IAA对愈伤组织的形成有促进作用。组培苗顶端前2节位展开叶片，切成4mm×4mm左右，远轴面接触培养基时的再生效果最好。切片在MS(1/2N)+ZT4.0mg/L培养基中的分化率和平均不定芽数分别为76.76％和1.26。
位于组培苗顶端前2节位的伸长无节茎段，切成约4mm左右，平铺于MS(1/2N)+ZT4.0mg/L+IAA0.1mg/L培养基中可以获得最高的分化率和平均不定芽数，分别为83.65％和2.25。ZT和基本培养基对分化率和平均不定芽数的影响显著高于IAA，
而I从对愈伤组织形成的作用明显高于ZT和基本培养基。
从成熟期的磨盘柿果实中克隆到2个ACC合成酶基因片段M一CSI和M一CsZ，
两者核昔酸与氨基酸的同源性分别为98%和99%;与DK一ACsl的核昔酸同源性分别
为69%和70%。二者均编码224个氨基酸，均含有其它植物ACC合成酶的氨基酸保
守区及不变氨基酸残基，在多肤水平上的同源性很高，与DK-ACSI的氨基酸同源性
分别是98%和99%。
Dormant buds formed in the same year of Mopan persimmon trees were taken as explants. Effects of explants form and basal media on primary culture were studied. Factors affecting subculture micropropagation were studied by using tube seedlings as materials- The suited ingredient of culture medium for subculture micropropagation of forepart and metaphase were established Some factors affecting adventitious shoot regeneration were studied. Adventitious shoot regeneration systems from in vitro leaves and stems without internode of tube seedlings were obtained. 2 ACC synthase gene fragments were successfully cloned from, mature Mopan persimmon fruit, Here all have been done is in order to improve the persimmon performance of storage and transportation and simplify the management of storage and transportation. The mean results were as follows:
Dormant bud explants taken from Mopan persimmon trees after being peelled squamas and 3 leaf primordia off were inoculated on MS, MS(1/2N) and 1/2MS all supplemented with 7.5 mg/L BA. Primary cultures were all obtained on the 3 media. The growth state of explants inoculated on MS(1/2N)+ BA 7.5 mg/L medium was the best. They then were transferred on MS (1/2N) +ZT 1.0 mg/L +IAA.04 mg/L+BA1.0mg/L medium. The mean multiplication reached 2.37 and the average height of tube seedlings reached 11.8 mm after being subcultured 2 times.
Tube seedlings having been cultured on MS(1/2N) + ZT 1.0 mg/L + LAA 0.1 mg/L + BA 1.0 mg/L medium for about 10-12 times then were transferred on MS + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium. The multiplication increased 44.4%, at the same time the average height of shoots increased 5 mm. The results showed that the effect of the 4 factors on subculture micropropagation was in the order of basal medium, ZT, IAA and BA. Compared with MS(1/2N), MS basal medium was more beneficial to forepart subculture micropropagation.
Tube seedlings having been cultured on MS + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium about 10~12 times then were inoculated on DKW + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium. The multiplication increased 43.1% , and the average height of shoots increased 6 mm. The results of R value showed that the DKW basal medium was
the most suitable medium to the metaphase subculture micropropagation of "Mopan" tube seedlings.
Effects of basal media, ZT and IAA on adventitious shoot regeneration were studied through orthogonal design trial L9(34). The results showed that the effect of ZT concentration was the greatest and the basal medium took the second place. But the concentration of IAA was more effective on the forming of callus than basal medium and ZT. The upper 2 expanding leaves of tube seedlings, cut into 4 mm X 4 mm and inoculated with abaxial layer downside may have the greatest regeneration efficiency. The differentiation percentage and the average adventitious shoot number per explants on MS(1/2N) + ZT 4.0 mg/L were 76.76% and 1.26 respectively.
The upper 2 extending stems without internode on tube seedlings, cut about 4mm in length and lay on the MS(1/2N) + ZT 4.0 mg/L + IAA 0.1 mg/L medium may have the greatest regeneration efficiency. The differentiation percentage and the average adventitious shoot number per explants were 83.65% and 2.25 respectively. The effect of ZT concentration was the greatest and the basal medium was greater than IAA. But the concentration of IAA was more effective on the forming of callus than basal medium and ZT.
2 ACC synthase gene fragments M-ACS1 and M-ACS2 were successfully cloned from mature Mopan persimmon fruit. The identities of nucleotide acid and amino acid were 98% and 99% respectively. The nucleotide acid identities of M-ACS1 and M-ACS2 with DK-ACS1 were 69% and 70% respectively. They both encoded 224 amino acid. They also have the conserved amino acid regions and invariant amino acid residues of ACC synthase existing in other plants, and with higher identity in polypeptide leveE The amino acid identities with DK-ACS1 were 98% and 99% respectively.
以剥离的休眠芽为外植体，在含BA7.5mg/L的MS，MS(1/2N)和1/2 MS培养基中均能建立初代培养。其中在MS(1/2N)+BA7.5mg/L中的外植体，生长状态最好，转入MS(1/2N)+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中，继代培养2代后，增殖系数达2.37，平均苗高为11.8mm。
组培苗在MS(1/2N)+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中继代培养10～12代，转入MS+ZT1.0mg/L中IAA0.1mg/L+BA1.0mg/L培养基中后，增殖系数较前者提高了44.4％，平均苗高增加5mm。方差分析结果显示，基本培养基对磨盘柿继代增殖的影响极为显著，ZT的作用次之，IAA作用较弱，BA最弱。与MS(1/2N)和1/2 MS相比，MS基本培养基更有利于组培苗早期继代增殖。
在MS+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中继代10～12代的组培苗，转入DKW+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基中，组培苗生长极为旺盛、健壮，增殖系数较MS基本培养基中提高了43.1％，平均苗高增加6mm。R值结果表明，DKW+ZT1.0mg/L+IAA0.1mg/L+BA1.0mg/L培养基为磨盘柿组培苗中期继代增殖的最佳培养基。
通过三因素三水平正交试验，对基本培养基、ZT及IAA对叶片不定芽再生的影响进行了研究。结果表明，ZT浓度对叶片不定芽再生的影响作用最大，基本培养基次之，IAA对愈伤组织的形成有促进作用。组培苗顶端前2节位展开叶片，切成4mm×4mm左右，远轴面接触培养基时的再生效果最好。切片在MS(1/2N)+ZT4.0mg/L培养基中的分化率和平均不定芽数分别为76.76％和1.26。
位于组培苗顶端前2节位的伸长无节茎段，切成约4mm左右，平铺于MS(1/2N)+ZT4.0mg/L+IAA0.1mg/L培养基中可以获得最高的分化率和平均不定芽数，分别为83.65％和2.25。ZT和基本培养基对分化率和平均不定芽数的影响显著高于IAA，
而I从对愈伤组织形成的作用明显高于ZT和基本培养基。
从成熟期的磨盘柿果实中克隆到2个ACC合成酶基因片段M一CSI和M一CsZ，
两者核昔酸与氨基酸的同源性分别为98%和99%;与DK一ACsl的核昔酸同源性分别
为69%和70%。二者均编码224个氨基酸，均含有其它植物ACC合成酶的氨基酸保
守区及不变氨基酸残基，在多肤水平上的同源性很高，与DK-ACSI的氨基酸同源性
分别是98%和99%。
Dormant buds formed in the same year of Mopan persimmon trees were taken as explants. Effects of explants form and basal media on primary culture were studied. Factors affecting subculture micropropagation were studied by using tube seedlings as materials- The suited ingredient of culture medium for subculture micropropagation of forepart and metaphase were established Some factors affecting adventitious shoot regeneration were studied. Adventitious shoot regeneration systems from in vitro leaves and stems without internode of tube seedlings were obtained. 2 ACC synthase gene fragments were successfully cloned from, mature Mopan persimmon fruit, Here all have been done is in order to improve the persimmon performance of storage and transportation and simplify the management of storage and transportation. The mean results were as follows:
Dormant bud explants taken from Mopan persimmon trees after being peelled squamas and 3 leaf primordia off were inoculated on MS, MS(1/2N) and 1/2MS all supplemented with 7.5 mg/L BA. Primary cultures were all obtained on the 3 media. The growth state of explants inoculated on MS(1/2N)+ BA 7.5 mg/L medium was the best. They then were transferred on MS (1/2N) +ZT 1.0 mg/L +IAA.04 mg/L+BA1.0mg/L medium. The mean multiplication reached 2.37 and the average height of tube seedlings reached 11.8 mm after being subcultured 2 times.
Tube seedlings having been cultured on MS(1/2N) + ZT 1.0 mg/L + LAA 0.1 mg/L + BA 1.0 mg/L medium for about 10-12 times then were transferred on MS + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium. The multiplication increased 44.4%, at the same time the average height of shoots increased 5 mm. The results showed that the effect of the 4 factors on subculture micropropagation was in the order of basal medium, ZT, IAA and BA. Compared with MS(1/2N), MS basal medium was more beneficial to forepart subculture micropropagation.
Tube seedlings having been cultured on MS + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium about 10~12 times then were inoculated on DKW + ZT 1.0 mg/L + IAA 0.1 mg/L + BA 1.0 mg/L medium. The multiplication increased 43.1% , and the average height of shoots increased 6 mm. The results of R value showed that the DKW basal medium was
the most suitable medium to the metaphase subculture micropropagation of "Mopan" tube seedlings.
Effects of basal media, ZT and IAA on adventitious shoot regeneration were studied through orthogonal design trial L9(34). The results showed that the effect of ZT concentration was the greatest and the basal medium took the second place. But the concentration of IAA was more effective on the forming of callus than basal medium and ZT. The upper 2 expanding leaves of tube seedlings, cut into 4 mm X 4 mm and inoculated with abaxial layer downside may have the greatest regeneration efficiency. The differentiation percentage and the average adventitious shoot number per explants on MS(1/2N) + ZT 4.0 mg/L were 76.76% and 1.26 respectively.
The upper 2 extending stems without internode on tube seedlings, cut about 4mm in length and lay on the MS(1/2N) + ZT 4.0 mg/L + IAA 0.1 mg/L medium may have the greatest regeneration efficiency. The differentiation percentage and the average adventitious shoot number per explants were 83.65% and 2.25 respectively. The effect of ZT concentration was the greatest and the basal medium was greater than IAA. But the concentration of IAA was more effective on the forming of callus than basal medium and ZT.
2 ACC synthase gene fragments M-ACS1 and M-ACS2 were successfully cloned from mature Mopan persimmon fruit. The identities of nucleotide acid and amino acid were 98% and 99% respectively. The nucleotide acid identities of M-ACS1 and M-ACS2 with DK-ACS1 were 69% and 70% respectively. They both encoded 224 amino acid. They also have the conserved amino acid regions and invariant amino acid residues of ACC synthase existing in other plants, and with higher identity in polypeptide leveE The amino acid identities with DK-ACS1 were 98% and 99% respectively.
引文
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