影响油桃（Prunus persica var.nectarina）茎尖培养和胚轴再生因子的研究
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摘要
桃[Prunus persica (L.) Batsch]以其丰富的营养、鲜美的风味而倍受人们欢迎,但在桃育种工作中,常规育种周期长、工作量大,且很难取得令人满意的结果。随着现代生物技术和基因工程的发展,可有目的地改良某些优良品种的不良性状,在较短时间内得到新品种,从而提高育种效果,而建立高效稳定的再生体系一直是桃基因工程发展所需要攻克的难题之一。
本文分别以油桃品种华光、曙光、秦光2#为试材,通过对影响茎尖培养的几个因子的研究,初步确立了茎尖培养的初代培养体系,并建立了以晚熟油桃秦光2#下胚轴为外植体的离体再生体系,在筛选培养基上培养秦光2#的有效愈伤组织,进行抗氧化酶的测定分析,并观察不同时期愈伤组织的发育状态,初步探讨不定芽诱导的再生途径。研究结果如下:
1.在茎尖培养中,油桃茎尖的消毒以茎尖在75%酒精30s+0.1%升汞5min+饱和次氯酸钠溶液8min中消毒效果较好,且成活率较高,为68.4%。采芽接种最适宜期为2月中旬,接芽状态应为试材为未萌动的1年生枝,室温下进行催芽,待芽萌出小叶后再接种,污染率最低,利于生长分化。
2.华光和曙光茎尖培养基为G+6-BA 0.25 mg/L+GA_3 1.00 mg/L+IBA 1.00 mg/L,诱导率分别为57.8%和42.6%;秦光为2#6-BA 0.50 mg/L+GA_3 0.50 mg/L+IBA 1.00 mg/L,诱导率较高为41.3%。
3.在秦光2#种胚萌发试验中,蔗糖质量浓度在30 g/L糖浓度中萌发最高,达到75.66 %;同时,种胚经过在1 600 mg/L的GA溶液中处理24 h萌发率较高,可达到62.3 %;剥去种皮,进行5℃低温处理75d,能够提高胚培养萌发率,使其达到93.33 %。
4.在秦光2#油桃的下胚轴再生试验中:下胚轴的上、中、下各部分再生能力逐渐下降;暗培养对胚轴再生的影响不显著;经过低温处理的种胚移至光下常温培养,7d时再生率最高,达到34.38%。
5.下胚轴再生的最适培养为QL培养基,再生率达到31.0%,平均再生不定芽数为1.63个;在QL培养基上以TDZ 2.0 mg/L+NAA 0.5 mg/L激素组合的下胚轴再生率最高,达到38.67%,平均再生不定芽数量最多,为2.50个。
6.在秦光2#油桃体细胞胚发生过程中, SOD活性先升高后降低,POD和CAT恰好与之相反,其活性先降低后又逐渐上升。
7.显微观察发现:在愈伤组织产生前期其细胞核小、细胞体积大、细胞质少、液泡化严重;接着细胞核变大,细胞排列分散;然后细胞变得比较松,可以看到有丝分裂;有不定芽点产生时,细胞中二细胞原胚产生和多细胞原胚产生;有不定芽产生时,细胞中开始有胚性细胞形成,产生球形胚。
Peach [Prunus persica (L.) Batsch]has been long enjoyed for its exuberantnutrition and delicious taste.But In the breeding work of the peach, conventional hybrid techniques are not always satisfied for its long period and large amount of work.. With the spring up of modern biological techniques and the applying of gene engineering, which made it possible for us to change the qualities of peach through gene engineering. But first of all, we need establish a high frequent and stable regeneration system of peach.
In order to set up regenerate system of peach, the nectarine cultivar Huaguang、Shuguang and Qinguang2# were used as materials, through studying the factors influencing shoot tip culture, we have developed the primary culture system of the shoot tip culture. And have developed a shoot organogenesis from hypocotyls procedure for later ripe nectarine cultuvar Qinguang2#。
Results are as follows:
(1)In the shoot tip culture,the 75% alcohol 30s and the 0.1% HgCl2 5min and the NaClO3 8min is the better for the nectarine shoot tip sterilization and the survival ratio is more high ,it is 68.4%.The mid February is better time for the gathering shoot tip, the 1 year no spring tress wattle was settled in the house, when it sprout and has 2-3 leaf, the pollute rate is lower and adapt to develop.
(2) The G+6-BA 0.25mg/L+GA_3 1.00mg/L+IBA 1.00mg/L is the better for the first culture for the Huaguang and the Shuguang, the inductivity is 57.8% and 42.6%. The 6-BA0.25mg/L+GA_30.50mg/L+IBA1.00mg/L is the better for Ginguang2#, the inductivity is 41.3%.
(3) In the embryo germination experimentation of the Qinguang2# nectarine, removed seed coats and cool(5℃) treatment are benefit to embryo’s germination, the germination rate is 93.33%; In the sugar moss concentration 30g/L ,germination rate is best highest, it is 75.66%; the 1600mg/L GA_3 24h treatment is benefit to germination , the germination rate is 62.3%.
(4) In the hypocotyls regermination experimentation of the Qinguang2# nectarine, the upper, middle and lower hypocotyls has significant difference; dark culture has no significant difference; and by the 7-days normal temperature and light beforehand train could get more stronger and younger hypocotyls, the regeneration rate reach 34.38 %.
(5) The QL culture medium is best for the hypocotyls, the regeneration rate is 31.0%,the per regeneration adventitious is 1.63. The hypocotyls regeneration performed best on the medium of QL+TDZ 2.0 mg/L+NAA 0.5 mg/L, reach 38.67 %, the maximum of 2.50 means number of shoots organogenesis per explants is obtained.
(6) In the process of Qinguang2# somatic cell develop, SOD is first rise and then lower, POD and CAT are opposition to SOD, the enzyme activity first lower and then rise gradually.
(7) Through observe under a microscope and found: before the generation the cell core is small and cytoplasm is few, the state of vacuole is serious; and then the cell bulk became bigger and cell arrangement was dispersible; then the cell became slack and have the process of mitosis; when the adventitious bud appereed, the cell continue division and fromed two cell embryo; then, when the callus obtain adventitious bud, the embryonic cell fromed and can see the sphere embryo in the cell.
本文分别以油桃品种华光、曙光、秦光2#为试材,通过对影响茎尖培养的几个因子的研究,初步确立了茎尖培养的初代培养体系,并建立了以晚熟油桃秦光2#下胚轴为外植体的离体再生体系,在筛选培养基上培养秦光2#的有效愈伤组织,进行抗氧化酶的测定分析,并观察不同时期愈伤组织的发育状态,初步探讨不定芽诱导的再生途径。研究结果如下:
1.在茎尖培养中,油桃茎尖的消毒以茎尖在75%酒精30s+0.1%升汞5min+饱和次氯酸钠溶液8min中消毒效果较好,且成活率较高,为68.4%。采芽接种最适宜期为2月中旬,接芽状态应为试材为未萌动的1年生枝,室温下进行催芽,待芽萌出小叶后再接种,污染率最低,利于生长分化。
2.华光和曙光茎尖培养基为G+6-BA 0.25 mg/L+GA_3 1.00 mg/L+IBA 1.00 mg/L,诱导率分别为57.8%和42.6%;秦光为2#6-BA 0.50 mg/L+GA_3 0.50 mg/L+IBA 1.00 mg/L,诱导率较高为41.3%。
3.在秦光2#种胚萌发试验中,蔗糖质量浓度在30 g/L糖浓度中萌发最高,达到75.66 %;同时,种胚经过在1 600 mg/L的GA溶液中处理24 h萌发率较高,可达到62.3 %;剥去种皮,进行5℃低温处理75d,能够提高胚培养萌发率,使其达到93.33 %。
4.在秦光2#油桃的下胚轴再生试验中:下胚轴的上、中、下各部分再生能力逐渐下降;暗培养对胚轴再生的影响不显著;经过低温处理的种胚移至光下常温培养,7d时再生率最高,达到34.38%。
5.下胚轴再生的最适培养为QL培养基,再生率达到31.0%,平均再生不定芽数为1.63个;在QL培养基上以TDZ 2.0 mg/L+NAA 0.5 mg/L激素组合的下胚轴再生率最高,达到38.67%,平均再生不定芽数量最多,为2.50个。
6.在秦光2#油桃体细胞胚发生过程中, SOD活性先升高后降低,POD和CAT恰好与之相反,其活性先降低后又逐渐上升。
7.显微观察发现:在愈伤组织产生前期其细胞核小、细胞体积大、细胞质少、液泡化严重;接着细胞核变大,细胞排列分散;然后细胞变得比较松,可以看到有丝分裂;有不定芽点产生时,细胞中二细胞原胚产生和多细胞原胚产生;有不定芽产生时,细胞中开始有胚性细胞形成,产生球形胚。
Peach [Prunus persica (L.) Batsch]has been long enjoyed for its exuberantnutrition and delicious taste.But In the breeding work of the peach, conventional hybrid techniques are not always satisfied for its long period and large amount of work.. With the spring up of modern biological techniques and the applying of gene engineering, which made it possible for us to change the qualities of peach through gene engineering. But first of all, we need establish a high frequent and stable regeneration system of peach.
In order to set up regenerate system of peach, the nectarine cultivar Huaguang、Shuguang and Qinguang2# were used as materials, through studying the factors influencing shoot tip culture, we have developed the primary culture system of the shoot tip culture. And have developed a shoot organogenesis from hypocotyls procedure for later ripe nectarine cultuvar Qinguang2#。
Results are as follows:
(1)In the shoot tip culture,the 75% alcohol 30s and the 0.1% HgCl2 5min and the NaClO3 8min is the better for the nectarine shoot tip sterilization and the survival ratio is more high ,it is 68.4%.The mid February is better time for the gathering shoot tip, the 1 year no spring tress wattle was settled in the house, when it sprout and has 2-3 leaf, the pollute rate is lower and adapt to develop.
(2) The G+6-BA 0.25mg/L+GA_3 1.00mg/L+IBA 1.00mg/L is the better for the first culture for the Huaguang and the Shuguang, the inductivity is 57.8% and 42.6%. The 6-BA0.25mg/L+GA_30.50mg/L+IBA1.00mg/L is the better for Ginguang2#, the inductivity is 41.3%.
(3) In the embryo germination experimentation of the Qinguang2# nectarine, removed seed coats and cool(5℃) treatment are benefit to embryo’s germination, the germination rate is 93.33%; In the sugar moss concentration 30g/L ,germination rate is best highest, it is 75.66%; the 1600mg/L GA_3 24h treatment is benefit to germination , the germination rate is 62.3%.
(4) In the hypocotyls regermination experimentation of the Qinguang2# nectarine, the upper, middle and lower hypocotyls has significant difference; dark culture has no significant difference; and by the 7-days normal temperature and light beforehand train could get more stronger and younger hypocotyls, the regeneration rate reach 34.38 %.
(5) The QL culture medium is best for the hypocotyls, the regeneration rate is 31.0%,the per regeneration adventitious is 1.63. The hypocotyls regeneration performed best on the medium of QL+TDZ 2.0 mg/L+NAA 0.5 mg/L, reach 38.67 %, the maximum of 2.50 means number of shoots organogenesis per explants is obtained.
(6) In the process of Qinguang2# somatic cell develop, SOD is first rise and then lower, POD and CAT are opposition to SOD, the enzyme activity first lower and then rise gradually.
(7) Through observe under a microscope and found: before the generation the cell core is small and cytoplasm is few, the state of vacuole is serious; and then the cell bulk became bigger and cell arrangement was dispersible; then the cell became slack and have the process of mitosis; when the adventitious bud appereed, the cell continue division and fromed two cell embryo; then, when the callus obtain adventitious bud, the embryonic cell fromed and can see the sphere embryo in the cell.
引文
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