甘蔗离体培养的胚性细胞团诱导与分化研究
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摘要
本论文以桂糖21号(G21)、桂糖28(G28)、新台糖22号(ROC22)和巴西固氮甘蔗RB86-7515(B8)四个甘蔗品种的嫩叶鞘进行离体培养,研究不同激素种类及组合对甘蔗愈伤组织及胚性细胞团的诱导、继代增殖、分化、生根的效应。同时,在离体培养过程中,对甘蔗愈伤组织和胚性细胞团的显微结果进行观察比较。主要试验结果如下：
1、甘蔗愈伤组织诱导试验结果：
(1)甘蔗愈伤组织诱导率最佳的激素组合是：MS+2,4-D3.0 mg/L + NAA0.2 mg/L +KT1.0mg/L。G21、G28、ROC22、B8的愈伤组织诱导率分别达到85.22%、79.21%、83.67%、78.63%。
(2)MS比White、N6培养基更适合甘蔗外植体愈伤组织的诱导。
(3)甘蔗外植体在诱导时加入5%椰子汁能提高愈伤组织诱导率,G21、G28.ROC22和B8的愈伤组织诱导率分别为87.33%,83.08%,84.13%和82.14%。
(4)添加丙氨酸能提高甘蔗外植体愈伤组织的诱导率,G21、G28、ROC22和B8的诱导率分别为85.17%,82.14%,84.10%和80.79%。
(5)在外植体诱导前期先暗培养后光培养能提高愈伤组织诱导率,G21、G28、ROC22和B8的诱导率分别为86.95%,81.11%,83.60%和77.67%。
(6)当培养温度为30℃时,四个甘蔗品种的愈伤组织诱导率均达到最高值,G21、G28、ROC22和B8诱导率分别为88.33%,83.33%,86.27%和81.24%。
(7)与其它季节相比较,在夏季取材,四个甘蔗品种愈伤组织诱导率均表现最高,G21、G28、ROC22和B8的诱导率分别达到84.00%,76.19%,83.59%和75.45%。
(8)甘蔗外植体进行低温预处理会降低愈伤组织诱导效果。
(9)不同季节甘蔗各品种之间的出愈时间早晚顺序为：G2春>秋>夏。
(10)使用MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L+KT 1.0 mg/L+AC 0.03 g/L+PVP1.5 g/L培养基及激素组合能有效的降低冬季甘蔗外植体的褐化率,G21、G28、ROC22和B8的褐化率分别为10.11%,17.74%,14.31%和20.04%。
2、甘蔗愈伤组织及胚性细胞团继代增殖的试验结果
(1)四种甘蔗品种均以MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L +KT 0.5 mg/L培养基激素组合对胚性细胞团的增殖效果最好,G2、G28、ROC22和B8的胚性细胞团增殖倍数分别达到41.47倍,32.28倍,38.80倍和30.35倍。
(2)胚性细胞团培养时间为15天且继代次数为第4代时,四个甘蔗品种胚性细胞团增殖倍数达到最大值。G21、G28、ROC22和B8的胚性细胞团增殖倍数分别达到46.23倍,41.22倍,44.80倍,37.33倍。
(3)与其它光照培养处理相比,先暗培养再光培养处理的愈伤组织及胚性细胞团增殖倍数最高。G21、G28、ROC22和B8的愈伤组织增殖倍数分别为4.45倍,3.33倍,4.13倍和3.13倍；而胚性细胞团的增殖倍数则分别达到37.45倍,30.00倍,35.05倍和28.89倍。
3、甘蔗愈伤组织及胚性细胞团的分化试验结果
(1)四种甘蔗品种均以MS+NAA 0.2mg/L+6-BA 2.0 mg/L +TDZ 0.04 mg/L培养基及激素组合对愈伤组织分化的效果最好,G21、G28、ROC22和B8的分化率分别达到86.83%,82.44%,88.89%和80.00%。
(2)胚性细胞团培养时间为15天,且继代次数为第4代时,四个甘蔗品种愈伤组织及胚性细胞团分化率均达到最大值。G21、G28、ROC22、B8愈伤组织的分化率分别达到88.11%,84.70%,90.97%,80.15%；而胚性细胞团的分化出芽率则分别达到92.28%,89.00%,95.72%,87.98%。
4、甘蔗芽增殖试验结果
培养基与激素组合：MS+NAA 0.2mg/L+6-BA 2.5 mg/L +PP3330.5 mg/L有利于甘蔗芽增殖及芽的生长。G21、G28、ROC22、B8四个甘蔗品种的芽增殖倍数分别为5.03倍,4.35倍,5.32倍,3.78倍。
5、甘蔗无菌苗生根试验结果
三因素激素组合：1/2MS+NAA 0.2mg/L+IBA2.0 mg/L +PP333 1.0 mg/L最有利于甘蔗无菌苗生根,四种甘蔗品种的生根率均达到100%。
6、甘蔗愈伤组织和胚性细胞团的显微结果分析
甘蔗胚性细胞团均有细胞结构紧凑、细胞小、细胞核相对较大、染色后颜色较深的特点；而愈伤组织的细胞结构较为疏松、细胞大、细胞核相对较小、染色后颜色较浅的特点。
The tender leaf sheath of four sugarcane varieties G21, G28, ROC22 and RB86-7515 (B8) were cultured in vitro to study the effects of different hormone treatment on the induction, multiplication, differentiation and root regeneration of the and embryogenic cell cluster During the process of culture in vitro, the microscopoc results of sugarcane callus and embryogenic cell cluster were observerd and compared in the test. The main test reasults were as follow:
1. The test results of sugarcane callus induction:
(1) The best hormone combination of sugarcane callus induction was MS+2,4-D3.0mg/L+NAA0.2mg/L+KT1.0mg/L. The induction rate of sugarcane varieties G21, G28, ROC22 and B8 reached to 85.22%,79.21%,83.67% and 78.63% respectively.
(2) MS medium was more suitable than White medium and Nf, medium on the callus induction of sugarcane explants.
(3) Adding 5% coconut milk to the induction of sugarcane explants could improve the callus induction rate. The callus induction rate of G21, G28, ROC22 and B8 were 87.33%, 83.08%,84.13% and 82.14% respectively.
(4) Adding Cys could improve its induction rate of sugarcane explants, the rate of G21, G28, ROC22 and B8 were 85.17%,82.14%,84.10% and 80.79% respectively.
(5) It could improve the rate of callus induction when it used the dark culture firstly and the used the linght culture in the early days of the explant induction, the induction rate of G21, G28, ROC22 and B8 was 86.95%,81.11%,83.60% and 77.67% respectively.
(6) The rate of callus induction of four sugarcane varieties reached the highest value when the cultural temperature was 30℃, the inductions rate of G21, G28, ROC22 and B8 was 88.33%,87.33%,86.27% and 81.24% respectively.
(7) Compared with other seasons, the rate of callus induction of four sugarcane varieties reached the best when sampled in summer. The induction rate of G21, G28, ROC22 and B8 reached to 84.00%,76.19%,83.59% and 75.45% respectively.
(8) The effect of callus induction could be reduce by the pretreatment with low temperature to the explants of sugarcane.
(9) The order of early and late time of the callus induction between the varieties of sugarcane in different seaaons was G21 spring> autumn> summer.
(10) Using MS+2,4-D 3.0mg/L + NAA 0.2 mg/L+KT 1.0 mg/L+0.03 AC+1.5 PVF hormone combination could effectively reduce the browing rate of sugarcane callus. The browing rate of G21, G28, ROC22 and B8 was 10.11%,17.74%,14.31% and 20.04% respectively.
2. The test reasults of subculturing multiplication of sugarcane callus and embryogenic cell cluster.
(1) The multiplication effect of four sugarcane varieties embryogenic cell cluster showed the best in the hormone combinations MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L+KT 0.5 mg/L, the multiplication multiple of embryogenic cell cluster of G21, G28, ROC22 and B8 reached to 41.47 times,32.28 times,38.80 times,30.35 times respectively.
(2) When the cuture time of embryogenic cell cluster was at the age of 15 days and subculturing time was the 4th generation, the multiplication multiple of embryogenic cell cluster of four sugarcane varieties reached the biggest value. The multiplication multiple of embryogenic cell cluster of G21, G28, ROC22 and B8 reached to 46.23 times,41.22 times, 44.80 times,37.33 times respectively.
(3) Compared to other treatments of illumination cultivation, the treatment used the dark culture firstly and then used the light culture showed the hightest multiple of multiplication of the callus and the embryogenic cell cluster. The multiplication multiple of the callus of G21, G28, ROC22 and B8 was 4.45 times,3.33 times,4.13 times and 3.13 times respectively, but the multiplication multiple of the embryogenic cell cluster reached to 37.45 times,30.00 times, 35.05 times and 28.89 times.
3.The test reasults of differentiation of sugarcane callus and embryogenic cell cluster
(1) The differentiation effect of the medium and hormone combination MS+ +NAA 0.2 mg/L+6-BA 2.0 mg/L+TDZ 0.04 mg/L to the callus and enmbryogenic cell cluster of four sugarcane varieties was the best, the differentiation rate of G21, G28, ROC22 and B8 reached 86.83%,82.44%,88.89% and 80.00% respectively.
(2) When the culture time of embryogenic cell cluster was 15 days and subculture time was the 4th generation, the differentiaion rate of the callus and embryogenic cell cluster of four sugarcane varieties reached the biggest value. The differentiation rate of callus was 88.11%,84.70%,90.97% and 80.15% respectively; but the differentiation rate of embryogenic cell cluster reached to 92.28%,89.00%,95.72% and 87.98% respectively.
4. The test reasults of sugarcane buds multiplication:
The medium and hormone combination MS+NAA 0.2mg/L+6-BA 2.5 mg/L+ PP3330.5 mg/L was benificial to the buds multiplication and growth. The buds multiplication of four sugarcane varieties G21, G28, ROC22 and B8 was 5.03 times,4.35 times,5.32 times and 3.78 times respectively.
5. The test reasult of sugarcane plantlet rooting:
Three factors hormone combination 1/2MS+NAA 0.2mg/L+6-BA 2.5 mg/L+ PP3330.5 mg/L was beneficial on rooting of sugarcane plantlets. The rooting rate of four sugarcane varieties all reached 100%.
6.Analysis of microscopoc results of sugarcane callus and embryogenic cell cluster:
Embryogenic cell cluster showed the following features:cell structure was compact, small cell, big nucleus, dyeing deeply; callus cell structure showed the following features:cell structure was loose, big cell, small nucleus,dyeing lightly.
1、甘蔗愈伤组织诱导试验结果：
(1)甘蔗愈伤组织诱导率最佳的激素组合是：MS+2,4-D3.0 mg/L + NAA0.2 mg/L +KT1.0mg/L。G21、G28、ROC22、B8的愈伤组织诱导率分别达到85.22%、79.21%、83.67%、78.63%。
(2)MS比White、N6培养基更适合甘蔗外植体愈伤组织的诱导。
(3)甘蔗外植体在诱导时加入5%椰子汁能提高愈伤组织诱导率,G21、G28.ROC22和B8的愈伤组织诱导率分别为87.33%,83.08%,84.13%和82.14%。
(4)添加丙氨酸能提高甘蔗外植体愈伤组织的诱导率,G21、G28、ROC22和B8的诱导率分别为85.17%,82.14%,84.10%和80.79%。
(5)在外植体诱导前期先暗培养后光培养能提高愈伤组织诱导率,G21、G28、ROC22和B8的诱导率分别为86.95%,81.11%,83.60%和77.67%。
(6)当培养温度为30℃时,四个甘蔗品种的愈伤组织诱导率均达到最高值,G21、G28、ROC22和B8诱导率分别为88.33%,83.33%,86.27%和81.24%。
(7)与其它季节相比较,在夏季取材,四个甘蔗品种愈伤组织诱导率均表现最高,G21、G28、ROC22和B8的诱导率分别达到84.00%,76.19%,83.59%和75.45%。
(8)甘蔗外植体进行低温预处理会降低愈伤组织诱导效果。
(9)不同季节甘蔗各品种之间的出愈时间早晚顺序为：G2
(10)使用MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L+KT 1.0 mg/L+AC 0.03 g/L+PVP1.5 g/L培养基及激素组合能有效的降低冬季甘蔗外植体的褐化率,G21、G28、ROC22和B8的褐化率分别为10.11%,17.74%,14.31%和20.04%。
2、甘蔗愈伤组织及胚性细胞团继代增殖的试验结果
(1)四种甘蔗品种均以MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L +KT 0.5 mg/L培养基激素组合对胚性细胞团的增殖效果最好,G2、G28、ROC22和B8的胚性细胞团增殖倍数分别达到41.47倍,32.28倍,38.80倍和30.35倍。
(2)胚性细胞团培养时间为15天且继代次数为第4代时,四个甘蔗品种胚性细胞团增殖倍数达到最大值。G21、G28、ROC22和B8的胚性细胞团增殖倍数分别达到46.23倍,41.22倍,44.80倍,37.33倍。
(3)与其它光照培养处理相比,先暗培养再光培养处理的愈伤组织及胚性细胞团增殖倍数最高。G21、G28、ROC22和B8的愈伤组织增殖倍数分别为4.45倍,3.33倍,4.13倍和3.13倍；而胚性细胞团的增殖倍数则分别达到37.45倍,30.00倍,35.05倍和28.89倍。
3、甘蔗愈伤组织及胚性细胞团的分化试验结果
(1)四种甘蔗品种均以MS+NAA 0.2mg/L+6-BA 2.0 mg/L +TDZ 0.04 mg/L培养基及激素组合对愈伤组织分化的效果最好,G21、G28、ROC22和B8的分化率分别达到86.83%,82.44%,88.89%和80.00%。
(2)胚性细胞团培养时间为15天,且继代次数为第4代时,四个甘蔗品种愈伤组织及胚性细胞团分化率均达到最大值。G21、G28、ROC22、B8愈伤组织的分化率分别达到88.11%,84.70%,90.97%,80.15%；而胚性细胞团的分化出芽率则分别达到92.28%,89.00%,95.72%,87.98%。
4、甘蔗芽增殖试验结果
培养基与激素组合：MS+NAA 0.2mg/L+6-BA 2.5 mg/L +PP3330.5 mg/L有利于甘蔗芽增殖及芽的生长。G21、G28、ROC22、B8四个甘蔗品种的芽增殖倍数分别为5.03倍,4.35倍,5.32倍,3.78倍。
5、甘蔗无菌苗生根试验结果
三因素激素组合：1/2MS+NAA 0.2mg/L+IBA2.0 mg/L +PP333 1.0 mg/L最有利于甘蔗无菌苗生根,四种甘蔗品种的生根率均达到100%。
6、甘蔗愈伤组织和胚性细胞团的显微结果分析
甘蔗胚性细胞团均有细胞结构紧凑、细胞小、细胞核相对较大、染色后颜色较深的特点；而愈伤组织的细胞结构较为疏松、细胞大、细胞核相对较小、染色后颜色较浅的特点。
The tender leaf sheath of four sugarcane varieties G21, G28, ROC22 and RB86-7515 (B8) were cultured in vitro to study the effects of different hormone treatment on the induction, multiplication, differentiation and root regeneration of the and embryogenic cell cluster During the process of culture in vitro, the microscopoc results of sugarcane callus and embryogenic cell cluster were observerd and compared in the test. The main test reasults were as follow:
1. The test results of sugarcane callus induction:
(1) The best hormone combination of sugarcane callus induction was MS+2,4-D3.0mg/L+NAA0.2mg/L+KT1.0mg/L. The induction rate of sugarcane varieties G21, G28, ROC22 and B8 reached to 85.22%,79.21%,83.67% and 78.63% respectively.
(2) MS medium was more suitable than White medium and Nf, medium on the callus induction of sugarcane explants.
(3) Adding 5% coconut milk to the induction of sugarcane explants could improve the callus induction rate. The callus induction rate of G21, G28, ROC22 and B8 were 87.33%, 83.08%,84.13% and 82.14% respectively.
(4) Adding Cys could improve its induction rate of sugarcane explants, the rate of G21, G28, ROC22 and B8 were 85.17%,82.14%,84.10% and 80.79% respectively.
(5) It could improve the rate of callus induction when it used the dark culture firstly and the used the linght culture in the early days of the explant induction, the induction rate of G21, G28, ROC22 and B8 was 86.95%,81.11%,83.60% and 77.67% respectively.
(6) The rate of callus induction of four sugarcane varieties reached the highest value when the cultural temperature was 30℃, the inductions rate of G21, G28, ROC22 and B8 was 88.33%,87.33%,86.27% and 81.24% respectively.
(7) Compared with other seasons, the rate of callus induction of four sugarcane varieties reached the best when sampled in summer. The induction rate of G21, G28, ROC22 and B8 reached to 84.00%,76.19%,83.59% and 75.45% respectively.
(8) The effect of callus induction could be reduce by the pretreatment with low temperature to the explants of sugarcane.
(9) The order of early and late time of the callus induction between the varieties of sugarcane in different seaaons was G21
(10) Using MS+2,4-D 3.0mg/L + NAA 0.2 mg/L+KT 1.0 mg/L+0.03 AC+1.5 PVF hormone combination could effectively reduce the browing rate of sugarcane callus. The browing rate of G21, G28, ROC22 and B8 was 10.11%,17.74%,14.31% and 20.04% respectively.
2. The test reasults of subculturing multiplication of sugarcane callus and embryogenic cell cluster.
(1) The multiplication effect of four sugarcane varieties embryogenic cell cluster showed the best in the hormone combinations MS+2,4-D 3.0 mg/L+NAA 0.2 mg/L+KT 0.5 mg/L, the multiplication multiple of embryogenic cell cluster of G21, G28, ROC22 and B8 reached to 41.47 times,32.28 times,38.80 times,30.35 times respectively.
(2) When the cuture time of embryogenic cell cluster was at the age of 15 days and subculturing time was the 4th generation, the multiplication multiple of embryogenic cell cluster of four sugarcane varieties reached the biggest value. The multiplication multiple of embryogenic cell cluster of G21, G28, ROC22 and B8 reached to 46.23 times,41.22 times, 44.80 times,37.33 times respectively.
(3) Compared to other treatments of illumination cultivation, the treatment used the dark culture firstly and then used the light culture showed the hightest multiple of multiplication of the callus and the embryogenic cell cluster. The multiplication multiple of the callus of G21, G28, ROC22 and B8 was 4.45 times,3.33 times,4.13 times and 3.13 times respectively, but the multiplication multiple of the embryogenic cell cluster reached to 37.45 times,30.00 times, 35.05 times and 28.89 times.
3.The test reasults of differentiation of sugarcane callus and embryogenic cell cluster
(1) The differentiation effect of the medium and hormone combination MS+ +NAA 0.2 mg/L+6-BA 2.0 mg/L+TDZ 0.04 mg/L to the callus and enmbryogenic cell cluster of four sugarcane varieties was the best, the differentiation rate of G21, G28, ROC22 and B8 reached 86.83%,82.44%,88.89% and 80.00% respectively.
(2) When the culture time of embryogenic cell cluster was 15 days and subculture time was the 4th generation, the differentiaion rate of the callus and embryogenic cell cluster of four sugarcane varieties reached the biggest value. The differentiation rate of callus was 88.11%,84.70%,90.97% and 80.15% respectively; but the differentiation rate of embryogenic cell cluster reached to 92.28%,89.00%,95.72% and 87.98% respectively.
4. The test reasults of sugarcane buds multiplication:
The medium and hormone combination MS+NAA 0.2mg/L+6-BA 2.5 mg/L+ PP3330.5 mg/L was benificial to the buds multiplication and growth. The buds multiplication of four sugarcane varieties G21, G28, ROC22 and B8 was 5.03 times,4.35 times,5.32 times and 3.78 times respectively.
5. The test reasult of sugarcane plantlet rooting:
Three factors hormone combination 1/2MS+NAA 0.2mg/L+6-BA 2.5 mg/L+ PP3330.5 mg/L was beneficial on rooting of sugarcane plantlets. The rooting rate of four sugarcane varieties all reached 100%.
6.Analysis of microscopoc results of sugarcane callus and embryogenic cell cluster:
Embryogenic cell cluster showed the following features:cell structure was compact, small cell, big nucleus, dyeing deeply; callus cell structure showed the following features:cell structure was loose, big cell, small nucleus,dyeing lightly.
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