百合属杂交育种的研究
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摘要
百合(Lilium spp.)是百合科(Liliaccae)百合属(Lilum)所有种的总称,在园林、盆花、特别是鲜切花等方面被广泛应用。我国是百合切花生产的大国,目前流通在我国花卉市场的百合切花全部为国外培育出来的品种,90%的种球依赖进口,加上百合易感染病毒,这些都大大限制了我国百合切花产业的发展。为了培育抗病、新颖的百合品种,本研究选取具有较强抗病及适应能力的野生百合作为父本进行了亚洲百合×野生百合的杂交育种。选取观赏及栽培性状互补的东方百合和亚洲百合作为亲本进行离体杂交育种。本研究进行了以下几方面的探讨:(1)辐射蒙导授粉、切割花柱授粉、添加含Ca2+杂交液辅助授粉等方法对百合杂交结实率的影响。(2)整合离体授粉、离体嫁接、子房切片培养、胚珠培养等方法对克服东方百合×亚洲百合远缘杂交障碍的影响。(3)对亚洲百合×野生百合组合的杂种胚进行培养,对‘科黛利亚’×毛百合、‘白天使’×毛百合两种组合的F1代幼苗的鳞片进行增殖培养,以期获得较适合的培养基。(4)利用ISSR分子标记的方法对‘白天使’×毛百合的杂交后代进行真实性鉴定。
本研究的结果如下:
(1)在亚洲百合×野生百合的杂交中,进行了9种组合,共杂交了1051朵花,收获种子6004粒,培育出F1代幼苗542株。在东方百合×亚洲百合的离体杂交中,进行了7种组合,共嫁接了358对花,收获胚珠1416粒,培育出F1代幼苗3株。
(2)采用混合、蒙导及先锋花粉授粉法在一定程度上克服了‘多安娜’×大花卷丹远缘杂交障碍。经10Gy或100Gy剂量60Co辐射的母本花粉自交均未获得种子,用10Gy和100Gy剂量辐射的母本花粉参与的蒙导花粉授粉最后分别获得9粒和5粒种子。
在‘科黛利亚’×毛百合杂交组合中,采用普通柱头授粉能够获得一些膨大果实和饱满种子,表明该杂交组合为亲合性组合。用经1000Gy或2500Gy剂量60Co辐射的母本花粉进行辅助授粉增大了座果率和饱满种子结籽数,有利于后续的选育工作。采用杂交液辅助授粉,座果率和饱满种子结籽数有所提高。而切割花柱授粉则减少了座果率和结籽数。
(3)外源Ca2+能促进毛百合花粉萌发率和花粉管生长,也能促进‘白天使’×毛百合杂交组合形成种子,其最适合的外源Ca2+浓度是4mol/L。同时直授花粉加杂交液比切割花柱授粉加杂交液的授粉方法更适合‘白天使’×毛百合这个组合。
(4)整合离体授粉、离体嫁接、子房切片培养、胚珠培养等方法在一定程度上能克服东方百合‘水晶布兰’×亚洲百合‘多安娜’远缘杂交障碍。从授粉后第7天开始做子房切片培养,同时在培养基中加入花药,有利于子房膨大。本试验中,离体杂交获得的F1代幼苗3株,成苗难度较高,原因可能是:杂交亲本之间亲缘关系较远;培养皿密合度不够好使得部分材料污染;胚珠培养的条件需进一步完善细化。
(5)本研究得到对于‘科黛利亚’×毛百合、‘白天使’×毛百合2种组合的杂种幼苗鳞片增殖培养的最佳培养基为:MS+NAA0.1mg/L + BA1.0mg/L。
(6)选择15种ISSR引物对‘白天使’×毛百合杂交组合的父本、母本、子代DNA片段进行扩增,杂交后代含有父母本的特征带,是真正的杂种。
Lily belongs to Lilum of Liliaccae, which is widely used in garden, potted flower and cut flower especially. China is the main country in producing cut flowers of Lily. However, all the varieties and 90% bulbs depend on importation and Lily is easy to catch virus and disease, which block the development of cut flower of Lily in China. Breeding for resistance against disease and novel varieties of Lily.,the research chose wild species of Lily which have the ability of resistance against disease as male parent for the combination of Asiatic hybrids×wild Lily and chose the complemantary pairs Oriental hybrids and Asiatic hybrids as parents to cross in vitro. The research contained the following aspects: (1)The effects of different pollination methods like‘mentor’and‘pioneer’pollination, cut-style pollination , pollination with Ca2+ in pollen germination liquor on fruitset and seedset for cross-breeding. (2) The effects of the method which integrated in vitro pollination, ovary slice culture and ovule culture for overcoming the barriers of Oriental hybrids×Asiatic hybrids.(3)Obtaining the best media for bullet propagation of the plantlets from the cross combinations.(4)Identification by ISSR proved that‘Navona’×Lilium dauricum was true hybrids.
Results of the research are as following:
(1)9 combinations were tested,1051 flowers were crossed, 6004 seeds were obtained, 542 young seedlings were planted in Asiatic hybrids×wild Lily. 7 combinations were tested, 358 pairs of flowers were grafted, 1416 ovules were obtained, 3 young seedlings were planted in Oriental hybrids×Asiatic hybrids
(2)The methods of‘mixture’,‘mentor’and‘pioneer’pollination using for overcoming lilium cross barriers of the‘Pollyanna’×lilium leichtlinii var. maximowiczii were studied.The result showed that the irradiation pollen of 10Gy and 100Gy lost pollination abilities completely and a small amount of seeds were produced by the application of 10Gy and 100Gy mentor pollen. By using the normal stigma pollination, a number of fruits and plump seeds were produced, which indicated the cross‘Cordelia’×lilium dauricum was a compatible cross. By applying the irradiation pollen’s assisting pollination methods (1000Gy or 2500Gy mentor pollen), fruitset and seedset were raised obviously,. The use of the cross chemical’s assisting pollination exhibited raised the fruitset and seedset to some extent. The application of the cut-style pollination deduced the fruitset and seedset .
(3)Ca2+ is able to promote the pollen energy and pollen tube length, and also can raised the seedset, the most suitable concentration of Ca2+ is 4mol/L.Meanwhile, normal stigma pollination with pollen germination liquor is better than cut-style pollination with pollen germination liquor.
(4)The method which integrated in vitro pollination, ovary slice culture and ovule culture was proved that can overcome the barriers of Oriental hybrids×Asiatic hybrids to some extent. Ovary slice development appeared to be positively affected by the presence of young anthers in 7 days after pollination.The number of hybrid plantlets was very limited, the reasons may as following: the genetic distance of the parents is not close, contamination hazards and ovule culture condition was not suitable, which need refinement.
(5) The research demonstrated that the best media for bullet propagation of the plantlets from the cross combinations of‘Cordelia’×Lilium dauricum and‘Navona’×Lilium dauricum is: MS+NAA0.1mg/L + BA1.0mg/L.
(6)15 kinds of ISSR primes were used, which showed the hybrid bands included both parental bands. The hybrid was true progeny of their parents.
本研究的结果如下:
(1)在亚洲百合×野生百合的杂交中,进行了9种组合,共杂交了1051朵花,收获种子6004粒,培育出F1代幼苗542株。在东方百合×亚洲百合的离体杂交中,进行了7种组合,共嫁接了358对花,收获胚珠1416粒,培育出F1代幼苗3株。
(2)采用混合、蒙导及先锋花粉授粉法在一定程度上克服了‘多安娜’×大花卷丹远缘杂交障碍。经10Gy或100Gy剂量60Co辐射的母本花粉自交均未获得种子,用10Gy和100Gy剂量辐射的母本花粉参与的蒙导花粉授粉最后分别获得9粒和5粒种子。
在‘科黛利亚’×毛百合杂交组合中,采用普通柱头授粉能够获得一些膨大果实和饱满种子,表明该杂交组合为亲合性组合。用经1000Gy或2500Gy剂量60Co辐射的母本花粉进行辅助授粉增大了座果率和饱满种子结籽数,有利于后续的选育工作。采用杂交液辅助授粉,座果率和饱满种子结籽数有所提高。而切割花柱授粉则减少了座果率和结籽数。
(3)外源Ca2+能促进毛百合花粉萌发率和花粉管生长,也能促进‘白天使’×毛百合杂交组合形成种子,其最适合的外源Ca2+浓度是4mol/L。同时直授花粉加杂交液比切割花柱授粉加杂交液的授粉方法更适合‘白天使’×毛百合这个组合。
(4)整合离体授粉、离体嫁接、子房切片培养、胚珠培养等方法在一定程度上能克服东方百合‘水晶布兰’×亚洲百合‘多安娜’远缘杂交障碍。从授粉后第7天开始做子房切片培养,同时在培养基中加入花药,有利于子房膨大。本试验中,离体杂交获得的F1代幼苗3株,成苗难度较高,原因可能是:杂交亲本之间亲缘关系较远;培养皿密合度不够好使得部分材料污染;胚珠培养的条件需进一步完善细化。
(5)本研究得到对于‘科黛利亚’×毛百合、‘白天使’×毛百合2种组合的杂种幼苗鳞片增殖培养的最佳培养基为:MS+NAA0.1mg/L + BA1.0mg/L。
(6)选择15种ISSR引物对‘白天使’×毛百合杂交组合的父本、母本、子代DNA片段进行扩增,杂交后代含有父母本的特征带,是真正的杂种。
Lily belongs to Lilum of Liliaccae, which is widely used in garden, potted flower and cut flower especially. China is the main country in producing cut flowers of Lily. However, all the varieties and 90% bulbs depend on importation and Lily is easy to catch virus and disease, which block the development of cut flower of Lily in China. Breeding for resistance against disease and novel varieties of Lily.,the research chose wild species of Lily which have the ability of resistance against disease as male parent for the combination of Asiatic hybrids×wild Lily and chose the complemantary pairs Oriental hybrids and Asiatic hybrids as parents to cross in vitro. The research contained the following aspects: (1)The effects of different pollination methods like‘mentor’and‘pioneer’pollination, cut-style pollination , pollination with Ca2+ in pollen germination liquor on fruitset and seedset for cross-breeding. (2) The effects of the method which integrated in vitro pollination, ovary slice culture and ovule culture for overcoming the barriers of Oriental hybrids×Asiatic hybrids.(3)Obtaining the best media for bullet propagation of the plantlets from the cross combinations.(4)Identification by ISSR proved that‘Navona’×Lilium dauricum was true hybrids.
Results of the research are as following:
(1)9 combinations were tested,1051 flowers were crossed, 6004 seeds were obtained, 542 young seedlings were planted in Asiatic hybrids×wild Lily. 7 combinations were tested, 358 pairs of flowers were grafted, 1416 ovules were obtained, 3 young seedlings were planted in Oriental hybrids×Asiatic hybrids
(2)The methods of‘mixture’,‘mentor’and‘pioneer’pollination using for overcoming lilium cross barriers of the‘Pollyanna’×lilium leichtlinii var. maximowiczii were studied.The result showed that the irradiation pollen of 10Gy and 100Gy lost pollination abilities completely and a small amount of seeds were produced by the application of 10Gy and 100Gy mentor pollen. By using the normal stigma pollination, a number of fruits and plump seeds were produced, which indicated the cross‘Cordelia’×lilium dauricum was a compatible cross. By applying the irradiation pollen’s assisting pollination methods (1000Gy or 2500Gy mentor pollen), fruitset and seedset were raised obviously,. The use of the cross chemical’s assisting pollination exhibited raised the fruitset and seedset to some extent. The application of the cut-style pollination deduced the fruitset and seedset .
(3)Ca2+ is able to promote the pollen energy and pollen tube length, and also can raised the seedset, the most suitable concentration of Ca2+ is 4mol/L.Meanwhile, normal stigma pollination with pollen germination liquor is better than cut-style pollination with pollen germination liquor.
(4)The method which integrated in vitro pollination, ovary slice culture and ovule culture was proved that can overcome the barriers of Oriental hybrids×Asiatic hybrids to some extent. Ovary slice development appeared to be positively affected by the presence of young anthers in 7 days after pollination.The number of hybrid plantlets was very limited, the reasons may as following: the genetic distance of the parents is not close, contamination hazards and ovule culture condition was not suitable, which need refinement.
(5) The research demonstrated that the best media for bullet propagation of the plantlets from the cross combinations of‘Cordelia’×Lilium dauricum and‘Navona’×Lilium dauricum is: MS+NAA0.1mg/L + BA1.0mg/L.
(6)15 kinds of ISSR primes were used, which showed the hybrid bands included both parental bands. The hybrid was true progeny of their parents.
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