柑橘花粉染色体加倍与越南柑橘三倍体育种的研究
摘要
柑橘属于芸香科(Rutaceae)、柑橘亚科(Aurantioideae),是世界最重要的果树树种之一。柑橘因其芳香味美,营养价值高,深受大众喜爱。但只有无核或少核的三倍体类型的柑橘是一个优良性状,这无论对于鲜食还是加工都具有重要意义价值。因此,培育柑橘三倍体无核品种是目前柑橘育种的主要目标之一。在多倍体的育种中,2n配子的形成具有很重要的作用。2n配子是由于减数分裂过程中某些因素导致形成具有体细胞染色体数的花粉或卵细胞,常被称为未减数配子,2n配子在传递杂合性和上位性方面具有特殊价值,因而2n配子在植物多倍体育种中的研究应用逐渐升温。
福泽柚和演柚(Citrus grandis L. Osbeck)是越南的两个重要的地方品种,风味独特,品质优良,唯其果实中种子太多,严重影响其商品价值。因此,本文在观察2个柑橘品种的生物学特征和2n花粉自然发生情况基础上,以秋水仙素为诱变剂对福泽柚和演柚2n花粉诱导进行了初步研究,并取得了一定的进展。本试验主要研究结果如下：
(1)2009-2010年连续两年,对福泽柚和演柚果实进行了观察。结果表明,福泽柚单果重指标数值均略高于演柚,但福泽柚的平均种子数高于演柚且皮厚,因此,演柚的可食率高于福泽柚；二者还有果实成熟期相差的特点,福泽柚的果实成熟期为9月中旬,演柚的果实成熟期为1月下旬。
(2)在红河平原地区,2010年福泽柚小孢子母细胞减数分裂开始于2月7日、演柚小孢子母细胞减数分裂开始于2月12日；当花蕾平均直径至0.3-0.4cm,小孢子母细胞减数分裂开始进入前期Ⅰ,4d左右减数分裂进行到四分体,前期Ⅰ持续时间较长。福泽柚和演柚的不同植株、不同花枝、不同花芽甚至不同花药的减数分裂进程都存在一定程度的差异,体现不同步性。
(3)采用不同浓度秋水仙素对福泽柚和演柚处于减数分裂前期Ⅰ的花蕾进行诱导,并成功的诱导出了2n雄配子。其中,秋水仙素溶液的最佳处理浓度为0.3%、0.5%,持续处理为4d、6d,可获得12.35和10.01%的福泽柚和演柚2n花粉比率,最高的处理达17.17和12.17%。
(4)福泽柚和演柚1n与2n花粉授粉后在柱头上都能正常萌发,但与1n花粉相比,2n花粉萌发较迟、2n花粉管伸长速度较缓慢。2n花粉参与受精能力比1n花粉弱,2n花粉在受精时间上竞争不过1n花粉。
(5)本试验对所获得的小粒种子和瘪粒种子进行拯救培养,获得的26株福泽柚苗和324株演柚苗进行Partec倍性分析仪检测,有3株福泽柚三倍体、4株演柚三倍体(染色体数为2n=3x=27)和3株演柚四倍体(染色体数为2n=4x=36)
Citrus fruit trees belong to the family of Rutaceae, subfamily Aurantioideae. Citrus fruit is one of the most important crops in the world. Seedless is a desirable horticultural attribute in Citrus and is positively associated with triploidy. Triploid seedless Citrus cultivar is one of the main objectives of Citrus breeding. In crop breeding, the frequency at which 2n gametes are formed is also very important, in that it may affect the rate of new polyploidy formation. Together with the endosperm,2n gametes have also been a major factor in the origin and evolution of polyploidy. The two main mechanisms of polyploid formation are somatic doubling of chromosomes in meristem tissues of sporophytes, zygotes, or embryo and meiotic nuclear restitution leading to 2n gamete production.
Phuc Trach and Dien pummelo are local varieties in Vietnam, good quality but there are too much seeds that will affect their commercial value. This research has studied on biology characteristics and carried out experiments on pollen chromosome doubling. These researches showed the results that include five points as follows:
(1) During the years of 2009-2010, the fruit quality of Phuc Trach and Dien pummelo were investigated, the results showed that:the weighted indexes of fruits in Phuc Trach pummelo are higher than Dien pummelo, but the seed number and fruit skin of Phuc Trach pummelo are higher than Dien pummelo. Therefore, the edible percentage of Dien pummelo is higher than Phuc Trach pummelo. There is a difference in fruit maturity between the two varieties, the Phuc Trach pummelo variety get mature in mid-September but the Dien pummelo variety get mature in late January.
(2) The PMCs meiosis of Phuc Trach and Dien pummelo begins at February 7 and February 12 in Red River Delta, when the flower buds a diameter of 0.3-0.4cm and PMCs at meiotic prophase I, by tetrads stage, about 4 days after meiotic prophase I. The duration of prophase I is very long. The synchronization of meiosis were greatly different in different clones, floral branches from the same clones, male flowers from the same floral branches and even anthers of the same flower of Phuc Trach and Dien pummelo
(3) At the meiotic prophase I, different concentrations of Colchicine were used, and successfully induced 2n pollen in Phuc Trach and Dien pummelo. The optimal concentration of Colchicine for treatment was 0.3%、0.5%. The highest average percentage of unreduced pollen by treating Phuc Trach and Dien pummelo flowers for 4 and 6 days with 0.3%、0.5% Colchicine was 12.35 and 10.01%, the highest proportion was 17.17 and 12.17%.
(4) The 1n and 2n pollen can germinated, but 2n pollen germinated later and pollen tube grew slower than 1n pollen in vivo in Phuc Trach and Dien pummelo, suggesting that the fertilizing ability of 2n pollen was weaker than that of 1n pollen and 2n pollen was less competent than 1n pollen.
(5) The developed small seeds and aborted seeds were rescued,26 seedlings of Phuc Trach pummelo and 324 seedlings of Dien pummelo were derived from these seeds with the aid of embryo rescue technique. Partec low cytometry analysis indicated that complex ploidy levels in these progenies, among which there were 3 triploid of Phuc Trach pummelo,4 triploid of Dien pummelo (triploid chromosome number is 2n=3x=27), and 1 tetraploid of Dien pummelo (tetraploid chromosome number is 2n=4x=36).
福泽柚和演柚(Citrus grandis L. Osbeck)是越南的两个重要的地方品种,风味独特,品质优良,唯其果实中种子太多,严重影响其商品价值。因此,本文在观察2个柑橘品种的生物学特征和2n花粉自然发生情况基础上,以秋水仙素为诱变剂对福泽柚和演柚2n花粉诱导进行了初步研究,并取得了一定的进展。本试验主要研究结果如下：
(1)2009-2010年连续两年,对福泽柚和演柚果实进行了观察。结果表明,福泽柚单果重指标数值均略高于演柚,但福泽柚的平均种子数高于演柚且皮厚,因此,演柚的可食率高于福泽柚；二者还有果实成熟期相差的特点,福泽柚的果实成熟期为9月中旬,演柚的果实成熟期为1月下旬。
(2)在红河平原地区,2010年福泽柚小孢子母细胞减数分裂开始于2月7日、演柚小孢子母细胞减数分裂开始于2月12日；当花蕾平均直径至0.3-0.4cm,小孢子母细胞减数分裂开始进入前期Ⅰ,4d左右减数分裂进行到四分体,前期Ⅰ持续时间较长。福泽柚和演柚的不同植株、不同花枝、不同花芽甚至不同花药的减数分裂进程都存在一定程度的差异,体现不同步性。
(3)采用不同浓度秋水仙素对福泽柚和演柚处于减数分裂前期Ⅰ的花蕾进行诱导,并成功的诱导出了2n雄配子。其中,秋水仙素溶液的最佳处理浓度为0.3%、0.5%,持续处理为4d、6d,可获得12.35和10.01%的福泽柚和演柚2n花粉比率,最高的处理达17.17和12.17%。
(4)福泽柚和演柚1n与2n花粉授粉后在柱头上都能正常萌发,但与1n花粉相比,2n花粉萌发较迟、2n花粉管伸长速度较缓慢。2n花粉参与受精能力比1n花粉弱,2n花粉在受精时间上竞争不过1n花粉。
(5)本试验对所获得的小粒种子和瘪粒种子进行拯救培养,获得的26株福泽柚苗和324株演柚苗进行Partec倍性分析仪检测,有3株福泽柚三倍体、4株演柚三倍体(染色体数为2n=3x=27)和3株演柚四倍体(染色体数为2n=4x=36)
Citrus fruit trees belong to the family of Rutaceae, subfamily Aurantioideae. Citrus fruit is one of the most important crops in the world. Seedless is a desirable horticultural attribute in Citrus and is positively associated with triploidy. Triploid seedless Citrus cultivar is one of the main objectives of Citrus breeding. In crop breeding, the frequency at which 2n gametes are formed is also very important, in that it may affect the rate of new polyploidy formation. Together with the endosperm,2n gametes have also been a major factor in the origin and evolution of polyploidy. The two main mechanisms of polyploid formation are somatic doubling of chromosomes in meristem tissues of sporophytes, zygotes, or embryo and meiotic nuclear restitution leading to 2n gamete production.
Phuc Trach and Dien pummelo are local varieties in Vietnam, good quality but there are too much seeds that will affect their commercial value. This research has studied on biology characteristics and carried out experiments on pollen chromosome doubling. These researches showed the results that include five points as follows:
(1) During the years of 2009-2010, the fruit quality of Phuc Trach and Dien pummelo were investigated, the results showed that:the weighted indexes of fruits in Phuc Trach pummelo are higher than Dien pummelo, but the seed number and fruit skin of Phuc Trach pummelo are higher than Dien pummelo. Therefore, the edible percentage of Dien pummelo is higher than Phuc Trach pummelo. There is a difference in fruit maturity between the two varieties, the Phuc Trach pummelo variety get mature in mid-September but the Dien pummelo variety get mature in late January.
(2) The PMCs meiosis of Phuc Trach and Dien pummelo begins at February 7 and February 12 in Red River Delta, when the flower buds a diameter of 0.3-0.4cm and PMCs at meiotic prophase I, by tetrads stage, about 4 days after meiotic prophase I. The duration of prophase I is very long. The synchronization of meiosis were greatly different in different clones, floral branches from the same clones, male flowers from the same floral branches and even anthers of the same flower of Phuc Trach and Dien pummelo
(3) At the meiotic prophase I, different concentrations of Colchicine were used, and successfully induced 2n pollen in Phuc Trach and Dien pummelo. The optimal concentration of Colchicine for treatment was 0.3%、0.5%. The highest average percentage of unreduced pollen by treating Phuc Trach and Dien pummelo flowers for 4 and 6 days with 0.3%、0.5% Colchicine was 12.35 and 10.01%, the highest proportion was 17.17 and 12.17%.
(4) The 1n and 2n pollen can germinated, but 2n pollen germinated later and pollen tube grew slower than 1n pollen in vivo in Phuc Trach and Dien pummelo, suggesting that the fertilizing ability of 2n pollen was weaker than that of 1n pollen and 2n pollen was less competent than 1n pollen.
(5) The developed small seeds and aborted seeds were rescued,26 seedlings of Phuc Trach pummelo and 324 seedlings of Dien pummelo were derived from these seeds with the aid of embryo rescue technique. Partec low cytometry analysis indicated that complex ploidy levels in these progenies, among which there were 3 triploid of Phuc Trach pummelo,4 triploid of Dien pummelo (triploid chromosome number is 2n=3x=27), and 1 tetraploid of Dien pummelo (tetraploid chromosome number is 2n=4x=36).
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