【作者】 云岚；

【导师】 云锦凤；

【作者基本信息】 内蒙古农业大学 ， 草业科学， 2009， 博士

【摘要】 新麦草(Psathyrostachys juncea (Fisch.) Nevski)是小麦族中少有的二倍体物种,因其低倍性,适宜用染色体加倍方法进行遗传改良。研究旨在利用离体组织培养并诱导同源四倍体对新麦草进行染色体加倍。首先以2品种新麦草为材料,建立新麦草高效再生体系。分别以幼穗、幼胚和成熟胚为外植体。结果表明,1～3cm幼嫩穗、授粉后14～17d长度为1mm的幼胚最适于愈伤诱导,但2品种有一定差异。幼胚需4℃预处理1～5d,成熟胚需10℃下浸泡48h。2,4-D在愈伤组织诱导阶段作用最显著。大于4cm幼穗和成熟胚需添加0.3～0.5 mg/LABA抑制花器官分化和胚萌发。CH可促进愈伤组织生长,但对外植体细胞分化方向没有决定作用。胚性愈伤容易分化和再生,而非胚性愈伤在添加低浓度2,4-D和6-BA的培养基上经2次以上继代改造可部分转化为胚性愈伤。愈伤组织分化再生过程中NAA和6-BA均起重要作用,胚性愈伤分化率均达80%以上。建立新麦草高效再生体系的关键步骤是外植体筛选、诱导愈伤组织形成和胚性愈伤组织的改造。用秋水仙素处理新麦草萌动种子、分蘖芽和愈伤组织诱导染色体加倍,处理后的植株用根尖压片法进行染色体倍性鉴定。结果表明,在30℃下,用1500mg/L秋水仙素和1.5%DMSO处理4h诱导萌动种子获得了混倍体植株,平均2n=28细胞突变率为24.4%;以500 mg/L秋水仙素和1.5%DMSO在25℃处理幼苗分蘖芽48h加倍诱导率更高,四倍体细胞平均比例为42.65%,此外还发现突变为单倍体、三倍体、非整倍体的细胞,总比例为8.88%;以100mg/L秋水仙素和1.5%DMSO、在25℃处理72h诱导愈伤组织加倍效率最高,平均四倍体细胞比例为53.58%,并得到8株四倍体细胞在80%以上材料。Pendimethalin处理后的愈伤组织没有得到再生植株。形态比较和气孔保卫细胞观察表明,新麦草加倍植株叶片长、宽均较二倍体有所增加。四倍体叶片气孔保卫细胞长度比二倍体增加13.52%,差异显著。气孔之间距离显著增大。比较诱导形成的同源四倍体新麦草光合作用和叶绿素荧光动力学。测定结果用直线回归和Farquhar模型进行拟合。结果表明,四倍体光补偿点、光饱和点、最大净光合速率高于二倍体,气孔导度值整体低于二倍体,表明四倍体对强光的适应性好于二倍体。四倍体CO2补偿点、CO2饱和点、最大羧化速率、最大电子传递速率和最大磷酸丙糖利用速率也显著高于二倍体,表明四倍体在高光强和高CO2浓度下比二倍体具有更大的光合潜力。叶绿素荧光观测结果表明,四倍体原初光能转化效率高于二倍体,干旱胁迫下四倍体PSⅡ实际光化学效率、光化学淬灭系数qP和非光化学淬灭系数qN低于二倍体,说明干旱胁迫下二倍体的光保护机制作用更明显。统计2个二倍体品种新麦草根尖细胞染色体,其中具14条染色体的细胞占统计细胞总数的86.25%。染色体顺次C-分带与45S rDNA分子原位杂交(FISH),结果表明,新麦草7对染色体可根据各自独特的带型而彼此区分开来。新麦草染色体组成为2n=2x=14=8m+6sm,核型对称类型属于2A。根据带型可以将7对染色体分为3组,同时也发现存在带型多态性。FISH结果显示45S rDNA在二倍体新麦草染色体上有6个主要分布位点,均位于染色体短臂端部,某些染色体中部和长臂末端也存在弱的杂交信号,可能属于次缢痕以外的rDNA分布位点。新麦草加倍细胞28条染色体上共存在12个主45S rDNA位点。顺次C-分带与原位杂交结果将3对45S rDNA位点初步定位于新麦草的Ⅰ、Ⅲ以及Ⅴ染色体短臂末端,推测这3对染色体可能是NOR染色体。更多还原

【Abstract】 Russian wild rye(Psathyrostachys juncea (Fisch.) Nevski)is a rare diploid species in Triticeae which is fit to be developed with chromosome doubling. polyploidy were induced via in vitro culture and chromosome were analyzed with C-banding and 45S rDNA FISH.Efficient regeneration system was established from three explants of two varieties, young spikes, immature and mature embryos. Young spikes with the length of 1~3cm and immature embryos after pollinated 14~17d were ideal for callus induction. Immature embryos was pretreatment at 4℃for 1~5d, mature embryos soaked at 10℃for 48h. 2,4-D had significant effect on callus induction. ABA in media was necessary when callus was induced from spikes with length longer than 4cm and mature embryos. CH in media promoted the growth of callus but had no impact on differentiation. A number of Non-embryogenic callus were transformed into embryogenic callus after subculture for more than 2 times. Differentiation rate of embryogenic callus reached 80% . NAA and 6-BA had significant effect on regeneration. The key points of efficient regeneration system of Russian wild rye is explants screening, callus induction and embryogenic callus transformation.Chromosome doubling were induced with colchicine from germinating seeds, buds and callus. Ploidy of colchicine treated plants were identified by chromosome counts in somatic cells. Mixploid plants were induced from seeds treated with 1500mg/L colchicine and 1.5%DMSO for 4 h. the percentage of tetraploid cells was 24.4%. The chromosome doubling efficient was higher when buds were treated with 500mg/L colchicine and 1.5%DMSO for 48h. the percentage of tetraploid cells was 42.65%. There were some haploid, triploid, aneuploid cells in colchicine treated plants, with percentage of 8.88%. Effect of treating callus with 100mg/L colchicine and 1.5%DMSO for 72h was the best for polyploidy induction. The tetraploid cell percentage was 53.58%, including 8 plants with 80% tetraploid cells. Callus can not regenerate after treated with Pendimethalin. Morphology comparation showed that the length and wide of tetraploid plant leaves were increased. The length of guard cells increased by 13.52% than diploid. The distance between stomotals increased significantly.The photosynthesis and chlorophyll fluorescence of induced autotetraploid and diploid P.juncea were compared. Light and CO2 response curves were estimated by linear regression and Farquhar model. Saturating and compensation light intensity and Maximum photosynthetic rate of tetraploid plant were higher than diploid, but stomatal conductance was lower than diploid. Saturating and compensation CO2 concentration was higher than diploid too. Photosynthetic limited factors (Vcmax, Jmax and TPUmax) of tetraploid is more efficient. Results showed that the photosynthetic system of tetraploid was more efficient in high light intensity and high CO2 concentrations than diploid. Fluorescence parameters were surveyed under water stress. Fv/Fm of tetraploid was higher, but qP and qN was lower than diploid. The photosynthetic apparatus of diploid was protected by exhausting the extra light energy through heat dissipation. Chlorophyll fluorescence parameters was sensitive to water stress. But the drought resistance of autotetraploid need further experiment.Chromosome number were counted in somatic cells of two varieties. Mean percentage of diploid cells (2n=14) was 86.25%. Chromosome was analyzed by Sequential C-banding and 45S rDNA FISH. Chromosome can be distinguished from each other according to their respective bandtype. Caryotype of P.junceais 2n=2x=14=8m+6sm, belongs to 2A. Chromosomes can be divided into 3 groups according the bandtype. C-banding pattern polymorphisms were observed among different individuals, and within homologous chromosome pairs. There were 6 main 45S rDNA sites located in the end of short arms of 3 pairs of diploid chromosome. They were chromosomeⅠ,ⅢandⅤwhich can be speculated as NOR chromosome. There were also weak hybridization signal on some long arm of chromosome. 12 main 45S rDNA sites were located on trtraploid chromosomes.更多还原