N~+、α粒子、~（60）Co-γ对陆地棉花粉的诱变效应研究及诱变后代鉴定
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摘要
诱变是棉花育种中创造变异的重要手段之一。棉花辐射诱变一般以种子为材料,但辐照种子长成的M1植株易出现嵌合体。花粉中含有精细胞,对辐射诱变敏感,突变的精细胞可通过受精遗传给后代,有助于提高选择效率和加速辐射育种进程。此外,辐照花粉也是创造非整倍体等遗传材料的有效方法。60Co-γ射线是棉花育种中应用最为广泛的诱变手段。离子束注入技术在植物、微生物诱变研究方面已取得丰硕成果。但是由于离子束穿透能力很弱,它能否进入细胞,一直存在争议。而且低能离子与生物体的相互作用涉及复杂的生物学效应,离子注入后对植物细胞的直接损伤及诱变效应方面的机理研究还很缺乏。a粒子属于直接电离粒子,具有较高的传能线密度,能引发细胞之间的辐射信号传导及其细胞间旁效应的产生。由于α粒子的穿透力较弱,a粒子的辐射生物学研究多集中在哺乳动物细胞方面,在植物上研究α粒子的生物效应尚少有报道,尚不清楚它是否能作为一种有效的植物诱变源。本研究以陆地棉为材料,分别用N+、α粒子和60Co-γ射线作为辐射源,从花粉粒表面结构、内部超微结构、花粉粒萌发率、授粉后萌发花粉管的数量和长度、花粉管微丝骨架、花粉管Ca2+浓度梯度以及N+注入后引起M1代DNA和RNA水平上的变异等方面研究N+、α粒子及60Co-γ射线辐射对花粉粒的诱变生物学效应,比较三种诱变源的诱变机理,为棉花诱变育种提供了新的方法。对60Co-γK射线诱变花粉后代的主要农艺性状的变异规律进行了研究,丰富了花粉诱变育种技术。研究内容和结果如下：
1.棉花雄性配子体发生和发育的检测方法
将荧光染料DAPI染色DNA、罗丹明标记的鬼笔环肽染色花粉管微丝、Fluo-3am染色花粉管中Ca2+、荧光显微镜和激光扫描共聚焦显微镜等方法应用到检测棉花雄性配子体的发生发育过程。发现陆地棉花蕾的大小与花粉母细胞及雄配子体的发育程度紧密相关,花粉母细胞减数分裂期形成雄配子时期的花蕾纵、横径大约在0.4-0.5、0.3-0.4 cm之间。处于减数分裂期的花蕾取样时间在夏季早晨5：00-7：00为宜。通过荧光染料DAPI染色,可直接观察减数分裂各时期特征及其变化过程；经过4%多聚甲醛溶液固定过的棉花成熟花粉粒,在10%次氯酸钠水溶液中,55℃水浴处理30 min,可脱去花粉粒外壁,棉花花粉粒萌发以前,其内部生殖核的分裂并不完全同步,同时存在单核花粉粒、双核花粉粒和三核花粉粒等三种不同类型的花粉粒；陆地棉花粉管内微丝主要以微丝束的方式沿花粉管连续纵向排列,花粉管顶端10-20μm的区域内,不存在微丝网络；花粉管内的游离Ca2+呈现顶端Ca2+浓度较近顶端高的极性分布。研究的方法和结果为开展与棉花花粉粒有关的生殖生物学研究提供了途径和依据。
2.N+、α粒子和60Co-γ对陆地棉花粉的诱变效应研究及诱变后代鉴定
2.1以10、20和30 keV的N+注入陆地棉花粉粒。发现N+通过刻蚀方式作用于花粉粒,对花粉内部结构、花粉活力以及花粉管的微丝骨架结构均产生程度不同的影响,影响程度与注入能量有关,能量越大,对花粉的损伤效应越明显。
2.2.通过对20 keV的N+注入后的花粉授粉后发育的M1(胚珠)DNA的SSR标记分析,在DNA水平上检测N+诱变后的多态性变化,结果表明,N+注入陆地棉花粉后再给雌蕊授粉,在一定程度上改变了花粉中精细胞的DNA序列,会对胚珠的DNA多态性产生影响。说明N+注入花粉,能产生可遗传的变异；通过抑制消减杂交技术,获得20 keV的N+诱变陆地棉花粉后M1代特异表达的cDNA片段,在mRNA水平上检测N+的诱变效应。已测序的有50个缩减cDNA克隆中,52%的序列在数据库中都有同源的棉属来源的EST。38个EST与其他物种已知基因部分区域的同源性为56%-100%,占总EST的76%；5条EST序列能在数据库中检索到同源性序列,但其功能尚不清楚；9个EST能在数据库中发现为推测蛋白；4个EST在GenBank中没有查到对应的同源序列(序列号分别为：GH291233；GH291234；GH291235和GH291236)
2.3.以陆地棉花粉为材料,研究不同注入机各参数(离子能量、总剂量、剂量率、脉冲剂量、间隔时间)对陆地棉花粉注入效应的影响。发现N+注入时的抽真空过程对陆地棉花粉活力无影响,离子能量、总剂量、剂量率、脉冲剂量和注入间隔时间等5个参数均在不同程度上影响注入结果。5个参数对注入效果的影响顺序为能量>剂量率>总剂量>脉冲剂量>间隔时间。因此,在实际的离子注入时,应根据实验目的综合考虑这些参数的效应,比如采用高能量-低剂量-长间隔时间可能是提高注入花粉活力较为有效的方法。
2.4.以20 Gy的60Co-,γ射线辐照陆地棉花粉粒,发现60Co-γ射线对花粉粒的表面结构无影响；但对花粉粒内部结构产生明显的破坏作用,内壁变薄,不规则且部分向内凹陷,内质网解聚,内含物增多；与对照(自然花粉)相比,花粉粒活力降低了38%,授粉后胚珠的DNA多态性明显增加；M1发芽率降低了41.03%。棉株的主根长,最长侧根长度,平均侧根长度,侧根数和株高等分别比对照下降了22.24%,18.93%,11.80%,28.02%和23.05%。雄性不育株占56.7%。铃数的变异系数最大,达119.79%,比对照增加103.21%,其次为衣分、茎粗、籽指、果枝数、最长果枝,变异系数分别比对照增加了74.59%、75.96%、69.83%、33.25%、29.62%；株高变异系数最小(20.15%),比对照增加了11.843%。M2代植株中的雄性不育株占24%。M2代铃数的变异系数最大(33.08%),较对照增加21.94%,其次为籽棉产量、果枝数、株高、单铃重,分别较对照增加了16.26%、3.83%、3.99%、7.25%。衣分的变异系数最小(4.90%),比对照增加0.50%。M2代各农艺性状的变异系数及变异幅度均小于M1代。M3代的株高、果枝数、铃数、产量和单铃重5个性状的均值皆低于对照,衣分均值高于对照,降低与升高的幅度皆小于M2代。在M1、M2、M3代等3个诱变后代中,M3代各农艺性状的变异系数最小,M1代的变异系数最大。M3代大多数的变异株系表现稳定,基本无分离,因此,在诱变后代选择上,M1代种子可混收,M2代再分单株收获。M3代可获得部分纯合株系。
2.5.以陆地棉成熟花粉粒为材料,结合超薄切片、荧光染色等技术。通过观察花粉的表面结构、内部结构、以及花粉萌发出的花粉管的生长和内部的微丝骨架结构的变化,分析α粒子及60Co_γ射线的诱变效应,以及诱变效应与辐照剂量间的关系,比较两种辐射源产生的辐射效应及机理方面的差异。结果表明,α粒子是通过刻蚀方式作用于花粉粒,不同于60Co-γ射线通过高能量射线穿透花粉粒作用于花粉粒。两种辐照方式均通过对花粉的内部结构产生损伤使花粉萌发率及萌发花粉管的数目下降,破坏花粉管微丝骨架。相同剂量时,60co-γ射线的损伤作用大于a粒子的作用。
2.6从诱变育种的角度来说,α粒子由于其发射仪器的限制,每次处理的花粉数量有限,不能在田间大量授粉；N+虽然对花粉的损伤效应明显,但田间授粉,后代变异不明显；20 Gy的60Co-γ射线是较为适宜的陆地棉成熟花粉育种的辐照剂量,花粉活力较高,后代性状变异明显；过高的剂量会抑制花粉管的萌发和生长,田间授粉不能收获种子。
Mutagenesis is the important means to create genetic variation in cotton breeding. A large number of energy rays are frequently applied in mutant breeding, such as X-,β-, and y-rays, neutrons, and protons. In most cases, seeds were used as mutation materials, but it is easy to obtain chimera in the M1 progeny. Pollen grains are haploid, which has a nutritional nucleus and two sperms. Pollen grain is sensitive to all kinds of mutagens. Pollination with irradiated pollen grains could directly transfer the mutation of sperms to the offspring of the pollinated plant. Furthermore, pollen irradiation is the effective means for creating dysploid. Gamma rays belong to the most efficient tools to create mutants in plants, with the advantages of convenient operation, short cycle, and high mutation quantity. Ion beams have been applied as a nuclear technique since the late 1950s, especially in the field of surface modification of materials. In the 1990s, this technology was first applied to improve crop cultivars by Chinese scientists, achieving great success in plant and microorganism. But the short path of ion-implantation was in doubt on whether the low-energy ions could enter plant cells. And the interaction between the organism and these ions is complicated. At present, the biological effect of ion-implantation is mostly emphasized on statistical analysis of phenotypic and genetic effects. Detailed information of interaction between ion beam implantation and the structure, vigor and skeleton system of cell is still insufficient. Alpha-particles, which are directly ionizing particles with a high linear energy transfer (LET), have a strong interaction with the target substance. Alpha-particles have been widely used as a mutagenic source in mammalian cells. However, low doses of ionizing particle radiation have beneficial effects on mammalian cells. Because plant cells have tough cell walls and because alpha-particles transfer energy to very limited regions, it is not known whether alpha-particles could be used as an effective mutagen on plant breeding. In this study, we used upland cotton as irradiation material to compare the effects induced by irradiation of y-rays, nitrogen ions and alpha-particles in the following:the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes. The effects of the law of genetic variation of their M1, M2 and M3 progeny induced by 60Co-γray were also studied. The results will be valuable not only for enriching the germplasm resources but also to the foundation of mutation material foreground for the location and clone of correlative genes. The main results were as follows:
1. The examination methods of development of upland cotton male gametophyte
We used DAPI labeling and fluorescence microscope to visualize the meiosis of the male gametophyte. The results showed that the size of bud is closely related with the development of pollen mother cell. The buds were harvested in the 7 am-9 am, whose glossy and elongated bell-shaped at the size of (0.4-0.5)×(0.3-0.4) cm were in meiosis. The difference in the stages of meiosis existed not only in different individual plant, different bud of the same in dividual inflorescence, but also in different cell of the same bud. The procedure of detaching the exine of Gossypium hirsutum L. pollen grain was established in this experiment. The pollen grain was fixated with 4% paraformaldehyde solution, oxidated with 10% sodium hypochlorite solution, heat shocked at 55℃for 30 minutes and pressed. After these treatments, the exine could be detached along the full de-exined pollen grains. The pollen grain and de-exined pollen grain were stained with DAPI. The nucleus of cotton pollen grain was not observed, but the nucleus of de-exined pollen grain was showed clearly. The inner wall had not fluorescence. The three nuclei of the pollen grain of Gossypium hirsutum L. were determinated firstly. We used TRITC-phalloidin labeling and laser scanning confocal microscope to visualize the F-actin cytoskeleton of the pollen tubes after germination in liquid medium. The results showed that F-actin bundles oriented along the longitudinal axis in the long pollen tube and extend only 10 to 20μm away from the tip of the pollen tube. Growing pollen tubes were loaded with the Ca2+ sensitive ratiometric dye Fluo-3 am, the result showed that the [Ca2+]i at the tip increased during the peaks of the Ca2+ oscillations, and that the Ca2+ gradient extended further down the shaft of the tube.
2. The study on mutation effects of N+、αandγrays on Gossypium hirsutum L. pollen grains and identification of mutation progeny
2.1 To study the radiobiological effects of low-energy nitrogen ions on plant cells, nitrogen ions of varied energies (10 keV,20 keV and 30 keV) were implanted in upland cotton pollen grains. Irradiation effects on pollen grains were assessed in terms of the ultrastructural changes in the exine and interior walls of pollen grains, the germination rate, the number of pollen tubes in styles after pollination, the extent of the tip-focused Ca2+ gradient and the ultrastructural changes in the F-actin of pollen tubes that developed from the treated pollen grains upon germination in liquid medium. The results showed that nitrogen ion passed through the pollen grains by etching and penetrating the exine and interior walls and destroying cell structures. More pollen grains were destroyed as the energy of the nitrogen ions increased. The pollen grain germination rate as well as the number and length of the pollen tubes after nitrogen ion implantation were decreased with increased ion implantation energy. Damaging effects were also observed for the tip-focused Ca2+ gradient and the F-actin of pollen tubes. The function and structure of pollen tubes in pollination and fertilization may be affected by the changes in the ultrastructure of pollen grains and the gene expression profile of pollen tubes.
2.2 The SSR-based molecular markers technique was used to determine the polymorphisms of ovule DNA. The ovule was developed after the pistils were pollinated by the pollen grains which had been implanted with nitrogen ions. The result showed that the ovule DNA polymorphisms was changed, which indicated that the DNA sequences of sperm cell were altered. The suppressive subtraction hybridization (SSH) technique was used to isolate the cDNA fragments that showed differential expression between the M1 progeny developed after the pistils were pollinated by the nature and pollen grains implanted with the nitrogen ions. Two differential expression cDNA libraries were constructed using the cDNAs of the treated-M1 progeny as driver and the cDNAs of untreated-M1 progeny as tester, vice versa. The results demonstrated that the insertion fragments of SSH library were ranging from 450 to 700 bp.50 clones were randomly picked for PCR and sequencing. After matching the result in GenBank dbEST and the BLASTN,50 sequences were obtained. Of them,38 had 56%-100% similarity with known genes, which was 76% of the total EST; 5 sequences had their homology with the sequences in GenBank with unknown function, which was 10% of the total EST; 9 were hypothetical protein, which was 18% of the total EST; 4 had no similarity with the sequences in GenBank, which might be new genes; or the gene could not search the similarity with the sequences of other species, because the sequences located in the easy-varied 3'-end, which was 8% of the total EST.
2.3 Effect of parameters of ion implantation machine, including ion energy, total dose, dose rate, impulse energy and implantation interval on the pollen grains of upland cotton implanted with nitrogen ions were studied. The best parameters were screened out. The results also showed that the vacuum condition before the nitrogen ion implantation does not affect the pollen viability. From the perspective of effects, the 5 parameters run as energy>dose rate >total dose> impulse dose> interval. Whereby the comprehensive considerations shall be given for the ion implantation based on the test objects. For instance, the high energy-low dose-long interval might be an effective option to improve the offspring vitality.
2.4 20 Gy 60Co-y ray irradiation effects on upland cotton pollen grains were tested in terms of the ultrastructural changes in the exine and interior of pollen grains, their germination rate, the single primer amplification reaction polymorphism of ovule developed after the pistils were pollinated with the pollen grains which had been irradiated with 60Co-y ray, and the law of genetic variation of their M1, M2 and M3 progeny. The results showed that 60Co-y ray had no effects on the exine wall of the pollen grains. The interior structure of pollen grain was destroyed significiantly. The interior wall became thin and irregular, and part of it concavitied to the inner. The endoplasmic reticulum depolymerized. The amount and the density of pollen grain inclusions increased. The number of pollen tubes in style decreased by 38%, compared with the control group. The single primer amplification reaction polymorphism of ovule increased. The germination rate of M1 progeny was decreased by 41.03%. In M1 progeny, the length of taproot, longest lateral root, average lateral root, the number of lateral root, and the height of seedling decreased by 22.24%,18.93%,11.80%,28.02%,23.05%, respectively., compared with the control group. The percentage of sterility plants was 56.7%. The coefficients of variations of boll number, lint percentage, perimeter of stem, seed index, fruit branch number, longissimus fruit branch and plant height increased by 103.206%,74.588%、75.96%,69.83%、33.25% and 29.624%,11.843%, respectively., compared with the control group. In M2 progeny, the percentage of sterility plants was 56.7%. And the coefficients of variations of boll number, cotton yield, fruit branch number, plant height, boll weight, and lint percentage increased by 21.944%,16.261%、3.827%.3.986%.7.25% and 0.497%, respectively. Compared with the control group, the coefficients of variations and change range of agronomic traits in M2 progeny were less than them in M1 progeny. In M3 progeny, the means of plant height, fruit branch number, boll number, cotton yield and boll weight were all less than that of the control group, respectively. And the lint percentage was more than that of the control group. The coefficients of variations of the agronomic traits in M3 progeny was least, compared with that in the progeny of M1 and M2. The mutants were inherited stably. Therefore, in the progeny selection, the M1 seeds are mixed harvested, and single-plant harvested in M1 seeds.
2.5 We compared the effects of irradiation by y-rays and alpha-particles on the structure and function of upland cotton pollen grains during pollination. We used mature pollen grains of upland cotton as irradiation material to observe changes in the following: the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains in culture medium, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes in medium. We found that alpha-particle and y-ray irradiation have different biological effects on the structure and function of cotton pollen grains.
2.6 Because the quantity limitation of irradiated pollen grains every time, alpha-particles do not apply to mutation breeding (Field pollination need a mass of pollen grains.). Nitrogen ions have obviously damage effect on the pollen grains, but the mutation progeny have no evident variation.20 Gy 60Co-γray is suitable for cotton mutation breeding. The pollen grains irradiated by 20 Gy 60Co-y ray were pollinated in the field, a large number of mutants was obtained in the mutation progeny.
1.棉花雄性配子体发生和发育的检测方法
将荧光染料DAPI染色DNA、罗丹明标记的鬼笔环肽染色花粉管微丝、Fluo-3am染色花粉管中Ca2+、荧光显微镜和激光扫描共聚焦显微镜等方法应用到检测棉花雄性配子体的发生发育过程。发现陆地棉花蕾的大小与花粉母细胞及雄配子体的发育程度紧密相关,花粉母细胞减数分裂期形成雄配子时期的花蕾纵、横径大约在0.4-0.5、0.3-0.4 cm之间。处于减数分裂期的花蕾取样时间在夏季早晨5：00-7：00为宜。通过荧光染料DAPI染色,可直接观察减数分裂各时期特征及其变化过程；经过4%多聚甲醛溶液固定过的棉花成熟花粉粒,在10%次氯酸钠水溶液中,55℃水浴处理30 min,可脱去花粉粒外壁,棉花花粉粒萌发以前,其内部生殖核的分裂并不完全同步,同时存在单核花粉粒、双核花粉粒和三核花粉粒等三种不同类型的花粉粒；陆地棉花粉管内微丝主要以微丝束的方式沿花粉管连续纵向排列,花粉管顶端10-20μm的区域内,不存在微丝网络；花粉管内的游离Ca2+呈现顶端Ca2+浓度较近顶端高的极性分布。研究的方法和结果为开展与棉花花粉粒有关的生殖生物学研究提供了途径和依据。
2.N+、α粒子和60Co-γ对陆地棉花粉的诱变效应研究及诱变后代鉴定
2.1以10、20和30 keV的N+注入陆地棉花粉粒。发现N+通过刻蚀方式作用于花粉粒,对花粉内部结构、花粉活力以及花粉管的微丝骨架结构均产生程度不同的影响,影响程度与注入能量有关,能量越大,对花粉的损伤效应越明显。
2.2.通过对20 keV的N+注入后的花粉授粉后发育的M1(胚珠)DNA的SSR标记分析,在DNA水平上检测N+诱变后的多态性变化,结果表明,N+注入陆地棉花粉后再给雌蕊授粉,在一定程度上改变了花粉中精细胞的DNA序列,会对胚珠的DNA多态性产生影响。说明N+注入花粉,能产生可遗传的变异；通过抑制消减杂交技术,获得20 keV的N+诱变陆地棉花粉后M1代特异表达的cDNA片段,在mRNA水平上检测N+的诱变效应。已测序的有50个缩减cDNA克隆中,52%的序列在数据库中都有同源的棉属来源的EST。38个EST与其他物种已知基因部分区域的同源性为56%-100%,占总EST的76%；5条EST序列能在数据库中检索到同源性序列,但其功能尚不清楚；9个EST能在数据库中发现为推测蛋白；4个EST在GenBank中没有查到对应的同源序列(序列号分别为：GH291233；GH291234；GH291235和GH291236)
2.3.以陆地棉花粉为材料,研究不同注入机各参数(离子能量、总剂量、剂量率、脉冲剂量、间隔时间)对陆地棉花粉注入效应的影响。发现N+注入时的抽真空过程对陆地棉花粉活力无影响,离子能量、总剂量、剂量率、脉冲剂量和注入间隔时间等5个参数均在不同程度上影响注入结果。5个参数对注入效果的影响顺序为能量>剂量率>总剂量>脉冲剂量>间隔时间。因此,在实际的离子注入时,应根据实验目的综合考虑这些参数的效应,比如采用高能量-低剂量-长间隔时间可能是提高注入花粉活力较为有效的方法。
2.4.以20 Gy的60Co-,γ射线辐照陆地棉花粉粒,发现60Co-γ射线对花粉粒的表面结构无影响；但对花粉粒内部结构产生明显的破坏作用,内壁变薄,不规则且部分向内凹陷,内质网解聚,内含物增多；与对照(自然花粉)相比,花粉粒活力降低了38%,授粉后胚珠的DNA多态性明显增加；M1发芽率降低了41.03%。棉株的主根长,最长侧根长度,平均侧根长度,侧根数和株高等分别比对照下降了22.24%,18.93%,11.80%,28.02%和23.05%。雄性不育株占56.7%。铃数的变异系数最大,达119.79%,比对照增加103.21%,其次为衣分、茎粗、籽指、果枝数、最长果枝,变异系数分别比对照增加了74.59%、75.96%、69.83%、33.25%、29.62%；株高变异系数最小(20.15%),比对照增加了11.843%。M2代植株中的雄性不育株占24%。M2代铃数的变异系数最大(33.08%),较对照增加21.94%,其次为籽棉产量、果枝数、株高、单铃重,分别较对照增加了16.26%、3.83%、3.99%、7.25%。衣分的变异系数最小(4.90%),比对照增加0.50%。M2代各农艺性状的变异系数及变异幅度均小于M1代。M3代的株高、果枝数、铃数、产量和单铃重5个性状的均值皆低于对照,衣分均值高于对照,降低与升高的幅度皆小于M2代。在M1、M2、M3代等3个诱变后代中,M3代各农艺性状的变异系数最小,M1代的变异系数最大。M3代大多数的变异株系表现稳定,基本无分离,因此,在诱变后代选择上,M1代种子可混收,M2代再分单株收获。M3代可获得部分纯合株系。
2.5.以陆地棉成熟花粉粒为材料,结合超薄切片、荧光染色等技术。通过观察花粉的表面结构、内部结构、以及花粉萌发出的花粉管的生长和内部的微丝骨架结构的变化,分析α粒子及60Co_γ射线的诱变效应,以及诱变效应与辐照剂量间的关系,比较两种辐射源产生的辐射效应及机理方面的差异。结果表明,α粒子是通过刻蚀方式作用于花粉粒,不同于60Co-γ射线通过高能量射线穿透花粉粒作用于花粉粒。两种辐照方式均通过对花粉的内部结构产生损伤使花粉萌发率及萌发花粉管的数目下降,破坏花粉管微丝骨架。相同剂量时,60co-γ射线的损伤作用大于a粒子的作用。
2.6从诱变育种的角度来说,α粒子由于其发射仪器的限制,每次处理的花粉数量有限,不能在田间大量授粉；N+虽然对花粉的损伤效应明显,但田间授粉,后代变异不明显；20 Gy的60Co-γ射线是较为适宜的陆地棉成熟花粉育种的辐照剂量,花粉活力较高,后代性状变异明显；过高的剂量会抑制花粉管的萌发和生长,田间授粉不能收获种子。
Mutagenesis is the important means to create genetic variation in cotton breeding. A large number of energy rays are frequently applied in mutant breeding, such as X-,β-, and y-rays, neutrons, and protons. In most cases, seeds were used as mutation materials, but it is easy to obtain chimera in the M1 progeny. Pollen grains are haploid, which has a nutritional nucleus and two sperms. Pollen grain is sensitive to all kinds of mutagens. Pollination with irradiated pollen grains could directly transfer the mutation of sperms to the offspring of the pollinated plant. Furthermore, pollen irradiation is the effective means for creating dysploid. Gamma rays belong to the most efficient tools to create mutants in plants, with the advantages of convenient operation, short cycle, and high mutation quantity. Ion beams have been applied as a nuclear technique since the late 1950s, especially in the field of surface modification of materials. In the 1990s, this technology was first applied to improve crop cultivars by Chinese scientists, achieving great success in plant and microorganism. But the short path of ion-implantation was in doubt on whether the low-energy ions could enter plant cells. And the interaction between the organism and these ions is complicated. At present, the biological effect of ion-implantation is mostly emphasized on statistical analysis of phenotypic and genetic effects. Detailed information of interaction between ion beam implantation and the structure, vigor and skeleton system of cell is still insufficient. Alpha-particles, which are directly ionizing particles with a high linear energy transfer (LET), have a strong interaction with the target substance. Alpha-particles have been widely used as a mutagenic source in mammalian cells. However, low doses of ionizing particle radiation have beneficial effects on mammalian cells. Because plant cells have tough cell walls and because alpha-particles transfer energy to very limited regions, it is not known whether alpha-particles could be used as an effective mutagen on plant breeding. In this study, we used upland cotton as irradiation material to compare the effects induced by irradiation of y-rays, nitrogen ions and alpha-particles in the following:the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes. The effects of the law of genetic variation of their M1, M2 and M3 progeny induced by 60Co-γray were also studied. The results will be valuable not only for enriching the germplasm resources but also to the foundation of mutation material foreground for the location and clone of correlative genes. The main results were as follows:
1. The examination methods of development of upland cotton male gametophyte
We used DAPI labeling and fluorescence microscope to visualize the meiosis of the male gametophyte. The results showed that the size of bud is closely related with the development of pollen mother cell. The buds were harvested in the 7 am-9 am, whose glossy and elongated bell-shaped at the size of (0.4-0.5)×(0.3-0.4) cm were in meiosis. The difference in the stages of meiosis existed not only in different individual plant, different bud of the same in dividual inflorescence, but also in different cell of the same bud. The procedure of detaching the exine of Gossypium hirsutum L. pollen grain was established in this experiment. The pollen grain was fixated with 4% paraformaldehyde solution, oxidated with 10% sodium hypochlorite solution, heat shocked at 55℃for 30 minutes and pressed. After these treatments, the exine could be detached along the full de-exined pollen grains. The pollen grain and de-exined pollen grain were stained with DAPI. The nucleus of cotton pollen grain was not observed, but the nucleus of de-exined pollen grain was showed clearly. The inner wall had not fluorescence. The three nuclei of the pollen grain of Gossypium hirsutum L. were determinated firstly. We used TRITC-phalloidin labeling and laser scanning confocal microscope to visualize the F-actin cytoskeleton of the pollen tubes after germination in liquid medium. The results showed that F-actin bundles oriented along the longitudinal axis in the long pollen tube and extend only 10 to 20μm away from the tip of the pollen tube. Growing pollen tubes were loaded with the Ca2+ sensitive ratiometric dye Fluo-3 am, the result showed that the [Ca2+]i at the tip increased during the peaks of the Ca2+ oscillations, and that the Ca2+ gradient extended further down the shaft of the tube.
2. The study on mutation effects of N+、αandγrays on Gossypium hirsutum L. pollen grains and identification of mutation progeny
2.1 To study the radiobiological effects of low-energy nitrogen ions on plant cells, nitrogen ions of varied energies (10 keV,20 keV and 30 keV) were implanted in upland cotton pollen grains. Irradiation effects on pollen grains were assessed in terms of the ultrastructural changes in the exine and interior walls of pollen grains, the germination rate, the number of pollen tubes in styles after pollination, the extent of the tip-focused Ca2+ gradient and the ultrastructural changes in the F-actin of pollen tubes that developed from the treated pollen grains upon germination in liquid medium. The results showed that nitrogen ion passed through the pollen grains by etching and penetrating the exine and interior walls and destroying cell structures. More pollen grains were destroyed as the energy of the nitrogen ions increased. The pollen grain germination rate as well as the number and length of the pollen tubes after nitrogen ion implantation were decreased with increased ion implantation energy. Damaging effects were also observed for the tip-focused Ca2+ gradient and the F-actin of pollen tubes. The function and structure of pollen tubes in pollination and fertilization may be affected by the changes in the ultrastructure of pollen grains and the gene expression profile of pollen tubes.
2.2 The SSR-based molecular markers technique was used to determine the polymorphisms of ovule DNA. The ovule was developed after the pistils were pollinated by the pollen grains which had been implanted with nitrogen ions. The result showed that the ovule DNA polymorphisms was changed, which indicated that the DNA sequences of sperm cell were altered. The suppressive subtraction hybridization (SSH) technique was used to isolate the cDNA fragments that showed differential expression between the M1 progeny developed after the pistils were pollinated by the nature and pollen grains implanted with the nitrogen ions. Two differential expression cDNA libraries were constructed using the cDNAs of the treated-M1 progeny as driver and the cDNAs of untreated-M1 progeny as tester, vice versa. The results demonstrated that the insertion fragments of SSH library were ranging from 450 to 700 bp.50 clones were randomly picked for PCR and sequencing. After matching the result in GenBank dbEST and the BLASTN,50 sequences were obtained. Of them,38 had 56%-100% similarity with known genes, which was 76% of the total EST; 5 sequences had their homology with the sequences in GenBank with unknown function, which was 10% of the total EST; 9 were hypothetical protein, which was 18% of the total EST; 4 had no similarity with the sequences in GenBank, which might be new genes; or the gene could not search the similarity with the sequences of other species, because the sequences located in the easy-varied 3'-end, which was 8% of the total EST.
2.3 Effect of parameters of ion implantation machine, including ion energy, total dose, dose rate, impulse energy and implantation interval on the pollen grains of upland cotton implanted with nitrogen ions were studied. The best parameters were screened out. The results also showed that the vacuum condition before the nitrogen ion implantation does not affect the pollen viability. From the perspective of effects, the 5 parameters run as energy>dose rate >total dose> impulse dose> interval. Whereby the comprehensive considerations shall be given for the ion implantation based on the test objects. For instance, the high energy-low dose-long interval might be an effective option to improve the offspring vitality.
2.4 20 Gy 60Co-y ray irradiation effects on upland cotton pollen grains were tested in terms of the ultrastructural changes in the exine and interior of pollen grains, their germination rate, the single primer amplification reaction polymorphism of ovule developed after the pistils were pollinated with the pollen grains which had been irradiated with 60Co-y ray, and the law of genetic variation of their M1, M2 and M3 progeny. The results showed that 60Co-y ray had no effects on the exine wall of the pollen grains. The interior structure of pollen grain was destroyed significiantly. The interior wall became thin and irregular, and part of it concavitied to the inner. The endoplasmic reticulum depolymerized. The amount and the density of pollen grain inclusions increased. The number of pollen tubes in style decreased by 38%, compared with the control group. The single primer amplification reaction polymorphism of ovule increased. The germination rate of M1 progeny was decreased by 41.03%. In M1 progeny, the length of taproot, longest lateral root, average lateral root, the number of lateral root, and the height of seedling decreased by 22.24%,18.93%,11.80%,28.02%,23.05%, respectively., compared with the control group. The percentage of sterility plants was 56.7%. The coefficients of variations of boll number, lint percentage, perimeter of stem, seed index, fruit branch number, longissimus fruit branch and plant height increased by 103.206%,74.588%、75.96%,69.83%、33.25% and 29.624%,11.843%, respectively., compared with the control group. In M2 progeny, the percentage of sterility plants was 56.7%. And the coefficients of variations of boll number, cotton yield, fruit branch number, plant height, boll weight, and lint percentage increased by 21.944%,16.261%、3.827%.3.986%.7.25% and 0.497%, respectively. Compared with the control group, the coefficients of variations and change range of agronomic traits in M2 progeny were less than them in M1 progeny. In M3 progeny, the means of plant height, fruit branch number, boll number, cotton yield and boll weight were all less than that of the control group, respectively. And the lint percentage was more than that of the control group. The coefficients of variations of the agronomic traits in M3 progeny was least, compared with that in the progeny of M1 and M2. The mutants were inherited stably. Therefore, in the progeny selection, the M1 seeds are mixed harvested, and single-plant harvested in M1 seeds.
2.5 We compared the effects of irradiation by y-rays and alpha-particles on the structure and function of upland cotton pollen grains during pollination. We used mature pollen grains of upland cotton as irradiation material to observe changes in the following: the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains in culture medium, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes in medium. We found that alpha-particle and y-ray irradiation have different biological effects on the structure and function of cotton pollen grains.
2.6 Because the quantity limitation of irradiated pollen grains every time, alpha-particles do not apply to mutation breeding (Field pollination need a mass of pollen grains.). Nitrogen ions have obviously damage effect on the pollen grains, but the mutation progeny have no evident variation.20 Gy 60Co-γray is suitable for cotton mutation breeding. The pollen grains irradiated by 20 Gy 60Co-y ray were pollinated in the field, a large number of mutants was obtained in the mutation progeny.
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