【作者】 孙守如；

【导师】 何松林；

【作者基本信息】 河南农业大学 ， 森林培育， 2011， 博士

【摘要】 南瓜（Cucurbita moschata D.）又名倭瓜,药用保健功效以及观赏价值高,具有“世界性蔬菜”的美称。目前我国观赏类南瓜的优良品种类型少,更新换代非常慢,相关的育种理论基础严重滞后。经南瓜未受精胚珠离体培养的单倍体育种途径,可以人工诱导南瓜的离体雌核发育,从而快速获得单倍体以及双单倍体植株,进而迅速获得纯合的系统,为观赏类南瓜优良自交系的选育提供有效的技术手段。未受精胚珠离体培养建立的再生体系也可以作为南瓜遗传工程的理想受体系统,使隐性基因得以顺利表达。此外,诱导南瓜的离体雌核发育所获得的再生胚囊植株群体,能够丰富南瓜种质材料的变异,获得的双单倍体（DH）植株,可以作为分子标记及遗传图谱构建的适宜群体。本论文以“试验南瓜1号”为材料,通过对南瓜（Cucurbita moschata D.）未授粉子房和未受精胚珠的离体培养,诱导了雌核发育,并获得了染色体数减半的胚囊植株。以MS为基本培养基,对影响诱导效果的黑暗热激预处理时间、2,4-D浓度、激素组合、碳源种类和浓度等以及KT预处理和AgNO3刺激对未受精胚珠诱导的影响进行了较为详细、系统的比较分析研究,同时应用石蜡切片技术观察了南瓜胚囊的发育过程并以改良苯酚品红压片法观察根尖染色体和用抗微管除草剂氟乐灵和秋水仙素对南瓜进行了加倍诱导研究,旨在为建立高效的南瓜未授粉子房和未受精胚珠的培养技术和探讨雌核发育的机理,为观赏类南瓜单倍体培养奠定重要的理论基础和实践支撑。研究结果如下:1.未受精离体雌核的诱导需要黑暗热激的预处理,南瓜未受精子房培养的黑暗热激处理以在35℃条件下热激处理6d为最佳,出胚数和出胚率的均值分别达到了4.8个和22%以上,而且与热激5d、7d相比,在0.01水平上差异显著;不同胚囊时期间的诱导结果分析结果显示没有差异,说明不同胚囊时期对于热激诱导的敏感程度是相似的。未受精胚珠直接培养以黑暗热激处理5d的转绿率和胚状体诱导率最高,分别达到了80%和15%以上,说明黑暗热激5d处理能够有效的诱导未受精胚珠的雌核发育,提高胚状体诱导率。总结两个黑暗热激实验,南瓜未受精子房的培养热激6天能够有效地起动离体雌核的发育,南瓜未受精胚珠离体培养则热激5d的诱导效果最佳。2.南瓜未受精子房离体培养的培养基筛选的第一步就是比较不同浓度的2,4-D对离体诱导的影响,结果显示:添加了3.5 mg·L^-1、4.0 mg·L^-1、4.5 mg·L^-1三个浓度的基本培养基能有效地刺激了离体雌核的发育。胚状体的诱导率在2,4-D浓度为3.5 mg·L^-1时达到最高,三个胚囊时期的诱导率都在4.5%以上,其次就是2,4-D浓度在4.5 mg·L^-1和4.0 mg·L^-1的诱导率;并在开花前0.5天（12小时）的未受精子房最终诱导出了子叶胚,三个浓度分别诱导出了4个、2个和1个。南瓜未受精胚珠离体培养在添加的2,4-D浓度为1.0 mg·L^-1和1.5 mg·L^-1的基本培养基上胚状体诱导率和转绿率较高,低浓度的和高浓度的诱导率都非常低。比较两个实验:较高的2,4-D浓度能够有效地启动南瓜未受精子房的雌核发育,抑制南瓜未受精胚珠胚状体的诱导;而低浓度的2,4-D能有效地诱导未受精胚珠的雌核发育。总之,基本培养基中添加的2,4-D应由培养方式的不同而进行选择不同的浓度水平。3.筛选培养基中最优激素组合的试验结果显示:诱导南瓜未受精子房培养出胚状体效果最好激素组合是4.0 mg·L^-12,4-D+0.5 mg·L^-1NAA+0.5 mg·L^-16-BA;南瓜未受精胚珠离体培养试验中诱导效果最好的激素组合是:1.0 mg·L^-12,4-D+ 0.25 mg·L^-1NAA+1.0 mg·L^-16-BA,诱导出子叶胚总数达到了6个。相比较而言,南瓜未受精子房离体培养需的激素组合为高浓度的2,4-D、NAA和低浓度的6-BA;南瓜未受精胚珠直接培养则需低浓度的2,4-D、NAA和高浓度的6-BA。4.蔗糖和葡萄糖都可作为南瓜未受精子房离体培养的碳源,诱导雌核发育。不同蔗糖浓度间的诱导效果比较,30g·L^-1蔗糖的胚状体诱导率最高;不同葡萄糖浓度间的诱导效果比较,40g·L^-1葡萄糖的胚状体诱导率最高;不同碳源浓度间的胚状体诱导率比较,蔗糖的最高胚状体诱导率为8.52%,葡萄糖的最高诱导率为4.44%,蔗糖的诱导效果好于葡萄糖的诱导效果。南瓜未受精胚珠离体培养的不同蔗糖浓度胚状体诱导率比较试验,结果以25g·L^-1的蔗糖作为碳源的诱导效果最好,胚状体诱导率为83.33%,子叶胚7个。南瓜未受精胚珠培养所需的蔗糖浓度与南瓜未受精子房培养诱导雌核发育所需的蔗糖浓度,处在低浓度水平。5.南瓜未受精胚珠胚状体的诱导以组合MS+蔗糖(30g·L^-1)+琼脂(6g·L^-1)+2,4-D(1.0mg·L^-1)+NAA(0.25mg·L^-1)+6-BA(0.5mg·L^-1)为宜,胚状体诱导率为3.3%。而此后的分化再生培养基则以MS+蔗糖(30g·L^-1)+琼脂(6g·L^-1)和MS+蔗糖(30g·L^-1)+琼脂(6g·L^-1)+活性炭(1g·L^-1)为佳。试验发现不定芽也能发育成正常植株,但比率很低,只有0.3%⁶.7%,而胚状体发育成正常植株的比率高达70%以上。AgNO3刺激对南瓜未受精胚珠不定芽的分化具有促进作用,最佳浓度为16mg·L^-1。KT预处理对南瓜未受精胚珠的不定芽的发生具有抑制作用,这种抑制作用在开花当天的未受精胚珠上最为明显,使其出芽率从50%降到了3.3%,对其他两个接种时期未受精胚珠的抑制作用稍减。另外KT预处理和AgNO3刺激均对未受精胚珠胚状体和小苗的诱导具有抑制作用。6.应用石蜡切片技术观察到南瓜胚囊发育的整个发育过程。得出了明确的结论就是:开花前3d的子房处于单核胚囊时期;开花前2d的子房处于双核胚囊时期;开花前1d的子房处于四核胚囊时期;开花当天的子房处于七细胞八核的成熟胚囊期,其胚囊为蓼型胚囊。处于成熟胚囊期也即开花当天的未受精胚珠,其胚状体诱导率、成苗率均高于开花前1d和开花前2d这两个时期的未受精胚珠。7.上午10:00取材,4℃冷水处理24h后用卡诺固定液固定24h,再用1mol·L^-1盐酸60℃水浴解离7min,最后用改良苯酚品红染色10min,镜检。用此方法处理的细胞染色体清晰,易于进行观察,计数。观察“试验南瓜1号”的染色体条数为2n=44条。8.通过不同浓度的氟乐灵和秋水仙素进行倍性诱导处理,浓度为0.006%的氟乐灵具有较好的加倍效果,可有效代替秋水仙素。和秋水仙素相比,氟乐灵使用的浓度低,价格低廉,效果好,且毒性小,对人和环境比较安全。并用流式细胞仪对南瓜诱导的幼苗进行了倍性分析鉴定。更多还原

【Abstract】 ABSTRACT:Pumpkin（Cucurbita moschata D.）, which has higher nutritioal and ornamental value is an important economic vegetable crop in our country. While the pumpkin varieties is a litter and very slow replacement. Haploid breeding has advantages because of the high purity of doubled haploid plants. These plants are ture breeding for all the characters and the selection efficiency is greatly enhanced. It requires only single selection generation thereby improving breeding efficiency. In order to obtain the best way to induce un-pollinated ovary and ovule in pumpkin, we used“NO.1”as test material which mainly studied the influencing factors of pumpkin in vitro gynogenesis, such as heat pretreatment time, 2,4-D concentration, hormone combination, KT pretreatment and AgNO3 stimulation, and while the process embryo sac development was observed by the technology of paraffin section. We expected to lay a theoretical and practical foundation on pumpkin in vitro gynogenesis.The main conclusions as follow:1.The heat pretreatment in dark was essential for in vitro gynogenesis. The best pretreatment was under 35℃for 6 days on pumpkin in vitro culture of unpollinated ovary, which had big differences compared with 5 days heat pretreatments and 7days pretreatment （p<0.01）. There showed no difference between different development embryo sac, it proved that different development embryo sac has similar sensitivity under heat-inducible. The result of in vitro culture unpollinated ovule in pumpkin showed that: 5 days under this treatment turning green ratio reached 80% and embryoid induction rate reached 15%.was the best heat pretreatment period. The result showed that at 35℃heat treatment for 6 days can effectively improve the embryoid rate. In a word, the best heat pretreatment time was 6 days on pumpkin in vitro culture unpollinated ovary or 5 days on pumpkin in vitro culture unpollinated ovule.2.The first step of inducting medium for pumpkin in vitro gynogenesis is to compare the influence of 2,4-D in different concentration to inducement in vitro, the result are as follows: 3.5mg·L^-1、4.0mg·L^-1 and 4.5mg·L^-1of the 2,4-D concentration can stimulate the growth of vitro gynogenesis effectively. Embryoid induction rate reached highest level when 2,4-D concentration was 3.5mg·L^-1, and the embryoid induction rates of the three stages embryo sac were above 4.5%. The next was successively 4.0mg·L^-1 and 4.5mg·L^-1. Cotyledon embryo was inducted on the medium with the three 2,4-D concentration at pre-flowering 0.5d, and the number was four, two and one. On pumpkin in vitro culture un-pollinated ovule, medium supplemented with 1.0 mg·L^-1 2,4-D and 1.5mg·L^-1 2,4-D can effectively induct gynogenesis. In a word, the amount of adding 2,4-D to vitro gynogenesis should be different according to different ways of growth of in vitro gynogenesis.3. We tried to screening the best hormone combination on MS basic medium. The result showed that: 4.0 mg·L^-1 2,4-D+0.5 mg·L^-1 NAA+0.5 mg·L^-1 6-BA was the optimal compounding for inducting unpollinated ovary in vitro gynogenesis. The best hormone was 1.0 mg·L^-1 2,4-D+0.25 mg·L^-1 NAA+1.0 mg·L^-1 6-BA, which induced 6 cotyledon embryos from unpollinated ovule.Comparatively speaking, the medium supplemented with higher concentration 2,4-D and NAA and lower concentration 6-BA can effectively induce embryo from pumpkin unpollinated ovary. But inducing embryoid from pumpkin nupollinated ovule needed the medium supplemented with lower concentration 2,4-D and NAA and higher concentration 6-BA.4. Sucrose and glucose could be used as carbon source of pumpkin un-pollinated ovary in vitro culture, and used to induct gynogenesis. The medium containing 30 mg·L^-1 sucrose inducted embryoid of the highest rate, comparing induced embryoid between difference sucrose concentrations. Between the induced effects of different glucose concentration, the best induced effect was 40 mg·L^-1 glucose. Comparing the highest induction rate between different carbon sources, 8.52 % was sucrose’s, 4.44 % was glucose’s. The inducted effect of sucrose was better than glucose’s. We studied the induct effect of in vitro culture pumpkin unpollinated ovule with the medium containing sucrose. The best sucrose concentration was 25 mg·L^-1, which embryoid induction rate was 83.3 % and 7 cotyledon embryo were inducted.5.The embryo sac development process was observed by technology of paraffin section. The result showed that: at preflowering 3d, embryo sac was at mononuclear stage; at preflowering 2d, embryo sac was at binuclear stage; it became tetranucleate embryo sac, at preflowering 1d; at pre-flowering 0.5d, eight nuclears embryo sac cell could be observed in ovule. AgNO3 stimulation on pumpkin unfertilized ovule promoted the occurre-nce of adventitious buds.16mg·L^-1 was the best concentration for the ovules. KT pretreatment on pumpkin unfertilized ovule inhibited the occurrence of adv-entitious buds. The inhibition was the most obvious in the current unfertilized ovule. The occurrence rate of adventitious buds was reduced from 50% to 3.3%. The inhibition on other two inoculation period of unfertilized ovule was lower than 0 day. KT pretreatment and AgNO3 stimulation had some inhibitio-n on the induction of unfertilized ovules and seedlings.6. The ovary of the three days before blossoming was in mononuclear embryo sac period;the ovary of the two days before blossoming was in binucleate embryo sac period;the ovary of the day before blossoming was in tetranuclear embryo sac period;the ovary of blossoming day was in mature embryo sac period with seven cells and eight nuclei. Unfertilized ovules of flower blossoming day （0d） was best stage for in vitro culture. Embryoid rate and seedling rate were the highest,and were higher than the other two periods （-2d,-1d）.7. Drawn the materials at 10 am,use Kano fixative to fix for 24h after 4℃cold water treating for 24h,and use 1mol·L^-1 hydrochloric acid in 60℃w-ater bath dissociation for 7 minutes,at last use modified carbol fuchsin to sta-in for 10min,microscope. The cell chromosomes treated by this method were clear and easy to observe and count. The number of chromosome were 44 by observing‘Shi Yan NO.1’.8. Trifluralin is a herbicide .Trifluralin have function of making double chromosomes. In the experiment the pumpkin shoot apices were treated with different concentration of triflualin and colchicine.The results showed that triflualin was an efficient agent for induction of pumpkin chromosome doubling with shoot apices, the suitable concentration was 0.006%.Colchicine could be efficiently replaced by trifleralin. Triflualin concentration for tetraploid induction is very low and effective. The price of trifleralin is also low. It is low toxicity and less harmful to human and environment. Flow cytometry was used to determine the ploidy of pumpkin plantlets induced after trifleralin and colchicine treatment in shoot apices.更多还原