观赏南瓜未受精子房、胚珠离体培养及其机理研究
|
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~(-1)2,4-D+0.5 mg·L~(-1)NAA+0.5 mg·L~(-1)6-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~(-1)2,4-D+ 0.25 mg·L~(-1)NAA+1.0 mg·L~(-1)6-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。
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~(-1)of 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.
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~(-1)2,4-D+0.5 mg·L~(-1)NAA+0.5 mg·L~(-1)6-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~(-1)2,4-D+ 0.25 mg·L~(-1)NAA+1.0 mg·L~(-1)6-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。
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~(-1)of 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.
引文
AbaK K, et al. In vitro colchicine application of haploid cucumber plant[J].Cucurbit Genetic Cooperative Report,1997,(20):21-23.
Bino R J. Flow cytometric deter mination of nuclear replication stages in tomato seeds during priming and germination[J].Annals of Botany,1992,69:231-236.
Caglar G, Abak K, Abak K, et al. Progress in the production of haploid embryos, plants and doubled hap loids in cucumber (C. sativus L. ) by gamma irradiated pollen, in Turkey [J]. Acta Horticulturae, 1999,492:31-322.
Caglar G, Abak K. Efficiency of haploid production in cucumber [J].Report Cucurbit Genetics Cooperative, 1996,19:36-37.
Caglar G, Abak K. In situ haploid embryo induction in cucumber (Cucumis sativus L.) after pollination by irradiated pollen [J ]. Turkish J of Agri and Fore,1999, 23 (1):63-72.
Caglar G, Abak K. Production of in vitro haploid plants from in situ induced haploid embryos in cucumber (Cucumis sativus L.) [J].Turk ish Journal of A griculture and Forestry, 1999, 23(3): 283-290.
Campion B,Alloni C.Induction of haploid plants in onion (Alliun cepa L.)by in vitro culture of unpollinated ovules[J].Plant Cell,Tissue and Organ Culture,1990,20(1):1-6.
Campion E,Perr M,Aszimonti T,et al. Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)[J]. Plant Breeding,1995,114(3):243-246.
Cao M Q, Li Y, Liu F.Influence of genotype and donor plant growth environment on embryogenesis from isolated microspores in Chinese cabbage (Brassica campestris ssp.Pekinensis) [J]. Acta Agricult. Boreali-Sin,1993,8(4):1-6.
Castillo A M, CistuéL. Production of gynogenic haploids of Hordeum vulgate L [J]. Plant Cell Reports,1993,12(3):139-143.
Chambonnet D, Dumas de Vaulx R. Obtention of embryos and plants from in vitro culture of unfertilizered ovules of Cucurbita pepo L[J]. Cucurbit Genet Coop Rpt,1985,(8):66.
Chaudhury A M, Ming L, Miller C, et al. Fertilization-independent seed development in Arabidopsis thaliana[J]. Proc Natl Acad Sci USA,1997,94(8):4223-4228
Choi M Y, Jeong H W, Lee S S, et al. Improvement of chloroplast observation technique in guard cells for ploidy detection of microspore derived plants in broccoli[J]. Journal of the Korean Society for Horticultural Science,1997,38(6):666-669.
Cohat J.Obtention chez l’ehalote (Allium cepa L.var.agregatum) de plantes haploides gynogenetiques par culture invitro de boutons fioraux[J].Agronomie,1994,14:299-304.
Cooper D C. Macrosporogenesis and the development of the macrogametophyte of LycopersiconExculentum[J]. American Journal of Botany,1931,18:739-748.
Deverna J W, Collins G B. Maternal haploids of Petunia axiollanis (Lam) B.S.P via culture of placenta attached ovules[J]. Theoretical and Applied Genetics,1984,69(2):187-192.
Dore C, Boulidard L, Sauton A, et al. Interest of irradiated pollen for obtaining haploid vegetables [J].Acta Horticulturae,1995,392:123-128.
Drews G N, Lee D, Christensen C A. Genetic analysis of female gametophyte development and function[J]. Plant Cell,1998,10(1):5-17.
Dumas D, Pochard V R E, Chambonnet D. Distribution of TMV-po susceptible and resistant doubled haploid lines from anther culture of heterozygous L/L’hybrids[J]. Capsicum Newsl,1982,1:52-53.
Estrada-Luna A A, García-Aguilar M, Vielle-Calzada J-Ph.Female reproductive development and pollen tube growth in diploid genotypes of Solanum cardiophyllum Lindl [J].Sexual Plant Reproduction,2002,17(3):117-124.
Faris N M, Nikolova V, Niem irowicz Szczytt K.The effect of gamma irradiation dose on cucumber (Cucumis sativus L.) haploid embryo production. Acta physiofogiae Plantarum,1999,21(4): 391-396.
Faris N M, Nikolova V, Niemirowicz S K. The effect of gamma irradiation dose on cucumber (Cucumis sativus L) haploid embryo production [J].Acta Physiofogiae Plantarum, 1999,21(4):391-396.
Fernando D, Javier M, Francisco J C. The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development [J].Planta,2001,213(3):352-360.
Ficcadenti N, Sestili S, Annibali S, et al. In vitro gynogenesis to induce haploid plants in melon (Cucumis melo L) [J]. Journal of Genetic & Breeding,1999,53:255-257.
Gemes J A, Venczel G P, Altman A, et al. Haploid plant induct ion in Zucchini (Cucurbita pepo L. ) [J].Euphytica,1997,97:249-254.
GémesnéJ A,Balogh P, Ferenczy A, et al. Effect of optimum stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber (Cucum issativus L.) [J].Plant Cell Reports,2002,21(2) :105-111.
Greenberg J T. Programmed cell death: A way of life for plants[J]. Proc Natl Acad,1996,93: 12094-12097.
Gu Z P, Zheng G C.In vitro induction of haploid plants from unpollinated ovaries of highland barley [J]. Acta Bot Sin, 1984, 26(5):549-551.
Guha S, Johri B M. In vitro development of ovary and ovule of Allium cepa L.[J]. Phyomorphology, 1966, 16:353-364.
Gurel S,Curel E,Kaya Z. Doubled haploid plant production from unpollinated ovule of sugar beet (Beta vulgaris L.) [J].Plant Cell Reports,2000,19(12):1155-1159.
Hansen N J P, Andersen S B. In vitro chromosome doubling potential of colchicine, oryzalin, trifluralin, and APM in Brassica napus microspore culture [J].Euphytica,1996,88(2):159-164.
Hoseman D, Bossoutrot D. Induction of haploid plants from in vitro culture of unpollinated beet (Beta vulgaris L.) [J]. Z. Pflanzenzucht,1983,91:74-77.
Hu S Y.Development of plant embryology in China [J].Acta Botanica Sinica,2002,44(9): 1022-1042. Huang B Q, Sheridan W F. Female gametophyte development in maize: microtubule organization and embryo sac polarity [J].Plant Cell,1994,6(6):845-861.
Jakge M, Bohanec B, Ihan A. Effect of media components on the gynogenic regeneration of onion (Alium cepa L.) cultivars and analysis of regenerants [J]. Plant Cell Reports,1996,15(12):934-938.
Kaya Z. Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.) [J]. Plant Cell Reports,2000,19(12):1155-1159.
Keller J. Culture of unpollinated ovules, ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Aliium cepa L) [J].Euphytica,1990,47:241-247.
Kojima A, Kawaguchi T. Dihaploids derived from unpollinated ovule culture of Chinese chives[C]. Proc 4th Album Symp Wellesbourne,1988:212-217.
Koltunow A M. Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules [J]. Plant Cell, 1993,5(10):1425-1437.
Kurtar E S, Sail N, Abak K. Obtention of haploid embryo s and plants through irradiated pollen technique in sqush (Cucurbita pepo L.) [J]. Euphytica, 2002,127(3):335-344.
Kwack S N, Fujieda K. Somatic embryogenesis in cultured unfertilized ovules of Cucurbita moschata [J]. Journal of the Japanese Society for Horticultural Science,1988,57:34-42.
Lacadena J R. Spontaneous and induced parthenogenesis and regenesis [A]. K Kasha. Haploids in Higher Plants. Advances and Potential[M].University of Guel Ph,1974:13-32.
Li R B, Pandey M P.Genetic studies on vitro unpollinated ovary culture in rice [J]. Oryza, 1999, 36(1):28-31.
Lotfi M, Alan A R, Henning M J,et al.Production of haploid and doubled haploid plants of melon (Cucumis melo L.) for use in breeding fo rmultiple virus resistanee [J]. Plant Cell Reports,2003,21(11):1121-1128.
Lotfi M, Kashi A, Onsinejad R, et al. Induction of parthenogenetic embryos by irradiated pollen in cucumber[J]. Acta Horticulturae,1999,492:323-328.
Lux H, Herrmann L,Wetzel C L. Production of haploid sugar beet (Beta vulgaris L.) by culturing unpollinated ovules [J].Plant Breeding,1990,104(3):177-183.
Maheshwari N. In vitro culture of excised ovules of Papaver somniferum L[J]. Phytomorphology, 1961, 11:307-314.
Metwally E I, Moustafa S A, El-Sawy B I, et al. Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo[J].Plant Cell,Tissue and Organ Culture,1998,52(3):117-121.
Morejohn L C, Bureau T E, Mole-Bajer T, et al. Oryzalin, a dinitroaniline herbicide, binds to planttubulin and inhibits microtubule polymerization in vitro [J].Planta,1987,172 (2): 252-264. Mukhambetzhanov S K. Culture of nonfertilized female gametophytes in vitro[J]. Plant Cell,Tissue and Organ Culture,1997,48(2):111-119.
Muren R C. Haploid plant induction from unpollinated ovaries in onion [J]. Hort Science,1989, 24(5): 833-834.
Musial K, Bohanec B, Przywara L. Embryological study on gynogenesis in onion (AIlium cepa L)[J].Sexual Plant Reproduction,2001,13(6):335-341.
Niemirowicz S K, Vaulx R D, Dumas V R. Preliminary data on haploid cucumber (Cucumis sa -tivus L.) induction[J]. Report Cucurbit Genetics Cooperative,1989,12:24-25.
Nikolova V, Rodeva V, Alexandrova M, et al. Effect of gamma irradiation for induction of gynogenesis in cucumber (Cucum is sativus L. ) [J]. Bulgarian J of Agri Sci,2001,7:415-420.
Nishi T, Mitsuoka S.Occurrence of various ploidy plants from anther and ovary culture of rice plant [J].Jap J Genet,1969,44(4):341-346.
Norio K, Surmio I W A I. Production of haploid plant of melon by pseudofertilized ovule culture [J]. Plant Tissue Culture,1993,10(10):60.
Przyborowski J, Niemirowicz S K. Main factors affecting cucumber (Cucumis sativus L. ) haploid embryo development and haploid plant characteristics [J ]. Plant Breeding,1994,112:70-75.
Roger C, Muren.Haploid plant induction from unpollinated ovaries in onion [J]. Hortscience, 1989, 24(5): 833-834.
Russell S D. The egg cell: development and role in fertilization and early embryogenesis [J]. Plant Cell, 1993, 5(10):1349-1359.
Sachar R C, Kapoor M. In vitro culture of ovules of Zaphyranthes. [J]. Phytomorphology,l959,9:147-15. Sachar R C, Kapoor M. Influence of kinetin and gibberellic acid on the test-tube seeds of cooperia pedunculata [J]. Herb Naturwissenschaften,1985,45 (5):52-53.
Sair N, Abak K. Comparison of ploidy level screening methods in watermelon: Citrulius Lanatus (Thunb) [J]. Scientia Horticulturae,1999,82:265-277.
San Noeum L H. In vitro induction of gynogenssis in higher plants[J].Proceedings of the Conference on Broadening Genetic,1978:327-329.
Sari N,Abak K,Pitrat M,et al.Induction of parthenogenetic haploid embryos after pollination by ir radited pollen in watermelon [J]. Hort Scienee,1994,29(10):1189-1190.San Noeum L H. Haploids d’
Hodemu vulgare L. paculture in vitro ovaries[J].Ann Aemlior Plant, 1976,24:751-754. Sauton A.Haploidisation chez les Cucurbitaceae.In cinquantenaire dela culture in vitro.Versailles (France) 24-25 October INRA,Paris,1990:325-326.
Savin F,Decomble V, Le Couviour M, et al. The X-ray detection of haploid embryos arisen in muskmelon (Cucumis melo L.) seeds,and resulting from a parthenogenetic development induced by irradiatedpollen[J].Cucurbit Genet Coop Rpt, 1988,11:39-42.
Shuizhang F E I , Paul E R, Terrance P R. Improvement of embryogenic callus induction and shoot regeneration buffalograss by silver nitrate[J].Plant Cell,Tissue and Organ Culture, 2000,60(3):197-203.
Sitbon M. Production of haploid Gerbera jamesonii plants by in vitro culture unfertilized ovules[J]. Agronomie, 1981,1:807-812.
Treng-Andre I. In vitro haploid plants derived from polinisation by irradiated pollen on cucumber [A]. Proceedings of the Eucarpia Meting on Cucurbit Genetics and Breeding. Avinon-Monfavet, 1988, 143-144.
Truong A. In vitro haploid plants derived from pollination by irradiated pollen on cucumber [J].Avignon Montfavet, 1988,1:143-144.
Uchimiya H, Kameya T K, Takahashi N. In vitro culture of unfertilized ovule in Solanum melongena and ovaries in Zea mays[J]. Japan Journal of Breeding,1971,21:247-250.
Van Geyt J, Speckrnann J G J, Halluin K D, et al. In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgans L.) [J].Theoretical and Applied Genetics, 1987, 73(6): 920-925.
Vladimir B, Michel H, Nathalie Q, et al. Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry [J]. Sexual Plant Reproduction,2003,15(6):311-320.
Walter T. A haploid tissue culture from the female gametophyte of Ginkgo biloba L[J]. Nature, 1964, 203(4940):94-95.
Yan H, Dong J, Zhou C, et al. Regulation of in vitro parthenogenesis and somatic proliferation in sunflower by several factors[J]. Journal of Integrative Plant Biology,1987,29(6):580-597.
Zhang X P, Rhodes B B, et al. Determination of watermelon ploidy level using flow cytometre[J]. Plant Cell Rep, 1993,12:61-65.
Zlesak D C, Thill C A, Anderson N O. Trifluralin-mediated polyploidization of Rosa chinensis minima (Sims) Voss seedlings [J].Euphytica,2005,141(3):281-290.
敖光明,赵世绪,李广华.从未受精的玉米子房诱导出单倍体植株[J].遗传学报,1982,(4):281-283.
蔡得田,陈冬玲,祝红,等.从“湖北光敏感核不育水稻”的未受精子房和花药培养出单倍体植株[J].实验生物学报,1988,21(4):401-407.
蔡得田,周嫦.由向日葵幼花或胚珠培养出单倍体小植株与胚状体[J].科学通报,1983,28(22):1399-1401.
蔡得田,祝虹,姚家琳.多胚水稻未受精子房和花药培养[J].华中农业大学学报,1991,10(3):228-232.
陈清华,彭庆务,黄涛,等.西瓜过氧化物酶、酯酶同工酶酶谱及其利用研究[J].广东农业科学,1998,(2):22-24.
陈瑞菊,王亦菲,孙月芳,等.玉米花粉高频再生培养技术程序研究[J].农业生物技术学报,2005,21(2):38-40.
陈小鹏,刘栓桃,孙小镭,等.黄瓜未授粉子房的胚状体诱导研究初报[J].西北农业学报,2005,14(2):148-151.
陈晓静,卢秉国,申艳红.番木瓜的大孢子发生与胚囊发育[J].福建农林大学学报(自然科学版),2005,34(4):446-449.
陈学军,邢国民,陈竹君.西葫芦未授粉胚珠离体培养和植株再生[J].浙江农业学报,2000,12(3):165-167.
陈祖铿.植物的生殖讲座(八)—植物生殖生物学研究进展[J].生物学通报,1996,31(10):29-31.
程井辰,王家训.油菜子房组织培养研究初报[J].华中师范大学学报(自然科学版),1980,(3):50-52.
刁卫平,陈劲枫,雷春,等.影响黄瓜未授粉子房培养胚发生因素的研究[J].南京农业大学学报,2008,3l(1):137-140.
杜立颖,冯仁青.流式细胞术[M].北京:北京大学出版社,2008.
杜胜利,韩毅科,丛颖,等.黄瓜离体雌核发育的过程及早期生化变化研究[J].南开大学学报,2003,36(2):27-30.
杜胜利,魏爱民,魏惠军,等.利用生物技术创造黄瓜育种新材料方法研究[J].天津科技,2001,(2):627.
杜胜利.利用生物技术创造黄瓜育种新材料方法研究[J].天津科技,2001,(2):627.
付金娥,覃斯华,李天艳,等.秋水仙素诱变薄皮甜瓜同源四倍体研究[J].中国瓜菜,2008,(2)11-15.
傅润民.果树瓜类生物工程育种[M].北京:农业出版社,1993, 89-104.
高盛香.遗传机理在单倍体育种上的应用[J].内蒙古师范大学学报(教育科学版),1997,(4):66-67.
葛志东,李海真,王惠林,等.氟乐灵诱导西葫芦染色体加倍研究[J].中国农学通报,2009,25(6):215-218.
葛志东.西葫芦未授粉子房离体培养研究[D].乌鲁木齐:新疆农业大学,2009.
谷祝平,郑国锠.百合未授粉子房的培养及其胚胎学观察[J].植物学报,1983,25(1):24-27.
官春云.油菜小孢子培养和双倍体育种研究[J].作物学报,1995,21(6):665-670.
郭永强,王建设.西葫芦胚囊再生植株倍性鉴定方法研究[J ].华北农学报,2004,19(3):80-83.
郭永强.西葫芦离体雌核发育途径诱导单倍体的研究[D].北京:首都师范大学,2004:21-22.
韩丽华,王建设,陈贵林.AgNO3对甜瓜离体胚囊发育的影响[J].河北农业大学学报,2005,28(2):48-50.
韩丽华.厚皮甜瓜未受精胚珠离体培养技术[D].保定:河北农业大学,2004.
韩毅科,杜胜利,王鸣,等.瓜类遗传育种中的染色体倍性操作[J].天津农业科学,2002,8(3): 30-34.
韩毅科,杜胜利,王鸣.黄瓜染色体制片及倍性研究[J].华北农学报,2003,18(1):72-74.
韩毅科,杜胜利,魏爱民,等.黄瓜离体雌核发育早期的特异蛋白研究[J].南开大学学报(自然科学版),2006,39(6):1-5.
韩毅科,杜胜利,张桂华,等.利用抗微管除草剂胺磺灵诱导黄瓜四倍体[J].华北农学报,2006,21(4):27-30.
何才平,杨弘远.水稻子房培养中助细胞无配子生殖的稳定性及其发育条件的探讨[J].武汉植物学研究,1988,6:203-206.
胡适宜.被子植物胚胎学[M].北京:人民教育出版社,1982.
黄国中,谷明光.玉米雌穗离体培养诱导孤雌生殖结实[J].遗传学报,1995,2(3):230-238.
黄群飞,杨弘远,周嫦.大麦未授粉子房培养的胚胎学观察[J].植物学报,1982,24(4):295-300.
姜华武,王琳.植物生殖生物学研究进展[J].湖北农学院学报,1998,18(2):178-184.
孔振兰,廖爱君,崔德珍,等.金柑胚珠与胚囊发育的观察[J].湖南农学院学报,1993,19 (6):570-573.
雷春,陈劲枫,张晓青,等.不同倍性黄瓜的形态和一些生理生化指标比较[J].植物生理学通讯,2005,41(4):471-474.
李光威,兰素缺,孙宝启.小麦游离小孢子培养的研究[J].华北农学报,2001,16(1):83-87.
李建欣,庞淑敏,方贯娜.瓜类作物单倍体育种研究进展[J].中国瓜菜,2007,(6):36-39.
李浚明,朱登云.植物组织培养教程[M].北京:中国农业大学出版社,2005:101.
李栒，官春云．油菜的花粉培养和双单倍体育种[J].作物研究,1994,8(3):1-3.
李友勇,王兴才,陈海燕.黑籽南瓜细胞学初步观察[J].河南职技师院学报,1991,19(1):40-43.
李赟,束怀瑞.流式细胞光度术用于草莓倍性鉴定的研究[J].西北农业大学学报,1998,26(4):45-48.
李正理,胡绍安.棉花胚胎学基础知识(二) [J].中国棉花,1978,4:32-34.
刘成洪,王亦菲.用气孔保卫细胞周长鉴定甘蓝型油菜植株倍性水平[J].上海农业学报,2002,18(3):35-38.
刘久东,徐涛,和兆荣,等.海桐大、小孢子发育及雌、雄配子体形成的研究[J].广西植物,2008,28(1):20-23.
刘穆.种子植物形态解剖学导论[M].北京:科学出版社,2001.
刘仁祥,黄莺.烟草花粉植株染色体倍性的早期快速鉴定[J].贵州农业科学,1998,26(6):4-7.
刘栓桃,赵智中,孙小镭,等.西葫芦未受精胚珠离体诱导植株再生的关键因素[J].华北农学报,2008,23(2):96-100.
刘用生.胚珠培养技术在园艺植物上的应用[J].河南职业技术师范学院学报,1990,18(2):32-36.
卢良恕.中国大麦学[M].北京:中国农业出版社,1996,255-259.
罗伯祥,沈俊国,赵苏海,等.南瓜属作物育种研究进展[J].种子,2006,25 (1):43-46.
马德伟,高锁柱,韩素江,等.甜瓜过氧化物酶同工酶研究初报[J].河北农业大学学报,1986,6(2):9-13.
马国斌,王鸣,郑学勤.甜瓜组织培养再生植株中的四倍体变异[J].园艺学报,1999,26(2):128-130.
潘莉,刘宗才,喜进安.外源激素对烟草未受粉子房培养的作用[J].河南农业大学,1998,36(2):167-175.
潘莉,杨铁钊.烟草未授粉子房胚状体诱导的研究[J].西北植物学报,2000,20(1):59-63.
潘莉,郑奇君.烟草未授粉子房单倍体诱导及影响因素的研究[J].河南农业大学学报,1999,33(l):48-52.
邱德勃,谢石文,孔维明.巴西橡胶树大孢子发生和胚囊发育过程的研究[J].热带作物学报,1991,12(1):1-9.
任宏,刘永胜,孙敬三.用共聚焦扫描显微镜检术观察水稻胚囊发育[J].植物学报,1998,40(9):786-789. 上海植物生理研究所细胞分化组.油菜花器培养中器官分化能力的研究[J].植物学报,1977,19(4):309-310.
上海植物生理研究所细胞生理室.棉花的组织培养[J].棉花学报,1977,(4):41-43. 申家恒,李伟,杨虹,等.辣椒大、小孢子发生与雌、雄配子体发育的研究[J].园艺学报,2007,34(6):1443-1452.
苏华,金宝燕,任华中.黄瓜单倍体育种的研究进展[J].农业工程学报,2005,21(增):13-15.
孙守如,翟庆慧,胡建斌,等.几种生理因素对子房未授粉南瓜胚形成的影响[J].植物生理学通讯,2009,45(10):977-980.
孙守如,翟庆慧,杨子琴,等.葫芦科作物离体雌核发育的研究进展[J].河南科学,2008,26(9):1046-1049.
唐宗祥,张怀琼,张怀渝,等.2,4-D、KT对小麦成熟胚愈伤组织形成、分化的影响[J].四川农业大学学报,2004,22(3):203-205.
陶自荣,刘敏颂,祝仲纯.马铃薯未传粉子房离体培养诱导双单倍体植株[J].遗传学报,1988,15:329-334.
田惠桥,杨弘远.韭菜未传粉子房培养中单倍体的胚胎发生和植株再生[J].实验生物学报,1989,22(2):139-141.
王敬驹,匡柏健.从大麦子房培养出单倍体植株[J].植物学报,1981,23(4):329-330.
王丽,赵桂仿.华山新麦草大孢子发生和雌配子体的形成[J].西北植物学报,2003,23(5):802-805.
王丽花,瞿素萍,杨秀梅,等.非洲菊未授粉胚珠的离体诱导和植株再生[J].植物生理学通讯,2007,43(6):1089-1092.
王丽莉,秦智伟.葫芦科植物单倍体离体诱导研究进展[J].中国农学通报,2007,23(9):85-89.
王萍,赵清岩.南瓜的营养成分药用价值及开发利用[J].长江蔬菜,1998,(7):1-3.
王日勇,肖旭峰.蔬菜作物游离小孢子培养研究进展[J].湖南农业科学,2003,(5):16-18.
王文和.草莓离体雌核发育的研究[D].沈阳:沈阳农业大学,2001.
王晓云,杜胜利,曹翠玲,等.黄瓜雌核发育早期过氧化物同工酶活性的变化[J].华化农学报,2004,19(4):55-58.
韦鹏霄,岑秀芬,吴丹红.水稻野栽杂交受粉子房离体培养研究[J].广西农业大学学报,1996, 15(4):291-298.
魏爱民,韩毅科,杜胜利,等.供体植株栽培季节和栽培方式对黄瓜未受精子房离体培养的影响[J].西北农业学报,2007,16(5):141- 144.
魏育国,蒋菊芳.氟乐灵诱导甜瓜四倍体研究初探[J].华北农学报,2006,21(增):73-76.
吴伯骥,郑国錩.从未授粉烟草子房诱导单倍体植株的细胞学和胚胎学研究[J].植物学报,1982,24(2):125-129.
吴克贤,徐妙珍.杨树未授粉子房离体培养诱导母系单倍体植株[J].遗传学报,1984,(1):47-51.
伍成厚,梁承邺,叶秀麟.被子植物未受精胚珠与子房离体培养的研究进展[J].热带亚热带植物学报,2004,12(6):580-586.
谢冰,王秀峰,樊治成.西葫芦未受精胚珠离体培养条件的优化及胚囊植株的再生[J].中国农业科学,2006,39(1):132-138.
谢冰.西葫芦的离体雌核发育及植株再生[D].泰安:山东农业大学,2005.
徐静.西葫芦雌核离体高效培养体系的建立[D].乌鲁木齐:新疆农业大学,2007.
许智宏.植物发育与生殖的研究、进展和展望[J].植物学报,1999,41(9):909-920.
阎华,吴燕,陈小民,等.向日葵未受精胚珠培养时胚状体发生的显微观察[J].植物学报,1985,27(1):13-18.
阎华,杨弘远.向日葵离体孤雌生殖的超微结构研究[J].植物学报,1989,31(1):1-6.
阎华,周嫦,杨弘远.向日葵为受精胚珠培养中各种影响因素的试验研究[J].武汉植物学研究,1988,6(4):319-326.
阎志红,刘文革,赵胜杰,等.利用二硝基苯胺类除草剂离体诱导西瓜四倍体[J].园艺学报,2008,35(11):1621-1626.
颜昌敬,赵庆华.水稻叶鞘和枝梗愈伤组织的植株再生[J].科学通报,1979,(20):943-947.
杨弘远,周嫦.被子植物离体受精与合子培养研究进展[J].植物学报,1998,40(2):95-101.
杨江义,李旭峰.植物雌性单倍体的离体诱导[J].植物学通报,2002,19(5):552-559.
杨学荣,叶银根,王建人,等.水仙外植体培养中的内、外源调节对细胞分化和形态发生的影响[J].华中师范大学学报(自然科学版),1981,(4):80-82.
杨艳琼,何丽萍.用气孔保卫细胞叶绿体计数法鉴定烟草染色体倍性方法初探[J].种子,2002,122(3):24-25,56.
原玉香,张晓伟,耿健峰,等.利用游离花粉培养技术育成早熟大白菜新品种‘豫新60’[J].园艺学报,2004,31(5):704-708.
翟庆慧.南瓜未受精子房和未受精胚珠离体培养研究[D].郑州:河南农业大学,2009.
张宝红,刘方,刘志红,等.外源激素对棉花体细胞胚胎发生及发育的调控作用[J].棉花学报,2000,12(1):17-21.
张福泉,郑思乡,刘逊,等.遗传工程水稻未传粉子房的培养[J].湖南农业大学学报,1999,25(4):275-278.
张伶俐,崔崇士,屈淑平.植物离体雌核发育的研究进展[J].东北农业大学学报,2009,40(11):133-136.
张雪梅.甘蓝型油菜未授粉子房离体培养的比较研究[D].雅安:四川大学,1987.
赵隽.黄瓜(Cucumis sativus L.)单倍体培养再生体系的建立[D].上海:上海交通大学,2004.
赵璘,刘文革,郭金丽,等.除草剂在植物离体染色体加倍上的应用[J].长江蔬菜,2008,(1):30-33.
郑泗军,蒋淑丽,洪彩霞,等.碳源和基因型对棉花未受精胚珠纤维离体发育的影响[J].棉花学报,1996,8(6):301-304.
周嫦,杨弘远,田惠桥,等.水稻的未传粉子房培养[J].武汉大学学报(自然科学版),1983,(4):146-154.
周嫦,杨弘远.从水稻未授粉的幼嫩子房培养出单倍体小植株[J].遗传学报,1980,7(3):287-288.
周嫦,杨弘远.水稻离体条件下未受精胚囊的胚胎发生[J] .植物学报,1981,23(3):176-180.
周嫦,杨弘远.水稻未受精胚囊的离体胚胎发生[J] .植物学报,1981b,(23):176-179.
周嫦,杨弘远.未传粉子房与胚珠的离体培养[J].武汉大学学报(自然科学版),1982,(3):61-72.
周嫦,杨弘远.诱导水稻胚囊产生愈伤组织的离体实验[J].遗传,1981,(5):10-12.
周毓君.祝光苹果大孢子发生和雌配子体的发育[J].河北大学学报(自然科学版),1992,(3):62-66.
周元昌,Kennedy S.抱子甘蓝花培苗倍性的快速鉴定[J].福建农林大学学报(自然科学版),2002,31(1):55-58.
朱惠琴,戴文新.烟草加倍单倍体群体的构建及其在遗传育种中的应用[J].青海大学学报(自然科学版),2003,21(6):20-23.
朱睦元,黄纯农,徐阿炳,等.大麦耐酸铝愈伤组织变异体的筛选及其特性研究[J].植物学报,1990,32(10):743-748.
朱献辉,巩振辉,王乾宏,等.辣椒未受精子房愈伤组织诱导的研究[J].西北农业学报,2008,17(5):310-316.
祝仲纯,刘振岳,吴海珊,等.离体培养未传粉烟草子房的胚状体发育[J].植物学报(英文版),1981,23(6):499-501.
祝仲纯,吴海珊,安庆坤.大叶烟草未传粉子房培养及其细胞学研究[J].遗传学报,1984,11 (4):281-287.
祝仲纯,吴海珊.从未授粉的小麦及烟草子房培养出单倍体植株[J].遗传学报,1979,6(2):181-183.
Bino R J. Flow cytometric deter mination of nuclear replication stages in tomato seeds during priming and germination[J].Annals of Botany,1992,69:231-236.
Caglar G, Abak K, Abak K, et al. Progress in the production of haploid embryos, plants and doubled hap loids in cucumber (C. sativus L. ) by gamma irradiated pollen, in Turkey [J]. Acta Horticulturae, 1999,492:31-322.
Caglar G, Abak K. Efficiency of haploid production in cucumber [J].Report Cucurbit Genetics Cooperative, 1996,19:36-37.
Caglar G, Abak K. In situ haploid embryo induction in cucumber (Cucumis sativus L.) after pollination by irradiated pollen [J ]. Turkish J of Agri and Fore,1999, 23 (1):63-72.
Caglar G, Abak K. Production of in vitro haploid plants from in situ induced haploid embryos in cucumber (Cucumis sativus L.) [J].Turk ish Journal of A griculture and Forestry, 1999, 23(3): 283-290.
Campion B,Alloni C.Induction of haploid plants in onion (Alliun cepa L.)by in vitro culture of unpollinated ovules[J].Plant Cell,Tissue and Organ Culture,1990,20(1):1-6.
Campion E,Perr M,Aszimonti T,et al. Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)[J]. Plant Breeding,1995,114(3):243-246.
Cao M Q, Li Y, Liu F.Influence of genotype and donor plant growth environment on embryogenesis from isolated microspores in Chinese cabbage (Brassica campestris ssp.Pekinensis) [J]. Acta Agricult. Boreali-Sin,1993,8(4):1-6.
Castillo A M, CistuéL. Production of gynogenic haploids of Hordeum vulgate L [J]. Plant Cell Reports,1993,12(3):139-143.
Chambonnet D, Dumas de Vaulx R. Obtention of embryos and plants from in vitro culture of unfertilizered ovules of Cucurbita pepo L[J]. Cucurbit Genet Coop Rpt,1985,(8):66.
Chaudhury A M, Ming L, Miller C, et al. Fertilization-independent seed development in Arabidopsis thaliana[J]. Proc Natl Acad Sci USA,1997,94(8):4223-4228
Choi M Y, Jeong H W, Lee S S, et al. Improvement of chloroplast observation technique in guard cells for ploidy detection of microspore derived plants in broccoli[J]. Journal of the Korean Society for Horticultural Science,1997,38(6):666-669.
Cohat J.Obtention chez l’ehalote (Allium cepa L.var.agregatum) de plantes haploides gynogenetiques par culture invitro de boutons fioraux[J].Agronomie,1994,14:299-304.
Cooper D C. Macrosporogenesis and the development of the macrogametophyte of LycopersiconExculentum[J]. American Journal of Botany,1931,18:739-748.
Deverna J W, Collins G B. Maternal haploids of Petunia axiollanis (Lam) B.S.P via culture of placenta attached ovules[J]. Theoretical and Applied Genetics,1984,69(2):187-192.
Dore C, Boulidard L, Sauton A, et al. Interest of irradiated pollen for obtaining haploid vegetables [J].Acta Horticulturae,1995,392:123-128.
Drews G N, Lee D, Christensen C A. Genetic analysis of female gametophyte development and function[J]. Plant Cell,1998,10(1):5-17.
Dumas D, Pochard V R E, Chambonnet D. Distribution of TMV-po susceptible and resistant doubled haploid lines from anther culture of heterozygous L/L’hybrids[J]. Capsicum Newsl,1982,1:52-53.
Estrada-Luna A A, García-Aguilar M, Vielle-Calzada J-Ph.Female reproductive development and pollen tube growth in diploid genotypes of Solanum cardiophyllum Lindl [J].Sexual Plant Reproduction,2002,17(3):117-124.
Faris N M, Nikolova V, Niem irowicz Szczytt K.The effect of gamma irradiation dose on cucumber (Cucumis sativus L.) haploid embryo production. Acta physiofogiae Plantarum,1999,21(4): 391-396.
Faris N M, Nikolova V, Niemirowicz S K. The effect of gamma irradiation dose on cucumber (Cucumis sativus L) haploid embryo production [J].Acta Physiofogiae Plantarum, 1999,21(4):391-396.
Fernando D, Javier M, Francisco J C. The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development [J].Planta,2001,213(3):352-360.
Ficcadenti N, Sestili S, Annibali S, et al. In vitro gynogenesis to induce haploid plants in melon (Cucumis melo L) [J]. Journal of Genetic & Breeding,1999,53:255-257.
Gemes J A, Venczel G P, Altman A, et al. Haploid plant induct ion in Zucchini (Cucurbita pepo L. ) [J].Euphytica,1997,97:249-254.
GémesnéJ A,Balogh P, Ferenczy A, et al. Effect of optimum stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber (Cucum issativus L.) [J].Plant Cell Reports,2002,21(2) :105-111.
Greenberg J T. Programmed cell death: A way of life for plants[J]. Proc Natl Acad,1996,93: 12094-12097.
Gu Z P, Zheng G C.In vitro induction of haploid plants from unpollinated ovaries of highland barley [J]. Acta Bot Sin, 1984, 26(5):549-551.
Guha S, Johri B M. In vitro development of ovary and ovule of Allium cepa L.[J]. Phyomorphology, 1966, 16:353-364.
Gurel S,Curel E,Kaya Z. Doubled haploid plant production from unpollinated ovule of sugar beet (Beta vulgaris L.) [J].Plant Cell Reports,2000,19(12):1155-1159.
Hansen N J P, Andersen S B. In vitro chromosome doubling potential of colchicine, oryzalin, trifluralin, and APM in Brassica napus microspore culture [J].Euphytica,1996,88(2):159-164.
Hoseman D, Bossoutrot D. Induction of haploid plants from in vitro culture of unpollinated beet (Beta vulgaris L.) [J]. Z. Pflanzenzucht,1983,91:74-77.
Hu S Y.Development of plant embryology in China [J].Acta Botanica Sinica,2002,44(9): 1022-1042. Huang B Q, Sheridan W F. Female gametophyte development in maize: microtubule organization and embryo sac polarity [J].Plant Cell,1994,6(6):845-861.
Jakge M, Bohanec B, Ihan A. Effect of media components on the gynogenic regeneration of onion (Alium cepa L.) cultivars and analysis of regenerants [J]. Plant Cell Reports,1996,15(12):934-938.
Kaya Z. Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.) [J]. Plant Cell Reports,2000,19(12):1155-1159.
Keller J. Culture of unpollinated ovules, ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Aliium cepa L) [J].Euphytica,1990,47:241-247.
Kojima A, Kawaguchi T. Dihaploids derived from unpollinated ovule culture of Chinese chives[C]. Proc 4th Album Symp Wellesbourne,1988:212-217.
Koltunow A M. Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules [J]. Plant Cell, 1993,5(10):1425-1437.
Kurtar E S, Sail N, Abak K. Obtention of haploid embryo s and plants through irradiated pollen technique in sqush (Cucurbita pepo L.) [J]. Euphytica, 2002,127(3):335-344.
Kwack S N, Fujieda K. Somatic embryogenesis in cultured unfertilized ovules of Cucurbita moschata [J]. Journal of the Japanese Society for Horticultural Science,1988,57:34-42.
Lacadena J R. Spontaneous and induced parthenogenesis and regenesis [A]. K Kasha. Haploids in Higher Plants. Advances and Potential[M].University of Guel Ph,1974:13-32.
Li R B, Pandey M P.Genetic studies on vitro unpollinated ovary culture in rice [J]. Oryza, 1999, 36(1):28-31.
Lotfi M, Alan A R, Henning M J,et al.Production of haploid and doubled haploid plants of melon (Cucumis melo L.) for use in breeding fo rmultiple virus resistanee [J]. Plant Cell Reports,2003,21(11):1121-1128.
Lotfi M, Kashi A, Onsinejad R, et al. Induction of parthenogenetic embryos by irradiated pollen in cucumber[J]. Acta Horticulturae,1999,492:323-328.
Lux H, Herrmann L,Wetzel C L. Production of haploid sugar beet (Beta vulgaris L.) by culturing unpollinated ovules [J].Plant Breeding,1990,104(3):177-183.
Maheshwari N. In vitro culture of excised ovules of Papaver somniferum L[J]. Phytomorphology, 1961, 11:307-314.
Metwally E I, Moustafa S A, El-Sawy B I, et al. Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo[J].Plant Cell,Tissue and Organ Culture,1998,52(3):117-121.
Morejohn L C, Bureau T E, Mole-Bajer T, et al. Oryzalin, a dinitroaniline herbicide, binds to planttubulin and inhibits microtubule polymerization in vitro [J].Planta,1987,172 (2): 252-264. Mukhambetzhanov S K. Culture of nonfertilized female gametophytes in vitro[J]. Plant Cell,Tissue and Organ Culture,1997,48(2):111-119.
Muren R C. Haploid plant induction from unpollinated ovaries in onion [J]. Hort Science,1989, 24(5): 833-834.
Musial K, Bohanec B, Przywara L. Embryological study on gynogenesis in onion (AIlium cepa L)[J].Sexual Plant Reproduction,2001,13(6):335-341.
Niemirowicz S K, Vaulx R D, Dumas V R. Preliminary data on haploid cucumber (Cucumis sa -tivus L.) induction[J]. Report Cucurbit Genetics Cooperative,1989,12:24-25.
Nikolova V, Rodeva V, Alexandrova M, et al. Effect of gamma irradiation for induction of gynogenesis in cucumber (Cucum is sativus L. ) [J]. Bulgarian J of Agri Sci,2001,7:415-420.
Nishi T, Mitsuoka S.Occurrence of various ploidy plants from anther and ovary culture of rice plant [J].Jap J Genet,1969,44(4):341-346.
Norio K, Surmio I W A I. Production of haploid plant of melon by pseudofertilized ovule culture [J]. Plant Tissue Culture,1993,10(10):60.
Przyborowski J, Niemirowicz S K. Main factors affecting cucumber (Cucumis sativus L. ) haploid embryo development and haploid plant characteristics [J ]. Plant Breeding,1994,112:70-75.
Roger C, Muren.Haploid plant induction from unpollinated ovaries in onion [J]. Hortscience, 1989, 24(5): 833-834.
Russell S D. The egg cell: development and role in fertilization and early embryogenesis [J]. Plant Cell, 1993, 5(10):1349-1359.
Sachar R C, Kapoor M. In vitro culture of ovules of Zaphyranthes. [J]. Phytomorphology,l959,9:147-15. Sachar R C, Kapoor M. Influence of kinetin and gibberellic acid on the test-tube seeds of cooperia pedunculata [J]. Herb Naturwissenschaften,1985,45 (5):52-53.
Sair N, Abak K. Comparison of ploidy level screening methods in watermelon: Citrulius Lanatus (Thunb) [J]. Scientia Horticulturae,1999,82:265-277.
San Noeum L H. In vitro induction of gynogenssis in higher plants[J].Proceedings of the Conference on Broadening Genetic,1978:327-329.
Sari N,Abak K,Pitrat M,et al.Induction of parthenogenetic haploid embryos after pollination by ir radited pollen in watermelon [J]. Hort Scienee,1994,29(10):1189-1190.San Noeum L H. Haploids d’
Hodemu vulgare L. paculture in vitro ovaries[J].Ann Aemlior Plant, 1976,24:751-754. Sauton A.Haploidisation chez les Cucurbitaceae.In cinquantenaire dela culture in vitro.Versailles (France) 24-25 October INRA,Paris,1990:325-326.
Savin F,Decomble V, Le Couviour M, et al. The X-ray detection of haploid embryos arisen in muskmelon (Cucumis melo L.) seeds,and resulting from a parthenogenetic development induced by irradiatedpollen[J].Cucurbit Genet Coop Rpt, 1988,11:39-42.
Shuizhang F E I , Paul E R, Terrance P R. Improvement of embryogenic callus induction and shoot regeneration buffalograss by silver nitrate[J].Plant Cell,Tissue and Organ Culture, 2000,60(3):197-203.
Sitbon M. Production of haploid Gerbera jamesonii plants by in vitro culture unfertilized ovules[J]. Agronomie, 1981,1:807-812.
Treng-Andre I. In vitro haploid plants derived from polinisation by irradiated pollen on cucumber [A]. Proceedings of the Eucarpia Meting on Cucurbit Genetics and Breeding. Avinon-Monfavet, 1988, 143-144.
Truong A. In vitro haploid plants derived from pollination by irradiated pollen on cucumber [J].Avignon Montfavet, 1988,1:143-144.
Uchimiya H, Kameya T K, Takahashi N. In vitro culture of unfertilized ovule in Solanum melongena and ovaries in Zea mays[J]. Japan Journal of Breeding,1971,21:247-250.
Van Geyt J, Speckrnann J G J, Halluin K D, et al. In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgans L.) [J].Theoretical and Applied Genetics, 1987, 73(6): 920-925.
Vladimir B, Michel H, Nathalie Q, et al. Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry [J]. Sexual Plant Reproduction,2003,15(6):311-320.
Walter T. A haploid tissue culture from the female gametophyte of Ginkgo biloba L[J]. Nature, 1964, 203(4940):94-95.
Yan H, Dong J, Zhou C, et al. Regulation of in vitro parthenogenesis and somatic proliferation in sunflower by several factors[J]. Journal of Integrative Plant Biology,1987,29(6):580-597.
Zhang X P, Rhodes B B, et al. Determination of watermelon ploidy level using flow cytometre[J]. Plant Cell Rep, 1993,12:61-65.
Zlesak D C, Thill C A, Anderson N O. Trifluralin-mediated polyploidization of Rosa chinensis minima (Sims) Voss seedlings [J].Euphytica,2005,141(3):281-290.
敖光明,赵世绪,李广华.从未受精的玉米子房诱导出单倍体植株[J].遗传学报,1982,(4):281-283.
蔡得田,陈冬玲,祝红,等.从“湖北光敏感核不育水稻”的未受精子房和花药培养出单倍体植株[J].实验生物学报,1988,21(4):401-407.
蔡得田,周嫦.由向日葵幼花或胚珠培养出单倍体小植株与胚状体[J].科学通报,1983,28(22):1399-1401.
蔡得田,祝虹,姚家琳.多胚水稻未受精子房和花药培养[J].华中农业大学学报,1991,10(3):228-232.
陈清华,彭庆务,黄涛,等.西瓜过氧化物酶、酯酶同工酶酶谱及其利用研究[J].广东农业科学,1998,(2):22-24.
陈瑞菊,王亦菲,孙月芳,等.玉米花粉高频再生培养技术程序研究[J].农业生物技术学报,2005,21(2):38-40.
陈小鹏,刘栓桃,孙小镭,等.黄瓜未授粉子房的胚状体诱导研究初报[J].西北农业学报,2005,14(2):148-151.
陈晓静,卢秉国,申艳红.番木瓜的大孢子发生与胚囊发育[J].福建农林大学学报(自然科学版),2005,34(4):446-449.
陈学军,邢国民,陈竹君.西葫芦未授粉胚珠离体培养和植株再生[J].浙江农业学报,2000,12(3):165-167.
陈祖铿.植物的生殖讲座(八)—植物生殖生物学研究进展[J].生物学通报,1996,31(10):29-31.
程井辰,王家训.油菜子房组织培养研究初报[J].华中师范大学学报(自然科学版),1980,(3):50-52.
刁卫平,陈劲枫,雷春,等.影响黄瓜未授粉子房培养胚发生因素的研究[J].南京农业大学学报,2008,3l(1):137-140.
杜立颖,冯仁青.流式细胞术[M].北京:北京大学出版社,2008.
杜胜利,韩毅科,丛颖,等.黄瓜离体雌核发育的过程及早期生化变化研究[J].南开大学学报,2003,36(2):27-30.
杜胜利,魏爱民,魏惠军,等.利用生物技术创造黄瓜育种新材料方法研究[J].天津科技,2001,(2):627.
杜胜利.利用生物技术创造黄瓜育种新材料方法研究[J].天津科技,2001,(2):627.
付金娥,覃斯华,李天艳,等.秋水仙素诱变薄皮甜瓜同源四倍体研究[J].中国瓜菜,2008,(2)11-15.
傅润民.果树瓜类生物工程育种[M].北京:农业出版社,1993, 89-104.
高盛香.遗传机理在单倍体育种上的应用[J].内蒙古师范大学学报(教育科学版),1997,(4):66-67.
葛志东,李海真,王惠林,等.氟乐灵诱导西葫芦染色体加倍研究[J].中国农学通报,2009,25(6):215-218.
葛志东.西葫芦未授粉子房离体培养研究[D].乌鲁木齐:新疆农业大学,2009.
谷祝平,郑国锠.百合未授粉子房的培养及其胚胎学观察[J].植物学报,1983,25(1):24-27.
官春云.油菜小孢子培养和双倍体育种研究[J].作物学报,1995,21(6):665-670.
郭永强,王建设.西葫芦胚囊再生植株倍性鉴定方法研究[J ].华北农学报,2004,19(3):80-83.
郭永强.西葫芦离体雌核发育途径诱导单倍体的研究[D].北京:首都师范大学,2004:21-22.
韩丽华,王建设,陈贵林.AgNO3对甜瓜离体胚囊发育的影响[J].河北农业大学学报,2005,28(2):48-50.
韩丽华.厚皮甜瓜未受精胚珠离体培养技术[D].保定:河北农业大学,2004.
韩毅科,杜胜利,王鸣,等.瓜类遗传育种中的染色体倍性操作[J].天津农业科学,2002,8(3): 30-34.
韩毅科,杜胜利,王鸣.黄瓜染色体制片及倍性研究[J].华北农学报,2003,18(1):72-74.
韩毅科,杜胜利,魏爱民,等.黄瓜离体雌核发育早期的特异蛋白研究[J].南开大学学报(自然科学版),2006,39(6):1-5.
韩毅科,杜胜利,张桂华,等.利用抗微管除草剂胺磺灵诱导黄瓜四倍体[J].华北农学报,2006,21(4):27-30.
何才平,杨弘远.水稻子房培养中助细胞无配子生殖的稳定性及其发育条件的探讨[J].武汉植物学研究,1988,6:203-206.
胡适宜.被子植物胚胎学[M].北京:人民教育出版社,1982.
黄国中,谷明光.玉米雌穗离体培养诱导孤雌生殖结实[J].遗传学报,1995,2(3):230-238.
黄群飞,杨弘远,周嫦.大麦未授粉子房培养的胚胎学观察[J].植物学报,1982,24(4):295-300.
姜华武,王琳.植物生殖生物学研究进展[J].湖北农学院学报,1998,18(2):178-184.
孔振兰,廖爱君,崔德珍,等.金柑胚珠与胚囊发育的观察[J].湖南农学院学报,1993,19 (6):570-573.
雷春,陈劲枫,张晓青,等.不同倍性黄瓜的形态和一些生理生化指标比较[J].植物生理学通讯,2005,41(4):471-474.
李光威,兰素缺,孙宝启.小麦游离小孢子培养的研究[J].华北农学报,2001,16(1):83-87.
李建欣,庞淑敏,方贯娜.瓜类作物单倍体育种研究进展[J].中国瓜菜,2007,(6):36-39.
李浚明,朱登云.植物组织培养教程[M].北京:中国农业大学出版社,2005:101.
李栒，官春云．油菜的花粉培养和双单倍体育种[J].作物研究,1994,8(3):1-3.
李友勇,王兴才,陈海燕.黑籽南瓜细胞学初步观察[J].河南职技师院学报,1991,19(1):40-43.
李赟,束怀瑞.流式细胞光度术用于草莓倍性鉴定的研究[J].西北农业大学学报,1998,26(4):45-48.
李正理,胡绍安.棉花胚胎学基础知识(二) [J].中国棉花,1978,4:32-34.
刘成洪,王亦菲.用气孔保卫细胞周长鉴定甘蓝型油菜植株倍性水平[J].上海农业学报,2002,18(3):35-38.
刘久东,徐涛,和兆荣,等.海桐大、小孢子发育及雌、雄配子体形成的研究[J].广西植物,2008,28(1):20-23.
刘穆.种子植物形态解剖学导论[M].北京:科学出版社,2001.
刘仁祥,黄莺.烟草花粉植株染色体倍性的早期快速鉴定[J].贵州农业科学,1998,26(6):4-7.
刘栓桃,赵智中,孙小镭,等.西葫芦未受精胚珠离体诱导植株再生的关键因素[J].华北农学报,2008,23(2):96-100.
刘用生.胚珠培养技术在园艺植物上的应用[J].河南职业技术师范学院学报,1990,18(2):32-36.
卢良恕.中国大麦学[M].北京:中国农业出版社,1996,255-259.
罗伯祥,沈俊国,赵苏海,等.南瓜属作物育种研究进展[J].种子,2006,25 (1):43-46.
马德伟,高锁柱,韩素江,等.甜瓜过氧化物酶同工酶研究初报[J].河北农业大学学报,1986,6(2):9-13.
马国斌,王鸣,郑学勤.甜瓜组织培养再生植株中的四倍体变异[J].园艺学报,1999,26(2):128-130.
潘莉,刘宗才,喜进安.外源激素对烟草未受粉子房培养的作用[J].河南农业大学,1998,36(2):167-175.
潘莉,杨铁钊.烟草未授粉子房胚状体诱导的研究[J].西北植物学报,2000,20(1):59-63.
潘莉,郑奇君.烟草未授粉子房单倍体诱导及影响因素的研究[J].河南农业大学学报,1999,33(l):48-52.
邱德勃,谢石文,孔维明.巴西橡胶树大孢子发生和胚囊发育过程的研究[J].热带作物学报,1991,12(1):1-9.
任宏,刘永胜,孙敬三.用共聚焦扫描显微镜检术观察水稻胚囊发育[J].植物学报,1998,40(9):786-789. 上海植物生理研究所细胞分化组.油菜花器培养中器官分化能力的研究[J].植物学报,1977,19(4):309-310.
上海植物生理研究所细胞生理室.棉花的组织培养[J].棉花学报,1977,(4):41-43. 申家恒,李伟,杨虹,等.辣椒大、小孢子发生与雌、雄配子体发育的研究[J].园艺学报,2007,34(6):1443-1452.
苏华,金宝燕,任华中.黄瓜单倍体育种的研究进展[J].农业工程学报,2005,21(增):13-15.
孙守如,翟庆慧,胡建斌,等.几种生理因素对子房未授粉南瓜胚形成的影响[J].植物生理学通讯,2009,45(10):977-980.
孙守如,翟庆慧,杨子琴,等.葫芦科作物离体雌核发育的研究进展[J].河南科学,2008,26(9):1046-1049.
唐宗祥,张怀琼,张怀渝,等.2,4-D、KT对小麦成熟胚愈伤组织形成、分化的影响[J].四川农业大学学报,2004,22(3):203-205.
陶自荣,刘敏颂,祝仲纯.马铃薯未传粉子房离体培养诱导双单倍体植株[J].遗传学报,1988,15:329-334.
田惠桥,杨弘远.韭菜未传粉子房培养中单倍体的胚胎发生和植株再生[J].实验生物学报,1989,22(2):139-141.
王敬驹,匡柏健.从大麦子房培养出单倍体植株[J].植物学报,1981,23(4):329-330.
王丽,赵桂仿.华山新麦草大孢子发生和雌配子体的形成[J].西北植物学报,2003,23(5):802-805.
王丽花,瞿素萍,杨秀梅,等.非洲菊未授粉胚珠的离体诱导和植株再生[J].植物生理学通讯,2007,43(6):1089-1092.
王丽莉,秦智伟.葫芦科植物单倍体离体诱导研究进展[J].中国农学通报,2007,23(9):85-89.
王萍,赵清岩.南瓜的营养成分药用价值及开发利用[J].长江蔬菜,1998,(7):1-3.
王日勇,肖旭峰.蔬菜作物游离小孢子培养研究进展[J].湖南农业科学,2003,(5):16-18.
王文和.草莓离体雌核发育的研究[D].沈阳:沈阳农业大学,2001.
王晓云,杜胜利,曹翠玲,等.黄瓜雌核发育早期过氧化物同工酶活性的变化[J].华化农学报,2004,19(4):55-58.
韦鹏霄,岑秀芬,吴丹红.水稻野栽杂交受粉子房离体培养研究[J].广西农业大学学报,1996, 15(4):291-298.
魏爱民,韩毅科,杜胜利,等.供体植株栽培季节和栽培方式对黄瓜未受精子房离体培养的影响[J].西北农业学报,2007,16(5):141- 144.
魏育国,蒋菊芳.氟乐灵诱导甜瓜四倍体研究初探[J].华北农学报,2006,21(增):73-76.
吴伯骥,郑国錩.从未授粉烟草子房诱导单倍体植株的细胞学和胚胎学研究[J].植物学报,1982,24(2):125-129.
吴克贤,徐妙珍.杨树未授粉子房离体培养诱导母系单倍体植株[J].遗传学报,1984,(1):47-51.
伍成厚,梁承邺,叶秀麟.被子植物未受精胚珠与子房离体培养的研究进展[J].热带亚热带植物学报,2004,12(6):580-586.
谢冰,王秀峰,樊治成.西葫芦未受精胚珠离体培养条件的优化及胚囊植株的再生[J].中国农业科学,2006,39(1):132-138.
谢冰.西葫芦的离体雌核发育及植株再生[D].泰安:山东农业大学,2005.
徐静.西葫芦雌核离体高效培养体系的建立[D].乌鲁木齐:新疆农业大学,2007.
许智宏.植物发育与生殖的研究、进展和展望[J].植物学报,1999,41(9):909-920.
阎华,吴燕,陈小民,等.向日葵未受精胚珠培养时胚状体发生的显微观察[J].植物学报,1985,27(1):13-18.
阎华,杨弘远.向日葵离体孤雌生殖的超微结构研究[J].植物学报,1989,31(1):1-6.
阎华,周嫦,杨弘远.向日葵为受精胚珠培养中各种影响因素的试验研究[J].武汉植物学研究,1988,6(4):319-326.
阎志红,刘文革,赵胜杰,等.利用二硝基苯胺类除草剂离体诱导西瓜四倍体[J].园艺学报,2008,35(11):1621-1626.
颜昌敬,赵庆华.水稻叶鞘和枝梗愈伤组织的植株再生[J].科学通报,1979,(20):943-947.
杨弘远,周嫦.被子植物离体受精与合子培养研究进展[J].植物学报,1998,40(2):95-101.
杨江义,李旭峰.植物雌性单倍体的离体诱导[J].植物学通报,2002,19(5):552-559.
杨学荣,叶银根,王建人,等.水仙外植体培养中的内、外源调节对细胞分化和形态发生的影响[J].华中师范大学学报(自然科学版),1981,(4):80-82.
杨艳琼,何丽萍.用气孔保卫细胞叶绿体计数法鉴定烟草染色体倍性方法初探[J].种子,2002,122(3):24-25,56.
原玉香,张晓伟,耿健峰,等.利用游离花粉培养技术育成早熟大白菜新品种‘豫新60’[J].园艺学报,2004,31(5):704-708.
翟庆慧.南瓜未受精子房和未受精胚珠离体培养研究[D].郑州:河南农业大学,2009.
张宝红,刘方,刘志红,等.外源激素对棉花体细胞胚胎发生及发育的调控作用[J].棉花学报,2000,12(1):17-21.
张福泉,郑思乡,刘逊,等.遗传工程水稻未传粉子房的培养[J].湖南农业大学学报,1999,25(4):275-278.
张伶俐,崔崇士,屈淑平.植物离体雌核发育的研究进展[J].东北农业大学学报,2009,40(11):133-136.
张雪梅.甘蓝型油菜未授粉子房离体培养的比较研究[D].雅安:四川大学,1987.
赵隽.黄瓜(Cucumis sativus L.)单倍体培养再生体系的建立[D].上海:上海交通大学,2004.
赵璘,刘文革,郭金丽,等.除草剂在植物离体染色体加倍上的应用[J].长江蔬菜,2008,(1):30-33.
郑泗军,蒋淑丽,洪彩霞,等.碳源和基因型对棉花未受精胚珠纤维离体发育的影响[J].棉花学报,1996,8(6):301-304.
周嫦,杨弘远,田惠桥,等.水稻的未传粉子房培养[J].武汉大学学报(自然科学版),1983,(4):146-154.
周嫦,杨弘远.从水稻未授粉的幼嫩子房培养出单倍体小植株[J].遗传学报,1980,7(3):287-288.
周嫦,杨弘远.水稻离体条件下未受精胚囊的胚胎发生[J] .植物学报,1981,23(3):176-180.
周嫦,杨弘远.水稻未受精胚囊的离体胚胎发生[J] .植物学报,1981b,(23):176-179.
周嫦,杨弘远.未传粉子房与胚珠的离体培养[J].武汉大学学报(自然科学版),1982,(3):61-72.
周嫦,杨弘远.诱导水稻胚囊产生愈伤组织的离体实验[J].遗传,1981,(5):10-12.
周毓君.祝光苹果大孢子发生和雌配子体的发育[J].河北大学学报(自然科学版),1992,(3):62-66.
周元昌,Kennedy S.抱子甘蓝花培苗倍性的快速鉴定[J].福建农林大学学报(自然科学版),2002,31(1):55-58.
朱惠琴,戴文新.烟草加倍单倍体群体的构建及其在遗传育种中的应用[J].青海大学学报(自然科学版),2003,21(6):20-23.
朱睦元,黄纯农,徐阿炳,等.大麦耐酸铝愈伤组织变异体的筛选及其特性研究[J].植物学报,1990,32(10):743-748.
朱献辉,巩振辉,王乾宏,等.辣椒未受精子房愈伤组织诱导的研究[J].西北农业学报,2008,17(5):310-316.
祝仲纯,刘振岳,吴海珊,等.离体培养未传粉烟草子房的胚状体发育[J].植物学报(英文版),1981,23(6):499-501.
祝仲纯,吴海珊,安庆坤.大叶烟草未传粉子房培养及其细胞学研究[J].遗传学报,1984,11 (4):281-287.
祝仲纯,吴海珊.从未授粉的小麦及烟草子房培养出单倍体植株[J].遗传学报,1979,6(2):181-183.