Creating New Germplasm by Distant Hybridization in Stone Fruits:Ⅱ-Embryo Rescue and Hybrid Identification Between Plum and Apricot

Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from three weeks after pollination. (2) The germinationand growth of embryos were different at different growth stages, which could germinateand grow with PF value>0.5, but failed with PF value<0.5. In embryo rescue system ofhybrids, the best germination and differentiation medium was MS+6-BA 2mgL-1+IAA 0.3mgL-1,the rate of germination and differentiation reached up to 80%, bud induction andmultiplication medium was MS+6-BA 1.5mgL-1+IAA 0.3mgL-1, rooting medium was 1/2 MS+IAA0.8mgL-1. Some hybrids were transplanted into the field successfully. (3) Leaf shapeinvestigation and identification by S allele-specific PCR and RAPDs showed that thehybrids were true ones.

Identification of the Sex of Earlier Embryos from Generic Hybrids of Chicken-Quail by Wpkci

In this study, a protocol was developed to identify the sex of earlier embryos of chicken （♂）-quail （♀） hybrids and successfully tested the sex proportion of each period （66-120 h）. We acquired cross bred eggs by artificial insemination, hatched them in the same batch according to the standard hatching condition of chicken, and collected earlier living embryos at 66, 72, 78, 84, 90, 96, 102, 108, 114, and 120 h randomly. We adopted RT-PCR protocol and multiple PCR, made the known sex quail as the external control, employed fl-actin as the internal control, and used primers that were designed according to conservative area of gene Wpkci of quail to identify the sex of earlier hybrid embryos. The results indicated that the primer of Wpkci can be used to identify the sex of hybrid embryos accurately; there were more male than female in earlier embryos, the sex proportion of earlier embryos compared with academic numerical value was significantly different （P〈0.01）, and there was no difference between different periods （P〉0.05）. In the present study, we concluded that a simple, fast, credible and stable protocol to identify the sex of earlier hybrids embryos had been established by using primer of Wpkci; in earlier embryos, the death rate of female was higher than that of male and there was no fluctuant peak.

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Optimization of Germinated Conditions for Somatic Embryos in Liriodendron Hybrids

The development of somatic embryos is a key technology in plant breeding. To gain the best cultural conditions,the factors influencing on germination and transplanting survival rate of the somatic embryos in Liriodendron hybrids were optimized,including the development period of somatic embryos,macro-element,Vitamin C,genotypes,plant hormones and transplant medium in this study. The results showed that the optimal development phase of germination was torpedo-shaped embryo,and the base cultural medium of 3/4 MS with vitamin C can keep normal form from somatic embryos. The germination rate of somatic embryo had obvious genotype difference,among which the hybrid 1 × 5002 was the most sensitive( 82. 46%),and the hybrid 1 × 4088 was the most insensitive( 13. 24%). The medium with 0. 1 mg/L of KT + 0. 1 mg/L of IBA can help promote the germination of somatic embryogenesis. In addition,the yellow soil was more suitable than mixed peat soil for transplanting Liriodendron hybrids seedlings. Therefore,the obtainment of the optimal conditions has important guidance for production practices of Liriodendron hybrids.

Hybridization between Gossypium herbaceum and Gossypium stocksii through embryo rescue

Nutrient media and culture conditions have been defined for ovules 3 and 5 d after pollination and embryos of Gossypium herbaceum respectively. The technique was then used to produce interspecific hybrids between a cultivated Gossypium herbaceum and a wild species, G. stocksii. The hybrid plants were transferred to field and they exhibited most of the characters of the pollen parent i.e.G. stocksii.

Pollination Methods and Embryo Rescue Techniques of Hybridization between Lilium Oriental Hybrids

The pollination methods and embryo rescue techniques of hybridization between I.ilium Oriental hybrids were studied in this paper. The results showed that ： （ 1 ） in the combination of ＇ Justina＇ x ＇ Shala＇, pollen tubes were observed to grow downward in styles and target to ovules in ovaries when normal sti^na 13o1- lination method was used; pollen tubes were found to grow directionless to a different degree at stylar cuts and in ovaries when cut-style pollination method was used; the longer the styles were left, the more pollen tubes toward to ovules were observed and more plump seeds were produced; the maximum plump seeds were obtained via normal stigma pollination. （2） Hybridization experiments were performed for the 56 combinations of Lilium Oriental hybrids with normal stigma pollina- tion, and one combination generated no plump ovule and three combinations generated fewer than 10 plump ovules while the other 52 combinations generated plenty of plump ovules, which preliminarily indicated that normal stigma pollination method could be used as an universal pollination technique for hybridization between Lilium Oriental hybrids. The experiment results of different embryo rescue techniques showed that using cut-ovule inoculation about 70 d after pollination had a fas- ter germination speed and a higher germination rate of hybrid embryo than normal ovule inoculation, and embryo rescue efficiency of the former method was obviously higher than ＂embryo isolated inoculation＂ on 40 d after pollination. The research suggested that ＂cut-ovule inoculation＂ about 70 d after pollination could be regarded as an universal embryo rescue technique for hybridization between Lilium Oriental hybrids.

芍药属组间杂交种皮开裂种子的解剖观察与胚培养

【目的】探究芍药属组间杂交种皮开裂种子的内部状态和胚培养成苗表现,为揭示种皮开裂种子的成苗潜力、提高组间远缘杂交育种效率提供参考。【方法】以24个芍药属组间杂交组合种皮开裂的种子为试验材料,解剖后观察并统计胚和胚乳的发育情况,通过离体培养观察胚的萌发和生长规律,分析不同组合种皮开裂种子发育和成苗差异。【结果】芍药属24个组间杂交组合中均出现了种皮开裂的杂交种子,平均种皮开裂率高达61.08%;1767粒种皮开裂种子中,有11.09%的胚乳已腐烂;胚乳未腐烂种子中,约90%以上种子的胚乳存在不同程度的损伤或病变,24.18%的种子内部未观察到可见胚。对解剖得到的1185个组间杂种胚进行离体培养发现,有24.73%的胚不萌发,63.47%的胚生长异常,仅有4.47%的胚能正常生长,得到完整植株。胚培养得到的完整植株具备良好的活力,移栽成活率达到86.79%。24个杂交组合种皮开裂种子通过离体胚培养共得到46株移栽成活的组间杂种苗,平均成苗率为2.60%,接近正常组间杂交种子的播种成苗率(2.75%)。【结论】种皮开裂现象在芍药属组间杂交种子中普遍存在,是组间远缘杂交障碍的重要表现之一,用离体胚培养技术能有效克服这类障碍,提高成苗率。

CLK2 mRNA在早期鸡胚中的表达及定位分析

为探究早期鸡胚(孵化后约48 h内)中蛋白激酶CLK2 mRNA的表达特点及部位,用qPCR法测定早期鸡胚中CLK2 mRNA的表达,利用分子克隆技术重组CLK2/pMD18-T质粒,制作CLK2 mRNA原位杂交探针并以地高辛标记,进而通过原位杂交技术测定不同时期鸡胚中CLK2 mRNA的表达和分布。结果表明,早期鸡胚中CLK2 mRNA在各时期均有表达但表达量有所不同;原位杂交结果显示,在鸡胚发育的各个阶段,CLK2 mRNA的表达有所差异,且在各个部位其表达的量也各不相同,头部和尾部的表达最为集中。综上,CLK2 mRNA在鸡胚中存在且表达,其表达分布提示CLK2可能与其头部和神经系统的发育密切相关。

猕猴桃远缘杂交胚胎挽救技术初探

为创新猕猴桃种质,以软枣猕猴桃栽培品种‘猕枣2号’为母本,以美味猕猴桃‘磨山3号’为父本进行远缘杂交,对杂交后的未成熟胚进行胚胎挽救,分析不同胚龄和培养基对胚胎挽救效果的影响.结果显示:授粉35d的未成熟胚在⑨号培养基中可同时诱导出丛生芽和胚性愈伤组织,且二者诱导率均为最高,分别为10.00%和13.33%.丛生芽在添加0.5mg·L^(-1)IBA的1/2 MS培养基上生根效果最佳.通过杂种未成熟胚的胚胎抢救,成功获得远缘杂交后代,创制了猕猴桃新种质,为猕猴桃品种选育提供了基础.

The Comparison in Tissue Culture Ability of Mature Embryo in Different Cultivars of Rice

In order to study the regeneration technology of mature embryos in different rice varieties,nine japonica,nine indica and eleven hybrid rice varieties of two line or three line or superiority combinations were selected as explants to study the callus induction,differentiation and regeneration rates on different media.The higher callus induction （61.7-89.2%） was observed in japonica rice,when cytokinin was added at lower concentration （0.3 mg L-1 6-BA） in M8 basal medium,supplemented with 30 g L-1 sucrose,8 g L-1 agar and 2 mg L-1 2,4-D.Further,the addition of two cytokinins （2 mg L-1 6-BA,0.5 mg L-1 KT） and 1 mg L-1 NAA in the M8 basal supplemented medium resulted in 9.1-100% of the callus induction in indica rice.The percent callus induction in hybrid rice varieties was 40-86.3% when addition of 1 mg L-1 6-BA and 1 mg L-1 KT was added,and the cytokinins was required by the japonica and indica rice varieties in the M8 basal supplemented medium.It was observed that when the 0.5 mg L-1 2,4-D and 1 mg L-1 6-BA were added in japonica rice,and 0.2 mg L-1 2,4-D and 0.5 mg L-1 6-BA were added in indica and hybrid rice in the MS different media,the regeneration rates were 9.2-59.5%,3.6-87.5% and 17.2-43.2% for japonica,indica and hybrid rice,respectively.Thus,the regeneration technology with higher output is established in the mature embryos of similar rice varieties.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

樱花远缘杂交胚抢救技术

【目的】为提高樱花杂交育种效率,对樱花远缘杂交胚离体培养进行胚抢救。【方法】以迎春樱桃为母本,华中樱桃、尾叶樱桃、钟花樱桃为父本进行远缘杂交,对不同时期的坐果率进行统计,确定杂交种胚败育时期;对不同时期杂交胚进行萌发实验,确定杂交胚抢救适宜时间。以1/2MS培养基为基本培养基,添加不同浓度的6-BA、KT和NAA混合物以研究植物生长调节剂对种胚萌发及幼苗生根的影响;将胚抢救苗分别接种到MS、1/2MS、WPM培养基中,研究其根系生长情况;以泥炭、珍珠岩(1∶1),椰糠、珍珠岩(1∶1),黄土、珍珠岩(1∶1)3种基质栽植胚抢救苗,筛选适合胚抢救苗移栽基质。【结果】以迎春樱桃为母本的杂交组合中,胚败育发生时间在授粉后15 d;胚抢救适宜的时间在授粉后25 d;诱导杂交胚萌发的适宜基本培养基为1/2MS培养基;诱导胚抢救苗生根的适宜培养基为WPM培养基;不同杂交组合的杂交胚萌发和胚抢救苗生根需要的植物生长调节剂配比不同;幼苗适宜移栽基质为泥炭、珍珠岩(1∶1),成活率95%以上。【结论】该技术对于樱花远缘杂交获得新种质资源有良好的效果,能明显提高樱花远缘杂交育种的成功率,对樱花育种意义重大。

石蒜与萱草远缘杂交育种研究

目的:为培育石蒜属植物新品种(系)提供参考依据。方法:以石蒜属(Lycoris)、朱顶红属(Hippeastrum)、萱草属(Hemerocallis)植物为试材,采用花粉离体培养、田间杂交、胚拯救等方法,研究了石蒜属植物在远缘杂交过程中的生殖隔离。结果:1、在花粉活力测定中,所得最适宜朱顶红花粉萌发的培养基组成为:70 g/L蔗糖+5 mg/L硼酸+0.01 g/L氯化钙+50 g/LPEG-4000;萱草组花粉的最适培养基组成为:100 g/L蔗糖+0.3 mg/L硼酸+0.4 mg/L氯化钙。2、以石蒜属植物作母本,设计8个杂交组合试验,结果表明子房膨大率随杂交组合的不同而呈现差异性,其中与四倍体萱草杂交组合所得平均子房膨大率为59.81%,高于二倍体萱草组(33.12%)。3、胚拯救初期,各组合获得胚珠0~44个,其中鹿葱×萱草‘金娃娃’组合最多,中国石蒜×萱草‘金娃娃’组合最少。胚珠膨大率随杂交组合的不同出现差异,7个杂交组合的胚珠均发生膨大,膨大率在1.79%~96.15%之间。其中鹿葱×'Custard Candy'组合胚珠膨大率较高(96.15%),稻草石蒜×'Custard Candy'组合的胚珠膨大率极低(1.79%)。4、石蒜属植物与多倍体萱草杂交后代胚拯救最适培养基配方为MS+1.0 mg/LNAA+2.0 mg/L 6-BA+60.0 g/L蔗糖和MS+2.0 mg/LNAA+1.0 mg/L 6-BA+60.0 g/L蔗糖,60 d后1个胚珠萌发,诱导率为0.709%,不定芽繁殖系数为4;5个胚珠存活未萌发。结论:石蒜与萱草存在严重生殖隔离,通过胚拯救,远缘杂交所得胚珠已萌动,还需进行杂种鉴定以及后续分析。

不同辣椒栽培种的杂交亲和性比较及胚胎拯救体系建立

为克服辣椒种间杂交受精胚后期发育障碍,提高杂种胚成活率,缩短育种周期,利用3个辣椒栽培种(Capsicum. annuum L.、Capsicum chinense L.、Capsicum frutescens L.)进行正、反交试验,比较人工授粉后坐果率和种子数;在胚胎拯救技术的基础上,统计胚萌发率、适宜的胚拯救时期、幼苗生根数、生根率、根长等指标。结果表明,不同栽培种正、反交组合坐果率为0~32.50%,C. annuum做母本时单果种子数最多,平均为20.84粒;胚拯救期间不同杂交组合胚平均萌发率为11.43%~25.00%,最优胚拯救体系为萌发培养基MF_0(MS+30 g·L^(-1)蔗糖+6 g·L^(-1)琼脂,pH=6.0),胚拯救时间为授粉后32~34 d,生根培养基为SG3(MS+0.5 mg·L^(-1)NAA+30 g·L^(-1)蔗糖+6 g·L^(-1)琼脂,pH=6.0)。

绣球属种间杂交胚拯救时期研究

以绣球属种间杂交膨大蒴果为材料,采用显微镜观察和组培的方法,研究了绣球属种间杂交胚拯救的时期。结果表明:胚和种子呈卵形,黄褐色或褐色,表面具有网状纹理结构;60 d胚龄的胚透明度明显降低,90 d时种脐区域颜色加深。种间杂交的胚个数随着胚龄的增长呈下降趋势。组合21-6的萌发数在胚龄为62~93 d时相对较高;组合21-7和{21-9}的萌发数在62~80 d左右时较高。组合21-6的萌发率随着胚龄的增长呈先上升后下降的趋势;组合21-7的萌发率在60~80 d时较高;组合21-9的萌发率在62~93 d时相对较高。综上,在62~80 d胚龄时,进行绣球属种间杂交胚拯救效果较好。

中熟桃品种黄妃杂交种胚培养的研究

针对中熟桃品种黄妃存在大量种子败育,影响育种效率和进程的问题,春季以黄妃做母本,黄金蜜1号为父本进行杂交,8月初果实成熟后收集杂交种子,研究了不同基本培养基中加入不同植物生长调节剂与浓度的培养基上接种种胚的培养效果。结果表明,杂交种胚在基本培养基WPM上加入1 mg/L 6-BA+0.1 mg/L IBA的培养基上具有最高的发芽率;在同浓度IBA条件下,6-BA浓度越高,种胚的发芽率越高,幼苗苗高越高而发根数减少;IBA浓度与根的发育成正相关。

黄鳝卵原细胞移植研究

【目的】明确黄鳝卵原细胞是否能迁移、整合到斑马鱼生殖嵴上,为建立黄鳝异体生殖途径打下研究基础。【方法】通过酶消化法制备黄鳝卵原细胞悬液,经PKH26标记卵原细胞后显微注射到受体斑马鱼胚胎中,在荧光显微镜下追踪受体斑马鱼胚胎中供体黄鳝卵原细胞的迁移、整合及增殖情况;采用组织形态学方法检测移植受体斑马鱼胚胎的发育情况,并以半定量PCR和荧光原位杂交检测受体斑马鱼胚胎中供体黄鳝卵原细胞标记基因nanos3的表达情况。【结果】黄鳝卵巢经蛋白酶消化后,分离得到的卵原细胞悬液浓度为2.4×10^(6)个/mL,以PKH26染色后在红色荧光下呈橙黄色。荧光追踪观察结果显示,在移植后0、12、24和48 hpt的受体斑马鱼胚胎中均能检测到供体黄鳝卵原细胞,且黄鳝卵原细胞迁移至受体斑马鱼胚胎的生殖嵴上进行整合及增殖,但72 hpt受体斑马鱼出膜后荧光信号丢失。组织形态学观察结果表明,黄鳝卵原细胞注入受体斑马鱼胚胎中48 hpt后其细胞质结构松散,生殖细胞由动物极向两端迁移而分化形成组织,72 hpt和30 dpt的移植受体斑马鱼与对照组斑马鱼无明显差别。半定量PCR检测结果显示,在48 hpt受体斑马鱼胚胎中能检测黄鳝nanos3基因表达,而在72 hpt和30 hpt受体斑马鱼中表达微弱。荧光原位杂交检测结果显示,48 hpt、72 hpt和30 dpt的移植受体斑马鱼均有黄鳝nanos3基因表达。【结论】黄鳝卵原细胞在48 hpt后仍存在于受体斑马鱼胚胎内,但72 hpt后在受体斑马鱼胚胎未检测到黄鳝卵原细胞,即黄鳝卵原细胞在斑马鱼受体中迁移并最终整合到生殖嵴上,但在斑马鱼出膜后无法继续存活。因此,为了提高黄鳝生殖细胞移植成功率应选择亲缘关系近的鱼种或构建性腺不育种质作为受体。

甜樱桃与中国樱桃杂种的胚抢救及杂种鉴定

以甜樱桃为母本 ,中国樱桃为父本 ,进行种间杂交 ,建立了杂种胚抢救技术体系 ,并对部分杂种进行了鉴定。结果表明 :(1)母本中 ,‘养老’坐果率最高 ,‘养老’在授粉 4周后胚发生败育 ,而‘那翁’与‘红灯’则是在第 3周后败育 ;(2 )最佳萌发与生长培养基为 1/ 2MS +BA 2mg/L +IAA 1mg/L +维生素C 10mg/L ,最佳继代增殖培养基为 1/ 2MS +BA 2mg/L +IAA 1mg/L +NAA 0 1mg/L +维生素C 10mg/L ,最佳生根培养基为 1/ 2MS +IBA 0 2mg/L +维生素C 10mg/L ;(3)S allelespecificPCR。

李、杏属间远缘杂交及杂种胚培养技术研究

以李为母本、杏为父本进行李、杏远缘杂交,在获得果实的5个杂交组合中,授粉后30d的坐果率为4%～30%,授粉后90～100d的坐果率为4%～16%。杂交后90～100d,取杂种胚进行离体培养,结果表明在S1:改良SH+6-BA1.0mg/L+IAA1.0mg/L和S2:改良SH+6-BA2.0mg/L+NAA0.2mg/L培养基上,杂种胚均可萌发。对萌发芽进行继代培养,结果在MS+6-BA1.0mg/L+NAA0.5mg/L培养基上增殖和生长良好。

小麦—偃麦草—簇毛麦—黑麦四属杂种的产生及其形态学、细胞学研究

通过胚培养产生了小麦－偃麦草－簇毛麦－黑麦四属杂种。用４个六倍体小偃麦，２个六倍体小簇麦，２个六倍体小黑麦及１个八倍体小偃麦配置了６个四属杂种组合。结果没有出现高度不亲和性。６个组合的杂交结实率分别为６．２％、９．４％、２．４％、９．５％、２５．０％和３．９％；胚培成苗率分别为１２．１％、２０％、３７．４％、４０％、１．１％和７．１％。结果表明，杂交结实率与胚培成苗率间没有相关性。杂种Ｆ１生活力旺盛，形态具有四个属的特征特性。杂种属于自交高不育与低育类型。５个低育类型自交结实率平均约１．２％。四属杂种体细胞染色体数目的变化为３６、３７、３８、３９、４０和４１。其中多数属于具有３８个左右染色体的植株。