番茄花粉管通道遗传转化方法

本研究针对授粉后的转化时机对遗传转化效果的影响,采用两种遗传转化方法(处理Ⅰ:授粉后24h切除花柱进行转化;处理Ⅱ:授粉后12h切除花柱进行转化),利用免疫组织化学技术对番茄花粉管通道遗传转化的途径进行研究。结果表明:处理Ⅰ在转化后26～36h,外源DNA从珠被的一侧进入胚囊;处理Ⅱ在转化后12～38h,外源DNA到达胚珠的珠柄处。外源DNA在番茄花柱的移动速度为1.507±0.105mm/h,其通过花柱的理论时间为4.5h。由此看出,不同的转化操作时机对转化率有影响,认为花粉管通道法转化番茄的适宜时期为授粉后24h进行切除整个番茄花柱的转化操作。     

小麦花粉管生长途径及受精过程经历时间的研究

应用常规石蜡切片和荧光显微镜技术对小麦花粉管生长途径及受精过程相应时间进行研究。结果表明，授粉后，花粉随即萌发，2个精子进入花粉管，营养核留在花粉粒内，不久解体。花粉管进入羽毛状柱头分支结构的细胞间隙，继续生长于花柱基部至子房顶部的引导组织的细胞间隙内。随后穿过子房壁，在子房壁与外珠被之间的缝隙中向珠孔方向生长。自花粉萌发至花粉管长入珠孔大约需要1 h。自花粉萌发并到达柱头分支结构中，花粉管均具明显的绿色荧光；但在引导组织区以及子房壁与外珠被之间生长的花粉管几乎看不到荧光。授粉后1 h花粉管经珠孔及珠心表皮细胞间隙进入1个助细胞，并释放精子。授粉后2～6 h精卵融合，授粉后16 h合子第一次分裂。授粉后2～3 h精子与极核融合，授粉后4 h初生胚乳核第一次分裂。授粉后2～16 h，即精卵融合至合子分裂前为花粉管通道法转化合适的时间，采用花柱横切滴加法转入外源DNA。     

蒙药植物蓝盆花的授粉亲和性

利用显微镜观察花粉和柱头形态,采用离体萌发法对花粉活力进行测定,采用压片法对自交授粉后花粉管生长动态进行观察,以探明蓝盆花自交是否存在授粉障碍,为今后蓝盆花人工栽培和杂交育种工作提供理论依据。结果表明:蓝盆花花粉饱满,具有1~3个萌发孔,二裂柱头,花柱细长通直,培养10 h花粉萌发率达86.06%,花粉活力高。授粉后4 h多数花粉萌发后花粉管进入花柱;授粉后10 h花粉管进入花柱中上部;授粉后12 h多条花粉管进入花柱并到达中部;授粉后48 h花粉管到达花柱基部进入子房,但通过观察发现花柱顶端有胼胝质及花柱内的花粉管交叉缠绕停止生长的异常现象,从而花粉管不能进入子房完成受精过程,这可能是导致果实空瘪的一个原因。由此推测,蓝盆花自交授粉存在受精前障碍,属于配子体自交不亲和类型。     

外源亚精胺对鸭梨自花授粉花粉管伸长及花柱蛋白表达的影响

为探讨外源多胺调控鸭梨自交不亲和的生理生化机制,分别以自花授粉和外源亚精胺处理后1~3 d的花柱为试材,测定了不同处理不同时期花粉管生长发育动态,利用双向电泳技术和质谱分析技术分析了外源亚精胺处理前后不同时期鸭梨花柱蛋白的差异表达。结果显示:与对照相比,不同浓度的Spd处理均能极显著促进花粉管生长,0.25 mmol/L Spd处理72 h后可使大量花粉管到达花柱基部;在自交花柱电泳图谱中发现了3种特异蛋白质,A蛋白质Mr A=28.0 k Da,p I=5.7;B蛋白质Mr B=28.5 k Da,p I=5.7;C蛋白质Mr C=22.0 k Da,p I=5.3,这3种特异蛋白质可能与自交不亲和性相关;质谱分析获得了2个较好的蛋白点,分别为推定和假定蛋白。     

DNA浓度及注射时间对苹果花粉管通道法基因转化率的影响

通过花粉管通道法，将携带CBF3和GUS基因的pWBVec10a质粒DNA导入苹果。坐果后70d左右采收种子，将收获的种子进行离体培养，培养基为MS+BA0.2mg/L+GA2.0mg/L，附加蔗糖50g /L，琼脂6 g/L，培养40d后对转化子叶进行GUS染色检测。结果表明，最佳注射时间为授粉后11~24h之间。随着注入外源DNA浓度的增加，坐果率逐渐降低，DNA浓度为500μg/ml时，坐果率可达3.1%；DNA浓度为1000μg/ml时GUS基因阳性表达率可达12.5%。     

麻疯树花粉管荧光染色及生长过程的研究

花粉管的生长是完成植物受精的重要生理过程,观察花粉管生长首先需要对雌蕊组织进行软化透明。本研究探索适合麻疯树雌蕊结构的透明方法,并利用苯胺蓝染色技术,在荧光显微镜下观察授粉后麻疯树花粉管的萌发和生长过程。结果表明:麻疯树雌蕊组织可采用NaOH溶液软化透明,较优的条件是2 mol·L-1的NaOH,50℃恒温箱内软化3~4 h;麻疯树授粉2 h后花粉管开始萌发,10 h后花粉管穿过花柱基部,11 h后在子房中观察到花粉管。麻疯树授粉后,花粉管进入子房至少需要11 h,完成受精则需要更长的时间。     

Bar基因的玉米花粉管通道法转化

通过花粉管通道法利用抗除草剂基因（bar）转化3个玉米自交系,D0代获得35株GUS阳性植株．其中17株PCR阳性植株,除草剂筛选获得抗性植株13株。研究发现DNA溶液浓度影响转化率,当浓度为100μg／ml时,转化率最高,为2．61％。DNA导入时间距人工授粉时间的延长,结实率升高,但转化率显著下降,人工授粉后1d导入DNA产生的转化株最多,为19株,平均转化率为1．35％。     

高粱受精过程及其经历的时间

应用常规石蜡切片技术, 对高粱花粉管生长途径、双受精过程及其经历的时间进行研究。结果表明, 高粱开花即授粉,花粉随即萌发, 花粉管进入羽状柱头分支结构的细胞间隙,向下生长于花柱至子房顶部引导组织的细胞间隙中;进入子房腔后,在子房壁与外珠被之间的缝隙中向珠孔方向生长;花粉管破坏一个助细胞后, 在卵细胞与中央细胞之间的空隙中释放连续的球状内容物;两个精核分别进入卵细胞和中央细胞分别与卵核和极核融合。受精过程经历的时间为开花后0.5~3 h左右, 花粉管破坏一个助细胞进入胚囊,并释放精子;1~3 h精核与极核融合形成初生胚乳核, 精卵融合形成合子;4 h左右初生胚乳核分裂;14 h左右合子分裂。4~14 h为合子静止期,该期为物理、化学诱变及花粉管通道法转基因技术实施的合适时间。     

利用花粉管通道法将BcWRKY2抗旱基因导入玉米的研究

利用花粉管通道法将含有PPT抗性筛选标记的BcWRKY2转录因子基因导入优良玉米自交系.采用正交设计对玉米品种、导入时间及DNA浓度等条件进行了优化.对T0代种子进行了除草剂抗性筛选,结果表明玉米品种龙抗11授粉后16h进行导入及DNA导入浓度为200ng/μL时效果最佳.对获得的除草剂抗性植株进行PCR及PCR-Southern检测,得到PCR阳性植株71株,初步证明了外源目的基因已整合到玉米基因组中.     

枣树不同杂交组合花粉萌发情况的荧光显微观察

以冬枣和雄性不育3号为母本,大叶无核枣等品种为父本进行品种间授粉,在荧光显微镜下观察人工授粉后柱头上花粉的黏附、萌发和花粉管生长情况。结果表明,不同授粉组合之间有花粉黏附柱头比率、单柱头平均花粉数、有萌发花粉的柱头比率和单柱头平均花粉萌发率差异显著。经不同品种授粉后,冬枣有花粉黏附柱头比率和有萌发花粉的柱头比率之间,及单柱头平均花粉数和单柱头平均花粉萌发率之间无相关性。冬枣×大叶无核枣、冬枣×无核小枣和冬枣×苹果枣三个组合的花粉萌发率高且花粉管更容易深入花柱,是较为理想的杂交组合。     

Study of morpho-physiological, phenological and reproductive behaviour in protogynous lines of Indian mustard [Brassica juncea (L.)]

Understanding the relationship between floral and reproductive traits are critical to understand the evolution of plant species in brassica and need for selecting lines as parental lines in hybrid seed prodcution. The protogyny nature of Brassica species are characterized based on the variations in floral morphology, protogyny interval, stigma receptivity, pollen biology, compatibility and seeds set. The variation in stigma morphology features (stigma exsertion prior to anthesis) and protogyny interval (ranged from 8 to 10 days) formed phenotypically distinct. Studies on stigma receptivity and pollen viability revealed significant variability and found to be at its peak up to 3 days after anthesis and reduced drastically thereafter. Compatibility crosses and post pollination events such as pollen deposition on the surface of the stigma and culminating with the entry of the pollen tube into the embryo sac, was rapid and completed within 2 days after pollination, whereas in in-compatible cross not even single pollen was germinated on stigmatic surface and consequently no pollen tube growth even after sixth day of pollination. In compatible crosses average seed set ranged from 3 to 8 seeds per pod from cross pollination and no seed set in self. The investigation revealed that variation exists for protogyny interval, stigma receptivity and compatibility in the protogynous lines of Indian mustard and this can be exploited for the production of hybrids without emasculation.     

Independence of self-compatibility and potentiality for self-pollination in peach x almond hybrids

Summary The almond of commerce (Prunus dulcis (Mill.) D.A. Webb) is self-incompatible (SI) and requires honey-bees to effect the transfer of pollen among cultivars that flower simultaneously. Four year old trees from the F₂ generation of several peach x almond hybrids were studied to determine whether self-compatibility (SC) and the potentiality for natural, i.e., abiotic, self pollination (NSP) are genetically related or are inherited independently. Both SC and the high potentiality for NSP are characteristic of peach (Prunus persica (L.) Batsch) but not almond. Forty percent of SC genotypes exhibited adequate NSP (SC, +NSP) for good fertility i.e., without insect-mediated pollination. The remaining 60% of SC genotypes (SC,-NSP) exhibited an average 61% reduction in fruit set on limbs bagged to exclude honeybees during anthesis relative to fruit set on open pollinated limbs. Our data are consistent with the concept that fertility is dependent upon the load of compatible pollen deposited on the stigma. Fruit set reduction on bagged limbs, compared with bagged and self-pollinated limbs, was presumably due to a) lack of/insufficient pollination for fertilization and/or b) post-zygotic abortion of genetically inferior recombinants. Selection following manual self-pollination may result in SC genotypes with or without the capacity for NSP. In contrast, significant fruit set on limbs enclosed during pistil receptivity necessitates that the genotype selected express both SC and the potentiality for NSP.     

不同柱头颜色向日葵三系亲和力研究

目的:为选育优质高产向日葵新品种及向日葵柱头颜色遗传规律研究提供依据.方法:以4种柱头颜色典型的食用向日葵亲本材料为试材,通过连续两年正反交试验,研究不同柱头颜色向日葵亲本传粉效应及亲和效应.结果:在相同的授粉条件下,4种柱头颜色向日葵自交系自交后柱头存活时间及结实率无明显变化,然而自然异交后两者均有明显差异.自交系间花粉数量(N)变化同样明显;三系杂交表现为亲本柱头颜色相差越大,A系柱头存活时间越短;传粉效应(C1)表现为C1黄 < C1红 < C1紫 < C1黑紫,亲和效应(C2)表现为双亲颜色相差越大,C2越大.结论:柱头颜色在影响结实率的诸多因素中有积极作用,且与亲本的传粉效应及亲和效应存在相关性.     

Overcoming pre-fertilization barriers in the wide cross between Chrysanthemumgrandiflorum (Ramat.) Kitamura and C. nankingense (Nakai) Tzvel. by using special pollination techniques

In our earlier study, pre-fertilization barrier was one of the main factors causing the failure of the wide hybridization between Chrysanthemum grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and seriously restricted the utilization of the excellent chrysanthemum germplasm, C. nankingense. In order to overcome the pre-fertilization barrier and obtain some hybrids, we carried out wide hybridization between them again in this study, and tentatively adopted three pollination techniques including mentor pollen, delayed pollination, and gibberellic acid treatment. The average number of pollen grains germinating on stigma at 24 h after pollination and the percentage of embryo were investigated with a temporary mount method and the technique of fluorescence microscopy. It was found that, compared with the control, delayed pollination, gibberellic acid treatment, and mentor pollen significantly increased the average number of pollen grains germinating on each stigma at 24 h after pollination by approximately 152, 235, and 182%, respectively. In addition, all the three pollination techniques significantly increased the percentage of normal embryos at different periods after artificial pollination in comparison to the control. Furthermore, seed sets of the crosses using the three special pollination techniques were 1.1, 1.4, and 2.0%, respectively, whereas we did not obtained any seeds in the control. Taken together, these results suggest that delayed pollination, gibberellic acid treatment, and mentor pollen are three useful pollination techniques for overcoming pre-fertilization barriers and increasing seed set in the wide hybridization between C. grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and they may be widely applied for increasing breeding efficiency in other chrysanthemum wide hybridization facing similar pre-fertilization barriers.     

Pollen viability and stigma receptivity in <Emphasis Type="Italic">Lilium</Emphasis> during anthesis

Pollen viability and stigma receptivity are prerequisites for successful pollination and seed set in flowering plants. In this study, the pollen viabilities and stigma receptivities of nine Lilium genotypes (six cultivars and three species native to China) were assayed by in vitro pollen germination and the benzidine-H₂O₂ method, respectively. Embryo sac development during anthesis was observed to further ensure the timing of controlled pollination. In addition, the relationship between stigma secretion and stigma receptivity was studied to estimate the pollination time based on phenotype. Anthers cracked on the day of flowering in all genotypes, but pollen germination during anthesis was not observed in Asiatic hybrids excepted for ‘Tiny pudhye’, which exhibited low pollen viability for a short period of time (from 0 to 1 day after anthesis). In the other genotypes, pollen germination rates were highest on anthesis (five of seven genotypes), 0–1 day after anthesis (L. sulphureum), or 0–2 days after anthesis (one Longiflorum hybrid), and then gradually decreased with days after anthesis. While, stigma receptivity first increased and then decreased during anthesis. For most genotypes, stigmas began to be receptive 1 day after anthesis, and all genotypes exhibited stigma receptivity at 2 days after anthesis. The durations of stigma receptivity and strongest stigma receptivity, were genotype dependent, and were 5–8 days and 1–4 days, respectively. Moreover, on the first flowering day, 6 of 7 genotypes had mature embryo sacs, and at the time at which stigmas began to be receptive, all tested genotypes had mature embryo sacs. Some Lilium genotypes showed stigma secretion, which can be a sign of stigma receptivity. Stigmas became receptive and reached highest receptivity within 1 day of the first appearance of secretion on the surface of the stigma and at peaking, respectively. The results of this study are valuable for the implementation of successful Lilium breeding programs.     

玉米三种不同花粉管通道法转化BcBCP1基因的初报

采用DNA柱头滴加法、花粉粒携带法和开苞叶导入法等3种不同的花粉管通道遗传转化方法,将抗逆基因BcBCP1转入3个玉米自交系合344、农大178和郑58中,对3种方法的部分影响因素进行了优化,旨在筛选出转化效率高的方法,为玉米转基因育种提供技术支持。结果表明:DNA柱头滴加法的DNA溶液浓度100g/mL,授粉后18h转化率最高。花粉粒携带法最佳处理时间为10min。开苞叶导入法在授粉后18h转化率最高,DNA浓度为100μg/mL。T0代共筛选出338株除草剂抗性植株,其中87株PCR呈阳性。DNA柱头滴加法的平均PCR阳性率最高,为1.99%;而花粉粒携带法的平均PCR阳性率最低,为0.23%;开苞叶导入法平均PCR阳性率居中,为0.32%。     

‘砀山酥梨’花柱可授性及蜜蜂授粉特性的研究

采用半离体培养法和联苯胺-过氧化氢法研究了‘砀山酥梨’花粉萌发特性和柱头可授性。通过对‘酥梨’的最适授粉时间以及7种授粉处理坐果率的研究,探讨其授粉特性。结果表明：开花后每天酥梨柱头活性最佳的时间是11时至15时,柱头最佳授粉时间为开花后第4~5天;‘酥梨’花粉最适萌发温度为25℃,花粉管生长最有利的温度为22~25℃,在相同温度下,花粉萌发率和花粉管长度随时间增加而增加。7种授粉处理‘酥梨’的花朵坐果率中,没有昆虫授粉的‘酥梨’坐果率为0.48%和0.44%,自然授粉和人工授粉坐果率分别为2.83%和13.05%,‘酥梨’ב雪花梨’组蜜蜂授粉的坐果率为32.95%,新鲜花粉和商品花粉坐果率分别为70.2%和13.05%。由此可见,‘酥梨’是异花虫媒果树,蜜蜂可以在‘酥梨’柱头活性最高时进行授粉,从而提高‘酥梨’的坐果率,同时通过对‘酥梨’花器官生理活性和坐果习性的研究,为‘酥梨’的授粉和生产提供基础资料。     

Pollen tube growth and fertilization after different in vitro pollination techniques of Aechmea fasciata

The effect of cut style and placental pollination on fertilization efficiency was studied for compatible Aechmea fasciata plants. These alternative in vitro pollination techniques resulted in lower penetration rates of the ovules by pollen tubes in comparison to pollination on the stigma. An explanation was found in the intervention of the normal pollination process,through which less pollen-pistil interactions were built up. After cut style pollination the percentage of ovule penetration by pollen tubes increased when a longer style part was left at the ovary. Probably fewer factors that control pollen tube growth are present in the lower style part. Pollen germinated on the ovules after placental pollination but only rarely penetration of the micropyle by a pollen tube occurred. Activation of the ovary, induced by in vivo prepollination for 6 hours, and pollination two days after anthesis did not increase the fertilization percentage. Grafting a style with active growing pollen tubes to ovules on the placenta (placental grafted style pollination) resulted in a higher fertilization percentage. Pollen tube growth through the style was essential for pollen tube guidance to the ovules and penetration of the micropyle. This revised version was published online in August 2006 with corrections to the Cover Date.     

狭瓣辣木的开花动态及花粉活力和柱头可授性

为了观测狭瓣辣木和多油辣木改良种PKM-1开花散粉时期花部形态、花粉萌发率和柱头可授性的变化规律,本研究采用琼脂培养基萌发法测定花粉萌发率,用联苯胺-过氧化氢法测定柱头可授性,结合田间花部性状随着时间变化的实际观测进行分析。结果表明,狭瓣辣木花朵开花之初,花药即完全打开并开始大量散粉,柱头直立,高于雄蕊;花朵开放后,柱头直立不变,高度逐渐低于雄蕊。PKM-1花朵开花之初,花药微微闭合,2 h后完全打开并开始大量散粉,柱头微微弯曲,高度低于雄蕊;花朵开放后,柱头逐渐弯曲,高度逐渐高于雄蕊。狭瓣辣木花粉萌发率在开花第2天达到最大值77%,呈现先上升后下降的趋势。PKM-1花粉萌发率在开花第1天达到最大值69%,之后迅速降低;狭瓣辣木和PKM-1柱头可授性均呈上升趋势,二者在开花第3天柱头可授性最强。在开花第1天,狭瓣辣木柱头具有微弱活性,而PKM-1柱头没有活性。在自然授粉条件下,狭瓣辣木花期柱头形态不利于其杂交授粉;在开花第2天采集花粉,在开花第3天柱头可授性最强时人工授粉,可以提高狭瓣辣木结实率和杂交成功率。本研究对于制定辣木杂交育种措施、提高辣木人工授粉效率和产量有重要意义。