Embryological Studies on In Vitro Pollination in Rice

n vitro pollination and its embryological studies were carried out in two japonica cultivars of rice (Oryza sativa L.), “Chunjiang 05" and “95046". N6 basic medium supplemented with different exogenous hormones was used for ovary culture after in vitro pollination. The main results were as follows: (1) both cultivars were induced to set kernels after in vitro pollination. The frequency of seedset was 52.1%, including 28.4%normal embryo development, 2.2% abnormal embryo development and 21.5% callus formation. (2) The processes of embryo and endosperm development after in vitro pollination were basically as normal as those in vivo , except there was some retardation in the first division of zygotes and primary endosperm nuclei as well as in their subsequent development. However, both kernels and plantlets could be produced finally. (3) A few abnormal embryos were observed, for instance, proembryos with elongated suspensor and vacuolated proembryos. (4) Two types of calli in the cultured ovaries appeared, namely, the compact callus and the fragile callus, which were able to differentiate into adventitious buds and roots.     

水稻离体授粉的胚胎学研究

采用2个水稻（Oryza sativa L.)品种“春江05”（早粳）和“95046”（晚粳）,对离体授粉过程中的胚胎学进行了详细的研究。结果表明：（1）2个品种均胡离体授粉结实,平均结实率为52．1％,其中28．4％胚胎发育正常,2．2％胚胎发育异常,21．5％愈伤组织化;（2）离体授粉时的胚胎发育途径和体内自然发育基本相同,中是合子和初生胚乳核首次启动分裂及以后的发育均较延缓,但最终也能萌发成幼苗。（3）观察到具细长胚柄的原胚及液泡化原胚等异常的胚胎类型;（4）子房内形成的愈伤组织分为致密和松散两种类型,二均可化出不定芽和不定根,还对离体授粉的方法,离体授粉中正常和异常的胚胎发育途径进行了讨论。     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

Genotype effects of Brassica napus on its reproductive behavior after pollination with B. juncea

To investigate the cause of variation in the interspecific crossability of Brassica napus, three different genotypes were studied in respect of their reproductive behavior after pollination with B. juncea. There were great differences among maternal genotypes in allowing foreign pollen to germinate on and penetrate into their stigmas, leading to a wide diversity of interspecific fertilization. The division of the hybrid primary endosperm nucleus and zygote appeared normal in all combinations of crosses. While the abundant free nuclei of the endosperm developed properly and never became cellular, the embryos degenerated as early as 10 days after pollination when the cultivar Rucabo, which had the highest fertilization record with species of B. juncea, was involved. When 81007 was used as female parent, the endosperm grew a little but the embryo halted at the heart-torpedo stage. Lack of nourishment might be responsible for the observed embryo abortion. Among the sic hybrid combinations, the cross 84014A x Changyang hunagjie was the only one where endosperm tissue was observable and an abnormal embryo occurred prior to cellular endosperm formation. Apart from the three typical embryological features, significant variation was also demonstrated among each of the cross combinations. Genetic diversity appears to exist not only between varieties, but also within cultivars. In addition, methods for developing interspecific crossable lines are discussed.     

内蒙古居群抗寒西伯利亚杏繁殖生态学研究

对内蒙古居群抗寒西伯利亚杏的12个种源同一树龄的102株进行雌蕊败育率、花粉量、花粉活力、柱头可授性及自交不亲和性进行研究,以明确西伯利亚杏主产地资源的花期生物学特性和繁殖特性,为杏杂交育种和资源利用提供理论依据。结果表明:(1)有86.27%的西伯利亚杏雌蕊败育率低于90%;73.53%的西伯利亚杏单朵花花粉量在1×104～5×104之间;86.27%的西伯利亚杏花粉活力低于50%,且寿命较短。(2)花苞在将开未开时,柱头已经具备一定的可授性,开花后1～4d内柱头可授性保持较高水平,第5天开始有下降趋势。(3)荧光显微观察结果显示,授粉后16h时,96.08%的杏花花粉管在花柱上部;授粉后24h时,74.51%的杏花花粉管仍在花柱上部;授粉后48h时,35.29%的杏花花粉管到达花柱中部;授粉后80h时,花粉管到达花柱下部的杏花约50%,但受精胚珠数为0。研究认为,西伯利亚杏基本为自交不亲和。     

Effect of pollination on floral longevity and costs of delaying fertilization in the out-crossing Polygala vayredae Costa (Polygalaceae)

Background and Aims

The effect of pollination on flower life span has been widely studied, but so far little attention has been paid to the reproductive consequences of delayed pollination in plants with long floral life spans. In the present study, Polygala vayredae was used to answer the following questions. (1) How does male and female success affect the floral longevity of individual flowers? (2) How does delaying fertilization affect the female fitness of this species?

Methods

Floral longevity was studied after experimental pollinations involving male and/or female accomplishment, bagging and open pollination. The reproductive costs of a delay in the moment of fertilization were evaluated through fruit set, seed–ovule ratio and seed weight, after pollination of flowers that had been bagged for 2–18 d.

Key Results

Senescence of the flowers of P. vayredae was activated by pollen reception on the stigmatic papillae, while pollen removal had no effect on floral longevity. Nonetheless, a minimum longevity of 8 d was detected, even after successful pollination and pollen dissemination. This period may be involved with the enhancement of male accrual rates, as the female accomplishment is generally achieved after the first visit. Floral life span of open-pollinated flowers was variable and negatively correlated with pollinator visitation rates. Delayed pollination had a major impact on the reproductive success of the plant, with fruit set, seed–ovule ratio and seed weight being significantly diminished with the increase of flower age at the moment of fertilization.

Conclusions

A strong relationship between pollination and floral longevity was observed. Flowers revealed the ability to extend or reduce their longevity, within some limits, in response to the abundance of efficient pollinators (i.e. reproductive fulfilment rates). Furthermore, with scarce or unpredictable pollinators, a long floral life span could maintain the opportunity for fertilization but would also have reproductive costs on production of offspring. Reduced female fitness late in the flower''s life could shift the cost–benefit balance towards a shorter life span, partially counteracting the selection for longer floral life span potentially mediated by scarce pollination services.Key words: Delayed pollination, endemic species, flower longevity, life span, pollen limitation, pollination, pollinator scarcity, Polygala vayredae, Polygalaceae, reproductive consequences, secondary pollen presentation     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs ofZea mays andTorenia fournieri

Summary Actin organization was observed inm-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs ofTorenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.     

Semi in vivo pollen tube growth of Aechmea fasciata

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l^–1 agar was better than that with 6 g l^–1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.     

Regulation of the maize ubiquitin (Ubi-1) promoter in developing maize (Zea mays L.) seeds examined using transient gene expression in kernels grown in vitro

Kernel culture was assessed for evaluating novel gene expression in developing maize (Zea mays L.) seeds by comparing the transient expression of maize ubiquitin (Ubi-1) promoter-driven β-glucuronidase (GUS) delivered by particle bombardment in kernels grown in culture with those grown in planta. With kernels from either source, GUS expression, as determined by histochemical staining, was widespread in young, actively growing kernels, but it diminished with kernel age and by 25 days after pollination was found only in the embryo. Transient expression of Ubi-1 in kernels grown in vitro was not affected by wounding, ethylene treatment, pathogen invasion, or heat shock. In contrast, the plant hormones indole-3-acetic acid and kinetin both stimulated transient Ubi-1 expression in the endosperm, particularly at the periphery. Transient gene expression in developing maize seeds grown in vitro should allow for facile and rapid evaluation of the tissue-specificity and environmental responses of novel gene constructs in developing maize seeds. Received: 31 July 1997 / Revision received: 13 October 1997 / Accepted: 31 October 1997     

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid,an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca²⁺), mPB1^- (modified PBS without Ca²⁺), mPB1⁺/EGTA (mPB1⁺ containing EGTA), mPB1^-/EGTA (mPB1^- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.     

Maternal haploids of Petunia axillaris (Lam.) B.S.P. via culture of placenta attached ovules

Summary Hybridization of Petunia axillaris and P. parodii with Nicotiana tabacum was attempted using the method of in vitro pollination and fertilization. Seedlings were produced when the Petunia species and N. tabacum were used as the maternal parents; however, most of these had the identical somatic chromosome complement of the maternal parent. With crosses involving P. axillaris as the maternal parent, a low frequency of haploids was also produced. Due to the potential of haploids in basic and applied genetic research, additional experiments were carried out to determine whether in vitro pollination was necessary to stimulate haploid production and to more closely define the optimal time for ovule excision and culture. Four treatments were applied to accomplish these objectives. They were: placentas cultured prior to the time of anthesis, with and without pollination, and placentas cultured after the time of anthesis, with and without pollination. In vitro pollination had no effect on the frequency of haploids produced. Placenta attached ovules cultured prior to the time of anthesis produced significantly more haploids than those cultured after anthesis. The preanthesis treatment produced a frequency of 6.5 haploids per 100 ovaries cultured. The culture of placenta attached ovules provides an alternative to anther culture as a means for haploid production.The investigations reported herein were supported by USDA/SEA/CRGO Project 59-2213-1-1-613-0 and the paper (No. 84-3-36) is published with the approval of the Director of the Kentucky Agricultural Experiment StationThe authors are Graduate Research Assistant and Professor, respectively, Department of Agronomy, University of Kentucky, Lexington 40546-0091. The research reported in this paper is in partial fulfillment of the PhD requirements for the senior author     

Maternal effects of general and regional specificity on embryos of Drosophila melanogaster caused by dunce and rutabaga mutant combinations

Summary The developmental patterns of embryos produced by female germ line cells homozygous for null-enzyme mutations of dunce and for dunce in combination with each of three different rutabaga mutations are compared with the normal pattern. At least three discrete developmental defects at progressive stages following fertilization can be identified and correlated with the loss of adenylate cyclase activity caused by rutabaga mutations, suggesting that the defects are caused by elevated cyclic AMP levels in female germ line cells. The earliest defect occurs soon after fertilization and affects DNA replication and mitosis, prevents nuclear migration, and leads to large polyploid nuclei. A later defect prevents cleavage nuclei from migrating into, or dividing in, the posterior region of the egg. The last affects the developmental behavior or fate of blastoderm cells. Some of these defects mimic those produced by previously described maternal-effect mutations.     

Late steps of egg cell differentiation are accelerated by pollination in Zea mays L.

 Egg cells were analysed cytologically during the female receptivity period in maize (Zea mays L., line A 188). Three classes of egg cell were distinguished: type A – small, non-vacuolated cells with a central nucleus; type B – larger cells with small vacuoles surrounding the perinuclear cytoplasm located in the middle of the cell; type C – big cells with a large apical vacuole and the mid-basal perinuclear cytoplasm. The less-dense cytoplasm of the vacuolated egg cells usually contained numerous cup- or bell-shaped mitochondria. The three egg types appear to correspond to three late stages of egg cell differentiation. The frequencies of each of the three egg types were monitored in developing maize ears before and after pollination. In young ears, with the silks just extending out of the husks, small A-type cells were found in about 86% of ovules. Their frequency decreased to about 58% at the optimum silk length, remained unchanged in non-pollinated ears, and fell to 16% at the end of the female receptivity period. However, after pollination and before fertilisation the frequency of these cells decreased to about 33%, and the larger vacuolated egg cells (types B and C) prevailed. At various stages of the receptivity period, pollination accelerated changes in the egg population, increasing the number of ovules bearing larger, vacuolated egg cells. Experiments with silk removal demonstrated that putative pollination signals act immediately after pollen deposition and are not species-specific. Received: 5 February 1999 / Accepted: 28 August 1999     

In vitro pollination of maize (Zea mays L.) — Proof of double fertilization

Isolated ovules from the maize homozygous recessive brown midrib (bm₃) were in vitro pollinated by pollen carrying the dominant allele — and the purple embryo marker (PEM). The colour characters of the embryos and kernels corresponded to the results of control pollination and confirmed the process of double fertilization. The question whether the method is useful for obtaining haploids is discussed.     

野菊与菊花杂交中花粉活力和柱头可授性及胚胎发育研究

应用石蜡制片、活体压片、光学显微镜及扫描电子显微镜观察等方法,研究了四倍体河南云台山野菊(Dendranthema indicum)与栽培菊花'钟山金山'(D.grandiflorum 'Zhongshanjinshan')种间杂交中父本花粉活力、花粉在柱头萌发、花粉管生长及胚胎发育情况等.结果发现,父本云台山野菊的花粉活力在授粉时为12%左右.人工授粉后的不同时间,在柱头上都观察到正常萌发的花粉粒,且花粉管都能进入柱头,其中,在授粉后0.5 h时,平均每柱头有5.9粒花粉萌发;12 h时,为59.9粒;而24和48 h时,则分别降为47.1和35.7粒.此外,在授粉后8、10、12和15 d时,分别在49.1%、40.8%、39.7%和38.5%子房内观察到正常发育的胚胎,最终杂交结实率为44.8%,而母本自然开放结实率为52.3%.研究表明,授粉前其多数母本雌蕊发育良好、授粉后多数花粉能在柱头正常萌发和花粉管正常生长,在受精后大部分胚胎发育正常是野菊与栽培菊种间杂交较高结实率的重要保证,而授粉前父本较低的花粉活力对杂交结实率影响不大.     

Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes

Immature oocytes were collected from immature female rats (60-65 g) 40 h after injection with 6 IU pregnant mare's serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5-20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development. The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.