Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Untersuchung der ersten Entwicklungsphasen von Embryo und Endosperm der Art Jasione montana L. an lebendem Material

Summary Growth of the zygote and the first phases of the endosperm development of Jasione montana L. in isolated intact ovules was studied. The zygote begins to grow simultaneously with the first division of the primary endosperm nucleus. It forms a long outgrowth into the embryo sac. A distinct oil droplet occurs in the basic part of the zygote, which disappears after the development of the embryo is advanced.The nucleus of the zygote shifts to the top of the outgrowth of the zygote before the prolongated growth of the zygote is completed. The first mitosis in the embryo takes plase in this position at the time when there are 8–16 cells in the endosperm.The endosperm division as it can be seen in the living material is described.     

Isolation of protoplasts from female gametophytes of Torenia fournieri

Protoplasts from the cells of mature embryo sacs (ES-protoplasts) of Torenia fournieri were obtained during incubation of ovules in an enzyme solution. Four protoplasts which arose from each embryo sac were connected together after isolation, or aggregates of the egg cell protoplast and two synergide protoplasts dissociated from the protoplast of the central cell. The ES-protoplasts stayed viable for 2 weeks in culture, but they did not regenerate cell walls.Abbreviations ES embryo sac - FAA fixative (formalin : acetic acid : alcohol = 1 : 1 : 18) - FDA fluorescein diacetate - PAS periodic acid Schiff reaction - 2,4-D 2,4-dichlorophenoxyacetic acid     

The Putative Factors Involved in the Induction of Embryogenesis in Angiosperms

We quantified various endogenous cytokinins during wheat (Triticum aestivumL.) and dandelion (Taraxacum officinaleWeb.) ovary development. Wheat ovaries were studied at the following developmental stages: the mature embryo sac with eight nuclei (stage 1), the interphasic zygote 12 h and 24 h after fertilization (stage 2), and the onset of zygote division (stage 3). The dandelion ovaries were studied at the stage of the mature embryo sac (stage 1), in the interphase of the parthenogeneticaly developing ovule (stage 2), and during its first division (stage 3). The material was analyzed by the method of competitive solid-phase immunoenzyme assay (ELISA) using peroxidase-labeled anti-rabbit antibodies. The onset of embryogenesis in wheat and dandelion ovules was accompanied by the substantial rearrangement of their hormonal complexes, which preceded the morphogenetic processes leading to seed formation. This implies that the hormonal system of the whole maternal plant is involved in the induction of embryogenesis. The final stages of embryogenesis depend on the hormonal systems in the flower, ovary, and ovule.     

The progamic phase, embryo and endosperm development in an intraspecific Tulipa gesneriana L. cross and in the incongruent interspecific cross T. gesneriana × T. agenensis DC.

 The development of the embryo and endosperm has been investigated in an intraspecific Tulipa gesneriana cross and in the incongruent cross T. gesneriana ×T. agenensis at intervals of 10 days, from 12 to 82 days after pollination (DAP). In both tulip crosses, the zygote gives rise to an apparently undifferentiated cell mass, the proembryonal cell mass, on which a suspensor then develops. Subsequently, a globular embryo is formed on top of the suspensor. This embryo finally elongates, giving rise to a spindle-shaped embryo. The cellular endosperm fills the whole embryo sac in mature seeds, except for a region immediately around the embryo. In both crosses, aberrant developments were found. In the intraspecific T. gesneriana cross, the pollen tubes did not open in a number of ovules. In other ovules, the pollen tubes seemed to have opened, but an embryo or endosperm was not found or only endosperm was observed. In the cross T. gesneriana ×T. agenensis, fewer pollen tubes entered the ovules than in the intraspecific T. gesneriana cross. The ovules with embryo and endosperm formation of the incongruent interspecific cross showed, in general, retarded development in comparison with the intraspecific T. gesneriana cross. The first globular embryos and spindle-shaped embryos were found at the later fixation dates and the relatively lower number of spindle-shaped embryos at 82 DAP had a shorter average length. The number of ovules with deformations in embryo and/or endosperm development was also higher in the cross T. gesneriana × T. agenensis in comparison with the intraspecific T. gesneriana cross. Between 87% and 100% of the ovules with embryo and endosperm development showed normal development in the intraspecific T. gesneriana cross, while in the incongruent interspecific cross, from 22 DAP, between 17% and 56% of the ovules showed normal development. Of those ovules with aberrations in embryo and/or endosperm formation, about 80% had a deformed endosperm, of which more than 50% also contained a deformed embryo. Embryos of the incongruent cross might be saved by the application of embryo rescue techniques. Received: 10 December 1996 / Revision accepted: 23 April 1997     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

葡萄胚胎发育与败育过程中胚珠的多胺含量变化

花后40~60d期间,种子败育型无核葡萄胚珠的3种多胺含量明显低于有核葡萄,据此认为无核葡萄胚珠中多胺含量急剧下降和较低的多胺水平可能是导致葡萄胚胎败育的重要原因。(Spd Spm)/Put和Spm/PAs比值变化与葡萄胚胎发育密切相关,较低的比值不利于胚胎的正常分化。     

Embryo sac development is affected in Petunia inflata plants transformed with an antisense gene encoding the extracellular domain of receptor kinase PRK1

 In a previous study of the function of a pollen-expressed receptor kinase of Petunia inflata, PRK1, it was found that transgenic plants carrying an antisense-PRK1 gene were unable to transmit the transgene through either the male or, unexpectedly, the female. In this report, the nature of this female phenotype was studied using one of the transgenic plants, ASRK-13. Electron and light microscopic examination of the embryo sac and seed development of ASRK-13 and a wild-type plant revealed that embryo sac development of approximately half of the ovules of ASRK-13 was abnormal. The development of the affected embryo sacs was arrested at the late stages of megagametogenesis. The majority of the affected embryo sacs completed three rounds of mitosis normally, but failed to progress through the maturation stages when cell expansion, nuclear migration, and differentiation take place. The remaining small number of abnormal embryo sacs were arrested at either the four- or eight-nucleate stages. The ovules containing the defective embryo sacs apparently failed to be fertilized, resulting in degeneration of half of the seeds produced by ASRK-13. RNA gel blot analysis suggests that the PRK1 gene is expressed in the ovary, albeit at a much lower level than in the anther. The possibility that the antisense PRK1 gene is responsible for the abnormal embryo sac development is discussed. Received: 25 April 1997 / Revision accepted: 25 June 1997     

The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.     

OVULE ABORTION IN QUERCUS (FAGACEAE)

This study deals with the occurrence and relative abundance of four different types of abortive ovules in three species of Quercus. It was found that, contrary to previous literature, fertilization does not always occur in the abortive ovules. The most common type of abortive ovule is the one in which a normal embryo sac develops, yet fertilization does not occur. The absence of embryo sacs and the occurrence of empty embryo sacs account for abortion in other ovules. All types of abortive ovules can occur in the same ovary. It is proposed that all of the ovules that develop a normal embryo sac are potential seeds, but the first one to be fertilized suppresses the normal development of the others.     

An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes × wild Chinese Vitis species

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3–28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4–55.4%) and plantlet development (11.15–44.6%) were all highest when embryos were cultured on Woody Plant Medium?+?5.7 μM indole-3-acetic acid?+?4.4 μM 6-benzylaminopurine?+?1.4 μM gibberellic acid?+?2% sucrose?+?0.05% casein hydrolysate?+?0.3% activated charcoal?+?0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes?×?wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.     

一叶萩大孢子发生和雌配子体发育

采用石蜡切片法对一叶萩(Flueggea suffruticosa(Pall.)Baill.)雌花大孢子发生及雌配子体的发育过程进行了观察。结果表明:一叶萩子房上位,中轴胎座,三室,每室2枚倒生胚珠,双珠被,厚珠心,具大戟科常见的珠心喙和珠孔塞。胚囊发育为蓼型,成熟胚囊结构为:卵器位于珠孔端,包括2个助细胞和1个卵细胞;中央是1个二核的中央细胞;合点端为3个反足细胞。本研究为大戟科植物生殖生物学和传粉生物学研究提供了基础资料,同时对大戟科植物系统分类研究有一定意义。     

Embryological Observation on the Apomictic Rice-ApⅢ (Shuang 13)

Since 1988s, the phenomenon of multiple seedlings and multiple embryoes of apomictic rice, Ap Ⅲ (Shuang 13) has been interestingly concerned. Embryological investigation of Ap Ⅲ has been carried out with the whole dissection and conventional sectioning technique. The main conclusions are as follows: 1.8. 9 % of 5000 matured caryopsises, contained one embryo in a caryopsis, from which single seedling arose, while 8.9 % twin embryoes and 1.2 % triplet embryoes of which developed two seedlings and triple seedlings respectively; 2. In over 700 young caryopsises, 90.0%～95.0% contained only one embryo, 5.0%～7.0% twin embryoes, except a few of the ovules with absorptive embryo sac or embryo and endosperm. Triplet embryoes caryopsis could not be traced further due to their low frequency; 3. Authors' materials showed that all the single, twin and triplet embryoes were situated at the micropylar end in one embryo sac of young ovule and no adventitious embryo was seen outside the embryo sac of ovules. From the aforementioned results, it can be infered that albeit single embryo is derived from the egg cell yet it could be possible twin or triplet embryocs may be developed from the egg cell and one or two of the synergids either or not through fertilization.     

STRUCTURE AND HISTOGENESIS OF THE EMBRYO OF ACER SACCHARINUM. I. EMBRYO SAC AND PROEMBRYO

Structure of the embryo sac and development of the proembryo of Acer saccharinum L. are described from paraffin sections. The embryo sac is monosporic and identical to the 8-nucleate Polygonum type in all respects. Cell, nuclear, and nucleolar sizes are constant within a narrow range and sharply distinctive for all components of the mature sac. Polar nuclei fuse before double fertilization. The longitudinal axis of symmetry of the egg, zygote, and proembryo is variously oriented with respect to the longitudinal axis of the embryo sac and is determined by the point of attachment of the presumptive egg cell to the sac wall. Subsequent development of the young embryo is responsive to aligning factors within the embryo sac and is collateral with the longitudinal axis of the sac. The first segmentation is transverse to the longitudinal axis of the zygote; the second and third are transverse in the basal cell and longitudinal in the apical cell. Descendants of ci form a short irregular suspensor; ca and m give rise to the apical and basal halves respectively of the embryo proper. The contribution of the proembryonic tiers to the older embryo differs in embryos of different initial orientation. Distribution and orientation of mitosis in the proembryo are shown in two accumulation maps.     

蓝猪耳卵细胞和合子的分离

蓝猪耳(Torenia fournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构.用解剖和酶解-解剖两种方法都能分离出生活卵细胞.用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰.在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%).用酶解-解剖方法也可分离出授粉14 h后的合子细胞.     

Embryo Sac Development in Almond [Prunus dulcis (Mill.) D. A. Webb] as Affected by Cross-, Self- and Non-pollination

Embryo sac development in ‘Nonpareil’ almond wasstudied following cross-, self- and non-pollination under fieldand greenhouse conditions. The embryo sac, which develops accordingto the Polygonum type, does not begin to differentiate untilanthesis in contrast to other Prunus spp. where a well-developedembryo sac is present at the time of flower opening. The developingmegagametophyte appears to be isolated from surrounding nucellartissue by the deposition of a ring of callose, which, as indicatedby aniline blue-induced fluorescence in the walls of nucellarcells, encloses the embryo sac during its elongation. Developmentand growth of the embryo sac following the different pollinationtreatments indicated that embryo sac development was stimulatedby the presence of compatible pollen tubes in the style andfinal elongation growth of the embryo sac was promoted by cross-pollination.Irregularities in megagametophyte development, including delayeddifferentiation of the megaspore mother cell, embryo sac abortionand lack of polar nuclei fusion and embryo sac elongation, werefrequently noted in ovules of self- and non-pollinated flowers. Almond, callose, embryo sac, megagametophyte, pollination, Prunus dulcis (Mill.) D. A. Webb.     

宁夏枸杞大孢子发生和雌配子体发育的细胞学研究

采用半薄切片技术和组织化学染色法对宁夏枸杞大孢子发生和雌配子体发育过程中的细胞结构变化及营养物质积累特征进行了观察。结果表明,(1)宁夏枸杞为中轴胎座,多室子房,倒生胚珠,单珠被,薄珠心类型。(2)位于珠心表皮下的孢原细胞可直接发育为大孢子母细胞,减数分裂后形成直线型大孢子四分体,合点端第一个大孢子发育为功能大孢子,胚囊发育类型为蓼型,具有珠被绒毡层。(3)初形成的胚囊外周组织中没有营养物质积累,成熟胚囊时期出现了大量的淀粉粒且呈珠孔端明显多于合点端的极性分布特征。(4)助细胞的珠孔端具有明显的丝状器结构,呈PAS正反应表现出多糖性质,成熟胚囊具有承珠盘结构。     

白桦雌花发育、大孢子发生及胚胎发育的解剖学观察

白桦雌花从开花到雌性器官的成熟需经历1个月左右的时间,解剖学观察表明：四月下旬越冬的雌蕊原基开始了活跃的分裂和分化。子房和柱头开始生长。四月末开花,五月初授粉。此后胚珠开始长大。五月中旬即分化形成珠被,珠心,珠被为单层珠被,胚珠为厚珠心胚珠,胚珠倒生,五月中下旬,珠心内产生大孢子母细胞,一周左右发育为成熟胚囊-七细胞八核胚囊,五月末完成双受精,白桦胚胎发育经过合子,原胚,球形胚,心形胚和鱼雷形胚等时期最后发育成熟,胚乳发育与胚胎同步,即受精的极核进行几次分裂后形成核型胚乳,胚乳核不断增多,在形成心形胚后胚乳细胞形成细胞壁。     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.