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.     

Nutrient and hormone levels in cotton ovules during embryony

In vitro zygotic and somatic embryogenesis protocols rely on nutrient and hormone levels from media to satisfy the physiological and developmental requirements of embryony. To better understand these requirements for cotton, we quantified levels of major and minor elements, carbohydrates, NH₄ ⁺, free amino acids and six hormones in whole cotton ovules (with fibers removed), nucelli (ovules with integuments removed), or ovule fluid (extracted from the endosperm region). Samples were collected from field-grown cotton at 1–18 days-past-anthesis (DPA) during each of three growing seasons. Replication across 2 years was obtained for carbohydrates, NH₄ ⁺, free amino acids and hormones from nucellus samples. The year effect was large primarily for hormones only. The most abundant minerals across tissue types and years were K, P, Mg and S. Potassium was the most abundant at 260, 600 and 1,660 mmol kg⁻¹ dry mass (DM) in nucelli, whole ovules and ovule fluid, respectively. Magnesium, Ca, Zn and Mn levels were 2–8-fold higher in ovule fluid compared to whole ovules or nucelli. In the free amino acid plus NH₄ ⁺ category, NH₄ ⁺, alanine, serine, glycine, asparagine (plus aspartic acid), glutamine (plus glutamic acid), leucine, threonine and arginine predominated in nucelli and ovule fluid, and levels tended to be higher in the older samples across years and tissue types. Fructose and glucose levels also increased with age with very high levels being found in late DPA ovule fluid. Arabinose, inositol and melibiose were also prominent sugars. Indole-3-acetic acid levels were similar between nucelli and ovule fluid and ranged from 10 to 80 μmol kg⁻¹ DM. An abscisic acid spike, from 15 to 400 μmol kg⁻¹ DM, occurred in nucelli and whole ovules from 2 to 8 DPA. Thereafter, abscisic acid levels remained between 5 and 10 μmol kg⁻¹ DM. Zeatin and zeatin riboside were the most abundant cytokinins, and levels of these hormones fluctuated between 1 and 4 μmol kg⁻¹ DM in both nucelli and ovule fluid.     

Exogenous Jasmonic Acid Inhibits Cotton Fiber Elongation

Jasmonic acid (JA) is a well-characterized phytohormone that acts in various ways to influence plant development. Its role in cotton fiber development, however, has not yet been thoroughly explored. In this study, JA was proven to be an inhibitor of ovule and fiber development in vitro. Continuous exogenous JA application inhibited fiber elongation. This effect was dependent on development stage and dosage. Fibers and ovules at three different stages of development and different JA dosages were compared. The most serious suppression was detected when ovules 1?day before anthesis (–1?DPA) were cultured in medium with 2.5?μM JA. Genes related to trichome and fiber development responded differently to JA treatment between –1?DPA and 1?day post anthesis (1 DPA). JAs (JA and JA-Ile) quantification showed that JAs content was sharply decreased from –1?DPA to 5?DPA ovules, which indicated that JA was negatively associated with fiber elongation in vivo. In addition, gene expression analysis showed the same trend. Our results demonstrate that there was a negative relationship of JA with fiber elongation in vitro and in vivo. These results are meaningful for uncovering the mechanism of fiber elongation in cotton.     

Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.)

 In many plant species with multiovulate ovaries, a considerable reduction in the number of ovules takes place. However, the underlying physiological causes are not clear. In Prunus spp., although flowers present two ovules, usually only one seed is produced. We have followed the development and degeneration of the two ovules in apricot (Prunus armeniaca L.) and examined the extent to which carbohydrates within the ovule might be involved in determining the fate of the ovule. While the primary ovule grows in the days following anthesis, growth of the secondary ovule is arrested. Starch distribution along the different ovular tissues exhibits several changes that are different in the two ovules. Primary ovule growth is inversely related to starch content and this growth takes place independently of pollination since it occurs in the same way in pollinated and unpollinated flowers. In the secondary ovule, starch disappears simultaneously from all ovular structures and callose is layered at the chalazal end of the nucellus. The size of the secondary ovule does not change significantly from anthesis to degeneration, and callose starts to accumulate 5 days after anthesis. Likewise, this process occurs independently of pollination. These results are discussed in terms of the implications of the starch content of ovules in fertilization success and ovule fate. Received: 26 August 1997 / Revision accepted: 17 December 1997     

小麦受精过程的细胞化学研究

小麦成熟胚囊卵细胞中存在较多围核分布的淀粉粒和少量散布的脂类颗粒;两个助细胞中积累很多脂类,未见有淀粉粒存在;中央细胞中存在中等量均匀分布的淀粉粒和脂类颗粒。受精时期,胚囊内各细胞中淀粉粒变化不大。精卵核融合时,卵细胞和中央细胞中的脂类分别存在一个积累高峰。合子与相应时期游离核胚乳中的脂类颗粒均较少。原胚初期,每个原胚细胞及胚乳原生质中均积累较多脂类。珠孔附近的内珠被细胞中脂类颗粒较多,并存在一个有规律的变化。在观察的所有发育时期的胚珠中,均未发现贮存蛋白质。胚珠中脂类的一系列变化可能与雌性细胞的营养、胚胎发育初期的养料及花粉管的定向生长等有关     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

小麦果皮绿色层中^1^4C标记同化物在胚珠内的分配与定位

小麦离体颖果饲喂~14CO_2后,经冷冻取代,于膜显微放射性自显影,追踪与检查了包括可溶性糖在内的~14C标记同化物在胚珠内的分配与定位。颖果绿色层细胞的高光合效率不仅保证了同化物的即时输出,而且还可以淀粉形式暂贮在叶绿体内。绿色层光合产物主要向腹沟处汇集,通过该处的维管束与珠心突起向胚乳方向迁移。~14C标记同化物以逆浓差梯度方式从珠心向珠心—胚乳交界处质外体空间输出并在胚乳内呈不均匀分布和富集于原胚端活跃增殖生长的区段内。绿色层同化物以多种方式进入原胚,主要以可溶态形式存在。同化物可能通过珠孔直接就近为原胚吸收。     

漆树胚,胚乳发育及花果生长的相关性研究

漆树为倒生胚珠,双珠被,厚珠心,具承珠盘及拟珠孔塞,胚囊发育为蓼型,核型胚乳,胚发育为柳叶菜型,后历经棒状形胚、心形胚、鱼雷形胚和成熟胚各期。花和果实生长与胚及胚乳发育有密切的相关性,胚内具原始的乳汁道系统为重要特征。一些胚珠内无胚或胚乳早期退化引起胚败育是造成种子空籽原因之一。     

Plant regeneration from ryegrass ovules cultivated on endosperm-derived feeder cells

An efficient method for the regeneration of zygote-derived plants via ovule culture is desirable for overcoming postzygotic cross incompatibility as well as for the development of certain methods for genetic manipulation. High-frequency plantlet regeneration from ovules of Italian ryegrass (Lolium multiflorum Lam.) and a hybrid Italian/perennial ryegrass excised 1 to 4 days post pollination was obtained by culture on endosperm-derived feeder cells. Ovules excised 3 or 4 days after anthesis and grown on feeder cells generally regenerated about twice as frequently as ovules grown directly on nutrient medium. In one of the genotypes tested, ovules excised 1, 2 and 3 days post pollination developed into plantlets at percentages of 38.1, 52.0 and 52.8, respectively, using the feeder-cell system.Abbreviations EM endosperm multiplications - OC ovule culture - R regeneration - 2,4-d 2,4-dichlorophenoxyacetic acid     

Variability in the developmental stage of apricot ovules at anthesis and its relationship with fruit set

The stage of ovule development at anthesis and its relationship with fruit set was studied in several apricot cultivars growing in Mediterranean climatic conditions. Although generally the ovule was immature at anthesis, a great variability was found in the stage of development of the ovules from different cultivars. Considering functional ovules to be those with an embryo sac with at least four nuclei at anthesis, the earliest flowering varieties frequently showed more than 50% of functional ovules. Though these results could suggest that there is an influence of the chilling requirement on the percentage of functional ovules at anthesis, data recorded from two cultivars, ‘Goldrich’ and ‘Colorao’, with high chilling requirements, contrast with this suggestion. These results indicate that in apricot flowers the development of the embryo sac at anthesis is genotype dependent. The fact that a high frequency of functional ovules at anthesis was found, in cultivars with more than 50% of fruit set, suggests that, to be fertilised, a certain level of development of the apricot ovules at this time is necessary.     

EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM UNFERTILIZED COTTON OVULES

Fertilization of cotton ovules was prevented by removal of styles and stamens on the morning of anthesis. Forty-eight hr later ovaries were harvested and ovules were aseptically transferred to liquid culture medium supplemented with various plant growth substances. In the absence of phytohormones, ovules browned and failed to increase in size or produce fibers. Indoleacetic acid and gibberellic acid provided for ovule growth and fiber development. Kinetin provided for ovule growth only. The ovule's capacity for indoleacetic acid- or gibberellic acid-stimulation of fiber development was reduced by high concentrations of kinetin or abscisic acid. Low concentrations of kinetin partially reversed the inhibitory effect of abscisic acid.     

四倍体双穗雀稗兼性无孢子生殖的研究

研究了四倍体双穗雀稗(Paspalum distichum L)无孢子生殖胚囊、胚胎发育以及假受精特点。当其大孢子母细胞发育至四分体阶段时,大多数情况下会发生四分体退化,同时有多个特化珠心细胞发育为1—3个无孢子生殖胚囊的现象。成熟无孢子生殖胚囊一般3核,包括1个卵细胞和2个极核。卵细胞在抽穗前就能自发分裂形成原胚团,而极核则在抽穗和传粉后参与假受精形成胚乳。当胚珠内存在多个无孢子生殖胚囊时,只是靠近珠孔端的1个无孢子生殖胚囊内的极核与精核结合,而其它的并不参与。种子成熟后出现很低频率的二胚苗。此外,还能观察到少量的有性生殖胚囊的发育以及有性生殖胚囊和无孢子生殖胚囊在同一胚珠中的发育现象,因此判断该类群为兼性无孢子生殖体。     

Chronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells

Cotton fibers are single elongated cells that develop from epidermal cells of the ovule. The chronology of fiber differentiation was investigated using cultured ovules. Epidermal cells differentiate into fiber cells approx. 3 d before anthesis. When ovules were cultured on a defined medium, fiber growth could be initiated on ovules any time between 2 d preanthesis and the time of anthesis by adding indole-3-acetic acid and gibberellic acid to the medium. In the absence of phytohormones, fibers did not grow, and when ovules between 2 d preanthesis and anthesis were cultured without hormones past the day of anthesis and hormones then added, most ovules failed to produce fibers. The results define the timing of fiber differentiation from epidermal cells, and also define a window of time when differentiated cells are capable of further development. During this window, fiber cells are latent awaiting appropriate stimulation which in the intact plant is apparently associated with anthesis.Abbreviations GA₃ gibberellic acid - IAA indole-3-acetic acid     

Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA₃) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA₃, which implied that fiber initiation could be promoted by them as additive.     

Simulating in ovulo osmotic potentials and O<Subscript>2</Subscript> tensions normalize growth and pigmentation of immature cotton embryos

Oxygen tensions and osmotic potentials are important physiological factors of plant growth and development. To optimize these variables for cotton (Gossypium hirsutum L.) embryo culture, we quantified dissolved O₂ (dO₂) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O₂ microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO₂ tensions were determined in integuments, nucelli and embryos. Ovular osmotic potentials and pH were determined from extracted ovule sap using vapor pressure osmometers and pH microelectrodes. Dissolved O₂ tensions near or in embryos decreased from 104 mmol m⁻³ at 5 days post-anthesis (DPA) to 83 mmol m⁻³ at 18 DPA. Osmotic potentials of ovule sap decreased from −0.70 megapascals (MPa) at 2 DPA to −1.12 MPa at 8 DPA but then increased to −0.84 MPa by 17 DPA. Ovule sap pH at 5–17 DPA varied inconsistently and ranged from 5.4 to 6.5. Based on these results, a factorial experiment with two osmotic and three O₂ treatments was designed. Immature embryos of cotton cultivar HS-26 were randomly assigned to the treatment combinations and cultured for 33 days. Oxygen treatments did not affect embryo growth, and there were no differences among treatments with regard to percentage of embryos that progressed to a more advanced stage of embryo development. However, cotyledons of embryos grown without osmotic adjustment were abnormally large, and embryos exposed to these treatments were abnormally brown. Browning was less severe for embryos exposed to low O₂ tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O₂ tensions and osmotic potentials that best simulated the observed in ovulo conditions.     

Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules

Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In fruits containing 4–6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 m M ), medium (350, 400 or 500 m M ) or high (0.7 or 1 M ) concentration of sucrose and/or mannitol. In pulse-labelling experiments with sucrose and α-aminoisobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules.
In our experiments, 350 m M appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmolarity of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in the seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the "sink strength" of developing seeds is turgor-sensitive.     

Ovule differences between single-kernelled and double-kernelled fruits in almond (Prunus dulcis)

In order to detect differences between almond cultivars that commonly produce one or two seeds, we compared ovule sizes and developmental stages at anthesis and three days after anthesis. The results showed that the presence of only small differences in size between primary and secondary ovules during flowering seemed to be a necessary but not sufficient condition for the production of double‐kernelled fruits. In contrast, differences in the developmental stage of both ovules did not prevent the production of double kernels. Large differences in ovule size at anthesis or in the following days resulted in fruits with single seeds. The behaviour of the secondary ovules in cultivars producing fruit with single seeds was very different, even in closely related cultivars. The degeneration of the secondary ovule seems to be genetically programmed and follows a genotype‐dependent pattern.     

Ovule development at anthesis in apricot (Prunus armeniaca) varieties in a Mediterranean climate

The stage of ovule development at anthesis has been studied in 2002 and 2003 in 35 apricot varieties and 4 new apricot cultivars grown in a Mediterranean climate. Significant differences were found among varieties as well as a high heterogeneity of the developmental stage of the ovules examined in each variety. In general, a relative delay of the ovule development was observed, the presence of mature ovules at anthesis being negligible. In the 2 years of the study, differences in the developmental stage of primary ovules at anthesis were not significant and correlation to flowering time was not observed, which emphasised the previously reported genetic determination of this trait.     

海南山薯雌花发育及胚胎发育的研究

通过研究山薯的雌花及胚胎发育,为山薯的胚胎学研究以及杂交育种奠定基础。结果表明:山薯大部分为雌雄异株,海南岛的山薯雌花花期约3个月,为9月初至11月末。子房3室,每室有2个倒生胚珠;胚珠具厚珠心,双珠被。珠孔一端表皮下的孢原细胞逐渐发育为大孢子母细胞。大孢子母细胞减数分裂形成4个呈线形排列的大孢子,其中只有1个可以发育为功能大孢子。成熟的胚囊为7胞8核胚囊,其胚囊发育类型为蓼型。卵细胞的受精属于有丝分裂前型。其胚的发育类型为柳叶菜型,经过二细胞原胚、倒T型原胚、棒状胚、球形胚和梨形胚这5个发育阶段。胚乳的发育为核型。