Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

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.     

Callus Induction by (2-Chloroethyl) Phosphonic Acid on Cultured Cotton Ovules

The response of cultured cotton (Gossypium hirsutum L.) ovules to the ethylene releasing growth regulator, (2-chloroethyl) phosphonic acid (ethephon), was measured. Control ovules grown on a complete liquid medium without hormones continued the differentiation normal for cotton, although at a slower rate than in vivo. When ethephon was added to the medium, normal organ and fiber development was inhibited but callus was induced from the micropylar end of the ovule. The callus was extremely friable and produced many free cells in the culture medium. Dry weight of the callused ovules increased over 4-fold and total cell number increased 56% over the controls. Apparently ethylene released from the ethephon stimulated both cell division and cell expansion in forming the callus.     

Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals

Human interferon-alpha 2b (IFN-α2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-α2b, Saccharomyces cerevisiae MF-α factor prepro sequence and a mutated α prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-α2b into the culture medium of P. pastoris. The native secretion signal of IFN-α2b did not secrete protein into the culture medium of P. pastoris. The α prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-α2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-α2b secreted by human lymphocytes. The full α prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-α2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full α prepro sequence was used for the secretion of IFN-α2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l^–1) combined with naphthylacetic acid (NAA; 0.1 mg l^–1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l^–1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.     

Effect of a pretreatment of seed coats with a low-osmolality solution on subsequent [14C] sucrose transport into attached empty ovules of pea

This paper discusses the question as to whether or not the seed coat tissues can‘adapt’to a treatment with a solution containing a low osmoticum concentration, representing an environment which is sub-optimal for assimilate transport into attached surgically modified ovules. Before the start of a pulse-labelling procedure, in experiments on [¹⁴C] sucrose transport into fruits of pea (Pisum sativum) with four empty ovules, two empty ovules were filled with a low-osmolality solution (a 200 mol m^?3 mannitol medium or a solution without mannitol) and the other two ovules were filled with a 400 mol m^?3 mannitol medium. Pretreatment with a low-osmolality medium, during a period of 2–3 h, enhanced subsequent transport of [¹⁴C] sucrose into empty ovules filled with a low-osmolality medium, in comparison with [¹⁴C] sucrose transport into empty ovules filled with a 400mol m^?3 mannitol medium during the pretreatment period. This partial recovery of sink strength of attached empty ovules can be explained as the result of a stimulation of solute efflux from seed coat cells at high cell turgor.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

IN-OVULO EMBRYO CULTURE OF VITIS VINIFERA L. C.V. ‘THOMPSON SEEDLESS’

In-ovulo embryo culture of Vitis vinifera L. c.v. 'Thompson Seedless' was investigated as a method to enable the hybridization of stenospermic seedless grapes. Fertilized ovules taken 68 days after anthesis were cultured on two basal media: 1) Cain's medium; and 2) Stewart and Hsu's (1977) medium, both tested with five separate addenda (IBA, Ergostim, tartaric acid, L-glutamine and L-cysteine). Addition of 10 mm L-cysteine to C medium promoted the growth of more embryos. Three months after culture, embryos were excised and subcultured. Thirty embryos were recovered by this method but only one embryo grew into a plant.     

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%). Received: 27 April 1999 / Accepted: 5 June 1999     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways

The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (secGFP) and secreted rat preputial β-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries. Maria Rosaria Leucci and Gian-Pietro Di Sansebastiano contribute equally to this work. Giuseppe Dalessandro and Gabriella Piro wish to dedicate this work to the memory of Professor Don Northcote.     

The effects of copper and zinc on <Emphasis Type="Italic">Spirulina platensis</Emphasis> growth and heavy metal accumulation in its cells

The effects of copper and zinc on Spirulina platensis (Nordst.) Geitl. growth and the capability of this cyanobacterium for accumulation of these heavy metals (HMs) were studied. S. platensis tolerance to HMs was shown to depend on the culture growth phase. When copper was added during the lag phase, its lethal concentration was 5 mg/l, whereas 4 mg/l were lethal during the linear growth phase. Zinc concentration of 8.8 mg/l was lethal during the linear but not lag phase of growth. HM-treated S. platensis cells were capable for accumulation of tenfold more copper and zinc than control cells. Independently of Cu²⁺ content in the medium and of the growth phase, cell cultures accumulated the highest amount of this metal as soon as after 1 h, which may be partially determined by its primary sorption by cell-wall polysaccharides. A subsequent substantial decrease in the intracellular copper content occurred due to it secretion, which was evident from the increased metal concentration in the culturing medium. When zinc was added during the linear growth phase, similar pattern of its accumulation was observed: the highest content after 1 h and its subsequent decrease to the initial level. When the initial density of the culture was low and the cells had much time to adapt to HM, zinc accumulated during the entire linear growth phase, and thereafter the metal was secreted to the medium. The mechanisms of S. platensis tolerance to HM related to both their sorption by the cell walls and secretion of metal excess into the culturing medium and its conversion into the form inaccessible for the cells are discussed.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 259–265.Original Russian Text Copyright © 2005 by Nalimova, Popova, Tsoglin, Pronina.This revised version was published online in April 2005 with a corrected cover date.     

The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l^-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.     

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     

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose‐deficient maize cells

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall‐bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 μM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight‐average relative molecular mass. In addition, polysaccharides underwent massive cross‐linking after being secreted into the cell wall, but this cross‐linking was less pronounced in habituated cells than in non‐habituated ones. However, when relativized, ferulic acid and p‐coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight‐average relative molecular mass, although cross‐linked, as a part of their strategy to compensate for the lack of cellulose.     

Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum) Ovules Grown in Vitro

Boron deficiency and phytohormone interactions have been studied in unfertilized cotton (Gossypium hirsutum) ovules grown in vitro. Such ovules required exogenous indoleacetic acid and/or gibberellic acid for fiber elongation. Boron also was required for maintenance of fiber elongation and normal morphogenesis throughout 14 days of culture. The amount of exogenous boron necessary for maximum fiber elongation varied among experiments, presumably in relation to endogenous boron levels at anthesis. Some ovular epidermal cells distant from the liquid medium could be induced to elongate into fiber even after 6 days in boron-deficient medium in response to the later addition of boron.     

Biosynthesis of Inositol by an Enzyme System in the Rice Seeds

As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the othe hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The preprosequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell. Integration of the fusion gene into the chromosome at a copy number of eight led to secretion of the fused protein in a larger amount than the case when the fusion gene was carried on a 2-µm plasmid with its copy number of a few hundreds, because the 2-µm derived plasmid containing the fusion gene was very unstable in the yeast cells. The secretion level was also improved by changing g the culture conditions. A maximum yield of hApoE part in the secreted fused protein was estimated to be 23.7 mg per liter and the amount of the fused protein was calculated to be 53.0 mg per liter.     

百合未授粉子房离体培养胚胎形成及植株再生

未受精子房离体培养是诱导雌核产生单倍体的技术之一。以1个野生种和3个杂种系共7个百合(Lilium)基因型为实验材料, 探讨了基础培养基、花蕾取样时期和外源激素等因素对百合未授粉子房离体培养胚胎形成的影响。结果表明, CBM、MS和BDS三种基础培养基均可诱导百合未授粉子房胚胎形成, 但以BDS培养基诱导效果最佳; 开花前1天的花蕾较适于百合未授粉子房离体培养; 2 mg·L^-1 2,4-D + 2 mg·L^-1 6-BA和2 mg·L^-1 2,4-D + 2 mg·L^-1 KT两种外源激素配方均适用于百合未授粉子房离体培养诱导胚胎形成。在培养过程中, 大多数胚性胚珠中只含有1个胚胎, 位于珠孔端、合子端或极核处, 少数胚性胚珠中含有双胚胎。通过百合未授粉子房离体培养, 从5个基因型材料中共获得146株再生植株。采用根尖染色体计数法对其中的62株进行了倍性测定, 其中43株与母体植株染色体倍性不同。     

Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author