Differential Regeneration Response in Two Cotyledon Types of Vigna radiata: Histomorphological Analysis and Effect of α-arabinogalactan

A unique differential regeneration response of the two cotyledon types, ‘Cot E’ (attached to the embryonal axis) and ‘Cot’ was reported earlier in Vigna radiata. The histological study revealed that there is a temporal difference in meristemoid development between ‘Cot E’ and ‘Cot’. The meristematic tissue differentiate directly from the epidermal/sub-epidermal layers of ‘Cot E’, whereas, callus mediated differentiation occurs in the ‘Cot’. It was observed that the frequency of shoot differentiation in ‘Cot’ explants increased distinctively when 20 mg l^?1 of β-Arabinogalactan (β-ABG) was supplemented in the medium and an altered mode of regeneration was noted like that of ‘Cot E’. While, under the same condition, the regeneration frequency decreased substantially in ‘Cot E’ and the explants became necrotic. The results indicate that the de-embryonated ‘Cot E’ grown in vitro contain more endogenous arabinogalactan proteins (AGPs) than ‘Cot’, presumably due to the wound formation during excision, as AGP is wound inducible. And perhaps due to the feed back inhibition, addition of β-ABG to the differentiation-inducing medium either resulted in necrosis of ‘Cot E’ or sharp decrease in regeneration efficiency. It was postulated that glycosylation of cytosolic peptides occurs when β-ABG was supplemented externally in the culture medium and resulted in AGP synthesis. Presence of AGP in the cotyledonary tissues was demonstrated through gel electrophoretic study and also verified by histochemical localization in these explant types. This is the first report showing presence of AGP in ‘V. radiata’. The dose dependent phytohormone like effect of β-ABG suggests its role as precursor for a novel ‘growth regulator’, AGP.     

Differential Protein Pattern of Two Cotyledon Explants of Vigna radiata During Induced In Vitro Differentiation: Probable Implication in the Conundrum of Differential Regeneration Response

Differential regeneration response in two cotyledon types (Cot and Cot E) of Vigna radiata was reported earlier. The Cot (one cotyledon) is easily detachable from the germinating embryonal axis, whereas, Cot E (the other cotyledon) remains firmly attached during seed germination or after imbibition. Shoots differentiated directly from the Cot E under the induction of in vitro differentiation, while, under the same milieu, shoot differentiation was preceded by callus differentiation from Cot. In this study, we present comparative analyses of protein profiles from these two explant types recorded at different point of time during induction of differentiation. Cot E always contained higher amounts of soluble protein than the Cot. Likewise, higher de novo protein synthesis was noted in Cot E than in Cot as revealed by ³⁵S methionine labeled study. Two polypeptides of ～37 and 84 kD disappeared earlier from Cot E than Cot and is presumed to be linked with shoot induction. Two marker proteins of ～88 and 158 kD synthesized during shoot differentiation were apparent. It was observed that the labeled protein synthesis initiated within 3h in Cot E under in vitro condition, while, no labeled protein was detected from Cot even at 12h. Irrespective of the mode of differentiation, a large amount of protein was hydrolyzed during the process of differentiation. However, in case of Cot, the process was delayed by a day than Cot E. In all probability, this is an indication of delayed cytokinin induced rejuvenation of Cot. Temporal difference in protein profile was also evident in these two explant types during in vitro differentiation. Yet, three major groups of proteins were consistently present in both the explants. The biochemical differences recorded between these two explants during induction of in vitro differentiation reflects the temporal difference in gene expression. Perhaps Cot E has the distinctiveness due to the temporal differences in certain key gene expression and proved its greater suitability over Cot for shoot regeneration purposes.     

Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata

Vigna radiata or mungbean belongs to the legume family of plants. Mature mungbean seeds are rich source of dietary proteins for human nutrition. The present study was aimed to analyze the comparative protein profiles of two cotyledon types, Cot and Cot E, prior to and during early time points of shoot morphogenic induction to understand the unique differential regeneration response in these two explant types which was reported earlier. These explants were grown separately in vitro on the shoot induction medium (SIM) containing Gamborg’s B5 basal nutrient composition supplemented with 15 μM N₆-benzyladenine. Isolation and characterization of the proteomes from Cot and Cot E explants at different time points, during early events of shoot differentiation, were performed using two dimensional gel electrophoresis following matrix assisted laser desorption-ionization tandem mass spectrometry. A total of 112 differentially identified proteins were classified according to their putative biological function. The differential control of protein synthesis between these explants under control condition, i.e. before in vitro culture, was also noted. In Cot E explants SIM induced prompt acquisition of competence for direct shoot morphogenesis probably through fast phytohormone signaling. Over accumulated proteins in Cot E indicated stimulation of several metabolic and associated pathways earlier than Cot explants. Abundance of stress and defense related proteins in Cot E explants was presumably to cope up with stressful cultural condition. Enhanced accumulation of folding-assisted proteins involved in organogenesis mediated cellular reprogramming in Cot E explants contributed further in rapid and efficient regeneration responsiveness.     

Structure of the zygotic embryos and seedlings of Butia capitata (Arecaceae)

Butia capitata, an endemic palm of the Brazilian savanna threatened by deforestation, demonstrates low germinability due to seed dormancy. The present study characterizes the structure of the zygotic embryo and describes germination and seedling development. Pyrenes were sown into sandy soil substrates to germinate, and their embryos were also cultivated in vitro in MS medium; structural evaluations were made during their development. Seedling growth through the endocarp germ pore culminates in the protrusion of the cotyledonary petiole, with the root and leaf sheaths subsequently being emitted laterally from its extremity. The embryos are composed of the cotyledon (whose proximal third has a haustorial function) and a diminutive embryo axis that is contained within the cotyledonary petiole. The protoderm, ground meristem, and procambium can be observed in their typical positions in the embryo axis and cotyledon. The development of the vegetative axis could be observed on the second day of in vitro cultivation, with elongation of the embryo axis and the beginning of the differentiation of the first eophyll. Elongation of the cotyledonary petiole and the differentiation of the parenchyma and tracheary elements were observed during the second to fifth day. Although the hypocotyl–radicle axis is less differentiated than the plumule, root protrusion occurs on the eighth day, and the leaf sheaths are only emitted between the twelfth and the sixteenth days; the haustorium atrophied during this stage. The embryonic structure of B. capitata does not impose limitations on seed germination as dormancy is of the non-profound physiological type, and the 50 % elongation of the cotyledonary petiole serves as a morphological indicator of germination.     

Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development

Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA-protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction.     

两种品质类型花生品质形成与子叶细胞超微结构的关系

在大田条件下研究了两种品质类型花生(Arachis hypogaea)品质形成的动态差异及其子叶细胞超微结构的差异。结果表明, 高蛋白品种‘XB023’的蛋白质含量在籽仁发育前期较高油品种‘鲁花9号’低, 后期显著高于‘鲁花9号’, 且成熟期籽仁8种必需氨基酸组分含量均高于‘鲁花9号’, 其中谷氨酸、赖氨酸和亮氨酸含量差异极显著; ‘XB023’脂肪含量在籽仁发育期一直低于‘鲁花9号’。‘XB023’各时期的籽仁可溶性糖含量和油酸/亚油酸(O/L)值均显著低于‘鲁花9号’。两品种在果针入土10天时子叶细胞即形成淀粉粒、脂体和蛋白体, 随后脂体、蛋白体的数量不断增加, 淀粉粒先增大后逐渐缩小解体。‘XB023’的脂体达到最大的时间早于‘鲁花9号’, 而‘鲁花9号’的脂体快速积累的时间比‘XB023’长。两品种蛋白体大小都在果针入土40天时达到最大值, ‘XB023’的蛋白体在籽仁发育后期数量增加较快。高蛋白品种较高的蛋白质含量由其子叶细胞中较大蛋白体的大小和较多的蛋白体数量决定, 而高油品种较高的脂肪含量是由其较多的脂体数量决定。     

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Maternal genotype influences pea seed size by controlling both mitotic activity during early embryogenesis and final endoreduplication level/cotyledon cell size in mature seed

When reciprocal crosses are made between different pea genotypes, there is a strong maternal influence on mature seed size of the reciprocal hybrids, i.e. their dry weights are similar to that of seeds obtained from their maternal parents. Reciprocal crosses between pea varieties having very different mature seed sizes were used to investigate how the maternal genotype controls seed development and mature seed size. The differences in dry seed weight between genotypes and reciprocal hybrids reflected differences in both cotyledon cell number and mean cell volume, and the maternal control on the establishment of these two traits was investigated. Using flow cytometry, data relative to endoreduplication kinetics in cotyledons during the transition between the cell division phase and maturation were obtained. The appearance of nuclei having an 8C DNA content indicates the initiation of the endoreduplication phenomenon and thus the end of the cell division phase. It was shown that the duration of the cell division phase was the same in the reciprocal hybrids, its value being intermediate between those recorded for their maternal parents. This result indicates that the timing of development of the embryo is not under maternal control, but depends on its own genotype. Consequently, maternal genotype must influence the mitotic rate during the cell division phase to achieve differences in cell number found in the cotyledons of mature F1-reciprocal hybrids. The final level of endoreduplication in cotyledons of mature seeds was also investigated. This study showed that there is a close relationship (r2 = 0.919) between the endoreduplication level in mature cotyledons and seed dry weight or mean volume of cotyledon cells, suggesting that both maternal and non-maternal factors could control the number of endoreduplicating cycles in the cotyledons and, hypothetically, the cotyledon cell size.     

Influence des corrélations entre organes sur la croissance et le développement floral des bourgeons cotylédonaires chez leScrofularia arguta Sol.

The importance of various correlative influences on growth and vegetative or floral development of cotyledonary buds inScrofularia arguta Sol. is shown. The terminal bud, on the one hand, inhibits growth of cotyledonary buds and, on the other hand, induces their early flowering. The cotyledon stimulates growth of its axillary bud, but has no action on its floral development. Leaves above the cotyledonary node have the same effect as the cotyledon. Finally, roots stimulate vegetative growth of cotyledonary buds and suppress floral expression, but only when apical dominance has been removed at an early stage of development.     

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco

Summary A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm. The mature seed thus consists of a seed coat of one rigid cell layer, and a degenerated embryo. The genetic ablation technique has thus contributed to the establishment of the sequence of events and elucidation of the role of different cell lineages and tissues in seed development.     

槐种子发育过程中子叶细胞显微结构的变化和过氧化物酶同工酶变化的动态

槐（SophorajaponicaL．）种子发育过程中,子叶细胞大量合成和积累储藏物质,光镜和电镜下用组织化学方法观察到开花后30d,叶肉细胞高度液泡化,其中分布着大量的质体、粗面内质网、一些线粒体和小脂体。淀粉粒开始积累。开花后60d,淀粉粒和脂体不断长大,数量明显增多,液泡中蛋白体开始形成。从花后90d开始,液泡中有大量蛋白体形成且不断长大,有些蛋白体变得不规则形。至种子成熟期,叶肉细胞几乎完全被蛋白体及脂体所填充。淀粉粒及大多数细胞器消失。基于上述观察,还对种子发育过程中子叶细胞过氧化物酶活力及同工酶港的变化进行了观察和分析。     

Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos

Variations in carbohydrates and proteins were monitored during avocado (Persea americana Mill.) zygotic embryo development and correlated with growth parameters in order to define specific markers characterizing distinct embryogenic phases. Hexose (glucose and fructose) levels were initially high and declined as embryo development advanced reaching the lowest levels in completely mature embryos. Sucrose and starch evolution showed an opposite trend with a progressive increase during embryo growth. The beginning of the maturation phase could be identified by a switch in the carbohydrate status from high hexose/sucrose ratio to low hexose/sucrose ratio. Storage protein accumulation began at early cotyledonary stages (7–8 mm), increasing significantly in the maturation phase where they represented 83% of total proteins. Mature embryos (38–40 mm) contained albumins, globulins and glutelins, albumins being the predominant and most heterogeneous fraction. Storage protein accumulation occurred in a sequential and specific way suggesting a possible role as indicators of embryo development. The complete maturation stage could be characterized by the synthesis and accumulation of a 49 kDa albumin.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Role of ethylene in cotyledon development of microspore-derived embryos of Brassica napus

Ethylene production during seed development in Brassica napus occurs first at 20 d after pollination (DAP), while a second greater peak occurs at 35 DAP. Because of the inaccessible location of the embryo within the maternal tissue, microspore-derived embryos (MDEs) of B. napus were used as a model for studying the role of ethylene during embryo development. The MDEs also produced a peak in ethylene evolution at 20 DAC (i.e. the early cotyledonary stage), dropping to minimal levels by 25-30 DAC. At 20 DAC the excised cotyledon evolved 85% of the ethylene found in the whole MDE. To determine the role of ethylene, MDEs were treated with aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis), CoCl(2) (an inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase), and silver thiosulphate (STS, an inhibitor of ethylene action). An inhibition in ethylene production or action at 20 DAC resulted in diminished lateral cotyledon expansion, due to a reduction in the lateral expansion of cells within the cotyledon. Recovery to 'control-type' levels of cotyledon cell expansion was achieved by application of ACC (the metabolic precursor of ethylene) to AVG-treated MDEs. Thus, ethylene production at 20 DAP likely controls cotyledon expansion during embryo development.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

The Ultrastructural Evidence on the Origin of Protein Bodies in the Rough Endoplasmic Reticulum of Developing Cotyledons of Soybean

The process of protein body formation from rough endoplasmicreticulum (RER) in cotyledon cells of soybean has been followed.From about 43 d after flowering (DAF), the lumen of some partsof RER cisternae was dilated and filled gradually with proteinaceousmaterial. This kind of dilated RER expanded further to formlong and irregularly shaped protein bodies (PBs), and the latterdivided into smaller spherical protein bodies in mature seedsat 63 DAF. From these results and our previous observations,we draw the conclusion that there are two pathways of proteinbody formation in developing cotyledon cells in soybean. Duringthe early stage, vacuoles in the cells were filled with proteinaceousmaterial and turned into protein bodies. During the late stage,some of the dilated RER with storage proteins developed intoPBs A few vacuoles can also form PBs at this late stage. Inaddition, some fibre structures, 7–8 nm in width, wereseen to be oriented in parallel in longitudinal sections ofRER cisternae in cotyledon cells at 45 DAF. Soybean, protein body, ER origin, storage protein