Patterns of somatic embryo formation in Siberian larch: embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Somatic embryogenesis from different tissues of Spanish populations of maritime pine

The somatic embryogenesis (SE) capacity of megagametophytes belonging to Continental and Mediterranean Spanish provenances of maritime pine (Pinus pinaster Aiton) was studied, noting factors (megagametophyte developmental stage and culture medium) that enhanced the induction and establishment of SE lines. In both provenances, initiation and establishment of embryogenic calli was higher on megagametophytes in which the dominant zygotic embryo had begun to develop. In the Mediterranean provenance, however, SE lines were also established from megagametophytes enclosing zygotic embryos with well-developed cotyledons. A modified Litvay medium (mLV) containing 9.9???M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4???M 6-benzyladenine (BA) was superior to DCR medium containing 13.6???M 2,4-D and 4.4???M BA for SE induction, but there were no differences between media in terms of the number of SE lines established after 4?months in culture (153 vs. 155 established SE lines, for mLV and DCR media respectively). Of the 26 embryogenic lines tested for maturation, 15 (58?%) produced cotyledonary somatic embryos and 75?% of these gave rise to plants on germination medium. SE-like cultures from adult maritime pine trees were also initiated, but embryogenic lines could not be established. This is the first report on the production of SE in maritime pine of Continental and Mediterranean origin. The micropropagation protocols presented here provide an important tool for the vegetative multiplication of selected families and breeding programs for maritime pines from Spain.     

Amino Acid Utilization in Seeds of Loblolly Pine during Germination and Early Seedling Growth (I. Arginine and Arginase Activity)

The mobilization and utilization of the major storage proteins in loblolly pine (Pinus taeda L.) seeds following imbibition were investigated. Most of the seed protein reserves were contained within the megagametophyte. Breakdown of these proteins occurred primarily following radicle emergence and correlated with a substantial increase in the free amino acid pool in the seedling; the majority of this increase appeared to be the result of export from the megagametophyte. The megagametophyte was able to break down storage proteins and export free amino acids in the absence of the seedling. Arginine (Arg) was the most abundant amino acid among the principal storage proteins of the megagametophyte and was a major component of the free amino acid pools in both the seedling and the megagametophyte. The increase in free Arg coincided with a marked increase in arginase activity, mainly localized within the cotyledons and epicotyl of the seedling. Arginase activity was negligible in isolated seedlings. Experiments with phenylphosphorodiamidate, a urease inhibitor, supported the hypothesis that arginase participates in Arg metabolism in the seedling. The results of this study indicate that Arg could play an important role in the nutrition of loblolly pine during early seedling growth.     

Growth and division of carrot cells in suspension culture

In a submerged culture of a strain of carrot cells, cellularmorphology and the mode of cell division were greatly affectedby growth factor(s) added to the medium. In the presence of2,4-D, cells showed two-dimensional growth and often formedtetrad-like structure after a set of two divisions. The sequenceof events was observed microscopically. Orientation of cellgrowth changed after the first division and the second cellplate formed at an oblique angle to the first. When IAA wasadded, instead of 2,4-D, cells showed one-dimensional growthand developed to a filamentous form. (Received June 1, 1970; )     

Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.     

Decline of dark coniferous stands in Baikal Region

The reasons for the decline in Siberian pine and fir in the Baikal Region (Khamar-Daban) were analyzed using remote sensing techniques, dendrochronology and GIS-technology methods, and in situ observations. It is found that a decrease in the value of the growth index (R ² = 0.69) and an decrease in the SPEI drought index (Standardized Precipitation–Evapotranspiration Index) (R ² = 0.72) has been observed since the 1980s. In the mid-2000s, the increase in aridity led to the division of Siberian pine trees into two cohorts: “survivors” and “decliners.” The spatial distribution of these cohorts is different: dead and declining stands are localized mainly on relief elements with increased risk of water stress (steep and convex slopes of southwestern exposure). The growth index of the trees is closely related to the dryness index in June (r ² = 0.55). Along with water stress, declining trees were also exposed to stem pests and plant pathogens. The primary cause of Siberian pine decline is water stress due to the increasing climate aridity. The weakened waterstressed trees were sensitized to pathogens. The synergism of climatic and biotic effects led to the decline of Siberian pine stands. On the whole, heavily damaged and declining stands (over 50% of dead and declining trees) within the Khamar-Daban ridge are 8–10% of the total area of dark coniferous forests.     

Loblolly pine arginase responds to arginine in vitro

Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.     

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.     

Pine embryogenesis: Many licences to kill for a new life

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes.Key words: conifer, developmental cell death, embryogenesis, megagametophyte, necrotic cell death, pine, seed development     

Effects of Culture Conditions on Cell Division and Composition of Regenerated Cell Walls in Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere grown in a liquid medium, and the effects of osmolarityand growth regulators on cell division and on the compositionof regenerated cell walls were investigated. The concentrationof mannitol optimal for cell division was 0.3–0.4 M. Thepresence of 2,4-D was essential for cell division, and 6-benzylaminopurine(BAP) enhanced cell division at concentrations of 0.03–0.1ppm. However, the composition of regenerated cell walls wasabnormal under suspension culture; the predominant sugar wasglucose, indicating that the regenerated cell walls consistedmostly of glucans, and that the other cell-wall components werereleased into the medium. Mannitol, 2,4-D, and BAP at variousconcentrations did not significantly affect the sugar compositionof the regenerated cell walls. Compared with liquid culture,cell division was stimulated when protoplasts were culturedon agar, and their regenerated cell walls had a compositionsimilar to that of the original culture. The importance of thephysical environment for the deposition of polysaccharide componentsin cell walls and the interrelationship between cell divisionand the regeneration of cell walls by protoplasts are discussed. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan. (Received June 10, 1981; Accepted December 12, 1981)     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.     

Variant maturity in seed structures of <Emphasis Type="Italic">Pinus albicaulis</Emphasis> (Engelm.) and <Emphasis Type="Italic">Pinus sibirica</Emphasis> (Du Tour): key to a soil seed bank,unusual among conifers?

The seeds of Cembrae pines are dispersed by nutcrackers (Genus Nucifraga), which cache seeds in soil during autumn. The dispersal and establishment of seedlings via this mutualistic relationship is highly successful. On the other hand, irregular quality of seed crops and lack of detailed knowledge on germination process of Cembrae pine seeds hamper effective seedling production in the nursery. Therefore we studied basic structures and maturity of whitebark pine (Pinus albicaulis Engelm.) and Siberian stone pine (Pinus sibirica Du Tour) seeds, as well as structural changes during a multi-step treatment of whitebark pine seeds, using field emission scanning electron microscopy, transmission electron microscopy and light microscopy. The most striking differences compared to many other conifer seeds were the solid surface structures, early structural differentiation of the embryo, clustering of the thin-walled megagametophyte cells, and great accumulation of starch in both the untreated and treated seeds. Protein bodies of the embryo were in early developmental stages, whereas in the megagametophyte their stages varied. The number, form and size of lipid bodies also varied within different parts of the seed, and lipids dissolved easily. Our results indicated that despite maturity of the seed coat and advanced differentiation of the embryo, the embryo and the megagametophyte were still immature. These morphological features and a notable proportion of storage reserves remaining in unstable form may, however, be advantageous for maintaining viability and reaching maturity within a soil seed bank. Well-controlled pre-treatment simulating natural conditions should result in improved germination.     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.     

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

Summary Embryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.     

Changes in mRNA populations during loblolly pine (Pinus taeda) seed stratification, germination and post-germinative growth

Changes in the patterns of gene expression were examined during loblolly pine ( Pinus taeda L.) seed stratification, germination, and post-germinative growth. In both the megagametophyte and the embryo, DNA contents remained relatively constant at all stages examined. RNA contents, however, increased in both tissues following seed germination, particularly in the embryo where a 7-fold increase in the RNA content was observed 5 days after germination. Poly(A)⁺ RNA, extracted from megagametophytes and embryos, was translated in vitro in a rabbit reticulocyte lysate cell-free system. Analysis of [³⁵S]-methionine-labelled translation products by two-dimensional electrophoresis/fluorography indicated that there were changes in the populations of mRNAs during all developmental stages examined. In both the megagametophyte and the embryo several distinct mRNA populations, including one constitutively present at all stages examined, were identified. One mRNA population, present in the mature seed, decreased during seed stratification. Another population, not present in the mature seed, rose during the period of stratification that coincided with an increase in seed germinability. A third population, which appeared during seed germination, increased steadily during post-germinative growth. Besides these similarities, specific differences between megagametophyte and embryo were noted. For example, one mRNA population, which was present in the megagametophyte of the mature seed and remained constant during the stratification period, disappeared immediately following seed germination. In the embryo, one set of messages was germination specific. In total, these results show that mRNA populations change in a temporal fashion that is consistent with the patterns of de novo protein synthesis known to occur in loblolly pine during the same developmental periods.     

石竹细胞悬浮培养研究

石竹细胞继代周期为 7d时 ,悬浮细胞培养系生长最快 ,生长率最高 ,而且培养物中胚性细胞较多 ,并能保持较快的分裂和生长 ,能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以1∶2较好 ,悬浮系细胞生长最快 ,生长率最高 ,以 1∶2和 1∶3的高倍稀释接种有利于胚性细胞的形成及产生小的胚性细胞团 ,对悬浮系添加椰乳和水解乳蛋白的混合物 ,可较大幅度地提高悬浮细胞系的生长速率 ,单独添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中 ,配方 2 (2 ,4 D 1 .5mg/L +NAA0 .5mg/L +6 BA 0 .5mg/L)最好 ,生长率最高。配方 5 (2 ,4 D 1 .5mg/L +NAA 0 .5mg/L +6 BA 1 .0mg/L)其次 ;配方 1 (2 ,4 D 1 .0mg/L +NAA 0 .5mg/L +6 BA 0 .5mg/L)次之。