Fatty acid composition of total lipids in embryogenic and nonembryogenic callus lines of larch

Fatty acid (FA) composition of total lipids in embryogenic and nonembryogenic calli of Siberian larch (Larix sibirica Ledeb.) was investigated by the method of GC-MS. We detected a high content of oleic acid in total lipids of embryogenic cell culture (32–56% by weight of total FA), which apparently depends on a high activity of acyl carrier protein (stearoyl-ACP-Δ9-desat-urase). At the same time, activity of Δ12-desaturase in the cells of embryogenic calli was considerably (2–3 times) lower than in nonembryogenic calli. We discuss a possibility that concentration of FA (oleic and linoleic acids) may be used as a marker of embryogenic potential when promising cell lines of Siberian larch are screened in the stage of early embryogenesis.     

宁夏枸杞器官发生和体细胞胚胎发生过程中DNA,RNA和蛋白质合成动？…

After the calli originating from the leaf explant of Lycium barbarum L. were selected and proliferated, the yellowish calli with same origin, similar state were transferred to O medium or E medium and the regenerative systems of organogenesis and somatic embryogenesis might form. By these systems, a comparative study on the synthetic activities of DNA, RNA and protein in the two in vitro regeneration pathways was carried out. The results were as follows: (1) Before meristemoid and embryogenic cells were formed, the synthesis of RNA was activated firstly, followed with the increase of synthesis rates of DNA and protein. During the formation of globular embryo, the synthesis rate of DNA increased quickly and then the activities of syntheses of RNA and protein reached the peak, while it was the contrary during germination of adventitious bud. (2) Components of soluble protein changed regularly. A peptide (153.6 kD) appeared during the initiation of both organogenesis and somatic embryogenesis. Several peptides disappearing gradually in the early-stage of differentiation could regenerate with the formation of shoot primordium and globular embryo. Corresponding to morphogenesis, both regenerative systems had specific peptides (84.9 kD, 46.3 kD and 44 kD, 36.2 kD) as molecular markers of its own development. In addition, the relation and mechanism of the two regenerative systems were discussed.     

Somatic embryogenesis and mass spectrometric identification of proteins related to somatic embryogenesis in Eruca sativa

Several different proteins expressed in embryogenic and nonembryogenic Eruca sativa calli were identified by combining one-dimensional SDS-PAGE protein mapping with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. By querying the widely recognized MASCOT search engine, it was found that three of the proteins that were particularly strongly expressed in the embryogenic callus represented sucrose synthase, phospholipase D, and enolase, respectively. RT-PCR analysis also confirmed that the gene coding for enolase was transcribed especially strongly in the embryogenic callus but not in nonembyogenic callus. Finally, the relationship between the three proteins and somatic embryogenesis is discussed.     

宁夏枸杞器官发生和体细胞胚胎发生过程中DNA、RNA和蛋白质合成动态的比较研究

宁夏枸杞(Lycium barbarum L.)叶外植体来源的愈伤组织经筛选、繁殖后,将来源相同、状态较为一致的淡黄色愈伤组织转移至O型或E型培养基上,可以诱导出器官发生和体细胞胚胎发生。利用该体系,对两条离体再生途径进行了比较研究。结果表明:(1)在拟分生组织和胚性细胞形成之前,RNA合成首先被激活,随后DNA、蛋白质合成加速;而球形胚形成期间,先是DNA合成的加快,接着RNA、蛋白质的合成高峰出现,在不定芽形成期间却正好相反;(2)可溶性蛋白组分发生规律性变化;器官发生和体细胞胚胎发生的启动阶段都有-153.6kD多肽出现,一些多肽分子在分化早期逐渐消失,而随芽原基或球形胚的形成又重新合成;与形态发生相对应,两种再生体系都有作为各自分子标记的特异多肽(84.9kD、46.3kD和44kD、36.2kD)的表达。此外,还对两种离体再生体系之间的关系和发生机制进行了讨论。     

枸杞体细胞胚发生中外源Ca2+的作用

脱分化的枸杞叶片外植体愈伤组织转入含有2,4-D的MS培养基上分化培养后有大量胚性细胞的分化和体细胞胚发生;加入一定量的外源Ca2 或45Ca2 ,明显地提高了胚性愈伤组织中体细胞胚发生的频率;加入Ca2 的鳌合剂EGTA则显著降低了体细胞胚发生频率;胚性愈伤组织中CaM的水平在多细胞原胚期和球形胚期显著升高,加入外源Ca2 后CaM含量几乎成倍增加;胚性愈伤组织中蛋白质组分与活性都远远多于或高于非胚性愈伤组织,加Ca2 后蛋白质组分种类也增加.     

Indole-3-acetic acid metabolism in hormone-autotrophic, embryogenic callus of Inmil® cherry rootstock (Prunus incisa×serrula'GM 9') and in hormone-dependent, nonembryogenic calli of Prunus incisa×serrula and Prunus domestica

Callus derived from the roots of Inmil® cherry rootstock ( Prunus incisa × serrula ) proliferates in a hormone-free solid medium. When transferred to a hormone-free liquid medium, such callus forms somatic embryos. On the other hand, leaf-derived callus of P. incisa × serrula and leaf- and root-derived calli of P. domestica require exogenous auxin for sustained growth and never form embryos. Levels of free and esterified indole-3-acetic acid (IAA) were similar in both types of calli grown on a solid medium, whereas the amide-conjugated IAA was higher in the root-derived embryogenic one. Transfer to a liquid medium did not affect the level of both free and conjugated IAA in the nonembryogenic callus, but in the embryogenic callus it decreased the level of amide-conjugated IAA. Isotopic dilution of 13 C-IAA taken up from a medium was faster in the embryoegenic than in the nonembryogenic calli, which shows that the rate of IAA metabolism was higher in embryogenic callus. Besides IAA, indole-3-ethanol and indole-3-acetyl-N-aspartate were detected in nonembryogenic calli and in in vitro-grown shoots of P. domestica , whereas in embryogenic callus and in in vitro - grown shoots of P. incisa × serrula indole-3-acetamide was detected.     

Identification of marker compounds for discriminating between embryogenic and nonembryogenic calluses of higher plants using pyrolysis gas chromatography mass spectrometry and genetic programming

When whole cells are subjected to pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) analysis, it provides biochemical profiles containing overlapping signals of the majority of compounds. To determine marker compounds that discriminate embryogenic calluses from nonembryogenic calluses, samples of embryogenic and nonembryogenic calluses of five higher plant species were subjected to Py-GC/MS. Genetic programming of Py-GC/MS data was able to discriminate embryogenic calluses from nonembryogenic calluses. The content ratio of 5-meyhyl-2-furancarboxaldehyde and 5-(hydroxymethyl)-2-furancarboxaldehyde was greater in nonembryogenic calluses than in embryogenic calluses. However, the content ratio of phenol, p-cresol, and¹H-indole in embryogenic calluses was 1.2 to 2.4 times greater than the ratio in nonembryogenic calluses. These pyrolysates seem to be derived from the components of the cell walls, which suggests that differences in cell wall components or changes in the architecture of the cell wall play a crucial role in determining the embryogenic competence of calluses.     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

Biochemical differences between embryogenic and nonembryogenic calli of conifers

Summary Embryogenic and nonembryogenic calli of loblolly pine (Pinus taeda), Eastern white pine (P. strobus), pond pine (P. serotina), white spruce (Picea glauca), and European larch (Larix decidua) were analyzed for biochemical parameters previously shown to be indicative of an embryogenic state in Norway spruce (Picea abies). Concentrations of glutathione and total reductants as well as rates of ethylene evolution and incorporation of radioactive leucine into protein in the two callus types were consistent with the Norway spruce observations. Embryogenic potential of loblolly pine and pond pine callus was predicted by biochemical analysis in advance of the appearance of somatic embryos. Other parameters such as isozyme patterns and SDS-PAGE of soluble proteins could also be used to distinguish embryogenic from nonembryogenic conifer callus. Among the species investigated, white spruce was the most difficult to sort by these methods.     

Variations in vinblastine production at different stages of somatic embryogenesis,embryo, and field-grown plantlets of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don,as revealed by HPLC

Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.     

多胺对宁夏枸杞愈伤组织器官发生和体细胞胚发生的影响

利用已建立的宁夏枸杞（Lycium barbarumL.)愈伤组织器官发生和体细胞胚发生体系,对多胺在其离体形态发生中的作用进行研究。通过检测内源多胺含量发现,在所研究的三种多胺中,Put是器官发生途径的主要多胺,而在体细胞胚发生途径Spd含量占优势。Put含量变化在两条途径中相似：在愈伤组织分化的早期迅速积累不仅又下降,随着芽原基和球形胚的形成含量又进一步上升。器官发生中Spd最高含量仅在培养的第一天后,Spd含量才开始上升,到第十天时达到最高值。三种外源多胺的添加均有有效地促进两种离体分化途径的形态建成：Spd(100 μmol/L）能显著增加不定芽数,而体细胞胚发生中Spd(100μmol/L）而Put（100μmol/L）的单独处理能最好地促进体细胞胚形成和进一步发育成苗;尽管Spm在离体形态发生中含量较低,但添加外源Spm也促进了不定芽形成和体细胞胚形成然后成苗。多胺生物合成抑制剂CHA处理阻碍不定芽形成和和体细胞胚的进一步发育;但是MGBG对器官发生途径中的形态建成没有影响,即降低体细胞胚的发生频率及再生苗数。添加Spd(50μmol/L)能部分逆转CHA、MGBG的抑制效应。以上结果表明,多胺对宁夏枸杞器官发生和体细胞胚发生途径的离体形态建成有一定影响。     

Single-cell origin and development of somatic embryos in Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce)

The origin and development of somatic embryos in calli initiated from immature zygotic embryos of Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce) was studied. Immature zygotic embryos cultured on callus induction medium produced two types of white calli that were phenotypically different from one another. The callus that proliferated from the hypocotyl region was white to translucent, glossy, mucilaginous and embryogenic. The callus mass which originated from the radicle end was reddish-white, nonmucilaginous and nonembryogenic. Whole mount preparations of the entire explant with two different types of calli showed the presence of embryogenic cells in the mucilaginous callus mass derived from the hypocotyl region of the zygotic embryo. The origin of somatic embryos in both Norway and white spruce could be traced to single cells of the hypocotyl callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Two-dimensional gel electrophoresis of nuclear proteins of different stages of B-chronic lymphocytic leukaemia

Two-dimensional polyacrylamide gel electrophoresis was used to compare the composition of nuclear proteins from normal and B-chronic lymphocytic leukaemia (B-CLL) mononuclear cells. Some differences in the electrophoretic behaviour of these proteins from normal and transformed cells, especially with molecular weights/pI of 14-16 kD/5.9-7.4; 28-32 kD/4.9-5.5; 38-39 kD/5.4-6.1; 44-46 kD/5.1-5.6; 47-52 kD/5.0-5.6; 64-69 kD/5.1-5.7, and 95-105 kD/5.2-5.5, were observed. The comparative analysis of nuclear proteins, obtained from mononuclear cells of patients with B-CLL at different stages of development, indicated that the expression of some protein components might be correlated with the progression of this disease.     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Correlation Between Appearance of Embryogenic Cells and the IAA Levels in Rice Somatic Cell Culture

Changes of endogenous IAA level and IAA action in cultured rice ( Oryza sativa L. ) somatic cells during the period from 7th to 15th day which was the transition from somatic to embryogenic cells were observed. The study was carded out in three experimental systems viz. mature caryopsis and young panicles (2 ~ 5 mm long) of rice cv. "Guangluai 4" under normal osmosis (3% sucrose), mature caryopses from rice cv. "Yanjing 2" or "Guangluai 4" under normal and higher osmosis (5% sucrose or 2.5 % sorbitol). During this period, endogenous IAA contents were greatly increased in young-panicle calli under normal osmosis and mature-caryoptic calli under higher osmosis but decreased in mature-caryoptic calli under normal osmosis. Exogenous IAA could induce the appearance of embryogenic cell from nonembryogenic callus at a lower frequency. And 2,3,5-tri-iodobenzoic acid could increase the frequency of embryogenic cell induction. From these results it could be concluded that accumulation of higher IAA level in the cultured rice cells was essential for induction of embryogenic cell appearance. Since 2,4-D was involved in all induction medium with the same concentration but exerted different effects on embryogenic cell induction, it was suggested that it might act through mediating the endogenous IAA metabolism.     

Biochemical differences between embryogenic and nonembryogenic callus of Picea abies (L.) Karst

Both embryogenic and nonembryogenic calli of Picea abies (L.) Karst. were initiated from the hypocotyl region of immature embryos. The two callus phenotypes were manually separated and subsequently maintained independently, but under identical culture conditions. Biochemical analysis of the two phenotypes revealed significant differences in ethylene evolution rate and in concentrations of glutathione and total reductants. Due to the constancy of the genetic background, age and growth conditions of the two callus types, differences in the measured quantities are not likely to be traceable to the genetic origin of the callus and serve to highlight biochemical changes associated with somatic embryogenesis in Norway spruce.Abbreviations GSH glutathione - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyl adenine - E embryogenic - NE nonembryogenic - NS Norway spruce     

Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63m mesh openings and the composition of each fraction was determined. Cellular units larger than 355 m were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 m. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 m fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 M 2,4-D. The 63–125 m fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.Abbreviations and definitions cellular units single cells, cell aggregates, and calli - cell aggregates discrete associations of cells - calli association of cell aggregates - embryogenic cell aggregates yellow aggregates of cytoplasmic cells which have the potential to produce embryogenic calli or embryos [3] - non-embryogenic cell aggregates white aggregates of vacuolated cells [3] - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Role of Abscisic Acid in Drought-Induced Freezing Tolerance,Cold Acclimation,and Accumulation of LT178 and RAB18 Proteins in Arabidopsis thaliana

Embryogenic tissues of Pinus caribaea Morelet var hondurensis produce extracellular proteins; among them germins have been identified. Two-dimensional electrophoresis followed by electroblotting onto a polyvinylidene difluoride membrane allowed isolation and N-terminal amino acid sequencing of extracellular GP111, which is present within the five embryogenic cell lines studied. The amino acid sequence showed strong homologies with the sequences of germins deduced from cDNA sequencing, starting at the same amino acid position but one, compared with other sequences of mature germins deduced from protein sequencing. Immunoblots of embryogenic and nonembryogenic extracellular proteins indicated that the polypeptide GP111 plus two others with similar relative molecular mass values are present in embryogenic cell lines but not in nonembryogenic ones. They were recognized by an antiserum raised against the nonglycosylated monomer of wheat germin. The cross-reaction between pine and wheat apoproteins was highly specific. An antiserum against the glycosylated pentameric germin-like protein (an oxalate oxidase) of barley cross-reacted with all three, as well as with several other glycosylated polypeptides.     

Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.)

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.