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.     

Organogenesis and somatic embryogenesis in Foeniculum vulgare: histological observations of developing embryogenic callus

Different NAA plus kinetin or BA combinations were tested on Francia Pernod fennel seedlings for callus induction and plant regeneration. Callogenesis from hypocotyls was obtained in all auxin/cytokinin-containing media. The organogenic response was observed especially in presence of NAA plus kinetin. The highest frequency of shoot regeneration was found when the auxin and kinetin were used at a 1:1 ratio. Moreover, a prolonged culture period increased shoot formation. Somatic embryogenesis was tested on several fennel populations. The results gave evidence of the genotypic importance. Two different protocols were used for somatic embryo induction. Using the first protocol among the different fennel genotypes tested, only Francia Pernod showed embryogenic capacity. In this case, from a primary non-embryogenic callus cultured for 12 months in presence of 2,4-D, an embryogenic secondary callus was produced. When transferred to the medium without 2,4-D (agarized or liquid), this gave embryogenic plants in high frequency. As far as the second embryogenic method is concerned, secondary embryogenic callus developed only in the presence of 2,4-D plus kinetin in Francia Pernod genotype. Thereafter, the replacement of those growth regulators by GA₃ into the medium greatly increased the somatic embryo development, especially in `Francia Pernod', but also in `Aboca erbe' callus, a population with a very poor embryogenic capacity. In Francia Pernod, the primary and secondary (embryogenic) calli showed different morphological and histological responses, either when the secondary callus was induced by 2,4-D alone or by 2,4-D plus kinetin. Ontogenetic processes leading to somatic embryo formation are described in this context. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acetylsalicylic acid and ammonium-induced somatic embryogenesis and enhanced plumbagin production in suspension cultures of <Emphasis Type="Italic">Plumbago rosea</Emphasis> L.

Summary Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog’s medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM). but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.     

Physiological and biochemical features of embryogenic and non-embryogenic peach palm (<Emphasis Type="Italic">Bactris gasipaes</Emphasis> Kunth) cultures

Both embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures arise during somatic embryogenesis induction, and both tissue types are often observed growing side-by-side from the same explant. To better understand why this occurs, samples from each tissue type were analyzed for their endogenous concentrations of indole-3-acetic acid (IAA), abscisic acid (ABA), polyamines, and amino acids with high-performance liquid chromatography and for total phenolics with spectrophotometry. Embryogenic cultures contained significantly higher concentrations of IAA, ABA, and total amino acids, whereas non-embryogenic tissue contained more total polyamines and phenolics. The greater IAA concentrations in embryogenic cultures supported the role of that hormone as a marker of embryogenic potential. Putrescine was especially prevalent in non-embryogenic cultures; however, the decreased putrescine/spermine + spermidine ratio in embryogenic cultures added support to the conclusions of previous studies in other species that this can serve as a marker of embryogenic competence. Though embryogenic cultures contained higher total amino acids, each culture type had different concentrations of specific amino acids.     

Relationships between endogenous hormonal content and direct somatic embryogenesis in Prunus persica L. Batsch cotyledons

Cotyledons of peach (Prunus persica L. Batsch cv. ZiseMay^®) were cultured in vitro on medium deprived of plant growth regulators. Two different lines varying in their embryogenic capacity were studied after 90 days in culture media. Endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), trans-zeatin (Z), trans-zeatin riboside (ZR), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA) were analyzed in embryogenic and non-embryogenic cotyledons. No significant differences were observed in total ABA, IAA, ZR, SA and JA concentrations between the embryogenic and non-embryogenic cotyledons. On the contrary, lower Z and ACC contents, and also a reduced balance between Z and IAA levels were related with the embryogenic capacity of the cotyledons. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic and non-embryogenic cotyledons is related to their endogenous Z contents, and that the endogenous hormonal balance between Z and IAA is an important index defining the embryogenic potential in peach cotyledons.     

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L^?1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L^?1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.     

Biochemical studies of embryogenic and non-embryogenic callus of Cardiospermum halicacabum L

Some physiological and biochemical changes were measured between embryogenic and non-embryogenic callus obtained from Cardiospermum halicacabum. Combination of auxin with cytokinin was more favourable for high amount of callus formation. 2,4-D played a key role in triggering somatic embryo formation. Embryogenic callus had more total carbohydrate and starch contents, total free amino acids, nucleic acids, phenols and ascorbic acid. Non-embryogenic callus exhibited high chlorophyll content, total soluble sugar, protein, ammonia and enzymes like peroxidase and polyphenol oxidase. Thus, the present study indicated that the process of somatic embryogenesis was characterized by some biochemical and physiological changes induced by plant growth regulators.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava

Direct somatic embryogenesis was successfully achieved from immature leaves of cassava (Manihot esculenta Crantz) cultured on induction medium containing 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid. Changing the duration of induction or changing plant growth regulators resulted in differences in regeneration of somatic embryos or adventitious shoots. The results showed that auxin was a key factor for inducing embryogenic cells. The embryogenic cells were mainly induced within 4–12 days. Only if the embryogenic cells were induced, the auxin enhanced formation of somatic embryo whereas 6-benzylaminopurine stimulated development of adventitious shoots. Histological examinations supported the conclusion.     

Detection of an Embryogenic Cell Antigen in Carrot

Monoclonal antibodies were raised against proteins in a microsomalfraction from carrot embryogenic cells. The presence of a 31-kDaembryogenic cell antigen detected by one antibody (ID 11) wasdemonstrated in embryogenic cells but not in other plant materials,such as non-embryogenic cells, somatic embryos, crown gallsand hairy roots. The antigen was also present in organ segmentsthat carried somatic embryos having been induced by exposureto various stresses. In non-embryogenic cells, the antibodyrecognized a small amount of a 32-kDa antigen. Both the 31-and the 32-kDa antigens accumulated in carrot seeds during theirdevelopment and then disappeared after germination. (Received April 16, 1990; Accepted July 16, 1990)     

Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.     

Characterization of a dehydrin-like phosphoprotein (ECPP-44) relating to somatic embryogenesis in carrot

Somatic embryogenesis in carrot can be induced by the treatment of shoot apices with various kinds of stress chemicals. Using this system, we previously identified a phosphoprotein (ECPP-44) that appears to be involved in the induction of somatic embryogenesis. We have also isolated and characterized a cDNA encoding ECPP-44. In this study, to further characterize ECPP-44, we performed Western blot and immuno-precipitation analyses. Western blot analysis revealed that ECPP-44 was present in embryogenic cells, stress- and non-stress-treated tissues, and somatic embryos but was absent in non-embryogenic cells. Furthermore, ECPP-44 was found in some parts of the carrot plant, such as tap roots, leaves, and flowers (18–26 days after fertilization) but not in mature dry seeds. Interestingly, we could detect phosphorylated ECPP-44 in embryogenic cells and somatic embryos but not in non-embryogenic cells, tap roots, and non-stress-treated shoot apices by immunoprecipitation analysis, even though the protein existed. Our results suggest that ECPP-44 may perform some role in the induction or maintenance of embryogenic competence.     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

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.     

The influence of growth regulators on membrane permeability in cultures of winter wheat cells

The effect of plant growth substances (IAA, 2,4-D, zeatin, kinetin, zearalenone) were studied on membrane properties of the cells of embryogenic (E) and non-embryogenic (NE) calli derived from immature inflorescences (inf) or embryos (emb) of winter wheat. Calli initiated from inflorescences show higher permeability. The ion leakage from cells of E calli was higher than from cells of NE calli. Growth regulators were used in concentrations of 2-30 mg/l (about 10-140 microM). All tested growth substances increased ion leakage from NE emb cells, IAA, zeatin and kinetin being most effective. In NE inf cells the effect of growth substances was similar as in NE emb, but much weaker. In E cells of both types (inf and emb) growth substances decreased ion leakage. Changes in the leakage of potassium and calcium ions were similar to those in total ion leakage. The uptake of labelled auxins (IAA and 2,4-D) was higher in NE cells (especially in NE inf) than in E cells. The endogenous level of IAA was higher in E cells than in NE cells and in inf cells than in emb cells. The importance of auxin in determining permeability of cell membranes is discussed.     

胡萝卜(Daucus carota L.)胚性细胞蛋白的分离研究

应用IEF/SDS-PAGE双向电泳技术,比较了胡萝卜胚性细胞、非胚性细胞和不同发育时期的胚状体中可溶性蛋白的双向电泳图谱,结果发现在胚性细胞中特异存在的胚性细胞蛋白在不同发育时期的胚状体中也存在,但在非胚性细胞中不存在。因此,推测体细胞胚胎发生所需的一些基因在胚性细胞中就早已表达了。我们还成功地分离和测定了ECP 45-2 N-末端和中央部分氨基酸序列。与已知氨基酸序列相比,ECP 45-2部分氨基酸序列与ECP 45-1 部分氨基酸序列具有较高比例的同源性。因此, ECP 45-1和45-2可能属于同一因基家族。     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.     

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。     

Embryo-specific Proteins in <Emphasis Type="Italic">Cyclamen persicum</Emphasis> Analyzed with 2-D DIGE

Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Lyngved and J. Renaut contributed equally to this work.