Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Short communication. Early changes in gene expression during direct somatic embryogenesis in alfalfa revealed by RAP-PCR

A procedure for direct production of somatic embryos from alfalfa leaf explants has been developed. Wounding followed by 2,4-D treatment induces competent cells in the leaf to produce embryos. Changes in gene expression following induction of direct somatic embryogenesis were investigated by RNA arbitrarily primed PCR (RAP-PCR) with a view to identifying genes involved in controlling the onset of somatic embryo development. Amongst the induced sequences identified were a calnexin-like sequence (which was induced within 2 d), and a novel plant homologue of the yeast SN12 multi-drug resistance protein (which did not appear until 10 d after induction). RT-PCR amplification of these sequences confirmed the expression patterns revealed by RAP-PCR and was used to show that the calnexin-like gene is induced by 2,4-D, but that the level of expression is enhanced by wounding. The technique of RAP-PCR has the potential to isolate genes of interest by identifying specific expression patterns during complex developmental processes.     

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.     

Induction of somatic embryogenesis using side chain and ring modified forms of phenoxy Acid growth regulators

The induction of somatic embryo development in cell cultures of alfalfa (Medicago sativa), celery (Apium graveolens), and lettuce (Lactuca sativa) was compared for 2,4-dichlorophenoxy-acetic acid (2,4-D) and various phenoxy acid growth regulators. Tests using a series of straight chain extensions to the phenoxy acid side chain indicate that phenoxybutanoic acid is active, whereas the phenoxypropanoic and phenoxypentanoic analogs are inactive for the induction of alfalfa embryogenesis. Side branching on the carbon adjacent to the phenoxy group results in optically active compounds. Racemic mixtures and the (+) enantiomers of the compounds are active for alfalfa embryo induction, whereas the (−) enantiomers are inactive and apparently do not inhibit embryogenesis in any way. Development of alfalfa embryos, as measured by plantlet formation from individual embryos, is improved by 4-(2,4-dichlorophenoxy)butanoic acid and with side branching at the carbon adjacent to the phenoxy group compared with induction with 2,4-D. Similarly, substituted phenoxy acids also enhance somatic embryo development in celery and lettuce when compared with 2,4-D. These results are discussed with reference to earlier studies on the structure activity of various synthetic auxins during cell elongation and with reference to the possible importance of auxin metabolism on subsequent somatic embryo development.     

Efficient protocols for in vitro regeneration of Pennisetum glaucum (L) Br

A system was developed for in vitro regeneration of Pennisetum glaucum through organogenesis and somatic embryogenesis. Mature embryo and leaf base explants of Pennisetum glaucum (L) Br. cv HH B60 (Poaceae) were cultured on Murashige and Skoog agar medium supplemented with 11.3 microM of 2,4-D for callus induction. Embryogenic calli were induced within eight weeks. Percentage of callus induction and somatic embryogenesis was significantly higher in mature embryo than leaf base explants. Maximum shoot regeneration was obtained via organogenesis on MS medium supplemented with 4.43 microM of BAP and 4.64 microM of kinetin from the calli of both the explants. The frequency of plant regeneration through somatic embryogenesis was comparatively lower than organogenesis. Regeneration frequency was higher in mature embryo explants than leaf base explants. The shoots regenerated via organogenesis were elongated and rooted efficiently on MS medium supplemented with IBA (0.49 microM). The rooted plantlets were hardened and transferred to soil.     

云南大叶茶体细胞胚发生及体细胞胚苗形成体系的建立

利用云南大叶茶(Camellia sinensis var.assamica Kitamura)胚性细胞系(CL_1)中悬浮培养物,建立了高频率同步化体细胞胚发生及体胚苗形成体系。以改良的MS为基本培养基,将CL_1中培养物由液体保持培养基(0.1mg/L 2,4-D 0.5mg/L 6-BA)继代转入液体诱导培养基(0.05mg/L 2,4-D 0.50mg/L6-BA),暗培养诱导28d,转入不含任何激素的液体分化培养基中再培养28d,获得了不同发育时期的体细胞胚,其发生频率为81.5％。不同发育时期的体细胞胚用不同目的细胞筛收集,在液体生长培养基(1/2 MS 1.0mg/L GA_3 0.5mg/L 6-BA)中培养发育成熟。ABA有利于高质量体细胞胚的形成。20～70月大小的体细胞胚在固体生长培养基中成苗转换率为75％。在液体悬浮培养条件下观察记录了体细胞胚发育过程,证实其过程与合子胚的形态发生过程相似。     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.     

Analysis of expression profile of selected genes expressed during auxin-induced somatic embryogenesis in leaf base system of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and their possible interactions

Somatic embryogenesis is a notable illustration of plant totipotency and involves reprogramming of development in somatic cells toward the embryogenic pathway. Auxins are key components as their exogenous application recuperates the embryogenic potential of the mitotically quiescent somatic cells. In order to unravel the molecular basis of somatic embryogenesis, cDNA library was made from the regeneration proficient wheat leaf base segments treated with auxin. In total, 1440 clones were sequenced and among these 1,196 good quality sequences were assembled into 270 contigs and 425 were singletons. By reverse northern analysis, a total of 57 clones were found to be upregulated during somatic embryogenesis, 64 during 2,4-D treatment, and 170 were common to 2,4-D treatment and somatic embryogenesis. A substantial number of genes involved in hormone response, signal transduction cascades, defense, anti-oxidation, programmed cell death/senescence and cell division were identified and characterized partially. Analysis of data of select genes suggests that the induction phase of somatic embryogenesis is accompanied by the expression of genes that may also be involved in zygotic embryogenesis. The developmental reprogramming process may in fact involve multiple cellular pathways and unfolding of as yet unknown molecular events. Thus, an interaction network draft using bioinformatics and system biology strategy was constructed. The outcome of a systematic and comprehensive analysis of somatic embryogenesis associated interactome in a monocot leaf base system is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterisation of the <Emphasis Type="Italic">Vitis vinifera</Emphasis>PR10 multigene family

Background  

Genes belonging to the pathogenesis related 10 (PR10) group have been studied in several plant species, where they form multigene families. Until now, such an analysis has not been performed in Vitis vinifera, although three different PR10 genes were found to be expressed under pathogen attack or abiotic stress, and during somatic embryogenesis induction. We used the complete genome sequence for characterising the whole V. vinifera PR10 gene family. The expression of candidate genes was studied in various non-treated tissues and following somatic embryogenesis induction by the auxin 2,4-D.     

枸杞体细胞胚发生中Ca^2＋和ATPase的超微结构定位研究

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca^2 含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca^2 和ATPase活性的时空分布动态。结果表明：2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca^2 沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca^2 在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca^2 呈弥散性分布。在此过程中,ATPase活性时空分布与Ca^2 的定位变化具有高度一致性,仅仅稍滞后于Ca^2 出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca^2 和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca^2 含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca^2 和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca^2 和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。     

5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels

DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8?%. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20?days, followed by an increase after 30?days (39.5, 36.2 and 41.6?%). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. Key message 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

枸杞体细胞胚发生中Ca2+和ATPase的超微结构定位研究

研究2,4-D诱导枸杞体细胞胚发生中的作用及其与Ca~(2+)含量和ATPase活性时空分布动态之间的关系,以探讨2,4-D诱导植物体细胞胚发生的作用机理。采用超微细胞化学定位的方法,跟踪分析了体细胞胚发生与发育的不同时期,Ca~(2+)和ATPase活性的时空分布动态。结果表明:2,4-D是诱导离体培养的枸杞体细胞进入胚胎状态的关键激素。在含有2,4-D和不含2,4-D的培养条件下,分别诱导枸杞体细胞脱分化后,再转入除去2,4-D的MS培养基上,进行分化培养,结果前者可分化形成体细胞胚,因而称为胚性愈伤组织。后者在相同条件却不能分化形成胚,故称为非胚性愈伤组织。在2,4-D诱导枸杞的胚性愈伤组织中,胚性细胞分化早期的细胞间隙和细胞壁上均有Ca~(2+)沉淀。随着胚性细胞的分化、分裂和多细胞原胚形成,这时Ca~(2+)在细胞内的分布主要集中在细胞膜和液泡膜上;球形胚期在细胞核中Ca~(2+)呈弥散性分布。在此过程中,ATPase活性时空分布与Ca~(2+)的定位变化具有高度一致性,仅仅稍滞后于Ca~(2+)出现的时间。而在胚性细胞分化早期,ATPase活性同样位于质膜上,随后在液泡和细胞核都可见ATPase活性分布。而在非胚性愈伤组织中,则未见Ca~(2+)和ATPase活性呈时空动态分布,而且随着非胚性细胞的液泡化,无论是Ca~(2+)含量,还是ATPase活性都呈逐渐降低的趋势。表明Ca~(2+)和ATPase活性变化与2,4-D诱导的胚性细胞分化和发育密切相关。并由此推测,Ca~(2+)和ATPase的时空分布对胚性细胞分化中的信息传递和调控相关基因表达起着关键性作用。     

Auxin pulse treatment holds the potential to enhance efficiency and practicability of somatic embryogenesis in potato

The objective of the current study was to simplify existing somatic embryogenesis systems in potato (Solanum tuberosum L.) cv. Desiree. The project targeted the agar-based induction phase of the potato somatic embryogenesis process as the key area for improvement. Experiments were established to ascertain the effect of a 2,4-D (2,4 dichlorophenoxyacetic acid) pulse, applied to the primary internodal section explant source and its subsequent effect on embryo induction. Parameters tested were the duration of the auxin pulse in a range from 0 to 300 min, and the concentrations of 2,4-D applied, in a range from 0 to 5,120 μM. The mean number of somatic embryos formed per explant was recorded after 4 and 8 weeks culture. Our findings indicated that the somatic embryogenesis in potato internodal segments could be evoked by an auxin (2,4-D) pulse treatment over a wide concentration and duration range. The results further suggested that a simple 20 μM 2,4-D pulse treatment could replace a lengthy 2 week induction phase in potato somatic embryogenesis and thus improve the system’s practicability for wider uptake.     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.