Temperature and genotype affect asparagus somatic embryogenesis

Summary Calluses from five asparagus genotypes G14, G32, G171, G203, and G447 and hybrid Jersey Giant (JG) were incubated at three temperature regimes (24, 27, and 30°C) on embryo induction medium to assess somatic embryo development and conversion to plantlets. The calluses from three genotypes (G14, G32, and G171) were not responsive, failing to produce somatic embryos at any temperature regime. For three responsive genotypes (G203, G447, and JG), both incubation temperature and genotype significantly affected the numbers of somatic embryos produced. The calluses produced the most and the least numbers of total, bipolar, and globular embryos when incubated at 27°C and 24°C, respectively. When incubated at 27°C, G203 produced the highest numbers of total and globular embryos, 178 g⁻¹ callus and 142 g⁻¹ callus, respectively while G447 produced the highest number of bipolar embryos, 77 g⁻¹ callus. Incubation temperature but not genotype significantly affected the conversion of somatic embryos to plantlets. The somatic embryos recovered from the three responsive genotypes incubated at 27°C also converted to plantlets at the highest frequencies, 60–63% of the bipolar embryos and 42–43% of the globular embryos converted to plantlets, while the somatic embryos recovered from the calluses incubated at 24°C converted to plantlets at the lowest frequencies.     

Long-term somatic embryogenesis and maturation of somatic embryos in Hevea brasiliensis

A high frequency of secondary embryogenesis was induced from isolated early cotyledonary-stage somatic embryos of Hevea brasiliensis. A long-term embryogenic line was established by the use of recurrent embryogenesis and maintained for 3 years on hormone-free medium by the transfer of selected proembryogenic masses every 10 days.

The addition of 234 mM sucrose as stress with sucrose and 10⁻⁵ M abscisic acid (ABA) to the culture medium enhanced the maturation of somatic embryos. Under these culture conditions, the embryo population was composed of 45% globular, 18% oblong and 37% torpedo-stage embryos. These somatic embryos had well-formed tissue structure, a well-defined epidermis, protein storage bodies, and a high accumulation of starch. The triglyceride content was five times as high in the torpedo-stage embryos that developed on medium supplemented with 234 mM sucrose and 10⁻⁵ M ABA as in embryos obtained on basal medium with 58 mM sucrose.     

Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

Commercial deployment of clonal trees via somatic embryogenesis (SE) could increase forest productivity over conventional tree breeding techniques. However, some technical advances need to be made to use SE in clonal forestry with Pinus radiata. For example, the conversion of embryonal mass (EM) into plants is at present a major bottleneck. For this reason, maturation experiments were carried out to determine the effect of the initial amount of EM, activated charcoal (AC) and the best combination of abscisic acid (ABA), sucrose and amino acid concentration in the maturation medium. Germination was evaluated on different media formulations with and without AC. When 100 mg of EM were suspended in liquid medium without AC, cotyledonary somatic embryos were obtained in all the maturation media tested. Maturation medium supplemented with 60 μM ABA, 6% sucrose, and embryo development medium amino acid mixture produced the highest number of cotyledonary somatic embryos, between 10 and 1,550 embryos per gram of EM fresh weight. Approximately half of the tested 25 lines produced more than 600 embryos per gFW. Embryo development was the best when somatic embryos were germinated in half strength modified Quoirin and Lepoivre medium supplemented with 2 g L⁻¹ AC. This protocol simplified and improved SE maturation and germination due to the elimination of subcultures, the large number of somatic embryos obtained from a very low amount of EM, and the elimination of pre-germination treatments, resulting in a significant saving of cost and labor.     

Invited review: Genetic regulation of somatic embryogenesis with particular reference to arabidopsis thaliana and Medicago truncatula

Summary Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Comparison of induction frequency, maturation capacity and germination of Abies numidica during secondary somatic embryogenesis

Efficiency of the method for improving repetitive somatic embryogenesis and plant recovery of Algerian fir (Abies numidica De Lann.) was investigated by evaluating of induction frequency, maturation capacity and germination. Individual zygotic embryos differed only slightly in induction frequencies (6.8 %) from somatic embryos of the first (5.7 %) and second cycle (5.5–9.0 %). The yield of mature embryos differed significantly among the cell lines of the same cycle and among cell lines of the different cycles. Percentage of abnormalities was lowest in the first cycle of somatic embryos, whereas the second and the third cycles of somatic embryos were branded by a higher frequency of abnormalities. The differences in germination of well developed somatic embryos depended on cell lines rather than on cycle of somatic embryos.     

细胞信号转导与植物体细胞胚发生

细胞信号转导主要是指胞间通讯的激素以及外界环境因子等作用于细胞表面（或胞内受体）后,如何跨膜传递形成胞内第二信使,以及其后的信息分子级联传递、诱导基因表达和引起生理反应的过程。植物体细胞胚发生的本质是基因的判别表达,因此细胞信号转导在此起关键作用。在植物组织培养过程中,外加的激素等因子通过细胞信号转导诱导基因差别表达,使体细胞转入胚胎发生过程,最终生长发育成完整植株。     

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 M N⁶-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 M 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 M -naphthaleneacetic acid and 5 M BA.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - ABA absicic acid - AC activated charcoal - NAA -naphthaleneacetic acid - QP Quoirin & Lepoivre (1977) - AE von Arnold & Eriksson (1981) - DCR Gupta & Durzan (1985)     

植物钙信号系统与体细胞胚发生

植物细胞离体培养中的体细胞胚发生受多种内外因素的调控,其中激素对细胞分化,发育和形态建成起着关键的作用。大量研究表明,在激素的作用过程中,Ca^2 信号系统可能是重要的介导者之一。Ca^2 和CaM在植物合子胚和体细胞胚发生中都具有重要作用,其作用机理可能是植物激素通过Ca^2 第二信号系统直接或间接地调控基因表达而实现的。     

Callus friability and somatic embryogenesis inHevea brasiliensis

The influence of plant growth regulators, sucrose, calcium and various macronutrient media on callus friability and somatic embryogenesis was investigated inHevea brasiliensis Müll. Arg. Friable and embryogenic calli were spontaneously formed in two rubber tree clones (PR 107 and RRIM 600) on the Medium for Hevea (MH), with 3,4-dichlorophenoxyacetic acid (3,4-d), kinetin and sucrose, while compact embryogenic calli were enhanced in three other clones (PB 260, PB 235 and GT1). Callus friability was enhanced in clone PB 260 when the concentration of one growth factor (3,4-d or kinetin) was reduced from 4.5 μLM to 0.45 μM during the first culture, or when high sucrose or calcium levels 351 mM and 12 mM, respectively) were maintained during subcultures. The different macronutrient media did not alter callus texture but only use of MH and Murashige and Skoog (MS) media led to somatic embryogenesis. Friable calli obtained by modifying the auxin/cytokinin balance lost their embryogenic potential. In contrast, those obtained on media with high sucrose or calcium concentrations were mainly composed of embryogenic cells embedded in a mucilaginous matrix. Such calli could be of potential interest for establishing embryogenic cell suspension cultures.     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Picloram induced somatic embryogenesis in Gasteria and Haworthia

Various leaf sections of Gasteria verrucosa Haw. and Haworthia fasciata Haw. were cultured on media to examine the effect of picloram (4-amino 3, 5, 6-trichloropicolinic acid) and 2, 4-D (2, 4-dichlorophenoxy acetic acid) on somatic embryogenesis. Picloram (0.5, 1.0, 2.0, 3.0 mgl^-1) outperformed 2, 4-D (0, 1.0, 2.0, 3.0 mgl^-1) as the auxin source of both earliness of callus and embryo induction and final yield of embryos produced at both kinetin levels examined (0.25, 1.0 mgl^-1). Embryos arose initially as a yellow, compact globular masses from the area just beneath the epidermis in linear pattern parallel with the main axis of the leaf and then developed a heartshaped appearance. Embryo formation was preceded by growth of callus almost crystalline in appearance on the cut surface. Subsequent shoot formation developed from green pigmented loci in crystalline callus derived from embryos. Shoot and root development in Gasteria was induced on a defined medium containing quarter strength MS or B5 salts with no hormonal supplementation.     

N-acetylglucosamine and glucosamine-containing arabinogalactan proteins control somatic embryogenesis

In plants, complete embryos can develop not only from the zygote, but also from somatic cells in tissue culture. How somatic cells undergo the change in fate to become embryogenic is largely unknown. Proteins, secreted into the culture medium such as endochitinases and arabinogalactan proteins (AGPs) are required for somatic embryogenesis. Here we show that carrot (Daucus carota) AGPs can contain glucosamine and N-acetyl-D-glucosaminyl and are sensitive to endochitinase cleavage. To determine the relevance of this observation for embryogenesis, an assay was developed based on the enzymatic removal of the cell wall from cultured cells. The resulting protoplasts had a reduced capacity for somatic embryogenesis, which could be partially restored by adding endochitinases to the protoplasts. AGPs from culture medium or from immature seeds could fully restore or even increase embryogenesis. AGPs pretreated with chitinases were more active than untreated molecules and required an intact carbohydrate constituent for activity. AGPs were only capable of promoting embryogenesis from protoplasts in a short period preceding cell wall reformation. Apart from the increase in embryogenesis, AGPs can reinitiate cell division in a subpopulation of otherwise non-dividing protoplasts. These results show that chitinase-modified AGPs are extracellular matrix molecules able to control or maintain plant cell fate.     

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation

Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence watermelon fruit ripening. However,the molecular mechanisms are unclear. Here,we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator ...     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.