Isolation and characterization of four somatic embryogenesis receptor-like kinase (<Emphasis Type="Italic">RhSERK</Emphasis>) genes from miniature potted rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> cv. Linda)

The isolation and expression analysis of four partial gene sequences from rose (Rosa hybrida cv. Linda) belonging to the receptor-like kinase gene superfamily are reported. These genes have been designated RhSERK1 to RhSERK4 (Accession No. EF631967 to EF631970) as they exhibit high sequence identities with genes from the somatic embryogenesis receptor-like kinase (SERK) family in other plant species. The RhSERK genes are differentially expressed in non-embryogenic callus, embryogenic callus, mature somatic embryos and a range of tissues from intact plants, indicating a broad role in plant growth and development. However, the expressions of RhSERK3 and RhSERK4 were approximately fivefold higher in embryogenic callus than in non-embryogenic callus, and they are even higher when compared to tissues from intact plants. In addition, RhSERK4 expression was approximately eightfold higher in somatic embryos than in embryogenic callus. These results suggest that the expression pattern of RhSERK3 and RhSERK4 may be used as a marker of somatic embryogenesis.     

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl₃, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca²⁺ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.     

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.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

铁皮石斛体细胞胚胎发生类受体激酶基因DoSERK的克隆和表达分析

为了解铁皮石斛(Dendrobium officinale)中的体细胞胚胎发生类受体激酶基因DoSERK 的功能,在转录组测序数据的基础上克隆了DoSERK 的全长cDNA。结果表明,DoSERK 与其他植物的SERK 高度同源,编码633 个氨基酸。生物信息学分析表明,DoSERK蛋白为亲水蛋白并定位于质膜,具有1 个信号肽、1 个富脯氨酸区域、1 个跨膜区、5 个富亮氨酸重复序列以及1 个保守的蛋白激酶活性结构域,属于SERK 蛋白家族成员。系统进化树分析表明,DoSERK 与同为兰科植物的文心兰(Cyrtochilum loxense)以及卡特兰(Cattleya maxima)的SERK 亲缘关系最近。组织表达分析表明,DoSERK 在铁皮石斛中广泛表达,以幼嫩小苗根部的表达量最高。这些说明DoSERK 除了可能参与铁皮石斛体胚发生过程以外,还参与其他生长发育过程。     

The stress kinase gene MtSK1 in Medicago truncatula with particular reference to somatic embryogenesis

Medicago truncatula, a model for legume genomics, can be regenerated by somatic embryogensis by the use of a suitable genotype and an auxin plus cytokinin. The stress response induced by explant wounding and culture is increasingly recognized as an important component of somatic embryo induction. We have cloned and investigated the stress kinase gene MtSK1 in relation to somatic embryogenesis in M. truncatula, using the highly embryogenic mutant Jemalong 2HA (2HA) and its progenitor Jemalong. The main features of the MtSK1 protein of 351 amino acids are an N-terminal kinase domain and a C-terminal glutamic acid-rich region, which is predicted to be a coiled-coil. MtSK1 is a member of the SnRK2 subgroup of the SnRK group of plant kinases. Members of the SnRK2 kinases play a role in stress responses of plants. MtSKI expression is induced by wounding in the cultured tissue independent of auxin or cytokinin. However, in both 2HA and Jemalong, as the callus develops in response to auxin plus cytokinin, MtSK1 expression continues to increase. MtSK1 responds to salt stress in vivo, consistent with its role as a stress kinase. The likely role of MtSK1 in stress-induced signaling will facilitate the relating of stress–response pathways to auxin and cytokinin-induced signaling in the understanding of the molecular mechanisms involved in the induction of somatic embryogenesis in M. truncatula.     

ABA对枸杞体细胞胚发生的调节作用

Using Enzyme Linked Immunosorbent Assay (ELISA) method, we determined the ABA contents of different stages in somatic embryogenesis. The results showed that endogenous ABA contents increased to maximum value twice during somatic embryogenesis. After first maximum value of ABA contents embryogenic cells were observed in callus, and simultaneously, there was a specific protein of somatic embryogenesis investigated by SDS-PAGE. This protein accumulates preferentially in embryogenic callus but not in transferred callus. So it is suggested that ABA could promote the expression of specific genes and the synthesis of embryogenic protein during somatic embryogenesis in Lycium barbarum L. and ABA play an important role in globular stage as well. In addition, treatment of non-embryogenic activity callus with 4 mumol/L exogenous ABA could stimulate somatic embryogenesis. And the ABA function mechanism in relation to somatic embryogenesis was discussed.     

Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1

We isolated the full-length cDNA of PgCDPK2DS1 gene whose expression was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain adult plants from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.     

Expression of the Daucus carota somatic embryogenesis receptor kinase (DcSERK) protein in insect cells

The Daucus carota somatic embryogenesis receptor kinase (DcSERK) gene serves as marker to monitor the transition from somatic into embryogenic plant cells. To determine the intrinsic biochemical properties of the DcSERK protein, a predicted transmembrane receptor, the kinase domain was expressed as a 40-kDa his-tag fusion protein in the baculovirus insect cell system. The kinase domain fusion protein was able to autophosphorylate in vitro. Phosphoamino acid analysis of the autophosphorylated DcSERK protein revealed that it was autophosphorylated on serine and threonine residues. This is the first evidence of the biochemical characterization of a transmembrane receptor kinase from embryogenic plant cell cultures.     

GhAGL15s,preferentially expressed during somatic embryogenesis,promote embryogenic callus formation in cotton (Gossypium hirsutum L.)

Somatic embryogenesis is a useful tool for gene transfer and propagation of plants. AGAMOUS-LIKE15 (AGL15) promotes somatic embryogenesis in many plant species. In this study, three homologous AGL15 genes were isolated from Gossypium hirsutum L., namely GhAGL15-1, GhAGL15-3, and GhAGL15-4. Their putative proteins contained a highly conserved MADS-box DNA-binding domain and a less conserved K domain. Phylogenetic analysis suggested that the three GhAGL15s clustered most closely with AGL15 proteins in other plants. Subcellular location analyses revealed that three GhAGL15s were localized in the nucleus. Furthermore, their expression levels increased following embryogenic callus induction, but sharply decreased during the embryoid stage. GhAGL15-1 and GhAGL15-3 were significantly induced by 2,4-D and kinetin, whereas GhAGL15-4 was only responsive to 2,4-D treatment. Over-expression of the three GhAGL15s in cotton callus improved callus quality and significantly increased the embryogenic callus formation rate, while GhAGL15-4 had the highest positive effect on the embryogenic callus formation rate (an increase from 38.1 to 65.2 %). These results suggest that over-expression of GhAGL15s enhances embryogenic potential of transgenic calli. Therefore, spatiotemporal manipulation of GhAGL15s expression may prove valuable in improving cotton transformation efficiency.     

9-kDa acidic and basic nsLTP-like proteins are secreted in the culture-medium conditioned by somatic embryogenesis in Cichorium

Direct somatic embryogenesis was induced in leaf fragments of the Cichorium ‘474’ genotype. Addition of glycerol to the induction medium allowed a relative synchronization of the first division of the embryogenic cells that only occurs after transfer at day 5 to a medium without glycerol. The abundant presence of 9-kDa extracellular proteins in the culture-medium conditioned by somatic embryogenesis is reported here. Such proteins were also secreted when embryogenesis was initiated in root but were never detected when a non-embryogenic genotype was used as control under the same conditions of culture. Among these proteins, one basic and one acidic isoform were separated through cation-exchange chromatography. Both proteins were recognized by an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). In addition, the partial N-terminal amino acid sequence of each isoform showed similarities with nsLTPs of different plant species. The presence of the acidic nsLTP-like protein was concomitant with the obtention of embryogenic cells during the induction step. The basic form was shown to have only accumulated during the expression step when first divisions of embryogenic cells have occurred. These results allowed us to report, for the first time, the secretion of a 9-kDa acidic nsLTP-like protein in the culture-medium conditioned by plant embryogenic cells.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

The expression of a new HD-Zip II gene, MSHB1, involving the inhibitory effect of thidiazuron on somatic embryogenic competence in alfalfa (Medicago sativa L. cv. Jinnan) callus

Homeodomain leucine zipper (HD-Zip) proteins play important roles in plant development. In this study, we not only identified and characterized a new HD-Zip II gene, designated as MSHB1 (HM114227), from alfalfa (Medicago sativa L. cv. Jinnan) callus treated with thidiazuron (TDZ) which reduced the embryogenic competence of the callus, but also presented the first evidence that MSHB1 is involved in the inhibitory effect of TDZ on somatic embryogenic competence in alfalfa callus. The full-length cDNA was 1,578 bp with an open reading frame of 1,023 bp, encoding a predicted protein of 340 amino acid residues, plus three introns. MSHB1 was strongly expressed in the callus treated with TDZ, but was only slightly detected in the leaf and petiole. TDZ treatment significantly decreased the frequency of somatic embryogenesis in the callus, but up-regulated MSHB1 expression during callus induction, callus maintenance and somatic embryo induction. These results suggest that the inhibitory effect of TDZ on embryogenic competence of alfalfa callus might be mediated by the regulation of MSHB1 expression.     

Somatic embryogenesis and plant regeneration in garden leek

High frequency plant regeneration via somatic embryogenesis has been induced from in vitro shoot-base cultures of seedlings of garden leek (Allium porrum L.). Four main steps are involved in the procedure using BDS medium:

- shoot multiplication with 17.6 mM benzyladenine;

- induction of nodular callus from the in vitro shoot base with 9 mM 2,4-dichlorophenoxyacetic acid;

- initiation of embryogenic callus from nodular callus with 9 mM 2,4-dichlorophenoxyacetic acid +7.6 mM abscisic acid;

- plant regeneration from embryogenic callus with 9.8 mM N⁶-(2-isopentenyl)adenine.

The presence of 2,4-dichlorophenoxyacetic acid in the medium and light conditions were shown to be essential for nodular callus induction and somatic embryogenesis. Abscisic acid was not a prerequiste for somatic embryogenesis, but it significantly increased the frequency.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3 %) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA₃ [5 mg/l]) and higher direct somatic embryogenic frequency (89.6 %). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA₃ (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.