Polyamines and somatic embryogenesis in two Vitis vinifera cultivars

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.     

Induction of somatic embryogenesis and plantlets in tendrils of Vitis vinifera L.

Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium. Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed. Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997     

Characterization of VvSERK1, VvSERK2, VvSERK3 and VvL1L genes and their expression during somatic embryogenesis of grapevine (Vitis vinifera L.)

Little is known about the genes expressed during grapevine somatic embryogenesis. Both groups of Somatic Embryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somatic embryogenesis in various plant species. Therefore, we identified and analysed the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2 and VvSERK3 are very similar to that of registered SERK proteins, with highest homologies for the kinase domain in the C-terminal region. The amino acid sequence of VvL1L presents all the domains that are characteristic for LEC1 and L1L proteins, particularly, the 16 amino acid residues that serve as signature of the B-domain. Phylogenetic analysis distinguishes members of subclass LEC1 and subclass L1L, and VvL1L is closely related to L1L proteins. Using semi-quantitative RT-PCR, we studied gene expression of VvSERK1, VvSERK2, VvSERK3 and VvL1L in calli and somatic embryos obtained from anther culture of Vitis vinifera L. cv Chardonnay. Expression of VvSERK2 is relatively stable during in vitro culture. In contrast, VvSERK1, VvSERK3 and VvL1L are expressed more 4 to 6 weeks after transfer of the calli onto embryo induction medium, before the visible appearance of embryos on the calli as seen by environmental scanning electron microscopy. Later on (8 weeks after transfer) VvSERK1 expression is maintained in the embryogenic calli and VvSERK3 in the embryos, whereas VvL1L expression is very low. All together, these data suggest the involvement of VvSERK and VvL1L genes in grapevine somatic embryogenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Paul Schellenbaum and Alban Jacques contributed equally to this work.     

Stimulation of somatic embryogenesis and plant regeneration from anther culture of Vitis vinifera cv. Cabernet-Sauvignon

Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera Cabernet-Sauvignon, a cultivar so far considered as recalcitrant to in vitro regeneration. Anthers enclosing microspores near the first pollen mitosis were found to be the most responsive. However, from a practical point of view anther length proved to be an easier criterium for determining the optimal physiological anther stage. Calli derived from the anther somatic tissues produced embryoids only when cultured on a medium supplemented with casein hydrolysate. Glutamine and adenine were found to stimulate this embryoid production. Evidence is presented that early removal of cotyledons increases the frequency of normal development of embryoids into plantlets.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - BA 6-benzylaminopurine     

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.     

Characterization of potential ABA receptors in Vitis vinifera

Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. The phytohormone abscisic acid (ABA) is a key endogenous messenger in a plant’s response to such stresses. A novel ABA binding mechanism which plays a key role in plant cell signaling cascades has recently been uncovered. In the absence of ABA, a type 2C protein phosphatase (PP2C) interacts and inhibits the kinase SnRK2. Binding of ABA to the PYR/PYLs receptors enables interaction between the ABA receptor and the PP2C protein, and abrogates the SnRK2 inactivation. The active SnRK2 is then free to activate the ABA-responsive element Binding Factors which target ABA-dependent gene expression. We used the grape as a model to study the ABA perception mechanism in fruit trees. The grape ABA signaling cascade consists of at least seven ABA receptors and six PP2Cs. We used a yeast two-hybrid system to examine physical interaction in vitro between the grape ABA receptors and their interacting partners, and found that twenty-two receptor-PP2C interactions can occur. Moreover, quantifying these affinities by the use of the LacZ reporter enables us to show that VvPP2C4 and VvPP2C9 are the major binding partners of the ABA receptor. We also tested in vivo the root and leaf gene expression of the various ABA receptors and PP2Cs in the presence of exogenic ABA and under different abiotic stresses such as high salt concentration, cold and drought, and found that many of these genes are regulated by such abiotic environmental factors. Our results indicate organ specificity in the ABA receptor genes and stress specificity in the VvPP2Cs. We suggest that VvPP2C4 is the major PP2C involved in ABA perception in leaves and roots, and VvRCAR6 and VvRCAR5 respectively, are the major receptors involved in ABA perception in these organs. Identification, characterization and manipulation of the central players in the ABA signaling cascades in fruit trees is likely to prove essential for improving their performance in the future.     

MiRNA expression analysis during somatic embryogenesis in Coffea canephora

Plant Cell, Tissue and Organ Culture (PCTOC) - The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton...     

AtLTP1 luciferase expression during carrot somatic embryogenesis

The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo.     

Differential gene expression for isozymes in somatic mutants of Vitis vinifera L. (Vitaceae)

Isozyme electrophoresis was used as a method to provide a measure of relationship among Italia, Rubi, Benitaka, and Brasil cv of Vitis vinifera traditionally grown in Marialva, a town in the northwestern region of the state of Paraná, southern Brazil. No allelic variation was observed for esterase (EST), malate dehydrogenase (MDH), peroxidase (POD), glutamate dehydrogenase (GTDH), alkaline phosphatase (AKP), acid phosphatase (ACP), and aspartate amino transferase (AAT). Tissue specific and variation in staining intensity of EST, MDH, POD, and GTDH isozymes indicate differential gene expression in colour grape varieties. Regulatory genes may be operative in determining the number of molecules of enzymes in a cell and determining the berry skin polymorphism in four cultivars. Change frequency for berry skin colour suggest the occurrence of somatic crossing-over in naturally cultivated plants and a periclinal chimerism in Brasil cv. The four grape colour cultivars seem to be clones of the same cultivar.     

An efficient leaf-disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.)

By manipulating hormone levels, light intensities and temperature, we have developed an efficient leaf-disc method for the regeneration of plants via embryogenesis and for transformation in four genotypes of Vitis vinifera L. In MS basal medium supplemented with 1 mg l^-1 6-benzylaminopurine (BAP) and 0.1 mg l^-1 2,4-dichlorophenoxyacetic acid, leaf discs cultured for 2 weeks under dark conditions produced calli in over 80% of the cultures. These subsequently differentiated into pro-embryos and embryos only if kept under conditions of low light intensity (15 µE m^-2 s^-1) for 2 weeks before being transferred to conditions of high light intensity (60 µE m^-2 s^-1). If the calli were directly transferred to high light intensity, the differentiation into embryos was blocked and the calli turned pink. The somatic embryos germinated at a frequency of about 10% on NN basal medium and about 32% on NN medium supplemented with 1 mg l^-1BAP and 0.1 mg l^-1 indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4°C for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 µE m^-2 s^-1), shoot buds were produced, whereas under conditions of low light intensity (15 µE m^-2 s^-1) secondary embryogenesis was induced. About 90-95% of the in vitro grown plantlets could be successfully transferred to soil. The above method was also applicable for developing transgenic embryos whose transgenic nature was monitored using #-glucuronidase as a reporter gene.     

A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless

To establish an efficient regeneration protocol for functional validation and variety resistance improvement, a long-term system that useful for embryogenic culture maintenance and transformation was developed through recurrent cycles of secondary embryogenesis from Vitis vinifera L. cv. Thompson Seedless. Three media and five types of somatic embryo in secondary embryogenesis were evaluated. Somatic embryos (SE) in the torpedo and mid-cotyledonary stages gave the best embryogenic responses with re-induction rates of about 80 %. Embryogenic callus, proembryonic masses and SE produced in the system, could be propagated for over 3 years and all proved competent for Agrobacterium-mediated transformation. Based on this system, different transgenic selection regimes were compared. Addition of kanamycin at 4 weeks after co-cultivation was optimal for embryo recovery. Plant conversion was improved by alternating culture on two media: one containing 0.2 mg l^?1 BA and the other 0.25 mg l^?1 kinetin. To further test the efficiency of the system, a ubiquitin ligase gene (VpPUB23) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. Of the 351 transgenic plants obtained, those overexpressing VpPUB23 exhibited decreased resistance to powdery mildew compared with non-transgenic plants.     

Characterization of competence during induction of somatic embryogenesis in alfalfa tissue culture

Systematic manipulations of the culture protocol leading to somatic embryogenesis in alfalfa petiole-derived callus were performed to study the competence phase during somatic embryogenesis in alfalfa. These demonstrated a requirement for the acquisition of competence prior to the induction of the embryogenesis pathway. Induction was triggered by a number of synthetic auxins including 2,4-dichlorophenoxyacetic acid (2,4-d). Competence could be acquired in the presence of these auxins as well as phenylacetic acid (PAA), an auxin that did not induce embryogenesis. Different degrees of competence were apparently acquired by exposure to 2,4-d and PAA. Some degree of competence was acquired in the absence of auxin treatment. The current understanding of the concept of competence is discussed.PRC contribution no. 1431     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.