A. Iantcheva

Regeneration of diploid annual medics via direct somatic embryogenesis promoted by thidiazuron and benzylaminopurine

Abstract The development of a simple and rapid procedure for direct somatic embryogenesis from wild Medicago spp. (M. truncatula, M. littoralis, M. murex, M. polymorpha) has exploited various explants including meristematic zones. Phytogel-solidified medium supplemented with thidiazuron or 6-benzylaminopurine at different concentrations effectively promoted this process. The first somatic structures emerged within 20 days of culture initiation. Histological analyses confirmed the nature of the directly formed embryos. Secondary embryogenesis was also observed. Cuttings of clusters of primary and secondary embryos were used for cyclic production of new embryo generations. Regenerated plants with well-developed root systems on medium with reduced levels of macroelements and sucrose were easily adapted to a greenhouse.

Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago.

To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.

Variable Leaf Epidermal Morphology in TNT1 Insertional Mutants of the Model Legume Medicago Troncatola

ABSTRACT In this report some typical leaf morphological characteristics of M. truncatula mutants generated by a Tnt1 retrotransposon insertion mutagenesis were evaluated and summarized. It was found that all the examined leaf epidermal parameters were strongly influenced in the Tnt1 mutant lines. Epidermal cells varied in shape and size, and diversified in the patterns of cell walls. Although the leaves of all mutant plants were amphistomatic, stomata were more abundant at the lower (abaxial) leaf surfaces than the upper (adaxial) leaf surfaces. On the other hand, the number of stomata on both leaf surfaces varied widely among different Tnt1 lines. Based on these observations, we conclude that most of the observed mutant phenotypes were caused by the Tnt1 insertions. In addition, the evaluated leaf epidermal features can be reliably applied for phenotypic profiling of M. truncatula mutant lines. Morphological variables in leaf epidermis in all the screened mutants demonstrated that Tnt1 is a very efficient mutagen, confirming that Tnt1 gene tagging strategy is one of the most valuable systems for legume functional genomics.

GENETIC TRANSFORMATION OF MEDICAGO TRUNCATULA USING SYSTEM FOR DIRECT SOMATIC EMBRYOGENESIS PROMOTED BY TDZ

ABSTRACT The efficient procedure for Agrobacterium mediated transformation of Medicago truncatula cv. R 108 1 was developed. Using the advantage of recycling embryo formation of system for direct somatic embryogenesis promoted by TDZ, transgenic plantlets were obtained for a short period of 60 days. Transformation was performed with bacterial strain—LBA 4404, containing binary vector pBI 121 carries two genes—uid A gene exprecing β- glucoronidase (GUS) and npt II gene for resistance to kanamicin. Different parameters of the gene transfer—pre-treatment of the embryo clusters, density of the bacterial suspension, time for inoculation and co-cultivation, type of selection were optimized. For evaluation of the transformation efficiency, histochemical GUS assay was performed and glucoronidase activity was detected in embryos, plantlets and seedlings of T1 progeny. Transgenic nature of plantlets was confirmed by npt II-specific PCR amplification and Southern hybridization. In the progeny transgenes segregated in Mendelian manner.

Is the auxin influx carrier LAX3 essential for plant growth and development in the model plants Medicago truncatula, Lotus japonicus and Arabidopsis thaliana?

The phytohormone auxin is transported by two distinct pathways in plants. Indole-3-acetic acid is mainly transported throughout the plant by an unregulated bulk flow in the mature phloem. The major auxin distribution is regulated via direct transport from cell to cell, known as polar auxin transport (PAT). PAT is maintained by the coordinated action of efflux (PIN) and auxin influx (AUX/LAX) carrier proteins. In this study, we examine, compare and localize the expression of a gene encoding an auxin influx carrier (MtLAX3) from Medicago truncatula in the model plants M. truncatula, Lotus japonicus and Arabidopsis thaliana. Transgenic plants with overexpression and down-regulation of MtLAX3, as well as with expressed promMtLAX3 transcriptional reporters, were constructed for the three model species, using Agrobacterium-mediated transformation. Histochemical and transcriptional analyses revealed the expression of MtLAX3 during various stages of somatic embryogenesis and plant development, as well as during formation of symbiotic nodules. The alteration of the MtLAX3 expression, as well as its overexpression in the analysed model species, results in various abnormal phenotypes and disturbance of leaf and root development. The reported results show that MtLAX3 plays an important role in proper plant growth and development, modelling of the root system and the number of formed nodules and seeds.

Somatic Embryogenesis of the Model Legume - Medicago Truncatula and other Diploid Medics

ABSTRACT This review outlines the information of somatic embryogenesis in model legume Medicago truncatula and diploid medics. Following the data considerable attention is paid on the factors affecting the process during the induction, development, maturation and conversion stages- genotype, explant choice and preparation, origin of somatic embryos and hormonal composition of culture media. A brief consideration is paid to phenotype and fertility of obtained regenerants.

High frequency plant regeneration of diploid Medicago coerulea through somatic embryogenesis

ABSTRACT A successful procedure for in vitro regeneration of M.coerulea (McG) via indirect somatic embryogenesis using different donor explants has been designed. Regenerable callus tissue from leaf, petiole, stem and root explant sources, was formed at good efficiency on MS or SH media, supplemented with BAP (0.2 mg/l) and 2,4 D in two concentrations (4 mg/l and 2 mg/l). However the petiole used as an initial explant, showed the best morphogenic response. Further development of the induced somatic embryos was promoted on free of hormones medium or after short term cultivation (2 weeks) on medium containing BAP (lmg/l) and NAA (0.01mg/l). At the applied culture conditions normal growth and conversion to plantlets of a part of primarily induced somatic embryos was blocked and secondary embyogenesis also takes place. This significantly increased the number of the obtained regenerants and allowed maintenance of the morphogenic culture for a long period.

DEVELOPMENT OF FUNCTIONAL GENOMIC PLATFORM FOR MODEL LEGUME MEDICAGO TRUNCATULA IN BULGARIA

ABSTRACT Legumes, because of the high protein content of their seedsin grain legumes, and leaves in forage legumes, are major crop plants used in human food and animal feed. They have the unique capacity among plants to associate with soil bacteria of the genus Rhizobium to form nitrogen-fixing nodules, thereby limiting the need for exogenous nitrate. The use of the model legume Medicago truncatula over the last 10 years has dramatically improved our understanding of genomic structure and gene function for legumes in general. Nevertheless, the development of new molecular and genetic tools is essential in order to optimise the exploitation of this model plant. For example, insertional mutagenesis is necessary to construct the large-scale mutant collections which will help to identify both key symbiotic and developmental genes, as well as genes of agronomical importance. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the M. truncatula 2HA Jemalong. This mini review comments utility and challenges of exploring forward and reverse genetic tools for functional genomic studies in M. truncatula and particular attention is paid on the use of tobacco Tnt1 retrotransposon as a tool for insertional mutagenesis in this model legume species. Current and future research activities of the AgroBioInstitute that concern this model species and the exploitation of Tnt1 insertional mutant collection will carry on in frame of the NSF funded DO02–105 “Centre for sustainable development of plant and animal genomics” project.

Flow Cytometric Analysis in Diploid Medicago Species from Algeria: Relationship Between Genome Size and Competence for Direct Somatic Embryo Formation

ABSTRACT The genome sizes of eight species from different wild population of diploid Medicago with origin from Algeria have been assessed by flow cytometry. This parameter vary from 2C=0.94 pg in Medicago orbicularis (the smallest genome) to 2C=1.80 pg in Medicago laciniata (the largest one). The relationship between genome size and competence for direct somatic embryo formation in liquid medium for four of the tested species (M. orbicularis Orbi DZ19 C 2C=0.94; M. truncatula Tru DZ 2B 2C=1.08; M.scutelata Scu 274 2C=1.11; M. arabica Ara 46 Ina 2C=1.22) was studied. It was found that Medicago orbicularis with the smallest genome size formed somatic embryos for a shortest period of time, with a high percent of reacting explants and number of somatic embryos per explant. It was followed by M. truncatula, M. scutelata and M. arabica.

PLANT REGENERATION VIA DIRECT ORGANOGENESIS AND SOMATIC EMBRYOGENESIS OF TWO NEW BULGARIAN SPRAY CARNATION CULTIVARS

ABSTRACT Conditions for plant regeneration of leaf expiant from Bulgarian spray carnation cultivars (Fea and Rossitza) were established. Solid MS medium supplemented with BAP and NAA was used for direct induction of adventitious shoots. The optimal medium for regeneration for both cultivars contained 0.9 mg/l BAP and 0.9 mg/l NAA. The mean number of regenerants per explant varied 7–8 for tested cvs. Regenerated plants possessed normal phenotype. At dark and light conditions induction of embryogenic callus were observed for both cultivars. Vigor somatic embryos were obtained on embryo formation medium supplemented with 0.05 mg/l BAP and 250 mg/l casein hydrolysate. They developed to plants with normal phenotype and rooted easily at in vitro condition.