Ana Sofia Duque

An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l −1 kanamycin and 500 mg l−1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l−1 usually used, we reduced the number of emerging false kanamycin-resistant (KanR) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T0) were regenerated within 3–4 months and those that were able to root in a 50 mg l−1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T0 transformants. Detection of β-glucuronidase (GUS) activity in Adc–Gus T0 plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per KanR embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T1 generation.

Abiotic Stress Responses in Plants: Unraveling the Complexity of Genes and Networks to Survive

Plants are often subjected to unfavorable environmental conditions – abiotic factors, causing abiotic stresses that play a major role in determining productivity of crop yields [1] but also the differential distribution of the plants species across different types of environment [2]. Some examples of abiotic stresses that a plant may face include decreased water availability, extreme temperatures (heating or freezing), decreased availability of soil nutrients and/or excess of toxic ions, excess of light and increased hardness of drying soil that hamper roots growth [3]. The ability of plants to adapt and/or acclimate to different environments is directly or indirectly related with the plasticity and resilience of photosynthesis, in combination with other processes, determining plant growth and development, namely reproduction [4]. A remarkable feature of plant adaptation to abiotic stresses is the activation of multiple responses involving complex gene interactions and crosstalk with many molecular pathways [5, 6].

A proteomics study of the induction of somatic embryogenesis in Medicago truncatula using 2DE and MALDI-TOF/TOF.

Medicago truncatula is a model legume, whose genome is currently being sequenced. Somatic embryogenesis (SE) is a genotype-dependent character and not yet fully understood. In this study, a proteomic approach was used to compare the induction and expression phases of SE of both the highly embryogenic line M9-10a of M. truncatula cv. Jemalong and its non-embryogenic predecessor line, M9. The statistical analysis between the lines revealed 136 proteins with significant differential expression (P < 0.05). Of these, 5 had a presence/absence pattern in M9 vs M9-10a and 22 showed an at least twofold difference in terms of spot volume, were considered of particular relevance to the SE process and therefore chosen for identification. Spots were excised in gel digested with trypsin and proteins were identified using matrix-assisted laser desorption ionization-time of flight/time of flight. Identified proteins indicated a higher adaptability of the embryogenic line toward the stress imposed by the inducing culture conditions. Also, some proteins were shown to have a dual pattern of expression: peroxidase, pyrophosphatase and aspartate aminotransferase. These proteins showed higher expression during the induction phases of the M9 line, whereas in the embryogenic line had higher expression at stages coinciding with embryo formation.

EFFICIENT SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM LONG-TERM CELL SUSPENSION CULTURES OF MEDICAGO TRUNCATULA CV. JEMALONG

SummaryPlants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.

Optimisation of a Selection Scheme using Kanamycin to Improve Transformation of Medicago truncatula cv. Jemalong

We developed an efficient method for in vitro selection of Medicago truncatula cv. Jemalong lines transformed with the nptII gene and subsequent confirmation of phenotype inheritance in these lines. For in vitro selection, the concentration of kanamycin inhibitory to embryogenic callus development and somatic embryo differentiation was identified by placing wounded leaves of non-transformed M. truncatula cv. Jemalong on Embryo Inducing Medium supplemented with 0, 85.8, 128.7, 171.6, 214.6, 257.5 and 343.3 µM of kanamycin. Differentiation of somatic embryos was inhibited with 171.6 µM of kanamycin but callus development was not altered. To confirm transgene inheritance, the kanamycin concentration to distinguish between resistant and non-resistant seedlings was identified by germinating non-transformed seeds of M. truncatula cv. Jemalong on 0.8% (w/v) water-agar plates containing 0, 171.6, 343.3, 514.9 and 686.6 µM of kanamycin. These concentrations did not impair seed germination since all the seedlings exhibited green cotyledons. The effect of kanamycin was only observed at 514.9 and 686.6 µM and on the first pair of leaves, which became white. Due to the high level of resistance to kanamycin of the seedlings the highest concentration is thought to be use to assure the selection efficiency. This optimised antibiotic selection scheme eliminates the regeneration of non-transformed escapes and discriminates between resistant and non-resistant seedlings, confirming the inheritance of the phenotype in transformed M. truncatula cv. Jemalong lines.

Reverse transcription-PCR assay to verify gene integrity within plasmid constructs for plant transformation

We have developed a rapid and simple RT-PCR based method to check the integrity of chimeric genes within plasmid constructs for plant transformation. It exploits the Agrobacterium tumefaciens-mediated transient expression of plasmid constructs in plant tissue. Total RNA was isolated from tobacco leaves co-cultivated for 3 days with Agrobacterium tumefaciens harbouring the plant transformation vectors constructed in our laboratory. First strand cDNA synthesis with oligo(dT) primers generate a pool of cDNA that was used for PCR amplification with primers specific for each of the genes present within the constructs. PCR amplification reactions were successful for all chimeric genes tested, thus confirming their intactness and suitability to be used for stable plant transformation.