Dmitry Miroshnichenko

Medium optimization for efficient somatic embryogenesis and in vitro plant regeneration of spring common wheat varieties

The efficiency of morphogenesis is investigated in an in vitro culture of common wheat varieties Andros, Lada, Lyuba, Noris, Taezhnaya, and Enita. Data of an analysis of the phytohormonal composition of the medium showed that, for efficient induction of somatic embryogenesis and plant regeneration, it is preferable to use auxin 2,4-D and Dicamba rather than 2,4,5-T and 4-CPA since they don’t have a sufficient stimulating effect on wheat tissues. Variation of the carbohydrate composition of the nutrient medium showed that the addition of various sources (sucrose, maltose, glucose) at a certain concentration can equally efficiently stimulate a morphogenic response in all studied wheat varieties.

Protocol for efficient regulation of in vitro morphogenesis in einkorn (Triticum monococcum L.), a recalcitrant diploid wheat species

Einkorn (Triticum monococcum L.) is A-genome diploid wheat that has a potential to become a useful model for understanding the biology and genomics in Triticeae. Unfortunately, the application of modern technologies such as genetic engineering, RNAi-based gene silencing and genome editing is not available for einkorn as there is no efficient in vitro tissue culture and plant regeneration system. In the present study an efficient and simple protocol for plant regeneration via direct or indirect somatic embryogenesis and organogenesis has been developed. Various auxins used as sole inductors in einkorn displayed low effect for morphogenesis (0–8%) and plant regeneration (1–2 shoots per explant). The addition of Daminozide, the inhibitor of biosynthesis of gibberellins, together with auxin significantly improved the formation of morphogenic structures, especially when Dicamba (51.4%) and Picloram (56.6%) were used for combination; furthermore, the simultaneous addition of cytokinin into induction medium significantly promoted in vitro performance. Among the tested cytokinins, the urea-type substances, such as TDZ and CPPU were more effective than the adenine type ones, BA and Zeatin, for the regulation of morphogenesis; especially, TDZ was more effective than CPPU for shoot formation (11.73 vs. 7.04 per regenerating callus). The highest morphogenic response of 90.2% with the production of more than 10 shoots per initial explant was observed when 3.0 mg/L Dicamba, 50.0 mg/L Daminozide and 0.25 mg/L TDZ were combined together. Along with the identification of appropriate induction medium, the optimal developmental stage for einkorn was found as partially transparent immature embryo in size of around 1.0 mm. Although in the present study the critical balance between plant growth regulators was established for einkorn only, we assume that further the proposed strategy could be successfully applied to other recalcitrant cereal species and genotypes.

GENETIC TRANSFORMATION OF RUSSIAN WHEAT CULTIVARS

ABSTRACT Wheat (Triticum aestivum L.) ranks first among cereal crops cultivated in Russia. During last years efficient genetic transformation protocol has been developed at the ‘Biotron’, for number of Russian spring cultivars. To produce wheat transgenic plants resistant to different biotic and abiotic environmental stress, researchers worked out on the biolistic transformation procedure, inheritance of transgenes in progenies and field evaluation of homozygous plants.

Agrobacterium-mediated transformation efficiency of carrot embryogenic callus using nptII and hpt selective genes

Experiments were carried out to compare the efficiency of embryogenic carrot calli transformation using selective genes nptII and hpt. Our data showed that it is more preferable to use kanamycin concentration 100 mg/L and hygromycin 20 mg/L for the selection of transgenic shoots. Under the same conditions, the use of kanamycin yielded a greater number of transgenic shoots. It is, therefore, more preferable to use vector constructs containing gene nptII for genetic transformation of the Nantskaya 4 carrot cultivar.

Genetic Engineering of Russian Wheat Genotypes for Abiotic Stress Resistance

The purpose of our investigations was the introduction of desirable genes into Russian wheat cultivars by biolistic transformation system. The ultimate yield of 47 independent transgenic lines of Andros variety, Russian spring wheat, with the herbicide resistance gene bar was produced. In 2004 field tests for herbicide resistance (1.0% Basta) of T3 homozygous offspring obtained from seven transgenic lines demonstrated that agronomic performance of treated transgenic and non-treated control plants were similar. Na+/H+ vacuolar antiporter hvnhx2 gene from barley genome was introduced into wheat under Ubi1 promoter so as to produce the salt- resistant plants. The total number of 14 transgenic T0 wheat plants was generated, and the RT-PCR confirmed the presence of high m-RNA concentration in extracts of 9 independent lines. Significant differences in plant growth were observed between the hvnhx2 transgenic and non-transgenic T1 progeny. The growth of the T1 transgenic plants in saline conditions was about 80% of the same in non-saline conditions, while the non-transgenic plants showed 50% delay in the growth