Fruit growing
and viticulture of South Russia

Annotation

In this study, we present the experimental data of optimizing the protocol for using different combinations of phytohormones (6-BAP, GA3) to increase in tea microshoots in vitro culture. Subculturing was done in regular intervals from four to 16 weeks (four subcultures). It was find that adding growth regulator BAP to the nutrient medium, according to the prescription of Murashige-Skoog, the concentration of 3 mg/l had a significant effect the number of producing shoots. With an increase dose up to 5 mg/l, due of callus tissue proliferation, the inhibition of reproduction were recording. In the variants of experiments, with combination of different concentrations of BAP and GA3, microshoots multiplication was observed, in comparison with the experiment when BAP was not presented. The best effect of the GA3 addition in the average number of producing microshoots was demonstrated in the fourth subculture BAP in concentration of 3 mg/l + GA3 0,5 � 1 mg/l (7-7,4 qt./expl.) At the same time, gibberellic acid supported not only increasing in the quantity of adventitious microshoots, but also its proliferation in height, and it can be used for further multiplication, and shortening of rhizogenesis stage. A decrease in the microshoots quantity from one explant, lengthening of their meristem, morphology changing, and the appearance of fasciation symptoms are associated with the use of BAP higher concentrations (5-7 mg/l). On the green mass from one explant, there was a distinction in the results between different combinations of growth regulators. The largest green mass weight (385 mg), on nutrient medium of MS + 5 mg/l BAP + 1 mg/l GA3 was obtained. The presence of BAP (7 mg/l) + GA3 (0,5-1 mg/l) supported to callus growth, which was the reason for the green mass weight decrease.

Annotation

The yield capacity of tea plantations, the quality of the collected raw materials directly depends on the amount of precipitation, especially in summer. In this regard, the efforts of scientists are aiming on studying the physiological, biochemical and molecular mechanisms of tea plants resistance to high temperatures. Stress induction in vitro is an alternative tool for studying these processes in depth. To select drought-resistant genotypes, we simulated moisture deficiency in vitro by adding a selective agent, polyethylene glycol (PEG � 30 g/l), to the nutrient medium. Osmotic stress was studying by conductometric method, in terms of the electroconductivity of tissues and the stability of cell membranes. Of the whole studied group of tea somaclones, only somaclone Sc-27 did not have an increase in the electroconductivity of leaf tissues and a reduce in the stability of cell membranes, which indicates its resistance to water deficiency. On these positions tissue damage in another plants, caused by the action of osmotic stress were observing. The research protocol also included the study of level expression the dehydrin 2 gene � DHN2, as the most informative genetic marker of drought resistance. Conducting PCR analysis showed that under water deficiency caused by the addition of PEG to the nutrient medium, the dehydrin 2 (DHN2) gene showed a high level of expression of Sc-27 somaclone. This confirms the direct relationship between the action of osmotic stress and the level of this marker expression. Significantly increased the expression of DHN2 gene with the addition of PEG, confirms the water deficiency in tea microplants, which is a trigger for the production of DHN2. Thus, during of osmotic stress inducted in vitro, among the studied somaclones of tea, Sc-27 demonstrated high adaptability to water deficit. The PCR analysis confirmed this by showing that the marker of drought resistance, the dehydrin 2 gene, were expressing much higher.

Annotation

The article presents the study of variability in vitro long-term cultivated somaclones, obtained through callus tea culture. Genetic diversity was tested using SSR and ISSR primers. The multilocal ISSR primers characterized by a higher level of polymorphism (40�52 %). The presence of genetic distances between somaclones and between calluses, from which they were obtained, was revealed, although the coefficient of differences was not so significant and amounted to 0,05�0,1. All somaclones had divided into three clusters. Some of the samples were identical, with zero differences. The highest variability of the whole group was detected in the sample �16. Analysis of somaclones population structure revealed that in the study group populations have not expressed by the allele frequencies. The variability induced from the tea callus is of a point nature. All alleles were distributed in the samples in approximately equal proportions. The average distance between samples in the population (expected heterozygosity) were about the same, and the frequency of alleles was 0,57, so all possible populations fell into the same cluster. Tea callus also showed a low level of DNA polymorphism, and a low percentage of genetic differences with somaclones, which confirms the presence of point variation. There was no correlation between the specific somaclon and callus, from which it was obtained � the clustering was of a random nature. SSR and ISSR analysis of full genome DNA showed, that the amplified SSR fragments differed in size within 10 base pairs; heterozygotes were not detected in the population. The structure of the population was homogeneous with an equal distribution of allele frequencies within the population. On the whole, there were a low percentage of genetic differences in the somaclonal variability of tea.