Study of the genetic variability of adventive microshoots of Picea pungens Engelm., using DNA markers and flow cytometry

Abstract

For the first time, the genetic variability/stability of blue spruce Picea pungens Engelm. microshoots obtained in vitro by direct regeneration from zygotic embryos under the influence of a synthetic growth regulator with cytokinin activity N-phenyl-N′-1, 2, 3-thidiazurol-5-urea or thidiazuron (TDZ) using ISSR and RAPD analysis, as well as by flow cytometry, was studied. Shoot formation was induced at ½ LV medium using two concentrations of TDZ (0.5 or 1 μM) for 28 days, then plant material was transferred to a hormone-free nutrient medium ½ LV for 35 days. The formed microshoots were separated from the explant’s tissues and cultured on the same hormone-free medium for another 35 days, than their genetic stability was evaluated. To evaluate the effect of each concentration of the growth regulator on the genome’s stability, microshoots formed by three different genotypes of donor plants were used. ISSR analysis revealed a slight polymorphism between the blue spruce microshoots formed by the same genotype. The genetic variability of regenerants in individual genotypes ranged from 1.29 to 1.58 %, while other genotypes have 100 % genetically stable microshoots. At the same time, RAPD analysis did not show differences between the analyzed samples; all studied primers initiated monomorphic spectra. Flow cytometry found that all the studied samples were diploid. The DNA content (2C) in microshoots ranged from (38.86 ± 0.55) – (40.35 ± 0.30) pg, and the genome size (1C) was (18 961.79 ± 256.91) – (19 933.27 ± 526.35) Mbp. The obtained results indicate a low somaclonal variability of blue spruce microshoots formed in vitro under as a result of direct regeneration from zygotic embryos under the influence of the studied concentrations in the TDZ .

Keywords

• in vitro culture
• microshoots regeneration
• thidiazuron
• issr and rapd analysis
• flow cytometry
• blue spruce