Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants.

Elena O Vidyagina,Nikolay N Kharchenko,Konstantin A Shestibratov

doi:10.3390/plants10010077

Abstract

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

Highlights

The extensive application of biotechnology, genetic engineering, genome editing and genome breeding in modern agriculture and forestry leads to creation of a wide variety of unique genotypes [1,2,3]
It was noted that using axillary buds after removal of the apical bud led to an increase in the regenerant survival by 8%, but the survival did not exceed 61% (Figure 2b)
Microplants cultivated on growth medium with 1% agar-agar were used, four-day-old axillary buds were extracted after removal of apical buds, and the explants were treated with osmotic fluid (OF)

Summary

Introduction

The extensive application of biotechnology, genetic engineering, genome editing and genome breeding in modern agriculture and forestry leads to creation of a wide variety of unique genotypes (breeding and hybrid forms, transgenic lines, candidate varieties and varieties) [1,2,3]. We need to take into consideration the fact that conservation of unique genotypes via regular in vitro subcultivation requires significant expenses, and has the risk of infecting the culture, accumulating somaclonal variations and, may lead to the loss of genotype [6,7]. This is the main reason why different ways of preserving unique genotypes are currently being considered. This method was successfully applied with woody plants [4,13,14,15]

Methods

Findings

Discussion

Conclusion