Abstract
In vitro culture of isolated cells from tissues and organs is sometimes used to preserve and reproduce unique genotypes of woody plants. The technique, however, requires regular subculturing which raises storage costs and creates risks for contamination and accumulation of somaclonal variations. We examined the effects of sugar composition of culture medium, the length of photoperiod, light intensity, and ambient temperature on the survival of plant material in vitro. The study was performed on 49 genotypes of Populus tremula (46 transgenic genotypes carrying GFP-, Xeg- and Gus-genes, and 3 control (wild-type) genotypes). It was shown that effective storage of plants was achieved through optimization of the combined effects of all storage parameters under study. Based on the experimental data, we developed a protocol for long-term in vitro storage of desirable genotypes without subculture and with a survival rate of up to 98%. The best results were obtained when the plant material was pre-cultured on a WPM medium containing 15 g/L sucrose, 7.5 g/L sorbitol and 7.5 g/L mannitol, and then stored at +4°C under a 24-hour light day cycle with only 8 hours of light per day and maximum light intensity of 2000 lux. Post-storage recovery was done by culturing on a medium containing 1 mg/L gibberellic acid. The developed method can be used for effective in vitro storage of the studied genotypes for up to 24 months without subculture.
Highlights
We examined the effects of sugar composition of culture medium, the length of photoperiod, light intensity, and ambient temperature on the survival of plant material in vitro
The study was performed on 49 genotypes of Populus tremula (46 transgenic genotypes carrying GFP, Xeg- and Gus-genes, and 3 control genotypes)
Based on the experimental data, we developed a protocol for long-term in vitro storage of desirable genotypes without subculture and with a survival rate of up to 98%
Summary
At near-zero temperatures plants can be stored in vitro for years without subcultures This storage method is suitable for many plants of temperate climate including berry and fruit trees [12] [13] [14] [15]. Its microshoots survived after one-year in vitro storage in the dark at 1 ̊C - 4 ̊C, with some losses for contamination [18] Such protocols, are not suitable for longer than a 12-month in vitro storage of aspen, a species often used in biotechnological manipulations [19]. Long-term in vitro storage of cultures without subculturing and creation of a genotype bank are viewed as a way to preserve plants with valuable properties
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