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.
One of the currently most promising methods for storage and recovery of plant material of valuable genotypes is in vitro culture of cells isolated from tissues and organs. The method can be used for long-term aseptic maintenance of plant material of certain genotypes and varieties, and thus can contribute to plant breeding and preservation of rare and endangered species. There are a lot of specialized in vitro collections of plants in the world, e.g. the in vitro Collection of Potatoes at NordGen (Nordic Genetic Resource Center, Sweden, Norway), in vitro Collection of Clones of Valuable Genotypes of Deciduous Woody Plants (All-Russian Research Institute of Forest Genetics, Breeding and Biotechnology, Russia), in vitro Collection of the Royal Botanical Gardens (Royal Botanic Gardens, Kew, UK), in vitro Collection of Selectively Bred Varieties of Aspen Populus tremula from Palencia province (Spain) and others. Each of the collections serves the aim of preserving valuable plant genotypes. However, in vitro maintenance of plants is quite costly and associated with the risk of somaclonal variations [
The choice of a storage method depends on the intended storage period. For short- and medium-term storage, growth inhibition is used to increase the subculture intervals. It is the simplest way to limit the growth of plant material in vitro. Culture growth can be slowed down in several ways, including by keeping the culture at low temperatures [
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 [
Aspen (Populus tremula) and its hybrids have long been widely used in biotechnology as a model for studying various aspects of forest woody plant genetics. This is due to a number of reasons: a wide distribution area, a relatively small and effectively transformable genome, rapid growth, easy clonal micropropagation and in vitro cultivation. A topical problem of today is to preserve a large panel of selectively bred and transgenic lines, each having its unique characteristics. These lines may be used as parents, donors of new features, or in fundamental research [
In-vitro cultures of aspen Populus tremula of the non-transgenic genotypes Pt, F2 and PtV22 (kindly provided by the Forest Institute of the National Academy of Sciences of Belarus, the Republic of Belarus), and 46 genotypes/lines of transformed plants carrying the genes GFP, Xeg or Gus and obtained from the Forest Biotechnology Group at the Branch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (
All selected plant lines were cultured in vitro on a WPM medium [
Four alternative sets and control set of long-term storage conditions and medium composition were tested to choose the optimal one (
The plants were cultured for 3 weeks on a 16-hour light: 8-hour dark cycle with a light intensity of 2000 - 2500 lux. After a 3-week growth, after root formation in microplants, the jars were placed into a refrigerator at + 4˚C, with a short photoperiod and a low light intensity. Explant survival was assessed at 6, 12 and 24 months. The obtained data were analyzed by ANOVA-1 using the Statistics 7.0 software.
After 6, 12 or 24 months of storage shoot tips were excised and transferred onto a recovery WPM medium supplemented with 30 g/L sucrose, 9 g/L agar-agar, MS vitamins, 1 mg/L gibberellic acid (GA). Microshoots were planted into 330-mL jars containing 50 mL of the medium each, 15 shoots per jar. The plants were cultivated for 4 weeks with a photoperiodic lighting at 2000 - 2500 lux, 16 hours of light:8 hours of dark. Upon completion of the 4-week period, cutting for multiplication was done.
Name of recombinant gene | Line name | Target effect of recombinant gene |
---|---|---|
GFP, gene encoding fluorescent protein (green fluorescent protein) | Pt III GFP 2b, Pt III GFP 3b, Pt III GFP 3c, Pt III GFP 5b, Pt III GFP 5c, Pt III GFP 6a, Pt III GFP 6c | Reporter gene |
Gus, gene encoding β-glucuronidase | f2 VII Gus 1a, f2 VII Gus 1b, f2 VII Gus 1c, f2 VII Gus 3a, f2 VII Gus 4a, Pt I Gus 1b, Pt I Gus 5a, Pt II Gus 1c, Pt II Gus 3a, Pt V Gus 2c, Pt V22IIGus 1a, Pt V22II Gus 1b, Pt V22II Gus 1c, Pt V22V Gus 14a | Reporter gene |
Xeg, gene of xyloglucanase sp-Xeg from Penicillium canescens | Pt XIV Xeg 1a, Pt XIV Xeg 1b, Pt XIV Xeg 1c, Pt XIV Xeg 4a, Pt XV Xeg 1a, Pt XV Xeg 1b, Pt XV Xeg 1c, Pt XV Xeg 2a, Pt XV Xeg 2b, Pt XV Xeg 2c, Pt XV Xeg 3a, Pt XV Xeg 3b, Pt XV Xeg 3c, Pt XV Xeg 4a, Pt XV Xeg 4b, Pt XV Xeg 4c, Pt XV Xeg 5a, Pt XV Xeg 5b, Pt XV Xeg 5c, Pt XVI Xeg 1a, Pt XVI Xeg 1b, Pt XVI Xeg1c, Pt XVI Xeg 5c, Pt XVI Xeg 8a, Pt XVI Xeg 8b | Changing the carbohydrate composition of wood, increasing biomass |
Test | WMP sugar content (g/L) | Light intensity (lux) | Light/dark cycle |
---|---|---|---|
Control | Sucrose, 30 | 4000 | 16/8 |
1 | Sucrose, 15 Sorbitol, 7.5 Mannitol, 7.5 | 4000 | 16/8 |
2 | Sucrose, 15 Sorbitol, 7.5 Mannitol, 7.5 | 2000 | 16/8 |
3 | Sucrose, 15 Sorbitol, 7.5 Mannitol, 7.5 | 2000 | 12/12 |
4 | Sucrose, 15 Sorbitol, 7.5 Mannitol, 7.5 | 2000 | 8/16 |
After the long-term storage without subculture, all studied aspen genotypes, both transformed and wild-type, showed similar survival rates under similar conditions. However, test 4 was the only one where plants survived the 24-month storage (
As seen during the tests, exposure to the temperature of +4˚C slowed down the growth, caused fall of leaves, with the apical bud alone remaining in the
Test | The number of survived plants (survival rate, %) | ||||||||
---|---|---|---|---|---|---|---|---|---|
6 months | 12 months | 24 months | |||||||
Wild-type | Transgenic | Wild-type | Transgenic | Wild-type | Transgenic | ||||
Control | 95 ± 4 (53) | 1434 ± 23 (52) | 0 | 0 | 0 | 0 | |||
1 | 119 ± 5* (66) | 1821 ± 34* (66) | 0 | 0 | 0 | 0 | |||
2 | 132 ± 4* (73) | 2017 ± 43* (73) | 42 ± 2# (23) | 631 ± 16# (23) | 0 | 0 | |||
3 | 178 ± 5* (99) | 2760 ± 11* (100) | 104 ± 7# (58) | 1572 ± 20# (57) | 0 | 0 | |||
4 | 180 ± 7* (100) | 2760 ± 15* (100) | 180 ± 4# (100) | 2940 ± 11# (100) | 176 ± 7$ (98) | 2706 ± 37$ (98) | |||
*,#,$Statistically significant difference from the results of control according to ANOVA-1, p ≤ 0.05 for all studied storage periods.
vegetative state. Judging by their appearance, the plants went dormant (
After 6, 12, 24 months of storage microshoots were cut and thansferred onto a recovery medium, and the culture jars were exposed to optimal growth conditions. Within a week, 100% of the explants produced green growing shoots from the apical and lateral buds. The leaves that remained after the storage period were not viable and died off. Upon further cultivation, the explants formed well-developed microplants (
According to the obtained results, the 4th set of storage conditions (Test 4) was chosen as the most suitable for in vitro storage of aspen explants and was used as a basis for the development of a protocol of long-term in vitro storage of aspen culture without subculture.
Our findings demonstrate the significance of the intergrated effect of ambient parameters and growth medium on the efficiency of long-term in vitro storage of aspen microplants at low-positive temperatures without subculture. They also show that a change in even one parameter can notably affect microplant survival during their long-term storage for up to 24 months without subculture (
by changes in the composition of incoming substances, light exposure, and effect of low temperatures [
Based on the results of the tests we have developed an effective method for long-term in vitro storage of aspen explants for up to 24 months without subculture and with high survival. The most suitable conditions were as follows: culture on a WPM medium supplemented with 15 g/L sucrose, 7.5 g/L sorbitol, and 7.5 g/L mannitol; 8:16 hours light:dark cycle with light intensity ca. 2000 lux. When transferred to recovery conditions after such storage, the survived explants developed into viable plants without abnormalities within 3 weeks. This method can be used for long-term in vitro storage of both transformed and non-transformed aspen genotypes.
This research was carried out under the state program of the Federal Agency of Scientific Organizations of the Russian Federation (topic “Modification of wood structure and phenotype of aspen plants by superexpression of xyloglucanes genesp-Xeg and inhibition of expression of 4-Coumarate:CoA Ligase gene,” No. 01201352438).
The authors declare no conflicts of interest regarding the publication of this paper.
Vidyagina, E.O. and Shestibratov, K.A. (2018) Efficient Technique for Long-Term in Vitro Storage of Transgenic Aspen Genotypes. American Journal of Plant Sciences, 9, 2593-2600. https://doi.org/10.4236/ajps.2018.913188