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  , therefore for most collections the researchers are looking for optimal conditions and develop protocols for longer in vitro storage of plants in order to reduce the costs and better preserve the genotypes     .
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   in the presence of growth regulators such as abscisic acid (ABA), by reducing sucrose levels or adding osmotically active substances (e.g., 3% mannitol) to the culture medium   . Exposure to low temperatures results in increased accumulation of unsaturated lipids on cell membranes and hence their thickening and slowing down of cell division and elongation. Cold storage of some species was done either in low light or in complete dark   . In the simplest case, reducing the culture temperature below levels needed for active growth is used to significantly increase subculture intervals. However, one should avoid low temperatures that can cause cold damage  or denaturation of active macromolecules  .
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     . Temperatures of 0˚C - 5˚C were shown to suit well for cold-resistant species such as apple (Malus domestica), plum (Prunus), and strawberry (Fragaria × ananassa)   . For example, the first reported successful cold storage of apple explants was that of the “Golden Delicious” variety. Its microshoots survived after one-year in vitro storage in the dark at 1˚C - 4˚C, with some losses for contamination  . Such protocols, however, are not suitable for longer than a 12-month in vitro storage of aspen, a species often used in biotechnological manipulations  .
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  . 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.
2. Materials and Methods
2.1. Plant Materials
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 (Table 1).
All selected plant lines were cultured in vitro on a WPM medium  without growth regulators, with the addition of 30 g/L sucrose, 9 g/L agar, and MS vitamins, and at pH of 5.6 - 5.8. After all the ingredients were added, the medium was autoclaved for 15 minutes at 1 atm and 120˚C. The plants were cultured under optimal growth conditions: at a temperature of +22˚C - 24˚C and on a 16-hour light/8-hour dark cycle with a light intensity of 2500 - 3000 lux.
2.2. Storage at Low Positive Temperatures
Four alternative sets and control set of long-term storage conditions and medium composition were tested to choose the optimal one (Table 2). Variants of medium composition were compiled on the basis of literature data   . Each set of conditions was tested on 60 microshoots of each genotype. The microshoots were planted in 330-mL jars, 15 per jar. Each jar contained 50 mL of medium. On average, about 2950 microplants took part in each storage condition.
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.
2.3. Culture Recovery after Long-Term Storage
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.
Table 1. List of transgenic aspen lines used in the study.
Table 2. Tests of alternative conditions for long-term storage of aspen explants without subculture.
3. Results and Discussion
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 (Table 3). In all other tests the 24-month storage survival was zero.
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
Table 3. Survival (mean ± SD) of aspen plants of all studied genotypes under different storage conditions at +4˚C.
*,#,$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 (Figure 1).
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 (Figure 2).
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 (Table 3). Noteworthy is that earlier methods of long-term storage mostly modified the composition of culture medium     . Yet, according to our findings, changing the culture medium composition alone is not enough for aspen plants. The life cycle of aspen, as well as of any temperate-climate perennial, consists of alternating periods of active vegetation and rest at low temperatures. Therefore, one might expect that lighting may also play an important role. The study has demonstrated that light intensity and cycle are also essential factors for successful long-term storage without subculture. Going dormant is associated with changes in the hormonal composition and properties of cell membranes, cell division rate and other physiological processes that allow plants successfully survive adverse conditions. In aspen, processes leading to dormancy are triggered
Figure 1. Aspen plantlets of the Pt XV Xeg 1b genotype on a WPM medium supplemented with 15 g/L sucrose, 7.5 g/L sorbitol, and 7.5 g/L mannitol after 24-month storage at +4˚C and a photoperiodic lighting (8 h of light:16 h of dark) with a light intensity ca. 2000 lux.
Figure 2. Aspen plantlets of the PtV22IIGus1a genotype at Week 3 of recovery phase, formed on microshoots after 24-month storage at +4˚C (on a WPM medium supplemented with 15 g/L sucrose, 7.5 g/L sorbitol, 7.5 g/L mannitol, exposed to photoperiodic lighting (8 h of light:16 h of dark) with a light intensity ca. 2000 lux). The pale chlorotic leaves persist after the storage phase.
by changes in the composition of incoming substances, light exposure, and effect of low temperatures  . Therefore, the conditions of Test 4, i.e. reduced content of easily metabolizable sugars in the medium, low light intensity and short photoperiod (Table 2), proved to be most suitable for long-term in vitro storage of aspen explants without subculture.
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
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