Cryopreservation as a Method to Preserve Some Fruit and Berry Crops and Wild Medicinal Plants

The cryopreservation eff ect on viability of black currant cuttings (Ribes nigrum L.), sweet cherry pollen (Cerasus avium L. Moench), Prunus rossica Erem. diploid plum and Prunus domestica L. garden plum pollen, as well as the seeds of wild medicinal plants: Syrian rue (Peganum harmala L.), tansy (Tanacetum ulutavicum Tzvel.) and sawwort (Serratula dissecta Ledeb.) was studied. The viability in 5 cultivars of R. nigrum L. was decreased after cryopreservation; in 7 cultivars of C. avium L. (17 cultivars totally) it was increased, in 6 cultivars there were no changes, in 4 ones it was decreased; in 3 varieties of P. rossica Erem. (9 cultivars totally) the one was increased, in 3 ones there were no changes, in 3 ones it decreased; in 6 cultivars of P. domestica L. (13 cultivars totally) this index was increased, in 3 ones there were no changes, in 4 ones it decreased; in P. harmala L. seeds it was unchanged; in the seeds of S. dissecta Ledeb. it was increased. For the seeds of three medicinal plants there were selected the optimal concentrations of cryoprotectants and thawing modes as follows: P. harmama L. – 100% glycerol, slow freeze-thawing; T. ulutavicum Tzvel. – 10% sucrose, slow warming; S. dissecta Ledeb. – 3% DMSO, rapid warming.

Цель работы -изучение влияния длительного воздействия сверхнизких температур (-183… -185℃) на показатель жизнеспособности черен-Сultured plants play a signifi cant role in biological diversity of our planet [4].Plant biodiversity is conserved in situ (in fi elds, forests, garden areas) and ex situ (in collection gardens and forest nurseries).The Experimental Plant-Breeding Station of the N.I.Vavilov Institute of Plant Genetic Resources (VIR) is an important participant of these activities.Unstable climate and environmental conditions entail a risk of the loss of the collection specimens.Cryopreservation under extremely low temperatures (-185 ... -196°C) allows to preserve the viability of gene pool of cultured fruit and berry plants for an unlimited time and thus it is an alternative method for conservation of vegetatively reproduced fruit and berry crops [9,24].Herewith, the plant material exposed to ultralow temperatures remains genetically stable [11].
Cryopreservation of fruit crop pollen is important not only for conservation of genetic resources, but also for the breeding programs [26,27].Weather conditions signifi cantly aff ect the formation of pollen of stone fruits.For example, extremely unfavourable weather conditions could result in a reduced viability of pollen and cryopreservation enables the use of the pollen with a high viability, selected in favourable years [28,33].
Cryopreservation of wild medicinal plant seeds is of great importance for establishing of the Kazakhstan national genetic bank of fl ora.The methods to freeze-thaw the seeds and store in liquid nitrogen were fi rstly developed in the late 80 s. [6,15].Based on the methodological developments of the VIR cryounit [13,21,29] the Kazakh scholars initiated the programs to conserve the seeds of valuable medicinal plants using long-term storage in liquid nitrogen [5,8].
Investigations in Ribes nigrum L. involved the cultivars suitable for growing in the northern regions, namely early ripening, high-yielding, diseaseresistant, having quality of dry pick off berries.To date there were no studies conducted in Cerasus avium L. pollen, collection material was assessed to work with Prunus domestica L. and Prunus rossica Erem.pollen.

Materials and methods
In the experiments the cuttings of Ribes nigrum L. black currant (5 cultivars), Prunus domestica L. garden plum pollen (13 varieties), Prunus rossica Erem.diploid plum pollen (9 cultivars) and Cerasus avium L. Moench sweet cherry pollen (17 cultivars) were used.All these samples were obtained from the collection of plant genetic resources of VIR.Seeds of medicinal plants were collected in Kazakhstan, cuttings of black currant were selected in the collection nursery of the branch of the Polar Experimental Station of VIR.
To cryopreserve Ribes nigrum L. cuttings, we used own method applied earlier for the apple buds [7].The cuttings of Ribes nigrum L. were collected in winter, their viability was evaluated by sprouting in water at 21°C under artifi cial lighting.Then, the cuttings were divided into small segments with 2-3 buds and dried at -4...-5°C to a residual humidity of 28-32%.Hereafter, the plant material was cooled according to the following procedure: the temperature in MDF-U442 (T) freezer (Sanyo Medical Freezer, Sanyo Electric Biomedical Co., LTD., Japan) was reduced every hour by 2°C within the range of -5...-32°С, then it was lowered by 4°С every hour and reached -50°С.Herewith prior to the beginning of the next hour the biomaterial was incubated at a fi xed temperature.Then the samples were placed into liquid nitrogen vapour for a long-term storage (-183…-185ºС).After 6 months, the cuttings were warmed in a water bath at 37°C for 4-5 min.They were planted in a fi eld within spring-summer period to test the viability [31].
Peganum harmala L. is a perennial herbaceous plant with a height of up to 50 cm, which occurs widely in lowland areas, excluding high mountains.Signifi cant thickets are found mainly in South Kazakhstan.Syrian rue is a valuable medicinal plant, whereas it contains a signifi cant amount of alkaloids, quinazoline and indole derivatives.
Serratula dissecta Ledeb. is a perennial plant, endemic, growing on rocky and gravelly mountain slopes, in the plain steppes of Central and Eastern Kazakhstan, as well as in the Dzungarian Alatau [16].Sawwort is a valuable medicinal plant, whereas it contains phytoecdysteroids with anabolic, lipid-lowering, anti-infl ammatory, adaptogenic, hemorheological properties [1].
Germination capacity and rate were investigated according to the method reported by M.S. Zorina and S.P. Kabanov [34], as well as M.V. Maltseva [12].The surface of the seeds was sterilized with 0.5% KMnO 4 and 75% calcium hypochlorite solutions under laboratory conditions, and then the seeds were germinated in a climatic chamber at 24°C.
The significance of differences of compared samples for three independent experiments was assessed using Student's t-test after testing for normality of distribution using the method of N.L. Udolskaya [25].

Results and discussion
Table 1 shows the indices of initial (prior to cryopreservation) viability of black currant cuttings (Ribes nigrum L.) and the index after cryopreservation.
The initial viability index of currant cuttings among cultivars was 80-90%; after storage in liquid nitrogen vapour, the viability of cuttings decreased signifi cantly for all the cultivars compared to the initial one: from 6.4 (Severnoye siyaniye) to 10.6% (Olesha) (Table 1).During spring-summer, the plants grew well, in summer they sprouted young healthy shoots and formed fruitful buds the following year.
Таким образом, при использовании нетоксичных криопротекторов и их смеси для хранения растительных объектов в низкотемпературных условиях повышался показатель их выживаемости.Кроме того установлено, что вариативность полученных результатов обусловлена сортовыми, особенностями и видом растительного материала.Similar results were obtained when using a mixture of glycerol (50%) and sucrose (10%) solutions as well as slow warming.The using of 10% sucrose and slow warming increased the germination of Tanacetum ulutavicum Tzvel.tansy seeds compared to the control almost in 2.5 times, however, with an increase in the concentration of sucrose up to 20%, seed viability was decreased.There was no germination of tansy Tanacetum ulutavicum Tzvel seeds after rapid warming in a water bath with 10 and 20% sucrose solutions and rapid.It could be supposed that in case of using this cryoprotectant only slow warming is recommended (Table 5).The protective properties of glycerol (100%) and safe concentrations of DMSO were investigated with sawwort Serratula dissecta Ledeb.seeds.The use of DMSO cryoprotectant in the studied concentrations even increased the germination of seeds if compared with the control, the highest rates were observed at a concentration of 3% (in 2.5 times).Glycerol as a cryoprotectant also signifi cantly increased seed germination if compared with the control (Table 5).
It should be noted that stratifi cation eff ect after deep freezing of dry seeds does not always appear; when germinating seeds after cryopreservation, it is necessary to carry out stratifi cation in a refrigerator at 4°C to overcome the dormant state.In particular, cryopreservation did not aff ect the duration of stratifi cation and seed germination of apple tree, Norway maple and Tatarian maple [3,21].It was unexpected to found stratifi cation in the seeds of Tanacetum ulutavicum Tzvel.and Serratula dissecta Ledeb.after cryopreservation.
Thus, when using non-toxic cryoprotectants and their mixtures to store the plants under low-temperature conditions, their survival rate was increased.In addition, it has been established that the variability of the obtained results is due to the varietal characteristics and the plant type.3.After cryopreservation of Prunus rossica Erem.diploid plum pollen (9 cultivars), its viability was increased (3 cultivars), there were no changes (3 cultivars), and was signifi cantly decreased (3 cultivars).In the Prunus domestica L. garden plum pollen (13 varieties) this index was signifi cantly increased (in 6 cultivars), remained unchanged if compared with the control (in 3 cultivars) and was signifi cantly decreased (in 4 cultivars).
4. The most eff ective method of cryopreservation without cryoprotectants for the seeds of Peganum harmala L. wild rue was cooling down to -50°C followed by immersion into liquid nitrogen and cooling down to -50°C without immersion into liquid nitrogen, and for Serratula dissecta Ledeb.sawwort cooling down to -50°C with the following immersion into liquid nitrogen.
5. Optimal concentrations of cryoprotectants and the thawing methods of seeds for three types of medicinal plants were selected.These were 100% glycerol and slow warming for Peganum harmala L., Syrian rue; 10% sucrose and slow warming for Tanacetum ulutavicum Tzvel tansy; 3% DMSO and rapid warming for Serratula dissecta Ledeb.sawwort.
Based on the obtained results, we suggest that cryopreservation enables preserving the fruit and berry crops and seeds of wild medicinal plants for a long time without signifi cant loss of viability.

Conclusions 1 .
The viability of cuttings of Ribes nigrum L. black currant for all the cultivars decreased by 6.4-10.6%after cryopreservation.The plants developed well under fi eld conditions, in the summer they sprouted healthy shoots and formed fruitful buds for the next year.2.After cryopreservation of Cerasus avium L. Moench sweet cherry pollen (altogether 17 cultivars), its viability was signifi cantly increased (in 7 cultivars), remained at the initial level, i. e. the diff erence was insignifi cant (in 6 cultivars), or was signifi cantly decreased (4 cultivars).проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 29, №/issue 1, 2019

Table 1 .
Viability indices of cuttings of Ribes nigrum L. black currant prior to and after storage in liquid nitrogen vapour saccording to the data of 2017 (M ± SE).

Table 3 .
Viability of pollen of Prunus rossica Erem.diploid plum and Prunus domestica L. garden plum prior to and after storage in liquid nitrogen vapour according to the data of 2017 (M ± SE) проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine