In vitro germplasm cold storage of fruit and berry plants of Kazakhstan

EurAsian Journal of BioSciences
                 Eurasia J Biosci 14, 1213-1219 (2020)

                 In vitro germplasm cold storage of fruit and berry plants
                 of Kazakhstan
                 Timur T. Turdiyev 1*, Irina Yu. Kovalchuk 2, Balnur Zh. Kabylbekova 2,
                 Nina I. Chukanova 2, Sergey N. Frolov 1
                  Institute of Plant Biology and Biotechnology, Almaty, KAZAKHSTAN
                  Kazakh Research Institute for Fruit & Vegetable Growing, Almaty, KAZAKHSTAN
                 *Corresponding author:

                     In vitro germplasm cold storage is an important part of the plant biodiversity conservation strategy,
                     which ensures long-term reliable conservation and use of fruit and berry crops. Screening of apple,
                     pear, cherry, raspberry, blackcurrant and strawberry plants for Cold Storage Duration on Murashige
                     and Skoog (MS) medium without plant growth regulators (PGRs). Murashige and Skoog (MS)
                     medium without plant growth regulators (PGRs) showed that the viability depends on the breed and
                     genotype, better preserved berry crops, worse fruit. The effect of plant growth regulators, carbon
                     sources and nitrate nitrogen on the storage duration in vitro of various crops in low positive
                     temperature and illumination conditions was studied. Many genotypes were well preserved at 4°C in
                     semi-permeable plastic tissue-culture bags for cultivation in MS medium with 3% sucrose or 2%
                     sucrose + 2% mannitol with or without PGRs. Reducing the nitrogen concentration increased the
                     storage duration of some crops (apple, pear), but abscisic acid did not improve the storage time. In
                     vitro cold collection is a reserve of the field collection and now has 196 samples of fruit and berry
                     crops stored in bags for cultivation on MS medium with Nitrate nitrogen at 50%, 0.5 mg·L-1 N6
                     benzylaminopurine (BAP), 0.1 mg·L-1 indole-3-butyric acid (IBA) with 3% sucrose and 0.75% agar at
                     pH 5.7.

                     Keywords: germplasm, genetic resources, fruit and berry crops, cold storage, in vitro, plant growth
                     regulators, carbohydrates

                     Turdiyev TT, Kovalchuk IYu, Kabylbekova BZh, Chukanova NI, Frolov SN (2020) In vitro
                     germplasm cold storage of fruit and berry plants of Kazakhstan. Eurasia J Biosci 14: 1213-1219.

                     © 2020 Turdiyev et al.
                     This is an open-access article distributed under the terms of the Creative Commons Attribution License.

   INTRODUCTION                                                      maintenance of collections is based only on vegetative
                                                                     reproduction. Using this approach is a reliable and cost-
    In Kazakhstan various varieties of fruit and berry               effective way to preserve genetic resources and allows
crops are cultivated and unique wild-growing forms are               to maintain the viability and regenerative ability of plants
grown, including apple tree (Malus sieversii (Ledeb.) M.             for a long time, protects collections from the impact of
Roem., Malus x domestica Borkh.), pear (Pyrus                        extreme environmental factors, minimizes the
communis L., Pyrus regelii Rehder), cherry (Prunus                   probabikity of genetic changes. Preservation of the gene
cerasus L., Cerasus tomentosa Thunb., Cerasus                        pool in vitro is carried out in two ways: a) Limiting the
fruticosa Pall.), raspberries (Rubus idaeus L.),                     growth of crops in conditions of low positive
blackcurrant (Ribes nigrum L.) and strawberries                      temperatures of 3-10°C (cold storage) and effective in
(Fragaria L., Fragaria x ananassa Duch.), adapted to                 the case of short and medium – from 1 to 10 years of
local environmental conditions. However, in recent                   storage. Against the background of cold storage,
decades, under the effect of environmental and                       additional methods of delaying metabolic processes are
anthropogenic factors, there has been a reduction in the             carried out – the use of growth inhibitors and the
areas occupied by these plants.                                      reduction of nutrients and hormones in media; b) Deep
    Currently, along with traditional methods of                     freezing and storage at an ultra-low temperature of -
preserving genetic resources (in nature reserves,                    196°C (cryopreservation) when the metabolism is
national parks, partial reserves, pomological and                    completely slowed down, it is used for long-term
botanical gardens), there is the most promising                      storage. If necessary, at any time, the material can be
approach to solving this problem – the creation of
germplasm bank using in vitro biotechnological                                                             Received: November 2019
methods, especially relevant for the conservation of fruit                                                    Accepted: March 2020
and berry plants, since their reproduction and                                                                    Printed: May 2020

EurAsian Journal of BioSciences 14: 1213-1219 (2020)                                                         Turdiyev et al.

extracted from the germplasm bank, propagated in vitro,         illumination 25 µmol m-2s-1, with a 16-h light photoperiod
and used in the selection process, the laying of                (25 µmol m-2s-1), then acclimated for one week at 10-h
plantations, or transferred to the natural environment to       light 23°C / 14-h dark 4°C photoperiod. The test involved
restore phytocenosis.                                           five plants of each genotype without repetitions. The
    The most common method of maintaining collections           condition of stored plants was evaluated every 3 months
of plant tissues in vitro, which allows to increase the shelf   on a scale from 0 to 5 points, in appearance: 1 - plants
life of plant material, is the use of low positive              are brown, sometimes yellow; 2 - plants are yellow-
temperatures (Blakesley et al. 1996). The success of the        brown; 3 - plants are etiolated, yellow-green; 4 - plants
method is determined by the genotype (Engelmann                 with slight etiolation, green; 5 - plants with bright green
1997), but it also depends on the storage temperature,          leaves and stems. Observations were made before the
illumination, nutrient composition, and precultivation of       complete death of plants.
plant tissues before laying for cold storage (Jouve et al.          Under the same conditions, experimental studies
2000, Mbulawa 2017).                                            were conducted to determine the optimal nutrient media
    To slow down the growth of aseptic plants, the              for each culture, increasing the duration of in vitro plant
composition of the nutrient medium is modified (Reed            cold storage. To determine the composition of the
1999). In particular, osmotically active substances such        nutrient medium that increases the shelf life of all the
as mannitol and polyethylene glycol are used (Chen and          studied crops, the effect of carbohydrates and their
Dribnenki 2004), reducing the concentration of basic            concentrations, plant growth regulators (0.5 mg·L-1 N6
media by 25% and 50% (Moriguchi and Yamaki 1989,                benzylaminopurine (BAP), 0.1 mg·L-1 indole-3-butyric
Mitrofanova et al. 2018.), add sucrose and a retardant,         acid (IBA) and nitrogen in MS medium was studied.
chlorcholinchloride, to the nutrient medium (Mitrofanova        Carbon sources – 3% sucrose, 2% or 3% mannitol, 2%
et al. 2018). In addition, they reduce the photoperiod          sucrose + 2% mannitol, nitrogen source - salts
(Reed 1999), use different materials for closing test           containing nitrogen nitrate (NO3) with a concentration of
tubes (Vysotskaya 2001), or store samples in plastic            100%, 50%, 25%, each variant was with or without plant
heat-sealed bags (Reed 1992). Low partial pressure              growth regulators. The effect of abscisic acid (ABA) in
oxygen storage is also used (Brindgen et al. 1981),             the concentration of 0.1; 0.5; 1.0 mg /l was studied.
under a layer of mineral oil (Calpin 1959), in low                  Each experiment was a randomized scheme with 5
illumination (Preil 1985), or in total darkness (Marino et      shoots in three repetitions for each genotype (n = 15).
al. 1985, Platonova et al. 2019).                               The condition of stored plants was assessed according
    Cold storage of genetic resources in in vitro               to the criteria given above. Plants with a rating >2 (out of
germplasm bank is an important part of the strategy for         5) were left for storage for another 3 months, plants with
preserving plant biodiversity, ensuring long-term reliable      a rating ≤2 were extracted and re-propagated (Reed et
conservation and use of the gene pool for scientific and        al., 2005). The data was analyzed by ANOVA with
industrial purposes. Many countries have germplasm              SYSTAT version 8.0.
banks of economically important plants. Kazakhstan is
also taking measures to preserve the genetic resources             RESULTS
of agricultural plants in the gene bank.
                                                                    Screening of Viability of Fruit and Berry
    The aim of this research was to study the effect of
                                                                Crops under Conditions of In Vitro Cold Storage
plant growth regulators (PGRs), carbohydrates and
                                                                    The duration of storage of 23 genotypes of apple, 9 -
nitrogen on the duration of in vitro storage of various fruit
                                                                pear, 59 - cherry, 32 - plum, 11 - cherry, 55 - raspberry,
and berry crops. As well as screening the longevity of
                                                                16 - black currant, 8 - strawberry, contained in a
storing genotypes of apple, pear, cherry, raspberry,
                                                                hormone-free MS medium, at a low positive temperature
blackcurrant and strawberry at a low positive
                                                                and low illumination was studied.
temperature and low illumination.
                                                                    The duration of storage depended on the genotype,
                                                                type of container in which plants are stored, as well as
                                                                temperature and illumination. The determining factor
     In vitro cold storage durability screening was             that slows down the metabolic processes of plants
performed for varieties, wild forms and hybrids of: apple,      during storage was the temperature of 3-4°C, as well as
pear, cherry, bird cherry, raspberry, blackcurrant and          the duration and degree of illumination – a 10-h
strawberry, contained at 3-4°C, illumination of 10 µm m-        photoperiod, illumination of 10 µmol m-2s-1. Keeping
2 -1
 s and 10-h photoperiod in glass jars and semi-                 plants in plastic air-permeable bags is more effective
permeable plastic tissue-culture bags (Star Pac Bags,           than in glass jars. Different viability of breeds and
PhytoTechnology Laboratories, Shawnee Mission,                  individual genotypes is observed during cold storage in
Kansas) on Murashige and Skoog (MS) medium without              conditions of forced rest. The storage time in plastic
plant growth regulators (PGRs). Previously, plants in           bags (until the complete death of plants), depending on
storage containers were kept for one week at 24°C,              the genotype is: apple trees from 12 to 21 months, pears

EurAsian Journal of BioSciences 14: 1213-1219 (2020)                                                          Turdiyev et al.



Fig. 1. Mean of storage condition ratings (0 low, 5 high) of in vitro shoots of apple (А) and pear (В) cultivars stored at 4°C
on medium in bags with carbon source treatments, with or without plant growth regulators (PGRs), for 33 months

- 15-24, cherries - 12-27, plums - 15-30; bird cherries -          mannitol + 2% sucrose. On such media, the cold storage
12-24, raspberries and strawberries - 21-30, black                 time for ‘Golden Delicious’ (rating of ≥ 2.0) was 30
currants - 18-21. Plants with an average rating of
EurAsian Journal of BioSciences 14: 1213-1219 (2020)                                                         Turdiyev et al.



Fig. 2. Mean of storage condition ratings (0 low, 5 high) of in vitro shoots of raspberry (А) and black currant (В) cultivars
stored at 4°C on medium in bags with carbon source treatments, with or without plant growth regulators (PGRs), for 18

remained viable (assessment ≥ 2) for 27 months on MS               time for ‘Marzhan’ (rating of ≥2.0 points) was 15 months,
medium with PGRs and 2% sucrose + 2% mannitol                      for ‘Dalnyaya’ - 12 months. The first dead plants in both
compared to 12 months for control and most other                   varieties were marked for 15 months of storage,
treatments. ‘Talgarskaya Krasavitsa’ is stored for only 6          individual plants remained viable for 21 months. On
months in control and most treatments, but storage can             media with 3% sucrose, but without PGRs, the cold
be extended to 18 months on MS with 2% sucrose + 2%                storage time with a rating of ≥2.0 points is only 12
mannitol without PGRs (Fig. 1B).                                   months, and the maximum viability is 15 months. On
    Storage time for three varieties of cherry                     media containing different concentrations of mannitol, as
‘Zhukovskaya’, ‘Moya Radost’, ‘Dolgozhdannaya’,                    well as combining mannitol and sucrose, plants were
increased in the nutrient medium with PGRs and with 3%             preserved worse, the first dead plants were noted for 3
sucrose or 2% sucrose + 2% mannitol (30 months). Only              months, the storage duration with a rating of ≥2.0 points
one mannitol was insufficient for all varieties and                was 6-12 months for ‘Marzhan’ variety, for ‘Dalnyaya’
provided storage for only 6-12 months.                             variety is 6-9 months (Fig. 2A).
    Two varieties of raspberries remain in good condition              Genotypes of black currant – ‘Orlovsky Vals’, ‘Minai
for the longest time on a medium with 3% sucrose in                Shmyrev’, hybrid 3-19-3, were preserved longer in the
combination with GPR. On this medium, the cold storage             nutrient medium with PGRs with 2% sucrose + 2%

EurAsian Journal of BioSciences 14: 1213-1219 (2020)                                                   Turdiyev et al.

mannitol (18 months). On the medium without PGRs             PGRs - 12 months. The effect on other varieties was
with 2% sucrose + 2% mannitol and in the control, the        small.
rating of ≥2.0 was only up to 12 months of storage (Fig.         The combination of several factors – optimal
2B).                                                         temperature and light conditions, a nutrient medium with
    Effect of Nitrogen and Plant Growth                      the best options for carbohydrate, nitrogen and
Regulators                                                   hormonal nutrition increased the cold storage time of all
    The effect of nitrogen and plant growth regulators       crops.
has a great influence on the preservation of apple tree          196 samples of fruit and berry crops are preserved in
microclones in conditions of low positive temperature. In    the in vitro germoplasm cold bank, including: 39 apple
media without PGRs, a reduction in nitrogen content of       trees, 31 pears, 7 cherries, 8 plums, 3 cherry plums, 3
up to 25 and 50% increased the duration of conservation      cherries, 42 raspberries, 32 strawberries, 28 black
of ‘Golden Delicious’ plants to 30 months, rating of ≥ 3.0   currants, 3 blackberries.
and 3.0, respectively (control of ≥1.2 and ≥2.0). In the
same period, the state of ‘Voskhod’ was assessed at ≥           DISCUSSION
2.0 and 1.8, and ‘Maksat’ at ≥1.6 and 1.4. On media with          Most researchers note that the duration of in vitro
PGRs and with 25 and 50% nitrogen, the condition of          cold storage of plants at a low positive temperature
plants was worse for 30 months, with ‘Golden Delicious’      depends on the genotype of the preserved plant, the
rating of ≥ 1.8 and 2.6, ‘Voskhod’ ≥1.2 and 1.8, and         components of nutrient media, especially the content of
‘Maksat’ ≥1.4 and 1.0.                                       carbohydrates, nitrogen and growth regulators, have a
    The effect of nitrogen concentration also varied         great influence.
depending on the cherry variety. Some experiments had             The results of our experiments are similar in many
a good rating without much difference when stored from       respects to the results of other researchers, but they
12 to 18 months. The rating of shoots when using a           differ in some positions of temperature and light modes,
standard MS medium with 100% nitrogen nitrate often          as well as hormonal and nutritional composition. In our
did not differ significantly from experiments with a         experiments, the combination of several cold storage
reduced nitrogen content. Many treatments were               factors – temperature regime of 3-4°C and 10-h
effective for ‘Dalnyaya’ variety, but the shoots were        photoperiod, optimal nutrient medium with the best
stored better – 30 months – with PGRs in packages with       options of carbohydrate and nitrogen nutrition, MS
100% nitrogen. ‘Moya Radost’ can be stored                   medium with 25-50% N03 and 3% sucrose или 2%
successfully for 30 months in many experiments with          sucrose + 2% mannitol without PGRs, increased the
100% nitrogen or 25% nitrogen in packages without            cold storage time of apple trees of ≥36 months. The
PGRs. ‘Zhukovskaya’ had a lower rating than the other        increase in the duration of pear cold storage was on MS
two varieties for 30 months with the best nitrogen           medium with a reduced nitrogen content and the
concentrations of 100% or 25% in packages without            addition of 2% sucrose + 2% mannitol and with or
PGRs. Reducing the nitrogen concentration in MS              without PGRs. Cherries were well preserved on MS
medium to 25% without PGRs increased the shelf life of       nutrient medium with 100% nitrogen content with 3%
‘Mramornaya’ pear to 36 months, compared with 18             sucrose or 2% sucrose + 2% mannitol and with PGRs.
months in the control, and ‘Talgarskaya krasavitsa’ is       Raspberry - on a medium with 3% sucrose in
viable for 18 months in this medium compared to 6            combination with PGRs. Black currant genotypes were
months for controls (Fig. 2B).                               preserved longer in a nutrient medium with 2% sucrose
    Effect of Abscisic Acid (ABA) Content                    + 2% mannitol with PGRs. Strawberries in our conditions
    The effect of abscisic acid (ABA) content in the         remain viable for up to 15-18 months on a hormone-free
experiments was insignificant. The cold storage time         agarized medium.
was significantly increased only for pears. Thus, the             The results of other researchers are somewhat
addition of 1 mg L-1 ABA with 3% sucrose to the medium       different from our own. Apple shoots of Golden Delicious
for ‘Mramornaya’ storage increased the storage duration      variety and 3 rootstocks showed 100% viability after
to 36 months compared to 18 months for control.              storage in the dark in a hormone-free medium at 2°C for
‘Talgarskaya Krasavitsa’ is stored for 18 months on MS       1.5-3.5 years (Druart, 1985). At 4°C and 16-h
medium with 1 mg L-1 and up to 21 months with 0.5 mg         photoperiod, 5 varieties of Malus domestica, M prunifolia
L-1 ABA in the medium. Some influence on the cold            (Willd.) Borkh., M. baccata (L.) Borkh. successfully
storage duration was observed at a concentration of 0.1      stored for 12-28 months in an agaric medium (Wilkins et
mg/l in ‘Golden Delicious’ apple tree, where the rating      al., 1988). Apple shoots were preserved for a year at +1
was ≥2.6 for 30 months of cold storage, in the same time     or + 4°C (Kundergan and Janick, 1979). For apple
period at a concentration of 0.5 mg/l-1.4, 1.0 mg/l – 0.5.   rootstock, only the standard content of 3% sucrose is
The raspberry variety ‘Dalnyaya’ at a concentration of       optimal. For apple varieties ‘Grushovka’, ‘Vernenskaya’
0.5 mg/l viability was the same as with 3% sucrose and       and wild form TM-6, it is also recommended, as in our

EurAsian Journal of BioSciences 14: 1213-1219 (2020)                                                     Turdiyev et al.

experiments, to reduce the nitrogen content to 25%, and      raspberries well preserved with 3% sucrose in
use 3% sucrose or 2% sucrose + 2% mannitol without           combination with GPRs (Reed et al. 2005).
PGRs as carbohydrates (Kovalchuk et al. 2009).                  At the All-Russian Institute of plant genetic resources
    The same temperature regime is recommended by            named after N. I. Vavilov (AIP) the germplasm of berry
Towill L. E. About 80% of pear shoots retained a healthy     and fruit crops is preserved on MS nutrient medium with
appearance and green color with normal morphology up         50% of the composition of mineral salts without
to 18 months of storage at 4°C (Towill 1981). And            phytohormones at 4°C, illumination 500 lux and a
Japanese pears, P. pyrifolia, survive best when stored       photoperiod of 8 h [Dunaeva et al. 2018).
at 1°C in the dark for 12 months (Moriguchi 1995). Pyrus        The results of our experiments and literature data
samples (n = 169) were stored in tissue culture bags in      show that the duration of medium-term in vitro
the dark at 4°C and had an average shelf life of 2.7 years   preservation of germplasm depends on the genotype of
(ranging from 8 months to 4.6 years) (Reed and Chang         the plant, temperature and light conditions, as well as
1997). According to literature data, the growth of pear      laboratory conditions for the maintenance of objects.
rootstocks slowed down when mannitol was added to
the culture medium (Lambardi et al. 2006).                      CONCLUSION
    There is evidence of storing cherry shoots for several
                                                                 Many genotypes of fruit and berry crops were well
months in a liquid culture medium at normal temperature
                                                             preserved at 4°C in bags for cultivation in MS medium
(Wanas et al. 1986). Positive effect on the viability and
                                                             with 3% sucrose or 2% sucrose + 2% mannitol with or
proliferation of wild cherry Prunus avium L in vitro
                                                             without PGRs. Reducing the nitrogen concentration
storage at 2°C on MS medium with the addition of 4.44
                                                             increased the storage time of some genotypes, but
mm BAP and 0.49 mm IBA (Murashige and Skoog
                                                             abscisic acid did not improve the storage time. The in
1962). Only the standard 3% sucrose content was
                                                             vitro collection is a reserve field collection for reliable
optimal for cherry rootstock (Ozudogru et al. 2017).
                                                             conservation of genetic resources and now has 196
Medium-term in vitro cold storage of raspberry
                                                             samples of fruit and berry crops stored in packages with
germplasm was studied using different temperatures,
                                                             50% nitrogen, 0.5 mg/l BAP, 0.1 mg/l IBA, and 3%
photoperiods and storage containers. Research data
showed that most Rubus germplasm can be preserved
                                                                 In the future, the conditions of medium-term in vitro
at 4°C and 12 h light day. Plastic bags are preferable to
                                                             germplasm conservation of other fruit and berry plants
glass jars. For cold-sensitive genotypes, an alternative
                                                             growing in the country will be studied, the cold collection
storage method is proposed at 25°C in a medium with a
                                                             will be replenished with other germplasm samples.
reduced nitrogen content (Reed 1993). Just as we have

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