In vitro germplasm cold storage of fruit and berry plants of Kazakhstan
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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
1
Institute of Plant Biology and Biotechnology, Almaty, KAZAKHSTAN
2
Kazakh Research Institute for Fruit & Vegetable Growing, Almaty, KAZAKHSTAN
*Corresponding author: turdievtt@mail.ru
Abstract
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
1213EurAsian 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
METHODOLOGY
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
1214EurAsian Journal of BioSciences 14: 1213-1219 (2020) Turdiyev et al.
(A)
(B)
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 ofEurAsian Journal of BioSciences 14: 1213-1219 (2020) Turdiyev et al.
(A)
(B)
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
months
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%
1216EurAsian 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
1217EurAsian 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
sucrose.
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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