ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

TERM PAPER ON: SOMACLONAL VARIATION AND GERM PLASM

CONSERVATION

BY: KASAHUN AMARE (PH.D. STUDENT)

SUMMATED TO DR. KERO JEMAL DEPARTMENT OF APPLIED

BIOLOGY, SCHOOL OF APPLIED NATURAL SCIENCE, ADAMA
SCIENCE AND TECHNOLOGY UNIVERSITY

JANUARY 2022
ADAMA, ETHIOPIA
 TABLE OF CONTENTS

PART I: SOMACLONAL VARIATION ................................................................ iii

SUMMARY ............................................................................................................. iii
1. Introduction .........................................................................................................1
1.2. Sources of Somaclonal Variations ...................................................................2
1.2.1. Pre-existing Variability (Genetic) .............................................................3
1.2.2. In-Vitro Induced Variability (Epigenetic) .................................................3
1.2.2.1. Explant Source.....................................................................................4
1.2.2.2. Medium Component ............................................................................4
1.2.2.3. Length of Time in vitro .......................................................................5
1.2.2.4. Effect of Genotype ..............................................................................5
1.2.2.5. Physical Factors ...................................................................................6
1.2.3. Physiological and Biochemical variation ..................................................6
1.3. Nomenclature of Somaclones .............................................................................6
1.4. Methods for Detecting Somaclonal Variation ....................................................6
1.4.1. Morphological Detection ..............................................................................7
1.4.2. Biochemical Detection of Somaclonal Variation .........................................7
1.4.3. Molecular Detection of Somaclonal Variation .............................................8
1.4.4. Cytological methods of somaclonal detection ..............................................8
1.5. Isolation of Somaclonal Variants ........................................................................8
1.5.1. Screening.......................................................................................................9
1.5.2. Cell Selection ................................................................................................9
1.6. Application of Somaclonal Variation ............................................................10
1.7. REFERENCES..................................................................................................13
Part II: GERMPLASM CONSERVATION ............................................................xv
SUMMARY .............................................................................................................xv

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 2.1. Introduction ....................................................................................................16
2.2. Conservation methods....................................................................................17
2.2.1. In situ conservation ..................................................................................18
2.2.2. Ex situ conservation .................................................................................18
2.3. In vitro conservation ......................................................................................18
2.3.1. Cryopreservation......................................................................................19
2.3.2. Slow Growth ............................................................................................23
2.3.3. Low pressure and low oxygen storage ....................................................25
2.4. ADVANTAGES OF IN VITRO GERMPLASM CONSERVATION............25
2.5. REFERENCES..................................................................................................26

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 PART I: SOMACLONAL VARIATION

SUMMARY

Plant tissue culture is the vegetative propagation of plant cells, tissues or organs that are
isolated from the plants and cultivated in vitro under aseptic conditions. Plants obtained in this
way are considered to be uniform and identical to the donor plant. However, cultured cells or
tissues are often subject to genetic changes. The genetic variability induced during tissue culture
in vitro is referred to as somaclonal variation. The variability can be genetically determined
(pre-existing) such as a change in chromosome number and structure or in vitro induced /
epigenetic due to different culture conditions, explant source, genotype and culture duration, etc.
The somaclonal variant can be used in. morphological, biochemical, molecular and cytological
levels are detected. The somaclonal variants can be isolated by screening and cell line selection
techniques. Screening involves observing large numbers of cells to detect individual variants and
is the only viable technique for isolating mutants for yield traits. Some examples of cell selection
are the selection of cells that are resistant to various toxins, herbicides, high salt concentrations,
and so on. Somaclonal variants are very important today in plant improvement to produce
genetically uniform, disease resistance, herbicide resistance and tolerance to environmental or
chemical stress and increased production of secondary metabolites. However, the somaclonal
variation, for example, has certain limitations; instability of variants, poor plant regeneration;
some somaclones have undesirable properties such as sterility, etc. Variation is usually not new
and unpredictability related to the nature of the somaclonal variation. Therefore, this section
briefly discusses the causes of somaclonal variation, methods of detection, and their application.

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1. Introduction

Plant tissue culture is essentially an extension of vegetative propagation, in which plant cells,
tissues or organs arc isolated from the plant and cultured in vitro under aseptic conditions(Oseni
et al., 2018). Plants obtained in this way are the product of asexual reproduction and should
therefore be uniform and identical to the donor plant from which the cultured cells were
obtained. In addition, the maintenance of elite genotypes, selected for their superior traits,
requires a high level of genetic uniformity among regenerated plants (Krishna et al., 2016).

However, recent studies have shown that cell or tissue cultures often undergo genetic changes
(polyploidy, aneuploidy, chromosome breaks, deletions, translocations, gene amplification and
mutations) and these are expressed on a biochemical or molecular level. Plant tissue cultures
therefore offer increased genetic variability relatively quickly and without the use of
sophisticated technology(Pullaiah, 2017). The genetic variability(somaclonal variations) that
occurred in tissue culture is due to gene mutation or changes in epigenetic marks(Krishna et al.,
2016). This phenomenon was given the name of ‗’somaclonal variation" by Larkin and
Scowcroft in 1981 and was advocated as a potential source of improvements for plant
breeding(Ozudogru et al., 2010).

In this context, somaclonal variations at different levels (morphological, cytological,

cytochemical, biochemical and molecular) have been reported in micropropagated plants (Leva
et al., 2012). Accordingly, the observed phenotypic variation among plant regenerants derived
from any form of tissue culture is defined as somaclonal variation (Soma = vegetative; clone =
identical copy) (Larkin & Scowcroft, 1981). Or it represents the genetic variability present in all
types of cells / plants obtained from cultures in vitro (Larkin & Scowcroft, 1981; Sahijram,
2015). Variants obtained using callus cultures are known as calliclones, while variants obtained
using protoplast cultures are known as protoclones.

Somaclonal variants are now very important in plant improvement for their control and possible
suppression with the aim of producing genetically identical plants and for their use as a tool for
creating genetic variability that will enable breeders to achieve genetic improvement. Traits for
which somaclonal mutants can be enriched during in vitro culture include resistance to disease or

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pathotoxins, herbicides and tolerance to environmental or chemical stress, and increased
production of secondary metabolites(Leva et al., 2012).

The main causes of these variations can be attributed to changes in karyotype (chromosome
number and structure), chromosome rearrangements, somatic crossing over, sister chromatid
exchanges, DNA amplification and deletion, transposable elements, and DNA methylation.
Somaclonal variation can be characterized based on morphological, biochemical (isozymes), and
DNA markers such as randomly amplified polymorphic DNA (RAPDs), restriction fragment
length polymorphism (RFLPs), and inter-simple sequence repeats (ISSR).

The variations could also occur in tissue cultures due to physiological changes induced by the
culture conditions. Such variations are temporary and are caused by epigenetic changes. These
are non-heritable variations and will disappear when the culture conditions are removed. In
general, there are several approaches (steps) to creating somaclonal variations, including:
 Growth of callus or cell suspension cultures for several cycles.
 Regeneration of a large number of plants from such long term cultures.
 Screening for desirable traits in the regenerated plants and their progenies. For example,
in vitro selection to select agronomically desirable somaclones for tolerance to various
biotic and abiotic stresses herbicides, high salt concentration and extremes of
temperature.
 Testing of selected variants in subsequent generations for desirable traits.
 Multiplication of stable variants to develop new breeding lines.
To be of commercial use, a somaclonal variant must fulfill certain basic requirements:
 It must involve useful characters.
 It should be superior to the parents in the character(s) in which improvement is sought.
 The improved character(s) must be combined with all other desirable characters of the
parent, and
 The variations must be inherited stably through successive generations by chosen means
of propagation.

1.2. Sources of Somaclonal Variations

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Sometimes, phenotypic variations may arise in the progeny of plants regenerated from culture,
but after the transplantation to the field, the plants exhibit the parental characteristic during their
further growth and development. Such variations are not considered as somaclonal variations.
The possible factors that contribute to the types and degrees of chromosome variation in cultured
tissues include: pre-existing cytological mutations in the explants, developmental status of the
explants, genotype; apparent development status of the cultured cells and culture
regime(Nwauzoma and Jaja, 2013; Orton, 1984).

1.2.1. Pre-existing Variability (Genetic)

Explants derived from a single clone, seed, or seedling are assumed to be genetically uniform,
permitting the conclusion that spontaneous genetic instability occurs in vitro(Orton,
1984).However, this assumption is not always valid, because explants may include several types
of cells, such as phloem, parenchyma, cortex, and xylem parenchyma. These cells may also vary
in their ploidy level. To account for the lack of uniformity in multicellular explants, Bright et al.
(1983) suggested that explants derived from sources other than protoplasts are called ―complex
cultures‖ in recognition of their multicellular origin(Bridgen et al., 1994)

Plant development generally involves a change in nuclear DNA, such as a change in

chromosome number and structure. Cells of apical meristems of plants such as root tips and
shoot tips are uniformly diploid in their genome. However, these variations occur due to DNA
duplication and reduplication. The different degrees of endoreduplication lead to a higher DNA
content. One can lead to polysomacy, in which cell division and redifferentiation results in a
change in genomic content and hereditary character throughout the plant. There is another pre-
existing variation known as aneusomacy. This occurs due to the aneuploid number of
chromosomes (Bairu et al., 2011; Kumari et al., 2019).

1.2.2. In-Vitro Induced Variability (Epigenetic)

The plant genome is under continuous flux which helps the plant adjust to changes in
environmental conditions. The tissue culture system is a stressful environment for the plant cells.
Under the stressful culture conditions, the plant cells are subject to genetic and epigenetic
changes. In plants, Epigenetic variability may be caused by DNA methylation, DNA
amplification, histone modification and activation of transposable elements (transposons). These

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may influence the gene transcription and finally leading to somaclonal variations(Azizi et al.,
2020; Mgbeze & Iserhienrhien, 2014). The triggering factors for mutations in tissue culture are
numerous including; wounding, exposure to sterilants during sterilization, tissue being
incomplete (protoplasts as an extreme example), imbalances of media components such as high
concentration of plant growth regulators (auxin and cytokinins), sugar from the nutrient medium
as a replacement of photosynthesis in the leaves, lighting conditions, the disturbed relationship
between high humidity and transpiration are acting as the major triggering agents for somaclonal
variations(Krishna et al., 2016). In addition, the propagation methods, explant source, genotype
and culture conditions and duration of subcultures are some of the factors that determine the
frequency of somaclonal variation in vitro (Bairu et al., 2011; Kumari et al., 2019).

1.2.2.1. Explant Source

When exploring a new species or cultivar for somaclonal variation, it is a good idea to use
several different explants and compare progeny from each. Not all explants should be assumed to
exhibit variation equally. The explant source largely influences genetic fidelity and nature of
somaclonal variation. The use of preformed shoots (axillary buds, shoot tips,) and meristematic
tissues (cambium, pericycle, and procambium) as explant reduces the possibility of variation.
However, highly differentiated tissues, that have no preformed shoot meristems, such as leaves,
roots, protoplasts, stems and roots generally produce more variants, probably due to the callus-
phase, than explants that have pre-existing meristems((Bridgen et al., 1994)). Therefore, the
source of ex-plant is very critical for somaclonal variations. Furthermore, preparation of many
explants from only one donor plant increases the possibility of variation in cultures. Pre-existing
somatic mutations in donor plant tissues can also induce somaclonal variation and first round of
somaclones yield higher variability than second generation, hence can be eliminated or
stabilised(Sarmah, 2017).

1.2.2.2. Medium Component

Unbalanced and high hormonal concentrations in culture media can be powerful agents for
inducing variation. Synthetic compounds used as growth regulators also have been linked with
somaclonal variation. Auxins increase genetic variation by increasing the DNA methylation rate
in cultures of unorganized calli or cell suspensions. For instance auxin hormones such as 2, 4-

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dichlorophenoxy acetic acid (2, 4-D) and naphthalene acetic acid (NAA) are frequently used to
achieve chromosomal variability. In addition, somaclonal variation can also be induced by rapid
disorganized growth(Sarmah, 2017). Chemical composition of the media to a great extent
influences cytological behavior of cultured cells. Besides, the physical conditions, such as
temperature and nature of media (liquid or semi-solid) also influence the rate of mutation.

1.2.2.3. Length of Time in vitro

The length of time that a culture has been maintained in vitro is among the most important
factors involved in inducing somaclonal variation(Bridgen et al., 1994). The rapid multiplication
of tissue and long term cultures affect genetic stability and somaclonal variation. It has been
reported that the number of subcultures and their duration are proportional to the frequency of
somaclonal variation in cell suspensions and callus cultures(Sarmah, 2017). To maintain clonal
stability, most commercial laboratories regularly sample their plants ex vitro in a field or
greenhouse to make sure that the characteristics of the particular cultivar are maintained. In
addition, most laboratories limit the number of subcultures that can be made from an explant. For
instance, after a year all cultures established from a particular explant will be harvested. The
clone will then be replaced with another explant taken from the original mother plant. To ensure
continuity in a commercial enterprise, new cultures are established regularly and older ones
eliminated on schedule(Bridgen et al., 1994). In general, for many plant cultures, somaclonal
variations are higher with increased duration of cultures. For example, it was reported that
genetic variability increased in tobacco protoplasts from 1.5 to 6% by doubling the duration of
cultures.

1.2.2.4. Effect of Genotype

The genotype component proves to be a vital factor for induction of somaclonal

variation(Kumari et al., 2019). Since, in vitro culture conditions impart different level of stress to
plant cells and different genomes respond differently to the stress-induced variation which finally
may initiate highly mutagenic(Sarmah, 2017). The amount of variation encountered in vitro is
not the same for all cultivars of a species. Some cultivars show excessive variation, others are
relatively stable. For instance, the variation rate among banana cultures is about 3%, but the rate
of variation for ‗Cavendish‘ bananas was up to 20 % (Bridgen et al., 1994).

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1.2.2.5. Physical Factors

In addition to the chemical composition, the physical condition of the medium also influences
the cytological behavior of the cultured cells. In Hevea brasiliensis/ rubber tree, polyploidy
increased when cells were cultured in suspensions but decreased when re-cultured as callus on
solid medium. The culture temperature can influence the mutation rate. Tobacco callus incubated
at 350 ° C remained predominantly diploid, while the same tissue cultured at 250 ° C showed
pronounced karyological instability and became mainly tetraploid. In Lilium longiflorum, the
incidence of albino seedlings from somatic embryos increased when the incubation temperature
was raised above 10-150 ° C.

1.2.3. Physiological and Biochemical variation

Physiological variations are temporary in response to stimulus and disappear when it is

removed. Several biochemical variations have been identified in various crop plants in tissue
culture. Examples of such biochemical variation include alterations in carbon metabolism
leading to lack of photosynthetic ability, starch biosynthesis, carotenoid pathway, nitrogen
metabolism, and antibiotic resistance(Jayasankar, 2018).

1.3. Nomenclature of Somaclones

Although different letters and symbols have been used, two symbols are generally used. Chaleff
(1981) named the plants regenerated from tissue culture as R or R0 plants and the self-fertilized
offspring of R0 plants as R1. Subsequent generations produced by self-fertilization are referred
to as R2, R3, R4, etc. Larkin and Scowcroft (1981) have designated regenerated plants as SC1 (=
R0) and subsequent self-fertilized generations as SC2, SC3, SC4 etc. for subsequent generations.

1.4. Methods for Detecting Somaclonal Variation

Somaclonal variation can be difficult to detect. Variation induced by physiological factors can be
identified quite early on and often without the aid of any tools(Jayasankar, 2018). All the
somaclonal variation is not obvious to the eye. Gross physical changes, such as leaf color may be
detectable in vitro, but most somaclonal variation won‘t be visible until the explants are removed

6
from tissue culture and grown in soil(Sarmah, 2017). In the field, such variants plants are
observed during their successive growth and development. Such qualitative and quantitative
phenotypic characters viz., plant height, maturity date, leaf size, flowering date, yield, seed
fertility, waxiness in different plant parts, flower morphology etc. are used as a parameter to sort
out the variants.

In this regard, an efficient and early detection of somaclonal variation is needed to spot variants
with useful agronomic traits. Overall, somaclonal variants can be detected using several
techniques, which are broadly categorized as morphological, physiological/biochemical, and
molecular detection techniques(Bairu et al., 2011).

1.4.1. Morphological Detection

Morphological traits have long been used to identify species, genera, and families in plants.
Variants can be easily identified by characteristics such as differences in plant stature, leaf
morphology, and pigmentation anomaly. Banana off-types, for example, can be visually
recognized during acclimatization in the greenhouse prior to transplanting into the field. In the
field it is also possible to identify dwarf off-types by observing the growth and leaf index (leaf
length / width) 34 months after establishment. In addition, the detection of variants on the basis
of morphological characteristics is often possible for fully established plants either in the field or
in the greenhouse. However, morphological traits are often heavily influenced by environmental
factors and may not reflect the true genetic makeup of a plant (Bairu et al., 2011).

1.4.2. Biochemical Detection of Somaclonal Variation

Compared to morphological detection, the use of physiological reactions and / or biochemical

tests to detect variants is faster and can be carried out in the youth stage in order to reduce the
possible economic loss. The response of plants to physiological factors such as hormones and
light can be used as a basis to distinguish between normal and variant somaclones. Gibberellic
acid, for example, regulates growth and influences various development processes such as stem
elongation and enzyme induction (Bairu et al., 2011).

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1.4.3. Molecular Detection of Somaclonal Variation

Molecular techniques are valuable tools used in analyzing the genetic fidelity of in vitro
micropropagated plants. At the molecular level, variations in tissue culture plants arise from
changes in the number or structure of chromosomes or from more subtle changes in DNA (Bairu
et al., 2011). Numerous molecular techniques are available to detect sequence variations between
closely related genomes, including differences between source plants and somaclones. These
techniques involve the use of molecular markers useful in comparing DNA from different
samples for differentiation in plants due to sequence variations by identifying random
polymorphisms (Bairu et al., 2011; Sarmah, 2017) and may include DNA markers such as z,
Random Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphism
(RFLPs) and Inter-Simple Sequence Repeats (ISSR) etc.

1.4.4. Cytological methods of somaclonal detection

Variations in ploidy or chromosome number and structure are direct and strong evidence of a
high probability of a change in the genetic makeup of an organism. Chromosomes, as well as
other nuclear components such as RNA and DNA content variations, are important somaclonal
variation detection techniques that are widely used.

1.5. Isolation of Somaclonal Variants

Mutants for several traits can be far more easily isolated from cell cultures than from whole plant
populations. This is because a large number of cells, say 106-109, can be easily and effectively
screened for mutant traits. Screening of as many plants would be very difficult ordinarily
impossible. Mutants can be effectively selected for disease resistance, improvement of
nutritional quality, adaptation of plants to stress conditions, e.g., saline soils, low temperature,
toxic metals (e.g., aluminium), and resistance to herbicides and to increase the biosynthesis of
plant products used for medicinal or industrial purposes. The various approaches to the isolation
of somaclonal variants can be grouped into two broad categories: screening and cell selection.

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1.5.1. Screening

It involves observing large numbers of cells or regenerated plants to detect individual variants.
This approach is the only viable technique for isolating mutants for yield and yield traits. In
general, R1 progeny (progeny of regenerated, Ro, plants) are scored for plant variant
identification and their R2 progeny lines are scored for confirmation. Screening has been used
profitably and widely for the isolation of cell clones that produce large quantities of certain
biochemicals. Computer-based automated cell sorters have also been used to screen up to 1000-
2000 cells / second from which desirable cell variants have been automatically separated
(Kumari et al., 2019).

1.5.2. Cell Selection

In the cell selection approach, a suitable selection pressure is exerted which enables preferred
survival / growth only of cell variants. Some examples of cell selection are the selection of cells
that are resistant to various toxins, herbicides, high salt concentrations, and so on. When
selection pressure only allows the mutated cells to survive or divide, this is called positive
selection. On the other hand, in the case of a negative selection, the wild-type cells divide
normally and are therefore blocked by a counter-selection agent, e.g. B. 5 BUdR or arsenate,
killed. The mutated cells are unable to divide, thereby escaping the counter-selection agent.
These cells are then rescued by removing the counter-selection agent. A negative selection
approach is used to isolate auxotrophic mutants (Kumari et al., 2019).The positive selection
approach may be further subdivided into four categories:
a. Direct selection,
b. Rescue method,
c. Stepwise selection and
d. Double selection.
In direct selection, cells that withstand the selection pressure survive and divide to form colonies;
the wild-type cells are killed by the selection agent. This is the most common selection method.
It is used to isolate cells that are resistant to toxins (produced by pathogens), herbicides, elevated
salt levels, antibiotics, amino acid analogs, etc. In the rescue method, the wild-type cells are
killed by the selection agent, while the variant cells remain alive, but usually do not divide due to

9
the unfavorable environment. The selection agent is then removed to harvest the variant cells.
This approach was used to recover cell variants with low temperature and aluminum resistance.
The selection pressure, e.g., salt concentration, may be gradually increased from a relatively low
level to the cytotoxic level. The resistant clones isolated at each stage are subjected to the higher
selection pressure. Such a selection approach is called stepwise selection. It may often favour
gene amplification (which is unstable) or mutations in the organelle DNA.

In some cases, it may be feasible to select for survival and/or growth on one hand and some other
feature reflecting resistance to the selection pressure on the other; this is called double selection.
An example of double selection is provided by the selection for resistance to the antibiotic
streptomycin, which inhibits chlorophyll development in cultured cells. The selection was based
on cell survival and colony formation in the presence of streptomycin (one feature) as well as for
the development of green colour in these colonies (second feature; only green colonies were
selected). This approach has been used for the selection of cells resistant to the herbicide
amitrole, 2, 4-D, tobacco mosaic virus (TMV) and aluminium.

1.6. Application of Somaclonal Variation

Somaclonal variants can be a source of increasing variability or genetic diversity needed for crop
improvement, particularly in the self-pollinated and asexually propagated systems. Somaclonal
variation cell selection and early rapid screening of regenerants together provide an effective
option for plant improvement(Pullaiah, 2017). It was most successful in plants with limited
genetic systems (e.g. apomicts, vegetative propagators) and / or narrow genetic bases. In the case
of ornamental plants, for example, the use of the variability generated in vitro has become
routine in many farms(Krishna et al., 2016).
Increased Genetic Variability for Agronomic Traits:
 Several useful somaclonal variants showing resistance to diseases, insects and
tolerance to herbicides have been isolated
In Vitro Selection:
 In vitro selection has been used to select agronomically desirable somaclones,
including tolerance to pathotoxins, herbicides and diseases. Some examples include:

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  Tobacco variants resistant to Pseudomonas syringae and Alternaria alternata
have been obtained following selecting protoplast-derived callus on medium
having pathotoxins.
 Somaclonal variants with increased resistance to Fusarium oxysporum in
celery have been obtained.
 Herbicide resistant variants have been obtained in cell culture
‘Elite’ Germplasm and Commercial Cultivars:
 Variants with improved traits have been obtained/developed giving rise to new useful
germplasm, as well as cultivars.
Improved Ornamental Plants:
 Somaclonal variation is known to be employed commercially in ornamental plants. A
wide range of somaclonal variation for flower size, plant morphology, plant height in
Begoniax elatior plants regenerated from callus have been reported. Flower variation
is reported in tissue culture – derived plants of carnation, Chrysanthemum and
Gerbera
Like any other technology, in vitro induced somaclonal variation has its own merits and
demerits, like the two sides of the same coin.
Advantages
Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time,
it is also more universally applicable and does not require ‗containment‘ procedures. It has been
most successful in crops with limited genetic systems and/or narrow genetic bases, where it can
provide a rapid source of variability for crop improvement. Tissue culture systems are available
for more plant species than can be manipulated by somatic hybridization and transformation at
the present time. It is not necessary to have identified the genetic basis of the trait, or indeed, in
the case of transformation, to have isolated and cloned it. Finally, it is used for increased
production of secondary metabolites(Karp, 1995, 2014).

Disadvantages
Serious disadvantage of somaclonal variation occurs in operations which require clonal
uniformity, as in the horticulture and forestry industries where tissue culture is employed for
rapid propagation of elite genotypes.
 Instability of variants.

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 Poor plant regeneration.
 Some somaclones possess undesirable features like sterility, etc.
 Variation is usually not new.
 Unpredictability associated with nature of somaclonal variation

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Leva, A. R., Petruccelli, R., & Rinaldi, L. M. R. (2012). Somaclonal variation in tissue culture: a case
study with olive. Recent Advances in Plant in Vitro Culture, 7, 123–150.
Mgbeze, G. C., & Iserhienrhien, A. (2014). Somaclonal variation associated with oil palm (Elaeis
guineensis Jacq.) clonal propagation: A review. African Journal of Biotechnology, 13(9).
Nwauzoma and Jaja, E. (2013). A review of somaclonal variation in plantain ( Musa spp ): mechanisms
and applications. Journal of Applied Biosciences, 5252–5260.

13
Orton, T. J. (1984). Somaclonal variation: theoretical and practical considerations. In Gene manipulation
in plant improvement (pp. 427–468). Springer.
Oseni, O. M., Pande, V., & Nailwal, T. K. (2018). A review on plant tissue culture, a technique for
propagation and conservation of endangered plant species. International Journal of Current
Microbiology and Applied Sciences, 7(7), 3778–3786.
Ozudogru, da SILVA, D. P. C., Ergun, K., Dradi, G., Paiva, R., & Lambardi, M. (2013). In vitro
conservation and cryopreservation of Nandina domestica, an outdoor ornamental shrub. Notulae
Botanicae Horti Agrobotanici Cluj-Napoca, 41(2), 638–645.
Ozudogru, E. A., Benelli, C., Dradi, G., & Lambardi, M. (2017). Effect of culture container and
carbohydrate content on in vitro slow growth storage of the cherry rootstock ‗Gisela® 5.‘ Acta
Physiologiae Plantarum, 39(4), 94.
Ozudogru, E. A., Previati, A., & Lambardi, M. (2010). In Vitro Conservation and Cryopreservation of
Ornamental Plants. 589. https://doi.org/10.1007/978-1-60327-114-1
Panis, B., Swennen, R., & Engelmann, F. (2000). Cryopreservation of plant germplasm. IV International
Symposium on In Vitro Culture and Horticultural Breeding 560, 79–86.
Paroda, R. S., & Arora, R. K. (1991). Plant genetic resources: conservation and management. Concepts
and approaches.
Pullaiah, and R. (2017). Plant Tissue Culture : Theory & Practicals 2nd Ed. SCINTIFIC PUBLISHERS
(INDIA).
Sahijram, L. (2015). Somaclonal Variation in Micropropagated Plants. Plant Genomics and
Biotechnology, II. https://doi.org/10.1007/978-81-322-2283-5
Sakai, A., Kobayashi, S., & Oiyama, I. (1991). Survival by vitrification of nucellar cells of navel orange
(Citrus sinensis var. brasiliensis Tanaka) cooled to-196 C. Journal of Plant Physiology, 137(4), 465–
470.
Sarmah, D. (2017). Somaclonal Variation and its‘ Application in Ornamentals Plants. International
Journal of Pure & Applied Bioscience, 5(2), 396–406. https://doi.org/10.18782/2320-7051.2762

14
 Part II: GERMPLASM CONSERVATION

SUMMARY

Germplasm is the sum of the genetic makeup of a species. Based on the source for collection and
conservation, germplasmas can be divided into advanced (elite) germplasm, improved
germplasm, landrace, wild or herbaceous relatives, and genetic stocks. Conservation includes
protecting the genetic diversity of plant crops from genetic erosion, which can either take place
ex situ or in situ. In situ conservation refers to the maintenance of germ plasm in ecosystems and
natural habitats and the maintenance and restoration of viable populations of species in their
natural environment. In vitro conservation is the storage of seeds or plant material under
artificial conditions in order to ensure longevity and availability efficiently and effectively. It is
mainly used in gene banks. It includes a number of methods of cold storage of seeds or
propagation, cryopreservation, field and pollen or DNA preservation. Cryopreservation at the
temperature of liquid nitrogen (-196C) offers the possibility of long-term storage with maximum
phenotypic and genotypic stability. This method is relatively convenient and economical; a large
number of genotypes and variants could be preserved. The technique comprises various steps:
selection of the plant material, preculture, cryoprotective treatment, freezing and storage,
thawing and recultivation. The germplasm preservation within the vitro technique has numerous
advantages, for example: entire sets of genetic material can be copied through regeneration
schemes, rapid proliferation is achievable; minimal space requirement; the number of materials
can be stored in a small area, disease-free plants can be produced and cared for, risks related to
environmental change are avoided, the plants are not at risk of change in government policy and
urban development.

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2.1. Introduction

As explained in the germplasm theory by Weismann (1834-1914); Germplasm is the sum of the
genetic make-up present in a cultivated species, including all related wild species. It can also be
defined as the genetic material that forms the material basis of inherited genes and can be passed
on from the germ cells from one generation to the next. Hence, germplasma collection and
conservation is one of the main tasks of many research institutions as a means of conserving
biological diversity.

The conservation of plant genetic resources has become an urgent task as these resources are
rapidly disappearing both in nature and in farmers' fields. In particular, cultivated plant wild
relatives and peasant varieties, also called land races, which are of particular interest for plant
breeding, are strongly threatened with extinction. Since the genetic resources of cultivated plants
are rather scattered regionally and even globally, efforts to preserve the germplasm require
regional and, in many cases, global coordination(Engels, 2021).

Germplasma conservation can be carried out by collecting and conserving genetic resources
through conventional methods in the form of seeds, vegetative propagation, etc. Based on source
to collection and conservation germplasms may be categorized into five major types: advanced
(elite) germplasm, improved germplasm, landraces, wild or weedy relatives, and genetic stocks.
This collection of plant seeds and propagation takes place in in vivo gene banks. Banks, in
which genetic resources are preserved by conventional methods, for example, seeds, vegetative
propagules, etc., are called in vivo gene banks whereas banks in which the genetic resources are
preserved by nonconventional methods such as cell and tissue culture methods are called in vitro
gene banks. Both these ensure the availability of valuable germplasms to a breeder to develop
new and improved varieties(Bhatia et al., 2015).

The conventional method of preserving germplasm has several limitations. Germ plasma cannot
be preserved for a very long time. The seeds are short-lived and have disadvantages in terms of
seed dormancy, seed-borne diseases, and germplasm requiring a lot of labor, space, and
cost(Bhatia et al., 2015). However, In vitro techniques have great potential for collecting,
exchange and conservation of: i) genetic resources of recalcitrant-seed and vegetatively

16
propagated species as well as of endangered species; ii) elite genotypes which are multiplied on a
large scale in production laboratories; iii) cultures with special attributes, eg., metabolite-
producing cell lines and genetically engineered material(Engelmann, 2004; Kartha &
Engelmann, 1994; Panis et al., 2000).

In vitro collecting techniques, which consist of introducing embryos or vegetative tissues, have
been developed for collection of germplasm of various problem species. In vitro cultures are
routinely used for exchange of plant genetic resources of a number of species, due to their
advantages in terms of phytosanitary status and reduced cost. Slow growth techniques have been
developed for medium-term conservation of numerous species but their routine use is still
restricted to a limited number of crop species. Routine use of cryopreservation is mostly
restricted to conservation of cell lines in research laboratories. However, simple and efficient
freezing protocols have been developed recently for apices and embryos, and can be considered
operational for an increasing number of species(Engelmann, 1997).

Current research priorities include: i) the development of in vitro collecting techniques for
additional species; ii) demonstration of the flexibility, simplicity and practicality of slow growth
storage to increase its utilization; iii) experimentation of existing cryopreservation techniques on
a large scale in a genebank context and the development of cryopreservation protocols for
additional species(Engelmann, 1997).

2.2. Conservation methods

Conservation includes protecting the genetic diversity of plant crops from genetic erosion, which
can either take place ex situ or in situ. In situ refers to conservation under natural habitats, which
requires the creation of biosphere or ecosystem resources in order to preserve endangered crops
or plants for future use. According to this method, both wild animals and natural biosphere are
preserved, which has the disadvantage that only a very small genotype area is covered for
individual species, it is a very expensive method and also requires a suitable management
system. An ex situ conservation germplasm is conserved in the form of a gene bank, which is a
very practical application that is used under laboratory conditions. This methodology enables the
preservation of all genetic diversity in one place. In addition, the process is inexpensive and easy
to use (Engels, 2021)).

17
2.2.1. In situ conservation

This type of conservation refers to the maintenance of germplasm in ecosystems and natural
habitats and the maintenance and restoration of viable populations of species in their natural
environment. For domesticated or cultivated species, this refers to their conservation in the
environment in which they developed their particular characteristics. This usually takes place in
protected areas, mostly to protect wild relatives, and in courtyard or home gardens to protect
cultivated species(Bhatia et al., 2015).

2.2.2. Ex situ conservation

This type of conservation is the storage of seeds or plant material under artificial conditions
(other than their natural environment), to efficiently and effectively guarantee its longevity
viability and availability. It is the type of conservation mostly used in genebanks. It covers a
range of methods suitable for various types of seeds or plant materials. It ranges from cold
storage of seeds or propagules, in vitro (tissue culture or cryopreservation), field, and pollen or
DNA conservation(Bhatia et al., 2015; Chauhan et al., 2019).

2.3. In vitro conservation

There are two basic approaches to maintaining germplasm conservation in vitro: (i) slow growth
and (ii) cryopreservation. Slow growth conditions for short to medium term storage can be
tracked in several ways: reduced temperature and or light; Incorporation of sublethal amounts of
growth inhibitors, induction of osmotic stress with sucrose or mannitol and maintenance of
cultures in a reduced nutritional state, in particular reduced carbon content, reduction of the gas
pressure above the cultures, dehydration and mineral oil overlay. The advantage of this approach
is that cultures can easily be returned to normal culture conditions to produce plants on demand.
However, the need for frequent subculture can be a major disadvantage, including contamination
of cultures and the imposition of selection pressures with subsequent alteration of genetic
makeup due to somaclonal variation(Chauhan et al., 2019; Paroda & Arora, 1991).

Cryopreservation at the temperature of liquid nitrogen (LN) (- 196° C) offers the possibility for
long-term storage with maximal phenotypic and genotypic stability. This method being relatively
convenient and economical, large number of genotypes and variants could be conserved and thus

18
 maximize the potential for storage of genetically desirable material(Chauhan et al., 2019; Paroda
& Arora, 1991).

2.3.1. Cryopreservation

Cryopreservation (Greek, karyos - frost) means preservation in the frozen state. The principle
involved in cryopreservation is to bring the plant cell and tissue cultures to zero metabolism or
non-dividing state by mean of storage of germplasm at a very low temperatures, (i) Over solid
CO2 (-79°C), (ii) Deep freezers (-80°C), (iii) in vapor phase nitrogen (-150°C), (iv) in liquid
nitrogen (-196°C)(Bhatia et al., 2015). The tissues can be stored at this temperature indefinitely
with the help of cryoprotectants such as glycerol, mannitol, dimethylsulfoxide, proline, etc.
Cryopreservation has proved to be an efficient conservation method for the long-term storage of
genetic stocks without any loss of viability and mutations(Colunga-GarcíaMarín & Zizumbo-
Villarreal, 2006; Engelmann, 2004; Kartha & Engelmann, 1994). The technique of
cryopreservation involves the following steps: Selection of plant material, Pre-culture,
Cryoprotective treatment, freezing and storage, Thawing, and Reculture(Bhatia et al., 2015)

i. Selection of plant material

Any totipotent tissue may be used for cryopreservation of clonal plants. The most commonly
used tissues are shoot tips, and to a lesser extent, somatic embryos and embryonic axes (shoot
and root axis of a seed). Embryonic axes derived from seed are used for seed-propagated crops
when the whole seed cannot be cryopreserved due to high oil content of the cotyledons, large
seed size, or general lack of cold tolerance(Jenderek & Reed, 2017).

ii. Pre-culture

In several cases, a brief culture of shoot apices for at least 48h at 4°C before freezing has proved
beneficial for consistently high frequency of survival of shoot apices after freezing in liquid
nitrogen. The other treatments include the application of additives that known to enhance plant
stress tolerance, for example ABA, proline, osmoticum (sucrose, mannitol), dimethylsulfoide
(DMSO, 1-5%). Sugars acts as osmotically effective agents, although they do not penetrate
inside the cells. Dehydration of cells/tissues occurs in the presence of sugars during the
preculture, which prevents lethal ice crystal formation during freezing. Proline may act by

19
 reducing the level of latent injury to the cells or it may actively participate in recovery
metabolism.

iii. Cryoprotective Treatment

There are two potential sources of cell damage during cryopreservation (1) Formation of large
ice crystals, inside the cells, leading to rupture of organelle and the cell itself, (2) intracellular
concentration of solutes increases to toxic levels before or during freezing as a result of
dehydration. Addition of cryoprotectants controls the appearance of ice crystals in cells and
protects these cells from the toxic solution effect. Cryoprotectants are categorized as: (a)
Penetrating, which exert their protective colligative action, (b) Non-penetrating, which affect
through osmotic dehydration. A large number of heterogeneous groups of compounds have been
shown to possess cryoprotective properties with different efficiencies, e.g. glycerol, DMSO etc.
Cryoprotectant depresses both the freezing and super-cooling point of water, i.e. the temperature
at which the homogeneous nucleation of ice occurs, thus, retarding the growth of ice crystal
formation in cells and protect cells from toxic effect. The cryo-protectants used in
cryopreservation are:
 Alcohols: Ethylene glycol, glycerol, propylene glycol, sorbitol, mannitol,
 Sulphur containing compounds: Amino acids, dimethyl sulphoxide (DMSO), sugar
(glucose, saccharose) and
 Polymers: Hydroxyethyl amidon, polyethylene glycol, polyvinyl pyrrolidine (Bekheet et
al., 2020).

A. Vitrification: At a sufficiently low temperature, highly concentrated aqueous solutions of

cryoprotective agents become so viscous that they solidify into an amorphous ―glassy‖ state,
without ice crystal formation (crystallization) at practical cooling rates, this phenomenon is
called vitrification. The significance of vitrification in cryopreservation of biological materials is
that the cells applied with highly concentrated solution of osmotially active compounds, are
protected from internal damage from ice crystal formation during freezing. This pretreatment
also causes dehydration of cells. The commonly used cryoprotectants are employed for
vitrification like DMSO(Panis et al., 2000; Sakai et al., 1991).

20
 B. Cryoprotective dehydration: If cells are sufficiently dehydrated they may be able to
withstand immersion in liquid nitrogen without further application of traditional cryoprotectant
mixtures. Dehydration can be achieved by growing the cultures in the presence of high
concentration of osmotically active compounds (sugars) and /or air desiccations in laminar-air-
flow cabinet or over silica gel. Dehydration reduces the amount of water available for the ice
formation.

C. Encapsulation and dehydration: This involves the encapsulation of tissues in calcium

alginate beads which are pre-grown in liquid culture media containing high concentrations of
sucrose. The beads are transferred to sterile airflow in a laminar cabinet and desiccated further.
After these treatments, the cells are able to withstand exposure to liquid nitrogen without
application of chemical cryoprotectants

iv. Freezing and Storage

The type of crystal water within stored cells is very important for survival of the tissue. Different
tissues have different sensitivities for cooling rates. In general, there are three different types of
freezing procedures:

A. Rapid freezing: The plant material is placed in vials, liquid nitrogen is poured directly in the
vial and dipping the vial into an open flask filled with liquid nitrogen. In this procedure, cooling
between -10°C and -70°C occurred at the rate of >1000°C/min. The quicker the freezing is done,
smaller the intracellular ice crystals are formed. In combination with desiccation or vitrification
pre-treatments, ultra rapid cooling is proved to be the most attractive method for
cryopreservation of plant materials. This method has been successfully used for the
cryopreservation of shoot-tips, somatic embryos and embryonal axes from zygotic embryos of a
number of plant species. The survival rate of cryopreserved tissues by this method is high and
when the desiccation pretreatment is applied even the cryoprotectants are not required.

B. Slow freezing: The tissue is slowly frozen at a slow cooling rate of 0.5-4°C/min from 0 to -
100°C, and then transferred to liquid nitrogen. Survival of cells frozen at slow freezing rates may
involve some beneficial effects of dehydration, which minimizes the amount of water that
freezes intracellularly. Slow cooling permits the flow of water from the cells to the outside,

21
 thereby promoting extracellular ice formation instead of intracellular freezing. It is generally
agreed that upon extracellular freezing the cytoplasm will be effectively concentrated and plant
cells will survive better when adequately dehydrated. This has been successfully employed for
cryopreservation of meristems of few plants and has proved especially successful with cells from
suspension cultures.

C. Stepwise freezing: Firstly, the material is cooled gradually (ca 1°C/min) or step-wise
(5°C/min) to an optimum intermediate temperature (-30°C to -50°C) for about 30 min, and then
rapidly cooled by dipping into liquid nitrogen. The method is highly favorable for freeze
preservation of shoot apices and buds. It is equally successful to cells from suspension
cultures(Ozudogru et al., 2010).

The initial slow freezing reduces the amount of intracellular freezable water by dehydrating the
cells. Early in the freezing process ice is formed first outside the cells, and the unfrozen
protoplasm of cells loses water due to the vapor pressure deficit between the supercooled
protoplasm and the external ice. This initial cooling, thus, acts as another pre-treatment for
dehydration of the cells.

D. Storage: Maintaining the frozen material at the correct temperature is as important as proper
freezing itself. Temperatures above -130°C may allow ice-crystal growth inside the cells and, as
a result reduce their viability. Long-term storage of the material frozen at -196°C, therefore,
requires a liquid nitrogen refrigerator. Generally, the frozen cells or tissues are immediately kept
for storage at temperature ranging from -70°C to -196°C. The storage is ideally done in liquid
nitrogen refrigerator at -150°C in the vapor phase or -196°C in the liquid phase. The temperature
should be sufficiently low for long term storage of cells to arrest all metabolic activities and to
prevent biochemical injury(Ozudogru et al., 2010).

v. Thawing

Rapid thawing of the material frozen at -196°C is achieved by plunging it into water at 37 to
40°C which gives thawing rate of 500-750°C/min. After about 90s, the material is transferred to
an ice bath and maintained there until re-cultured or its viability is tested. The transfer is
necessary because the cells might get damage if it is left long in the water bath 37-45°C. Rapid

22
 thawing protects the cells from the damaging effects of ice crystal formation, which may occur
during slow warming.

vi. Re-culture

The material after thawing should be washed several times to remove the cryoprotectant which
may otherwise be toxic to the cells. A gradual dilution of the cryoprotectant is desirable in-order
to avoid any deplasmolysis injury to the cells. The plant material frozen at -196°C may need
some special requirements for better survival when re-cultured. Shoot-tips from frozen seedlings
of tomato directly developed into plantlets only if the medium was supplemented with GA3. In
its absence, apices callused, followed by the differentiation of adventitious shoots.

2.3.2. Slow Growth

Slow-growth (also known as mid-term conservation) is based on the species to be conserved and
the methodology used; in vitro techniques when applied for short to medium term conservation
allow the storage of germplasm from a few months to 2–3 years without sub-culturing
(Ozudogru et al., 2013). The slow growth conservation strategies are consistently used for a wide
variety of plant species, including various threatened species, both tropical and
temperate(Chauhan et al., 2019). The objective of slow growth is to reduce the sub-culture
interval to a critical level which does not impose a long-term deleterious effect on the
germplasm, or the stability of regenerated plants. In addition, the objective is to reduce cost of
consumables and labor, besides mitigating risk of contamination at each subculture. Slow growth
storage is the simplest and most effective way of restricting growth and development of in vitro
stored germplasm and is usually applied to meristem, shoot and plantlet cultures, but rarely used
for storing root, callus and somatic embryo cultures. However, slow growth treatments incur
some level of stress and it is essential to optimize regimes for each species for timing of sub-
culture and regeneration (Ozudogru et al., 2010, 2017). Varied physicochemical methods used to
achieve slow growth storage.

i. Reduction of Temperature

Incubating normal tissue cultures under suboptimal culture room temperatures has been found to
reduce the growth rate of plants in several species. Low temperatures reported for medium-term

23
 conservation usually ranges from 4 to 20°C. Tropical spe cies tend to be more cold-sensitive
and are required to be stored in the range of 15–20°C or even higher, depending on their
sensitivity. Temperature-dependent medium-term conservation can be thus divided into three
classes: very low (20°C). Temperate crops like apple, hazel nut, pear and olive are normally
stored at very low temperatures (usually at 4°C), whereas crops like banana and ensete require
storage temperature around 15°C.

ii. Uses of Growth Retardants or Growth Regulators

Incorporation of different concentrations of various growth retardants in culture media is also

one of the common methods to reduce the growth of in vitro conserved plants. Maleic hydrazide
(MH), abscisic acid (ABA), cycocel (CCC) and n-dimethyl amino succinamic acid (DSA) are the
frequently used growth retardants. These growth retardants are generally used at 5–50 mg/l
concentration. In pineapple, addi tion of paclobutrazol (PBZ) at a concentration of 0.5 mg/l
reduced the number of subcultures (Canto et al. 2004). Addition of choline chloride to the culture
media may be useful for maintaining shoot quality for prolonged periods in cool storage. In
jojoba, 55% female and 37% male cultures could be conserved up to 270– 480 days depending
upon the genotype on 10μM BAP added to culture medium (Tyagi and Prakash 2004).

iii. Use of Osmotic Regulators

Several osmotic regulators are useful in reducing growth and prolonging the subculture period.
Mannitol, sorbitol and sucrose are the common osmotica used in in vitro conservation of PGR.
Osmotic regulators are metabolically inert, hence are frequently used in culture media for
suppression of culture growth. The combined use of alar, maleic hydrazide and mannitol resulted
in significant delay in bud sprouting, least number of shoots, and reduced shoot length and
storage period of 273 days in gladiolus shoot buds (Patil et al. 2005). Different kinds of sugars
(ribose, fructose, glucose, sucrose and lactose) were tested in Musa spp., and ribose was found to
be the most effective in prolonging storage duration up to 21 months with 83% survival (Ko
et al. 1991). However, in cedar shoot cultures, addition of 6% mannitol negatively affected
survival and storage.

24
2.3.3. Low pressure and low oxygen storage

In this method, the atmospheric pressure (below 50 mmHg reduces plant tissue growth) and the
concentration of oxygen surrounded by plant material are reduced, resulting in reduced
availability of O2, reduced CO2 production and thus reduced photosynthetic activity, which
leads to the Inhibits plant tissue growth and dimensions. These conditions can lead to slow and
reduced growth of the plant material, which helps to extend the shelf life of many fruits,
vegetables and flowers. Therefore, the preservation of germplasm by conventional methods has
several limitations such as dormancy, short-lived seeds, seed-borne diseases, and high cost and
labor. These modern techniques such as cryopreservation (freezing cells and tissues at 196 ° C)
and cold storage help overcome these problems (Bhatia et al., 2015).

2.4. ADVANTAGES OF IN VITRO GERMPLASM CONSERVATION

Germplasm preservation through in vitro culture is advantageous because: 1) entire sets of

genetic materials can be copied through regeneration schemes, 2) rapid proliferation is
achievable; 3) minimal space is required; hence large amount of materials can be stored in a
small area, 4) disease free plants can be produced and maintained, Being free from known
viruses and pathogens, the clonal plant material could be sent from country to country, thus,
minimizing the obstructions imposed by quarantine systems on the movement of live plants
across national boundaries, 5) risks related to environmental changes are avoided, or protection
from natural hazards, 6) Under special storage conditions the plants do not require frequent
splitting and pruning and 7) the plants are not exposed to the threat of changing government
policies and urban development.

25
2.5. REFERENCES
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Mgbeze, G. C., & Iserhienrhien, A. (2014). Somaclonal variation associated with oil palm (Elaeis
guineensis Jacq.) clonal propagation: A review. African Journal of Biotechnology, 13(9).
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Orton, T. J. (1984). Somaclonal variation: theoretical and practical considerations. In Gene manipulation
in plant improvement (pp. 427–468). Springer.
Oseni, O. M., Pande, V., & Nailwal, T. K. (2018). A review on plant tissue culture, a technique for
propagation and conservation of endangered plant species. International Journal of Current
Microbiology and Applied Sciences, 7(7), 3778–3786.
Ozudogru, da SILVA, D. P. C., Ergun, K., Dradi, G., Paiva, R., & Lambardi, M. (2013). In vitro
conservation and cryopreservation of Nandina domestica, an outdoor ornamental shrub. Notulae
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