Ebook Plant biology and biotechnology (Volume II: Plant genomics and biotechnology): Part 2

Applications of Triploids
in Agriculture

19

Ashwani Kumar and Nidhi Gupta

Abstract

Triploid hybrids have one of the most important traits, seedlessness, which
is the characteristic for the fresh-fruit market. Triploid embryos are found
in small seeds that do not germinate. Hybridization-based extensive breeding programmes require very efficient methodologies for embryo rescue
and evaluation of ploidy. Biotechnology provides powerful tools for plant
breeding. Triploid plants raised from endosperm are generally sterile.
Endosperm-ploidy levels and its applications in plant breeding have been
discussed here. Endosperm-raised triploid plants are of commercial value,
e.g. timber-yielding plants, edible fruit plants or ornamentals propagated
vegetatively and multiplied mainly through micropropagation. Illustration
cases of many successful endosperm cultures are described here.
Keywords

Triploids • Embryo rescue • Plant tissue culture • Biotechnology •
Polyploidy breeding

19.1

Introduction

In a fertilization process, the egg fuses with one
of the male gametes to form a zygote, which

A. Kumar
Department of Botany, University of Rajasthan,
Jaipur, Rajasthan 302004, India
N. Gupta (*)
Department of Biotechnology, C.C.S. University,
Meerut, Uttar Pradesh 250004, India
e-mail:

afterward forms the embryo. The other male
gamete fuses with the central cell containing two
haploid nuclei. This second fusion is actually a
double fertilization and triple fusion which often
results in a triploid structure, the endosperm, and
found to be present in all angiosperm families
except
Orchidaceae,
Trapaceae
and
Podostemaceae. Such endosperm-raised triploid
plants are generally sterile, but this seedlessness
does not affect commercial utility of such plants,
e.g. edible fruit plants, timber-yielding plants or
ornamentals which are multiplied mainly through
micropropagation or propagated vegetatively.
The growth of triploids is generally higher than

Bir Bahadur et al. (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics
and Biotechnology, DOI 10.1007/978-81-322-2283-5_19, © Springer India 2015

385

A. Kumar and N. Gupta

386

respective diploids (Thomas and Chaturvedi
2008). Also, triploids are more vigorous than diploids (Morinaga and Fukushima 1935).
Rather than the typical pair of chromosomes,
a cell having three complete sets of chromosomes
is called triploid. To produce viable offspring,
chromosomes need to occur in pairs. But due to
chromosomal number three, the triploid plants
are sterile as the odd numbers of chromosomes
are unable to pair up properly. Such plants do
flowering and bear fruits, but flowers cannot be
fertilized and fruit is sterile. Some of the examples of triploid crops are:
• Seedless watermelons (Citrullus vulgaris)
produced due to cross between tetraploid
females and diploid males. These are commercially cultivated in Japan.
• Triploid sugar beets (Beta vulgaris) produce
larger roots with more sugar content.
• TV29 of tea produced by Tea Research
Association of India is cultivated in North
India. It produces larger shoots and leaves and
is tolerant to drought.
• Cultivated banana (Musa paradisiacal) produces larger and seedless fruits.

19.2

Endosperm and Origin
of Triploids

Endosperm is a natural and unique triploid tissue
in its origin, ploidy level and nature of growth. It
is the triploid stage of the flowering plant which
is produced by fusion of three haploid nuclei; two
from the female gametophyte and one from the
male gametophyte (Thomas et al. 2000). It lacks
histological differentiation. Lampe and Mills’
(1933) first report on endosperm culture was on
maize, whereas La Rue (1949) first reported the
establishment of tissue cultures in maize from
immature endosperm. Since then, mature and
immature endosperm of various species has been
shown to form continuously growing calli
(Bhojwani and Razdan 1996). Johri and Bhojwani
(1965) demonstrated totipotency of endosperm
for the first time. They also demonstrated direct
shoot formation from cultured mature endosperm
of cherry ballart (Exocarpos cupressiformis). By
the time, embryo/shoot/plantlet regeneration

Haploid (N)

Diploid (2N)

Triploid (3N)

Tetraploid (4N)

Fig. 19.1 Haploid (single), diploid (double), triploid
(triple) and tetraploid (quadruple) sets of chromosomes

from endosperm has been reached to dozen of
species (Bhojwani and Razdan 1996).
In tissue culture, endosperm tissues provide
natural material for regenerating plants with triploid chromosome number, and thus, regeneration
of plants from this tissue offers a direct method to
produce triploids. A number of successful regeneration reports of organogenesis and somatic
embryogenesis are available. Endosperm culture
(Johri and Bhojawani 1977), reviews on endosperm (Cheema and Mehra 1982; Bhatnagar and
Sawhney 1981), micropropagation (Driver and
Kuniyuki 1984), walnut tissue culture (Mc
Granahan et al. 1987), embryo rescue (Mc
Granahan et al. 1986), somatic embryogenesis
(Tulecke and McGranahan 1985), triploids in
woody perennials (Lakshmi Sita 1987), Hordeum
vulgare (Sehgal 1974; Sun and Chu 1981),
Triticum aestivum (Sehgal 1974) and Oryza
sativa (Bajaj et al. 1980; Nakano et al. 1975) are
already in records (Fig. 19.1).

19.3

Production of Triploids

Triploids can be produced by crossing an induced
tetraploid plant with normal diploid plant.
Tetraploids can be produced by treating the terminal buds of diploid plants with chemicals such

19 Applications of Triploids in Agriculture

387

b
a
Triploid
Parent creates

Diploid
Sperm

together
produce
Triploid
Offspring

Triploid
Parent

creates

Triploid
Ovule

produces

Triploid

Offspring

Diploid
Parent

creates

Haploid
Ovule

Fig. 19.2 (a) Asexual triploid reproduction via parthenogenesis. (b) Triploid-diploid sexual reproduction

as colchicine, oryzalin, pronamide, amiprophos
methyl and trifluralin (Wan et al. 1991). However,
such crosses are not always fortunate as it results
in reduced seed setting compared to cross
between two diploids (Sikdar and Jolly 1995).
Moreover, seedling survival and seed germination are also very low. Still, triploids play an
important role in biomass and soil conservation
and thus represent a significant importance in
shrubs and trees. They help in preserving vast
amounts of photosynthetic energy and thus promote vegetative growth. Similarly, seedlessness
is used to increase the quality of several fruits,
like banana, papaya, grapes, apple, etc. In some
plants, like Miscanthus sinensis, seed-sterile triploids have been grown to prevent seed dispersal
in the environment (Petersen et al. 2002)
(Fig. 19.2).

19.4

Examples of Triploid Plants

Triploid seedless trait has been described in many
crops, especially in fruits. Artificially, triploid
fruits are produced by first developing tetraploids
using above-mentioned chemicals, which are
then crossed with respective diploids. Such fruits
are then commercially used.

19.4.1 Watermelon (Citrullus
vulgaris)
When tetraploid females are crossed with diploid
males, seedless watermelons (Citrullus vulgaris)

are produced. Native African vine Citrullus
lanatus (syn. C. vulgaris) derived modern varieties of the watermelon that are unable to produce
viable gametes during meiosis, and thus, their
ripened melons are seedless. Wild populations of
C. lanatus var. citroides are common in Central
Africa and give rise to domesticated watermelons
var. lanatus (Robinson and Decker-Walters
1997). Wild, ancestral watermelons (var. citroides) have a spherical, striped fruit and white,
slightly bitter or bland flesh and are commonly
known as the citron or citron melon (Fig. 19.3).
Japan commercially grows seedless watermelons which are produced by crossing tetraploid
female with diploid male lines. Reciprocal cross
was also tried but was not successful. Seeds produced by triploid plants are not true seeds; they
are small in size having white rudimentary structures like that of cucumber (Cucumis sativus)
seeds. However, a few normal sized seeds may
occur, but they are generally empty. It is also to

be noted that all cultivated triploid watermelons
do not have red pulpy flesh. They may have seedless yellow, sweet flesh (Fig. 19.4).

19.4.2 Little Gourd (Coccinia grandis)
Babu and Rajan (2001) developed a triploid variety of Coccinia grandis, fruit of which is used as
a vegetable. It was also produced by crossing a
normal diploid parent with colchicine-induced
tetraploid. 2.4 % of seeds per fruit were observed.
Morphologically, the triploid plants were somewhat resembled to the diploid, but the substantial


A. Kumar and N. Gupta

388

Fig. 19.4 Triploid watermelon having red flesh

Fig. 19.3 Citron melon

features were its vigorous growth, increased fruit
size, lower astringency and higher yield.
However, these triploid fruits were tastier with
good amount of vitamin A, vitamin C and iron
and had less polyphenols; hence, they could be
used as a salad crop. This plant also has many
medicinal properties against diabetics, skin infections and bronchitis (Table 19.1 and Fig. 19.5).

19.4.3 Citrus
Citrus fruits are the most extensively and primarily produced fruit tree crop in the world (FAO
2009) for the fresh-fruit market, especially in the

Mediterranean area. Area-wise, Spain is the main
producer which covers a surface of 330,000 ha
and produces about 6.3 million tons of citrus.
Diploids are the available genetic resources
for citrus fruit, and their naturally produce seeds
include polyploid individuals. These natural
polyploid plants can give rise to interesting characteristics in citrus fruit; thus, they are very useful for genetic breeding projects. CIRAD (French
Agricultural Research Centre for International
Development) has developed genetic breeding
programmes for citrus fruit in the Mediterranean
Basin to create triploid varieties of sterile and
seedless fruit and tetraploid rootstocks resistant

to abiotic constraints, such as water deficiency or
salinity, both from predominantly diploid genetic
resources that would meet agronomic constraints,
market expectations and consumer demand
(Fig. 19.6).

19.4.4 Mandarin
As per increasing consumer demand, seedless
citrus fruits are the basic requirement for the
fresh market. Mandarin triploid hybrids have this
seedlessness trait as its one of the most important
characteristics. The availability of a number of
high-quality seedless varieties in mandarins is
very low; thus the production and recovery of
new seedless triploid hybrids of mandarin varieties have a high priority for many citrus industries
worldwide (Fig. 19.7).
Citrus triploid hybrids can be recovered by

2x × 4x (Esen and Soost 1971b; Oiyama et al.
1981; Starrantino and Recupero 1981), 2x × 2x
(Cameron and Frost 1968; Esen and Soost 1971a;
Geraci et al. 1975) and 4x × 2x (Cameron and
Burnett 1978; Esen et al. 1978; Aleza et al. 2009)
sexual hybridizations as a consequence of the
formation of unreduced gametes at low frequency
(Aleza et al. 2010).
For the first time, Esen and Soost (1971a)
indicated that triploid embryos were mainly
found in between one third and one sixth smaller
seeds than normal seeds that do not germinate in
conventional greenhouse conditions. However,
still at relatively low germination percentages,


19 Applications of Triploids in Agriculture

389

Table 19.1 Comparative evaluation of diploid, tetraploid and triploid of Coccinia grandis (Source: Babu and Rajan
2001)
Characters
Days to flowering
Flower size
Fruit size
Fruit length (cm)
Fruit girth (cm)
Fruit weight (g)
Polyphenol per gram of fruit (fig)

Fruit colour
Leaf size
Leaf thickness
Fruit yield/plant/year (kg)

Diploid
40
Medium
Medium
6.59
7.40
15.20
0.300
Green white strips
Medium
Medium
10.32

Tetraploid
42
Large
Small
4.49
8.20
14.50
0.311
Green with white strips
Large
Very thick
9.34

Triploid
38
Medium
Large
7.50
11.60
44.20
0.090
Green with white
Medium
Medium
15.25

Fig. 19.6 Seedless lemon (Citrus limon)
Fig. 19.5 Coccinia grandis

Fig. 19.7 (a) Mandarin plant having flowers and fruits. (b) Seedless mandarin


A. Kumar and N. Gupta

390

the in vitro culture of whole seeds with their
integuments can improve germination rates
(Ollitrault et al. 1996). In rare cases, triploid
hybrids can be found in conventional greenhouse
seedlings, as in ‘A-12’ mandarin (Bono et al.
2004) and ‘Winola’ mandarin (Vardi et al. 1991).

19.4.5 Neem (Azadirachta indica)
Because of the arising use of neem and its products in medicine, agriculture, cosmetics and animal health care, it is an important and economic
tree of India. Triploid plants of neem were
obtained from immature endosperm culture
(Chaturvedi et al. 2003). Over 66 % of the plants
were triploid with chromosome number 36. A
characteristic feature of the shoots of endosperm
origin is the presence of a large number of multicellular glands. The selected triploids, expected
to be sexually sterile, can be bulked up by micropropagation (Fig. 19.8).

19.4.7 Shanin (Petunia violacea)
Gupta (1983) reported the formation of haploid,
diploid and triploid plants by direct pollen
embryogenesis in Petunia violacea. In certain
species, especially in Petunia, an almost exclusive production of androgenic triploids has been
reported which is useful in ornamental plants for
the introduction of vigorous foliage and flowers
(Fig. 19.10).

19.4.8 Triticale

Garg et al. (1996) describe somatic embryogenesis and triploid plant regeneration from immature endosperm cultures of Acacia nilotica, an
important leguminous tree species suitable for
afforestation of arid and marginal lands
(Fig. 19.9).

Triticale, first bred in laboratories during the late
nineteenth century, is one of the most successful
synthetic allopolyploids produced by crossing

tetraploid wheat or hexaploid wheat with rye.
The grain was originally bred in Sweden and
Scotland; however, now it is being grown commercially in many parts of the world, e.g.
Germany, Canada, France and Poland (the largest
area), covering an area of around 2.6 million
hectares with an annual production of 8 million
tons. Triticale high-yielding ability and grain
qualities of wheat combined with tolerance ability for adverse environment of rye provide its
important and desirable features. In more than
15 years, the yielding ability of triticales has been
increased to about 90 %. However, in Sweden,
the raw triticales yielded about 50 % of the standard varieties of wheat.

Fig. 19.8 Neem

Fig. 19.9 Acacia plant having flower and fruit

19.4.6 Acacia (Acacia nilotica)


19 Applications of Triploids in Agriculture

391

Fig. 19.10 Shanin flower
Fig. 19.11 Triticale

India have released three varieties of triticales:
TL419, DT46 (amber colour grains) and TL1210.
Although TL1210 grain yield is comparable to

that of the best wheat varieties, its deep grain
colour represents its chief drawback, thus mainly
grown as a fodder crop in Punjab. To overcome the
problem, Indian breeders have developed ambercoloured triticales by using white-seeded rye as
one of the parents of the triticales (Fig. 19.11).
Some other examples of allopolyploids are
Raphanobrassica, the triploid (AAC) produced
by crossing B. campestris (AA) with B. napus
(AACC), Festuca-Lolium hybrids, allopolyploid
clovers and some species hybrids in Rubus and
jute (Corchorus sp.).

19.4.9 Sugar Beet (Beta vulgaris L.)
The triploid varieties of sugar beet are mixtures
of diploid, triploid and other ploidy level plants.
As compared to diploids, triploid sugar beets produce more sugar and larger roots and 10–15 %
higher yields per unit area, while tetraploids produce smaller roots and lower yields.
Commercially, Japan and Europe produce triploid varieties of sugar beet, but their popularity is
declining rapidly. As the beet flower is small in
size, triploid sugar beet seed production is quite
difficult (Fig. 19.12).
Triploid sugar beet seed may be produced by
using any of the following two ways: (1) using 4x

plant as male and 2x as female or (2) using 4x
plants as female and 2x as male. The first cross
provides higher seed yield but a lower proportion
of triploids, while the second gives lower seed
yield but a higher proportion of triploids.
Commercially, interplanting 4x and 2x lines in

the ratio 3:1 is used for producing triploid sugar
beet seed, and finally, seeds from both 4x and 2x
plants are harvested. These harvested seeds consist of about 75 % triploid (3x) seeds.

19.4.10 Cassava (Manihot esculenta)
Cassava, commonly known as poor man’s crop,
is an important root crop to be cultivated in tropical countries and propagated by stem cuttings. It
has become a subsidiary food in many countries.
It is also used as cattle feed and its raw material
for starch-based industries. Cultivated cassava is
highly heterozygous and cross-pollinated, having a diploid number of chromosomes (2n = 36).
Among artificially produced polyploids, cassava
triploids have higher starch potential and a
higher yield (Jos et al. 1987; Sreekumari and Jos
1996).
The first triploid variety of cassava named
Sree Harsha was released in 1996 (Sreekumari
et al. 1999) and was produced by crossing
induced tetraploid plants with natural diploid.
The use of a 2x female plant yielded better results
than reciprocal crosses. Many features of triploid


A. Kumar and N. Gupta

392

Fig. 19.12 Sugar beet: (a) seed, (b) flower, (c) root

cassava make it superior than its diploid. These

include higher harvest index, rapid bulking,
higher yield, early harvestability, increased dry
matter and starch content in the roots, shade
tolerance and tolerance to cassava mosaic virus
(Fig. 19.13).

19.4.11 Tea (Camellia sinensis)
Tea Research Association, India, has recently
released a triploid clone of tea (Camellia sinensis
var. assamica) for its commercial cultivation in
northern parts of the country. This triploid cultivar, TV29, produces larger shoots and, thereby,
biomass yields more cured leaf per unit area and
is more tolerant to drought than the available diploid cultivars. The quality of the triploid clone is
comparable to that of diploid cultivars used for
making CTC (curl-tear-cut) tea (Fig. 19.14).

19.4.12

Mulberry (Morus alba L.)

Being an exclusive source of feed for silkworms,
mulberry is an indispensable crop for the sericulture industry. Both natural and in-vivo-induced
mulberry triploids have been reported (Das et al.
1970; Katagiri et al. 1982; Dwivedi et al. 1989).
Many of the triploid lines are superior to its diploids (Thomas et al. 2000), in cold and disease
resistance (Hamada 1963) and in yield and nutritive qualities of leaves (Seki and Oshikane 1959).
The endosperm callus differentiated shoots,
which could be rooted, and full triploid plants
have already been established in soil (Fig. 19.15).

19.4.13 Ornamental
Excised cultures of endosperm from immature
fruits having zygotic embryo have been used at


19 Applications of Triploids in Agriculture

393

Fig. 19.13 Cassava: (a) flower and (b) root

Fig. 19.15 Mulberry plant with fruit
Fig. 19.14 Tea leaves

early dicotyledonous stage to produce triploid
annual phlox or Drummond’s phlox (Phlox
drummondii Hook.) ornamental plants (Razdan
et al. 2013). It was reported that over 70 % of
annual phlox plants were triploid with a chromosome number of 2n = 3x = 21. The growth of
triploids is generally higher than respective
diploids (Thomas and Chaturvedi 2008). These
triploid plants have greater size of leaves, stem,
flowers and/or foliage with higher number of
pollen and larger stomata as compared to naturally occurring diploid plants (Miyashita et al.
2009). Moreover, triploid plant flowers showed

enlarged central eye and bright colour, adding
to their ornamental value (Razdan et al. 2008)
(Fig. 19.16).

19.4.14 Pomegranate (Punica
granatum L.)
Pomegranate is one of the oldest known
fruit trees of the tropics and subtropics, cultivated
for its delicious edible fruits. In addition,
the tree is also valued for its pharmaceutical
properties.


A. Kumar and N. Gupta

394

References

Fig. 19.16 Phlox drummondii flowers

19.5

Discussion

Endosperm is a unique tissue in its origin, ploidy
level and nature of growth. It is mostly formed by
the fusion product of three haploid nuclei, one
from the male gametophyte and two from the
female gametophyte, and is, therefore, triploid.
Traditionally, triploids are produced by crossing
induced superior tetraploids and diploids. This
approach is not only tedious and lengthy (especially for tree species), but in many cases, it may
not be possible due to high sterility of autotetraploids. In contrast, regeneration of plants from

endosperm, a naturally occurring triploid tissue,
offers a direct, single-step approach to triploid production (Bhojwani and Razdan 1996; Kumar
2010; Kumar and Roy 2006, 2011; Kumar and
Shekhawat 2009; Neumann et al. 2009).

19.6

Conclusion

In conclusion, gametic embryogenesis hold
great promise for making a significant, low-cost
and sustainable contribution to plant breeding,
aimed at increasing farm productivity and food
quality, particularly in developing countries and
in an environmentally friendly way, helping to
reduce the proportion of people suffering from
chronic hunger and from diseases due to
malnutrition.

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Improving Secondary Metabolite
Production in Tissue Cultures

20

Ashwani Kumar

Abstract

Plant cell and tissue culture has been suggested as an alternative means
for year-round production of secondary metabolites with an added
potential of increasing yields by culture selection and manipulation,
genetic transformation, hairy root cultures, and use of bioreactors for
mass production. Secondary metabolite pathways and genes involved in
those pathways have been identified, and regulation of transcription and
transcription factors has been determined by studying functional genomics in conjunction with data-mining tools of bioinformatics. Besides this,
advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or
more, heterologous genes taken from various other species. As an alternative, the metabolic pathways of useful secondary metabolites have
been modified to improve their productivity via genetic transformation.
However, there is a need to understand metabolic pathways of secondary
metabolism at the molecular level. Plant hairy roots offer a novel and
sustainable tissue-based system that preserves multiple specialized cell
types believed to be important in maintaining a better consistency in
synthesis of bioactive secondary molecules. This paper will review stateof-the-art reports on improving production of secondary metabolites in
tissue cultures in various plant species.
Keywords

Secondary metabolites • Alkaloids • Saponins • Terpenoids • Nicotine

20.1
A. Kumar (*)
Department of Botany, University of Rajasthan,
Jaipur, Rajasthan 302004, India
e-mail:

Introduction

Plant cell and tissue culture technology has been
known as a possible tool for studying both the
biosynthesis and the production of plant secondary metabolites (Kong et al. 2004, 2013, 2014).

Bir Bahadur et al. (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics
and Biotechnology, DOI 10.1007/978-81-322-2283-5_20, © Springer India 2015

397


A. Kumar

398

It has been used for production of large number
of secondary metabolites. The degree of cellular
differentiation and organization of the tissue,
which is implied in a culture of this nature,
favors the accumulation of these secondary
compounds (Flores 1992; Wu et al. 2003, 2005;
Kim et al. 2004; Kong et al. 2004; Thwe et al.
2012). An increased secondary metabolite production is correlated with a slow cell division
rate in cell suspension cultures (Lindsey and
Yeoman 1983; Sharma et al. 2011). Similarly,
secondary metabolite production at the stationary phase of growth has been related with tissue
organization (Tabata et al. 1972), morphological
differentiation (Ramawat et al. 1985; Sharma
et al. 2009), and low growth rates (Lindsey and
Yeoman 1983).
Controlled transcription of biosynthetic genes

achieved by specific transcription factors is one
major mechanism regulating secondary metabolite production in plant cells (Bopana and Saxena
2010). Transcription factors are sequencespecific DNA-binding proteins that interact with
the promoter regions of target genes and modulate the initiation of mRNA synthesis by RNA
polymerase II. These proteins regulate gene transcription depending on tissue type and/or in
response to internal signals. The two well-studied
secondary pathways are the phenylpropanoid
pathways and its flavonoid branch and the terpenoid
indole alkaloid biosynthetic pathway (Neumann
et al. 2009).
Hairy root cultures provide novel opportunities
for production of valuable phytochemicals that are
synthesized in roots. Hairy roots are developed
by infecting plant leaf or stem tissue with
Agrobacterium rhizogenes that transfers genes that
encode hormone biosynthesis enzymes into the
plants. Hairy root cultures have several advantages
over undifferentiated plant suspension cell cultures. Hairy roots are genetically stable and grow in
hormone-free culture media. Hairy roots show
rapid growth and promote the synthesis of phytochemicals whose biosynthesis requires differentiated cell types. Hairy root lines producing valuable
phytochemicals have been developed from various
plant species (Dehghan et al. 2012; Cardillo et al.
2013). Recently, have been used to hairy root cul-

tures to improve secondary metabolism compounds in Hyoscyamus niger (Zhang et al. 2004)
and p-hydroxybenzoic acid (pHBA) glucose ester
production in hairy roots of Beta vulgaris (Rahman
et al. 2009), express foreign proteins or vaccine in
tobacco (Shadwick and Doran 2007). Several
TIAs’ biosynthesis genes have also been overexpressed in C. roseus hairy root cultures (Zhao et al.

2012a, b). Kochkin et al. (2013) demonstrated for
the first time the presence of large amounts of ginsenosides malonyl-Rb1, malonyl-Rc, malonylRb2, and malonyl-Rd in a suspension culture of
Panax japonicus var. repens cell.
Due to an increased appeal of natural products
for medicinal purposes, metabolic engineering can
have a significant impact on the production of pharmaceuticals and help in the design of new therapies
(Bender and Kumar 2001; Kumar and Roy 2006,
2011; Kumar and Sopory 2008, 2010; Neumann
et al. 2009; Kumar and Shekhawat 2009; Kumar
2010; Fernandez et al. 2010; Sharma et al. 2011;
Kumar 2014). According to Bailey (1991), metabolic engineering is “the improvement of cellular
activities by manipulation of enzymatic, transport,
and regulatory functions of the cell with the use of
recombinant DNA technology.” Application of
recombinant DNA methods can improve production of metabolite and protein products by altering
pathways and regulate release process in downstream processing. In many cases, this approach
relies on the identification of limiting enzyme
activities after successful pathway elucidation and
metabolite mapping (metabolomics) (Neumann
et al. 2009). Some of the important metabolites
being produced in tissue culture and some technologies to improve their production are presented
in this review paper.

20.2

Hairy Root Cultures

20.2.1 Azadirachtin from Azadirachta
indica A. Juss. (Neem) Cultures
Azadirachtin (C35H44O16), obtained from

Azadirachta indica (neem), is a high-value
secondary metabolite commercially used as a
broad-spectrum biopesticide. Neem (Azadirachta


20

Improving Secondary Metabolite Production in Tissue Cultures

indica A. Juss.) plant tissue and cell culture have
been used to obtain year-round production of
azadirachtin and other neem metabolites with the
added potential of increasing yields by culture
selection and manipulation. Allan et al. (2002)
established hairy root cultures from stem and leaf
explants of Azadirachta indica A. Juss (neem)
following infection with Agrobacterium rhizogenes. Transformation was confirmed using
polymerase chain reaction analysis. Srivastava
and Srivastava (2013) reported batch cultivation
of Azadirachta indica hairy roots in different
liquid-phase bioreactor configurations (stirred
tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disk
as root supports) to investigate possible scale-up
of the A. indica hairy root culture for in vitro
production of the biopesticide, azadirachtin. The
hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column).
They reported batch cultivation of A. indica hairy
roots in modified bubble column reactor (with
polypropylene mesh support). The incorporation
of a PUF disk as a support for the hairy roots

inoculated inside the bubble column reactor
facilitated increased biomass production and
azadirachtin accumulation in hairy roots
(Srivastava and Srivastava 2013).

20.2.2 Tropane Alkaloids
from Nicotiana tabacum
Hairy root cultures of Nicotiana tabacum are a
better alternative for tropane alkaloid production
than cell suspension cultures, mainly because
they are stable, both genetically and in alkaloid
production during long subculture periods
(Maldonado-Mendoza et al. 1993). The utility of
hairy root cultures to produce valuable phytochemicals could be improved by repartitioning
more of the desired phytochemical into the spent
culture media, thereby simplifying the bioprocess
engineering associated with the purification of the
desired phytochemical. The majority of nicotine
produced by tobacco hairy root cultures is retained
within roots, with lesser amounts exuded into the
spent culture media. Reduced expression of the

399

tobacco nicotine uptake permease (NUP1), a
plasma membrane bound transporter, results in
significantly higher nicotine accumulation in the
media. Thus, NUP1-reduced expression lines provide a genetic means to repartition more nicotine
into the culture media (Dewey and Xie 2013;
Murthy et al. 2014).

20.2.3 Diosgenin from Trigonella
foenum-graecum L.
(Fenugreek)
Plant tissue cultures (in vitro techniques) offer an
opportunity to improve the plant properties via
genetic engineering, and recently it has been used
as a tool for genetic transformations. Trigonella
foenum-graecum L. (in Arabic, Hulabah) is also
employed as a herbal medicine in many parts of
the world. Diosgenin provides about 50 % of the
raw material for the manufacture of cortisone,
progesterone, and many other steroid hormones
and is a multibillion-dollar industry. However, the
supply of diosgenin cannot currently satisfy the
demands of the ever-growing steroid industry,
and therefore new plant species and new production
methods, including biotechnological approaches,
are being researched (Verpoorte 2000; Neumann
et al. 2009).

20.2.4 Terpenoid Indole Alkaloids
(TIAs)
Terpenoid indole alkaloids (TIAs) are very
important pharmaceutical compounds. The
monomeric alkaloids serpentine and ajmalicine
are used as a tranquilizer and to reduce hypertension, respectively. The dimeric alkaloids vincristine and vinblastine are potent antitumor
drugs. The biosynthesis of TIAs is highly regulated and depends on tissue-specific factors as
well as environmental signals. Low productivity
is the main hindrance toward commercial application of the production of secondary metabolites by plant cells in suspension culture.

Different strategies have been developed to
overcome this problem (Kumar and Roy 2006;


A. Kumar

400

Kumar and Sopory 2008, 2010; Kumar and
Shekhawat 2009; Kumar 2010; Bopana and
Saxena 2010; Sharma et al. 2011).

20.2.5 Ginsenoside Saponin
Han et al. (2013) established dammarenediol-II
production via a cell suspension culture of
transgenic tobacco overexpressing PgDDS.
Dammarenediol-II is a biologically active tetracyclic triterpenoid, which is a basic compound of
ginsenoside saponin and is a useful candidate
with potentially biologically active triterpenes.
Transgenic tobacco plants overexpressing
PgDDS (AB122080) under the control of the
CaMV35 promoter were constructed.

20.2.6 Benzylisoquinoline Alkaloids
(BIAs)
This group of alkaloids is derived from aromatic
amino acid tyrosine. BIAs include the narcotic
analgesic morphine, the cough suppressant
codeine, the muscle relaxants papaverine and
(+)-tubocurarine, the antimicrobial compound

sanguinarine, and the cholesterol-lowering drug
berberine (Kong et al. 2004; Kong and von
Aderkas 2007). Recently, efforts have been made
to assemble BIA biosynthetic pathways in microorganisms through the heterologous expression
of multiple alkaloid biosynthetic genes (see
Kumar and Sopory 2010; Nakagawa et al. 2011).
Farrow et al. (2012) suggested that species-specific metabolite accumulation is influenced by
the presence or absence of key enzymes and
perhaps by the substrate range of these
enzymes. They provided a valuable functional
genomics platform to test these hypotheses
through the continued discovery of BIA biosynthetic enzymes.
BIA noscapine is cough suppressant and
promising anticancer agent (Dumontet and

Jordan 2010), BIA biosynthetic enzymes from a
number of related plant species have been characterized using EST (Farrow et al. 2012). The
integration of transcript and metabolite profiles
predicts the occurrence of both functionally redundant and novel enzymes.

20.2.7 Hypericin from Hypericum
perforatum L.
Hypericin is a traditional medicinal plant for the
treatment of depression and wound healing, and
hypericin is one of the main effective active
substances. To optimize the culture system for
producing hypericin in adventitious root, Wu
et al. (2014) reported the use of balloon-type airlift bioreactors. They investigated the effect of
air volume, inoculation density, indole-3-butyric
acid (IBA) concentration and methyl jasmonate

(MeJA) concentration on hypericin content,
and productivity during adventitious root culture.
MeJA efficiently elicited the hypericin synthesis
of H. perforatum adventitious roots (Wu et al.
2014).

20.2.8 Anthraquinones
Baque et al. (2013) reported improve root growth
and production of bioactive compounds such as
anthraquinones (AQ), phenolics, and flavonoids
by adventitious root cultures of Morinda citrifolia. They studied effects of aeration rate, inoculum
density, and Murashige and Skoog (MS) medium
salt strengths using a balloon-type bubble
bioreactor.

20.2.9 Isoflavone Production
Isoflavones have an affinity to estrogen-β receptors
in humans and are reported to exhibit numerous
health-promoting effects, including the allevia-


20

Improving Secondary Metabolite Production in Tissue Cultures

tion of menopausal symptoms, the prevention of
osteoporosis and cardiovascular diseases, and
the lowering of risk of breast cancer (Patisaul
and Jefferson 2010). The growing demand for
isoflavonoid derivatives resulted in numerous

research projects focused on the in vitro cultures
of selected plant species of Fabaceae which is
rich in isoflavones. Some of the best known
phytoestrogens are genistein, genistin, daidzein,
and puerarin (Patisaul and Jefferson 2010).
Biotechno-logical production of isoflavones,
mainly from the family Fabaceae, is based on suspension cultures of Pueraria sp. (Goyal and
Ramawat 2008a, b; Sharma et al. 2009; He et al.
2011), Psoralea sp. (Shinde et al. 2009a, b), and
Glycine max (Federici et al. 2003; Terrier et al.
2007). Calycosin, formononetin, and pseudobaptigenin are also present in the more widespread
legume Trifolium pratense (Kokotkiewicz et al.
2013).

20.3

Air volume promoted the hypericin production
of adventitious roots (Wu et al. 2011). Higher
efficiency of genetic transformation resulted not
only from greater target tissue yield, but there
was also evidence of improved transgenic event
production with the tissue produced with airlift
bioreactors than tissue produced in shaken flasks
(Kong et al. 2013).
Numerous studies have applied bioreactors in
plant cell (Huang and McDonald 2012) and organ
(Srivastava and Srivastava 2012) culture to obtain
specific metabolites. Modern bioreactor culture
systems provide a more advanced technology to
produce higher secondary metabolites from plant

cell, tissue, or organ using artificial nutrients with
MeJA. Yu et al. (2002) found that the ginsenoside
content was obviously enhanced by the addition
of 100 μM MeJA during adventitious root culture
of Panax ginseng; Donnez et al. (2011) examined
that 0.2 mM MeJA was optimal for the efficient
production and high accumulation of resveratrol
in grape cell.

Adventitious Root Culture
Versus Hairy Root Culture
20.5

Culture of adventitious roots in bioreactors offers
several advantages such as faster growth rates,
tremendous quantities of metabolite accumulation, and stable production year-round. This can
also reduce production costs and time and final
product quality can be more easily controlled
(Lee et al. 2011). Baque et al. (2013), using largescale bioreactors, raised adventitious root culture
as an efficient and attractive alternative to cell,
hairy root, or whole-plant cultivation for biomass
and metabolite production.

20.4

401

Bioreactors for Secondary
Metabolite and Plant
Propagules

Recent advances with large-scale production
have successfully produced ginseng roots in a
10,000 l bioreactor establishing the feasibility of
the root system to accommodate industrial
processes (Sivakumar et al. 2006). A suitable air
supply inside a bioreactor is an important factor.

Propagules in Bioreactors

Besides this, a considerable number of researchers have cultured plant propagules in bioreactors
to produce high-quality seedling (Zhao et al.
2012a, b). It is clear from these studies that
temporary immersion bioreactor culture systems
are appropriate for shoot multiplication and
regeneration and the continuous immersion
system is suitable for the proliferation of propagules (without leaves) such as bulblets (Kim et al.
2004), PLBs (Yang et al. 2010), and rhizomes
(see Gao et al. 2014). Kong et al. (2013) constructed and tested airlift bioreactors (ALBs) for
their potential to enhance chestnut embryogenic
tissue proliferation for genetic transformation
and mass propagation.

20.5.1 Ginsenoside Production
Panax ginseng roots have been widely used in
Chinese traditional medicine since ancient times
owing to their stimulating and tonic properties.


A. Kumar

402

The pharmacological activities of ginseng or its
crude extracts are based on the presence of a
mixture of triterpenic saponins referred to as
ginsenosides. The two major groups of ginsenosides are the Rb and Rg groups, which have
proto-panaxadiol and protopanaxatriol, respectively, as the sapogenins (Mallol et al. 2001). Rb
group includes the ginsenosides Rb1, Rb2, Rc,
and Rd, while Rg group includes the Re, Rf, and
Rg1 ones as the main compounds. Among all
these ginsenosides, Rb1 and Rg1 are the most
effective compounds (Tanaka and Kasai 1984;
Mallol et al. 2001). Ginsenosides accumulate in
the root of the plant, but the agricultural production of roots is expensive. Therefore, the production of ginsenosides, by means of different
biotechnological alternatives, has been extensively
studied by a number of researchers, using callus
tissues (Mallol et al. 2001), cell suspensions
(Mathur et al. 1994; Kochkin et al. 2013), and
root cultures (Yoshikawa and Furuya 1987); nevertheless, the productivity obtained so far has
been low because of the low growth rates of cultures. The induction and establishment of hairy
roots after the infection of Panax ginseng rhizomes with Agrobacterium rhizogenes has been
successfully performed (Washida et al. 1998).
These roots grow more rapidly and produce
higher levels of saponins than the ordinary cultured roots obtained by hormonal control. In the
case of agropine-type strains (such as A. rhizogenes A4), two T-DNA fragments (TL-DNA and
TR-DNA) are separately transferred into the
plant material (Jouanin 1984). The integration of
the TL-DNA into the plant genome is essential
for developing transformed roots; three genes of

this fragment, known as rolA, rolB, and rolC, are
responsible for the full hairy root syndrome
(Palazon et al. 1998). Although the TR-DNA is
not essential for hairy root formation, it has been
shown that the aux1 genes harbored in this T-DNA
segment provide to the transformed cells with an
additional source of auxin. Aux genes play a significant role in the morphology and alkaloid production of transformed roots of Datura metel and
Duboisia hybrid (Mallol et al. 2001).

20.5.2 Resveratrol 1
These plant polyphenols have received considerable interest based upon a number of associated
health benefits (Baur and Sinclair 2006; Delmas
et al. 2006). Most notably, the significant levels
of resveratrol 1 in red wine have been credited to
the phenomenon known as “the French Paradox,”
wherein low incidence of heart disease is
observed among a population with a relatively
high-saturated-fat diet and moderate wine consumption (Frankel et al. 1993). Over the past two
decades, numerous health benefits impacting
cardiovascular disease, various cancers, atherosclerosis, and aging have been linked with resveratrol 1 (reviewed; Baur and Sinclair 2006;
Roupe et al. 2006). The majority of resveratrolcontaining dietary supplements are composed of
unknown/unidentified botanical components
wherein resveratrol 1 and resveratrol derivatives
only make up a small fraction of the product.
While chemically synthesized resveratrol 1 may
address this issue, natural sources often contain
derivatives, cofactors, and other phytonutrients
that provide added or synergistic benefits to the
nutraceutical product and are often preferred by
the consumer (Wallace 1998). Recent studies

showing antiaging benefits of resveratrol 1 (Baur
et al. 2006) further accelerate interest in a natural, food-grade source of enriched resveratrol/
resveratrol derivatives that delivers a more
defined and consistent product composition and
ensures a stable supply chain, and several biotic
production strategies targeting recombinant
plants, yeast, and bacteria have been advanced
(Watts et al. 2006).

20.5.3 Camptothecin (CPT)
Camptothecin (CPT), a monoterpene indole alkaloid,
has been found in several plant species including
Camptotheca acuminata, Nothapodytes foetida,
and Ophiorrhiza pumila (Saito et al. 2001;
Lorence and Nessler 2004). Since it possesses
topoisomerase I poisoning properties, its semi-


20

Improving Secondary Metabolite Production in Tissue Cultures

synthetic derivatives, topotecan and irinotecan,
have been developed to be clinically used as
anticancer drugs. Previously, we have established a hairy root culture of O. pumila which
has already been shown to be a desirable experimental system to study the biosynthesis of
camptothecin, since the culture produces a high
level of CPT and excretes it into the culture
medium (Sirikantaramas et al. 2007).

20.5.4 Catharanthus roseus
(Madagascar Periwinkle)
As an important medicinal plant, Catharanthus
roseus (Madagascar periwinkle) produces a
large amount of terpenoid indole alkaloids
(TIAs). Among them, vinblastine and vincristine are important antitumor bisindole alkaloids.
However, these two anticancer compounds are
produced at a very low level in C. roseus leaves,
about 5.8 μg/g for vinblastine and 0.9 μg/g
(fresh weight) for vincristine, leading to their
high price in the market (Favretto et al. 2001).
The lack of vinblastine and vincristine in C.
roseus hairy roots has been ascribed to an
absence of vindoline (Bhadra et al. 1998). This
may be due to the undetectable expression of
the D4H and DAT genes in the transgenic hairy
roots. Very recently the DAT gene, which is
responsible for the terminal step of vindoline
biosynthesis in C. roseus, was overexpressed in
C. roseus hairy roots. Interestingly, overexpression of DAT did not increase vindoline production but improved the accumulation of another
monoterpenoid indole alkaloid, horhammericine (Magnotta et al. 2007). Biotechnological
methods may provide an efficient alternative
for producing natural products since a number
of genes involved in the TIAs’ biosynthetic
pathway have been cloned (Pasquali et al.
2006; Wang et al. 2010; Zhou et al. 2011).
Deacetylvindoline-4-O- acetyltransferase
(DAT) is a key enzyme for the terminal step of
vindoline biosynthesis. In this research, the
DAT gene promoter was cloned, sequenced,

and analyzed.

403

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Somaclonal Variation
in Micropropagated Plants

21

Leela Sahijram

Abstract

Plants generally exhibit cytogenetic and genetic variations that are helpful
to plant breeders for crop improvement. When such variants arise through
the cell and tissue culture process, using any plant portion as an explant
material, these are termed ‘somaclonal variations’ (SV). Variants obtained
using callus cultures are referred to as ‘calliclones’, while variants obtained
using protoplast cultures are known as ‘protoclones’. On the other hand,
‘gametoclonal variation’ refers to variations arising in cell cultures of
gametic origin, as in pollen and microspore cultures, to distinguish them
from somatic cell-derived regenerants. Somaclonal variation is a doubleedged sword whereby its presence in micropropagation programmes is
inimical, while it can be gainfully exploited to create stable variations, e.g.

disease resistance, where other methods fail or are cumbersome.
Keywords

Somaclonal variation • Genetic stability • Gametoclonal • Detection of
variants • Useful variants

21.1

Introduction

Somaclonal variation (SV) is a phenotypic variation either genetic or epigenetic in origin displayed among somaclones (soma=vegetative;
clone=identical copy) and occurs among plants
regenerated from tissue culture. A general term

L. Sahijram (*)
Division of Biotechnology, Indian Institute of
Horticultural Research (IIHR), Hessaraghatta Lake
Post, Bangalore, Karnataka 560 089, India
e-mail:

‘somaclonal variation’ was proposed to describe
genetic variation in plants regenerated from any
form of cell cultures. Accordingly, plants derived
from cell and tissue cultures are termed ‘somaclones’. Somaclonal variation has come to represent genetic variability present among all kinds
of cells/plants obtained from cultures in vitro. SV
can be problematic during micropropagation and
in vitro conservation and in genetic transformation of crop plants, although it may be put to
good use as a tool in plant breeding. Plants regenerated from tissue and cell cultures show heritable variation for both qualitative and quantitative

Bir Bahadur et al. (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics

and Biotechnology, DOI 10.1007/978-81-322-2283-5_21, © Springer India 2015

407


L. Sahijram

408

traits. Several useful somaclonal variants too
have been obtained in a large number of plant
species such as potato, sugarcane, banana,
tomato, etc. Somaclonal variation is well documented in the widely commercialized tissue
culture-raised fruit crop, banana (Musa spp.).
Variants obtained using callus cultures are
referred as calliclones, while variants obtained
using protoplast cultures are known as protoclones.
Larkin and Scowcroft (1981) proposed a general
term ‘somaclonal variation’ to describe genetic
variation in plants regenerated from any form of
cell cultures. Accordingly, the plants derived from
cell and tissue cultures are termed as somaclones,
and the plants displaying variation as ‘somaclonal
variants’. However, generally the term somaclonal
variation is used for genetic variability present
among all kinds of cell/plants obtained from cell
cultures in vitro. Plants regenerated from tissue and
cell cultures show heritable variation for both qualitative and quantitative traits. Several useful somaclonal variants have been obtained in large number
of plant species such as potato, sugarcane, banana,
tomato etc. Chaleff (1981) labelled plants regenerated from tissue cultures as R0 generation and their

successive sexual generations as R1, R2, etc.
SV can be problematic during micropropagation and in vitro conservation and in genetic
transformation of crop plants, although it may be
put to good use as a tool in plant breeding. These
changes are heritable. Early detection of SV is,
therefore, very useful. Shoot-tip culture preserves
genetic stability much better than callus or cell
suspension cultures, yet somaclonal variation
appears to be widespread among plants regenerated from banana shoot-tip cultures. Off-type frequencies are reported to vary from 1 to 74 %.
In banana, a globally important fruit crop that
is extensively micropropagated, it is even more
pertinent to study SV as the crop is especially
prone to this phenomenon. To date, somaclonal
variation affecting in vitro propagated banana is
not well understood, suggesting a complex genetic
cause of this phenomenon. A molecular biologybased approach of analysis would help throw light
on causes and detection of variants to cure this
scourge of the banana micropropagation industry.
In vitro conditions can induce mitotic instability.
Labile portions are known to exist in the genome

rendering it susceptible. These portions get modulated when cells undergo ‘stress’ in tissue culture,
resulting in higher rearrangement and mutation
rates than other portions of the genome.
Occurrence of hotspots of mutation and recurring
menus of alternative alleles is consistent with this
response being limited to a sub-fraction of the
genome. In banana, where production of somaclonal variants is substantial, only those plants
that show side shoots as well with the same type
of variation are considered as ‘variants’.

21.2

Gametoclonal Variation

Another term for variations arising due to the tissue culture process is gametoclonal variation for
variations arising in cell cultures of gametic origin, like in pollen and microspore cultures, to distinguish them from somatic cell-derived
regenerants.
When gametic cells are cultured under in vitro
conditions and variations observed in such cultures, these are called gametoclonal variations.
Products obtained from gametoclonal variations
are termed gametoclones. In gametoclonal variation, gametes (being products of meiotic division)
possess only half the number of parent chromosomes. Gametoclones can be developed by culturing male or female gametic cells. Anthers or
isolated microspores are widely used for developing gametoclones. A large number of plants have
been regenerated from gametoclonal variations
like Oryza sativa, Nicotiana tabacum, Brassica
napus and Hordeum vulgare. Improvements have
been made in several plant species through gametoclonal variation, e.g. rice, wheat and tobacco.
There are three major reasons that can cause
genetic variations in gametoclones:
• The technique used in cell culture may induce
genetic variation(s).
• Doubling of haploid chromosomes may generate variation(s).
• Heterozygosity in diploids may induce genetic
variation(s).
• Variations may result from segregation and
independent assortment.
Gametoclones differ from somaclones in three
distinct ways: (1) Gametoclones regenerate into

21 Somaclonal Variation in Micropropagated Plants

haploid plants in comparison to somaclones which
develop into diploid plants. (2) The recombination
process occurs by meiotic crossing over in gametoclonal variation. (3) Gametoclones can be stabilized by doubling their chromosome number.

21.3

Factors Contributing
to Occurrence of Somaclonal
Variation

• Genotype
Obvious chromosome breakages or aberrant
number of chromosomes are found even in
conventional sucker-grown plants. However,
these defects get magnified in plants grown in
tissue cultures.
• Ploidy level
In general, more incidence of SV is observed
with increase in ploidy.
• Number of subculture cycles
Restricting the number of subculture cycles to
five to eight is considered safe. SV is known to
increase to 2.9 and 3.8 % at 9th and 11th subcultures, respectively, i.e. mutation rate is
higher in prolonged culture. Almost all somaclonal variants produce poor quality bunches.
• Overdosing with hormones in vitro
• Starter material: sword suckers vs. water
suckers

The basic cause of these variations may be
attributed to changes in karyotype (chromosome
number and structure), chromosome rearrangements, somatic crossing over, sister chromatid
exchange, DNA amplification and deletion,
transposable elements and DNA methylation.
Somaclonal variation can be characterized based
on morphological, biochemical (isozymes) and
DNA markers such as random amplified polymorphic DNA (RAPDs), restriction fragment
length polymorphism (RFLPs) and inter-simple
sequence repeats (ISSR).
The variations could also arise in tissue culture 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 disappear when the
culture conditions are removed. Changes in DNA
methylation pattern have been implicated.

409

There are different approaches (steps) to create somaclonal variations, which include:
1. Growth of callus or cell suspension cultures
for several cycles
2. Regeneration of a large number of plants from
such long-term cultures
3. 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
4. Testing of selected variants in subsequent
generations for desirable traits
5. Multiplication of stable variants to develop

new breeding lines
To be of commercial use, a somaclonal variant
must fulfil certain basic requirements:
1. It must involve useful characters.
2. It should be superior to the parents in the
character(s) in which improvement is sought.
3. The improved character(s) must be combined with all other desirable characters of
the parent.
4. The variations must be inherited stably
through successive generations by chosen
means of propagation.

21.3.1 Identification of Variants
There are identifiable and predictable DNA
markers for early diagnosis of SV. DNA methylation has been recognized to cause
SV. Representational difference analysis (RDA)
has been employed to isolate unique fragments
(‘difference products’) between visible off-types
and ‘normal’ tissue culture (TC)-derived plants.
Various other molecular techniques are available
to detect sequence variation between closely
related genomes such as those of source plants
and somaclones, viz. RAPD, AFLP (including
MSAP – methylation-sensitive amplification
polymorphism), microsatellites, etc.
Somaclonal variants have also been shown to
have gibberellic acid profiles different from those
of normal TC plants. Overdosing with cytokinins
and culture frequency or number (or both) have
all been shown to cause SV. However, genome is