Annals of Horticulture 5(1) : 1-6 (2012) Hi-tech Horticultural Society

ROLE OF BIOTECHNOLOGICAL TOOLS FOR GENETIC IMPROVEM

-ENT IN HORTICULTURAL CROPS- A REVIEW

B. Singh1 , R.P.S Dhaka2, Navneet Kumar 3, Anshu Dhaka3 and Pushpendra kumar4
1
Horticulture, Sardar Vallabh Bhai Patel, University of Agri. & Tech., Meerut.
2
Department of Biotechnology, RCP College Roorkee (Uttrakhand).
3
DN College Meerut (U.P).
4
Ag. Biotechnology, Sardar Vallabhbhai Patel University of Agri. Tech. Meerut.

ABSTRACT
There is need to integrate the biotechnology with classical breeding as an aid to
molecular marker assisted selection of potential parents, germplasm
conservation and micropropagation, reduction of the duration of breeding
programme and early production of homozygous lines by making use of tissue
culture technique which also creates the new genetic variation for achieving
useful varieties. It widens the area of hybridization through embryo rescue,
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culture and somatic hybridization. The protoplast fusion not only expands the
possibility for increasing genetic divergence but also assists in inducing male
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sterility and self incompatibility for controlling pollination and production of

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hybrids. Transgenic plants have been developed by recombinant DNA

techniques for resistance to diseases, insect pests, drought, salinity, herbicide,
nutritional quality and other desirable traits i.e altered flavor and colour of
produce and regulation of flowering, ripening and plant architecture etc. This
review describes overview of the opportunities extended by the integration of
plant biotechnology into improvement efforts mostly for horticultural crops.

Keywords: Somatic hybridization, Embryo rescue and culture, Cryopreservation, Somaclonal

variation.

The release of new improved genotypes
by classical breeding is too slow to cope with the
demands of agriculture and horticultural produce
and is considerably limited by lack of
appropriate "natural" genes that can be
integrated by traditional genetic crosses and the
problems arising in distant hybridization. Hence
the food production for both quality and quantity
in developed and developing countries around
the globe now depends on effective merging of
classical breeding with modern biotechnology
and novel tools it provides indicating the need
for integration i.e, combining biotechnology
with classical physiology and breeding.
The plant biotechnology comprised only a few
applications of tissue culture, recombinant DNA
technology and monoclonal antibodies Today
transformation and marker aided selection and
breeding are just a few of the examples of the
applications of biotechnology in crop
improvement. Conventional plant breeding
consists of attermpting crosses between desirable
plants followed by selection of promising
recombinants in subsequent segregating
generations for producing lines breeding true to
type which requires repeated cycles of selection
taking 6 to 8 years. One of the way out to
minimise this time is to produce plants from the
 2 Role of Biotechnological Tools for Genetic Improvement in Horticultural Crops- A Review
pollens of F1 hybrids and doubling the number
of chromosomes of the haploids so produced in
order to develop a fertile homozygous diploid
plant. Thus the use of biotechnology is
beneficial to agriculture and horticultural crop
improvement programmes in reducing the
duration of breeding programme by making use
of tissue culture which also creates new genetic
variation (somaclonal variation), resulting the
production of useful new varieties and the
production of transgenic plants engineered for
resistance to diseases, pests and herbicides by
using the recombinant –DNA techniques.
Keeping in view the imortance and
impact of biotechnology in crop improvement,
initiatives were taken for starting National
Horticulture Mission, annouced by Hon'ble
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Prime Minister on 15th August, 2003. Crop
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diversification has become an imperative for
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India’s agroeconomy. To address this need and
to give further impetus to encouraging growth in
cultivation of fruits, vegetables, flowers species
etc. a National Horticultural Mission has been
launched for doubling India's horticultural
production by 2010. Subsequently national
horticultural mission was launched during tenth
plan 000 2005-06 for holistic development of
horticultural crops having competitive
advantages.
The practical application and impacts of
biotechniques and their future prospects for
genetic improvement of horticultural crops are
being explained under te following aspects:
Somaclonal variation:
In vitro regenerated plants can show some
modifications called somaclonal variation as a
result of mutagenic effect of the culture or the
chimeric nature of the cultured tissue. Useful
traits have been obtained in variants derived
from tissue somaclonal variation and developed
cultivars DNAP-9 and DNAP- 17 of
Lycopersicum esculentum for high solids in
fruits and fusarium race-2 resistance respectively
and chlorosulfuron and imidezilinone resistance
Brassica napus develped through direct
selection. Genetic variability in tissue culture
derived matrial called somaclonal variation (14)
is specially prevelant if the material is kept in a
rapidly dividing, non- differented state (callus
or cell suspension) for extended period. Infact
the frequency of somaclonal variation can be
10,000 times higher than spontaneous mutation
rate in whole plants (14).
Genotypic selection and identification:
The difficulties arise in selecting plants
that express recessive alleles, genes that have
only minor effects on phenotypes or genes
whose expression is strongly modified by the
growth environment creating hinderance in plant
improvement. The utilization of marker assisted
selection may allow plant breeders to overcome
some of these problems. Differences
(Polymorphisms) in isozyme, DNA restriction
fragment and polymerase chain fragment
patterns are useful as molecular markers and
have been shown to be genetically linked to both
simple and complex traits such as disease
resistance and fruit composition in crop plants (1
& 13).
The addition of metabolites and selective
agents in the media makes it possible to identify
resistant cells which will develop resistant plant.
The in vitro selection is potentially more
effective and cheaper than whole plant selection
as a single flask of cells can have the same
number of selection units as 30,000 plants in a
three hectare nursery. This method has been
particularly effective for selecting herbicide and
disease resistant plant in tomato,(2).
Somatic hybridization:
The development of somatic hybrids
through protoplast fusion is a promising
approach leading to production of a polyploid
variety, overcoming sexual incompatibility
existing between two species or even two genera
 Singh et al.
and transferring cytoplasmic male sterility for
exploiting hybrid vigour. Protoplast are released
from the plant tissue after incubation in cell wall
degrading enzymes (2). Several procedures
including incubation in PEG (9) or treatment
with electrical pulses (24) can induce protoplasts
form different plants to fuse. Somatic hybrid
plants can be generated from cultures of these
fusion products. Protoplast fusion has been used
to achieve very wide combination between
distantly related species that are not possible by
conventional crossing procedures like tomato
and potato (15) and to bring about limited gene
transfer for example disease resistance from a
wild relative of potato (Solanum brevidens) to
potato (S. tuberosum) (6).
Cytoplasmic male sterility and atrezine
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resistance characters were developed in Brassica
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napus through somatic hybridization whereas
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virus resistance and tobacco mosaic virus
resistance was developed in Solanum tuberosum
plan Lycopersicum esculentum respectively
through gene transfer. The vegetable crops for
which transformation and regeneration systems
available are carrot, tomato, pepper and egg
plant.
Embryo rescue and culture:
In vitro culture of zygotic embryos
(embryo rescue) has enabled us to overcome
barriers to a number of interspecific crosses
from zygotic failure. Several hybrids have been
retried which would otherwise have been
aborted, employing this technology such as
hybrid of Musa acuminate × M. balbiciana,
Carica papaya × C. cauliflora, interspecific
crosses in pine apple and seedless × seedless
grape varieties. In India protocols for production
of syn-seeds, have successfully been developed
in mulberry, banana & mango.
New plants can be obtained through
somatic embryogenesis form pedicels, stems,
leaves, roots, and other explants. This technique
3
makes possible the production of "artificial
seed". There are some examples of somatic
embryogenesis in some vegetables eg. Daucus
carota, Solanum melongena, Brassica oleracea
plain. botrytis, Cucurbita pepo and cucumis
sativus through in vitro culture of roots,
hypocotyl, leaf, cotyledons and hypocotyl and
anthers (Somatic tissue) respectively.
Germplasm conservation:
There are two approaches which are
followed for genetic conservation i.e in situ &
ex-situ. The in situ involves conservation in
natural habitats either in reserves, sanctuaries or
protected areas or within the traditional farming
system, while in vitro conservation covers
artificial culture and storage of different plant
germplasm which requires less space and
germplasm can be effectively stored for longer
duration. Tissue culture is a means of preserving
species that are rare and threatened and
providing an alternative source of plants for
commercial horticultural and traditional trade
since it is aseptic culture of plant protoplast
cells, tissue or organs and reduces the labour
associated with line maintenance and germplasm
conservation.
In cryopreservation the live plant
material is stored at ultra low temperature in
liquid nitrogen (-1960C) at which all metabolic
activities are ceased hence material can be stored
for indefinite period. Cryopreservation of live
tissues involves dehydration followed by
immersion of liquid nitrogen. The material is
also given pretreatments with cryoprotectants
like DMS, PEG and glycerols to avoid chilling
injury before immerging the plant tissue in liquid
nitrogen. Techniques such as minimal growth in
tissue culture and cryopreservation have been
used to store plant materials from a wide variety
of species (10 & 23). These techniques are
particularly important for plant species that are
vegetatively propagated.
 4 Role of Biotechnological Tools for Genetic Improvement in Horticultural Crops- A Review
Micropropagation:
Micropropagation is the most effective
for producing large number of uniform, disease
free and healthy food crops. It is fast, requires
little space for multiplication and used routinely
to generate large number of high quality clonal
agricultural plants including ornamentals and
vegetable species and in some cases also
plantation crops, fruits and vegetable species.
Plant cloning is a method that has been used
with success by horticulturist and ornamentalists
for many years to rapidly propagate desired
genotypes. The multiplication of fruit varieties
by bud grafting is an example of vegetative
propagation. Vegetative propagation can be
greatly accelerated by utilizing tissue culture
procedures because small piece of tissue can be
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used to produce hundreds of plants. This
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procedure has been called micropropagation and
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has been applied to a wide variety of plant
species (12). In addition, the material that is
produced in vitro is disease free which facilitates
international plant exchange.
Geneticstabilization/development of homozy
-gous diploid:
The most potential use of biotechnology.
in crop breeding could be early achievement of
homozygous diploid plants through in vitro
androgenesis which involves use of anthers or
microspores. This technique has perfected in
custardfd, apple, papaya, citrus, pomegranate,
grapes etc. Homozygosity is required for many
crop varieties to ensure their uniformity in the
field as well as their stability from year to year.
Haploid plants are obtained by culturing gamete
cells and they are subsequently doubled by a
treatment with colchicine to produce
homozygous lines (8&11). Lines produced from
anther culture of hybrids are obtained in less
time and show greater variability than those
obtained by self pollination. Mango cultivar has
been produced for agronomic character using
maternal haploid.
Genetic engineering:
The crop improvement through breeding
is time taking and also not very precise. The
genetic engineering creates plants with specific
changes in the background of a proven cultivar
without disturbing their genetic constitution.
Expression of undesirable genes can be blocked
by the application of antisense technology. The
major constraints limiting the production of fruit
crops are diseases caused by several fungi,
bacteria and viruses. Conventional breeding fails
to solve the problem due to non- availability of
resistant genes in gene pool of a particular crop.
In fruit crops, the coal protein mediated
approach to engineer virus resistance has been in
application to introduce resistance against
disease like PuLM. Pox virus (PPV), Papaya
Ring Spot Virus (PRSV), Citrus Tristeza virus
(CTV), Grape Fan Leaf virus (GFLV), etc. For
importing bacterial and fungal resistance genes –
Chitinase, Glucanase, Attacin, Osmotin,
Cercopin etc are being attempted at world over.
The large number of insect species attack
plant and cause damage to yield. The
bioinsecticidal -endotoxin gene (Bt gene)
isolated from Bacillus thuringiensis is currently
in use to make the plants resistant to insect pests.
The most commonly used gene employing are
cow pea trypsin inhibitor (CpTi) and insecticidal
crystal protein isolated form B.thuringiensis.
However, most of attempts are restricted to
vegetable crops. Transgenic plants of over 45
species that contain genes from other plants
species, bacteria, viruses and animals are
currently available (18 & 5). The foreign genes
expressed in transgenic plants confer on them a
variety of important agronomic traits including
herbicide resistance, insect, virus and microbial
resistance, altered macromolecular composition,
modified reproductive capacity and delayed
senescence (21,22,7).
 Singh et al.
Proprietary protection:
long time, investment of money and labour.
Hence the Proprietary protection is needed to
establish the ownership by describing the
varietal identification for preventing its
unauthorized multiplication and sale which is
accomplished by application of biotechniques.
Isozyme, restriction enzyme and PCR fragment
polymorphism have been used to describe the
uniqueness of the varieties. The latter two
techniques can be used to obtain fingerprints of a
plant that are based on the sequences in DNA
itself
Future prospects:
There are various uses of
biotechnological tools which can form the
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second phase of fruit crop revolution. These
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advances are production of edible vaccines in
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banana, value addition, altered flavor and color
of produce, delayed ripening, regulation of plant
architecture etc. Alternate bearing or erratic
flowering is a serious problem observed in
cultivation of mango, avocado, litchi, olive and
apple. The development of regular bearing
cultivars through conventional breeding is
cumbersome and time consuming job as the trait
for regularity in bearing is governed by recessive
allels.
The major targets in flower development
are color, scent and senescence which includes
over and under expression of color i.e.
anthocyanin and carotenoids and scent
compounds. Important objective for controlling
fruit development include growth, ripening and
senescence, color, scent and metabolic control of
sugar, acids and other flavour compounds. The
biotechnological strategies are also helpful for
the production of seedless fruits via
parthenocarpy either by over production of auxin
or pollen destruction to prevent fertilization or
arrest of embryo development. The efforts are
required in future to work out the biotechniques
5
The development of a variety requires
for overcoming these problems encountered in
cultivation of fruit trees and flowering plants.
Molecular biology has been used to
identify some of genes (3,4) that determine self
incompatibility and there is some hope that this
information can be used to develop hybrid seed
production systems for crops that are now sold
as inbreds or homozygous varieties. Protoplast
fusion has been used to transfer organelles
particularly mitochondria from one species to
another. This approach has been used to
introduce cytoplasmic male sterility in crop
plants for controlling pollination and hybrid seed
production (20,19) which may also be tried in
vegetable hybrid seed production.
Artificial genetic male sterility system
has been created by transforming plants with a
RNAase gene from bacillus amyloliquefaciens
under the control of a promoter that is specific
for cells i.e tapetal cells that surround the pollen
sacs (16). In these transgenic plants the tapetum
is destroyed which do not produce pollen and
behaves as female parent and helpful in hybrid
seed production system. The male parents are
transgenic for a RNAase inhibitor gene which is
also under the control of the tapetum promoter
(17). This approach could be applicable in
inducing genetic male sterility system for
producing hybrid seeds in other vegetable crops.
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Received : January, 2011, Revised : 2011 July,
Accepted : March, 2012.