Biotechnological Interventions in Forage Crops-A Review

Abstract

Fodder crops are the plant species that are cultivated and harvested for feeding the animals in the form of forage, silage and hay. The average cultivated area in India under fodder production is only 4.4 % of the total cultivated area, but in Punjab, the situation is somewhat better with approximately 10 % of the total cultivated area under fodder crops which is not sufficient. There is urgent need to improve productivity of fodder and pasture lands by developing new fodder varieties and increased use of wasteland for fodder production. To improve the genetic potential of fodder crops required variability is not many a time available in the same species but has to be looked for in other species or genera. The genetic engineering/transgenics technology may be the remedy to above listed problems. Genetic engineering has greatly contributed to breakthroughs in plant improvement and led to the development of widely grown cultivars in major cash crops. The modern plant improvement technologies developed for genetic manipulation of various forage, turf and bioenergy species have opened up new opportunities for breeding these crops, which may help make them more valuable to cropping systems and hence more likely to become a component of them, bringing along their multifunctional benefits. By efficient incorporation of novel germplasm into applied breeding programmes, transgenic cultivars have the potential to play a critical role in fulfilling the increasing demand for animal products and renewable fuels in the 21st century.

Full text

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Review Article https://doi.org/10.20546/ijcmas.2018.707.148
Biotechnological Interventions in Forage Crops-A Review
Rahul Kapoor1*, Tarvinder Pal Singh2 and Gaurav Khosla1,2
1Department of Plant Breeding and Genetics, Punjab Agricultural University
Ludhiana-141004, India
2Office of Director (Seeds) Punjab Agricultural University Ludhiana-141004, India
*Corresponding author
A B S T R A C T
Introduction
Fodder crops are the plant species that are
cultivated and harvested for feeding the
animals in the form of forage (cut green and
fed fresh), silage (preserved under anaerobic
condition) and hay (dehydrated green fodder).
In India, the total area under cultivated fodders
is 8.3 million ha on individual crop basis.
Sorghum amongst the Kharif crops (2.6
million ha) and berseem (Egyptian clover)
amongst the Rabi crops (1.9 million ha)
occupy about 54% of the total cultivated
fodder cropped area. The area under
permanent pastures has been declining over
the years and the trend could well continue in
the future. Green fodders have an important
role in ushering the white revolution in the
state. Farmers are cultivating fodder crops in
traditional crop rotations which lead to
irregular supply of fodder round the year
hence the success of dairy industry lies greatly
on the availability of green fodder throughout
the year, as green fodders are the cheapest
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 7 Number 07 (2018)
Journal homepage: http://www.ijcmas.com
Fodder crops are the plant species that are cultivated and harvested for feeding the animals
in the form of forage, silage and hay. The average cultivated area in India under fodder
production is only 4.4 % of the total cultivated area, but in Punjab, the situation is
somewhat better with approximately 10 % of the total cultivated area under fodder crops
which is not sufficient. There is urgent need to improve productivity of fodder and pasture
lands by developing new fodder varieties and increased use of wasteland for fodder
production. To improve the genetic potential of fodder crops required variability is not
many a time available in the same species but has to be looked for in other species or
genera. The genetic engineering/transgenics technology may be the remedy to above listed
problems. Genetic engineering has greatly contributed to breakthroughs in plant
improvement and led to the development of widely grown cultivars in major cash crops.
The modern plant improvement technologies developed for genetic manipulation of
various forage, turf and bioenergy species have opened up new opportunities for breeding
these crops, which may help make them more valuable to cropping systems and hence
more likely to become a component of them, bringing along their multifunctional benefits.
By efficient incorporation of novel germplasm into applied breeding programmes,
transgenic cultivars have the potential to play a critical role in fulfilling the increasing
demand for animal products and renewable fuels in the 21st century.
Ke ywor ds
Biotechnological
interventions,
Forage crops,
Fodder crops
Accepted:
08 June 2018
Available Online:
10 July 2018
Article Info

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1230
source of carbohydrates, proteins, vitamins
and minerals for dairy animals. Hence by
providing sufficient quantities of green fodder
instead of costly concentrates and feed to the
milch animals the cost of milk production can
be substantially reduced.
The average cultivated area in India under
fodder production is only 4.4 % of the total
cultivated area, but in Punjab, the situation is
somewhat better with approximately 10 % of
the total cultivated area under fodder crops
which is not sufficient. This gap between
demand and supply is likely to widen further
due to increased pressure to grow more food
grains, oilseeds, pulses and commercial crops
resulting into a possibility of no further
increase in area under forage crops.
Forage production is an important not only for
augmenting feed availability but for
maintaining the natural resource base through
soil stabilization, preventing soil erosion and
contributing to soil fertility enrichment. But
still, the present fodder supply of 27
kg/animal/day is far from satisfactory as for
proper feeding about 40 kg green fodder per
animal should be supplied daily. This gap
between demand and supply is likely to widen
further due to increased pressure to grow more
food grains, oilseeds, pulses and commercial
crops resulting into a possibility of no increase
in area under forage crops. Therefore there is
urgent need to improve productivity of fodder
and pasture lands by developing new fodder
varieties and increased use of wasteland for
fodder production.
To improve the genetic potential of fodder
crops required variability is not many a time
available in the same species but has to be
looked for in other species or genera. Thus
genetic engineering/transgenics technology
may be the remedy to above listed problems
because it allows the introduction of foreign
genes from unrelated species and the down
regulation or up-regulation of endogenous
genes, moreover it offers the opportunity to
introduce novel genetic variation into plant
breeding programmes which otherwise is not
possible with intra-specific hybridization and
for extending the range of genetic variability
beyond that of a single species. The progress
and status of the work on genetic
engineering/transgenic technology is
presented in the following text.
Genetic engineering successes in forages
Since the production of the first transgenic
forage-type tall fescue plants (Wang et al.,
1992), tremendous progress has been made in
genetic engineering of forage, turf and
bioenergy crops in the last two decades. Some
of the achievements in genetic engineering of
grasses and legumes have been reviewed by
Wang and Ge (2006) and Ko¨ lliker et al.,
(2010). In brief, transgenic approaches have
been employed to improve these species in the
following aspects: significant improvement of
in vitro dry matter digestibility in alfalfa, tall
fescue and perennial ryegrass (Guo et al.,
2001; Chen et al., 2003, 2004; Reddy et al.,
2005; Tu et al., 2010); enhanced drought
tolerance in alfalfa, white clover, creeping
bentgrass and bahiagrass (Paspalum notatum
Flugge) (J.-Y. Zhang et al., 2005, 2007; Fu et
al., 2007; Jiang et al., 2009, 2010; Xiong et
al., 2010); increased phosphorus acquisition in
white clover and alfalfa (Ma et al., 2009,
2012); enhanced salt tolerance, cold tolerance
or freezing tolerance in perennial ryegrass, tall
fescue and creeping bentgrass (Hisano et al.,
2004; Hu et al., 2005; Wu et al., 2005; Li et
al., 2010); delay or inhibition of floral
development in red fescue (Festuca rubra)
(Jensen et al., 2004); development of hypo-
allergenic perennial and Italian ryegrasses
(Petrovska et al., 2004); enhanced aluminium
tolerance in alfalfa (Tesfaye et al., 2001;
Barone et al., 2008); delay of leaf senescence
in alfalfa (Calderini et al., 2007; C. Zhou et

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al., 2011); virus-resistant perennial ryegrass
and white clover (Xu et al., 2001; Ludlow et
al., 2009); increased disease resistance in tall
fescue and creeping bentgrass (Fu et al., 2005;
Dong et al., 2007, 2008; M. Zhou et al.,
2011); improved turf quality in bahiagrass
(Agharkar et al., 2007; H. Zhang et al., 2007);
accumulation of sulphur-rich protein in
subterranean clover (Trifolium subterraneum
L.) and tall fescue (Rafiqul et al., 1996; Wang
et al., 2001); production of
polyhydroxybutyrate in switchgrass (Somleva
et al., 2008); increased sugar release in alfalfa
and switchgrass (Chen and Dixon, 2007;
Jackson et al., 2008; Fu et al., 2011a, b;
Saathoff et al., 2011); increased biomass yield
in switchgrass (Fu et al., 2012); and a large
improvement in bio ethanol production in
switchgrass (Fu et al., 2011a). Genetic
engineering has greatly contributed to
breakthroughs in plant improvement and led to
the development of widely grown cultivars in
major cash crops (Park et al., 2011). The
adoption of transgenic crops in the last 15
years has experienced an 87-fold increase
since biotech crops were first commercialized
in 1996, making biotech crops the fastest
adopted crop technology in history. The
accumulated growth areas from 1996 to 2010
exceeded 1 billion hectares (James, 2011). The
number of countries planting biotech crops
reached 29 in 2010 and the top ten countries
each grew more than 1 million hectares. The
United States remains the biggest adopter of
transgenic crops, with 66.8 million hectares
planted in 2010, which represent 45% of the
global biotech area (James, 2011). Despite the
wide adoption and the beneficial economic
and environmental impacts of transgenic
crops, it has been extremely difficult to
deregulate and commercialize new transgenic
cultivars. The situation is even more
complicated in transgenic forage, turf and
bioenergy species. One enduring lesson from
agricultural biotech is that it is a huge mistake
to underestimate bio safety concerns (Stewart,
2007). In this paper, we focus our discussions
on the deregulation process of transgenics in
the US only. Specific successful and
unsuccessful examples will be given to
illustrate the process and the complications
involved in deregulation of forage and turf.
Gene flow studies in forages
Pollen is an important vector of gene flow in
out crossing species. A simple pollen
germination medium was used to assess in
vitro viability and longevity of tall fescue and
switchgrass pollen (Wang et al., 2004a; Ge et
al., 2011). Weather conditions have a large
impact on pollen longevity. Under sunny
atmospheric conditions, viability of transgenic
and nontransgenic tall fescue pollen declined
to 5% in 30 min, with a complete loss of
viability in 90 min. Under cloudy atmospheric
conditions, viability of tall fescue pollen
declined to 5% after 150 min, with a complete
loss of viability in 240 min (Wang et al.,
2004a). Similarly, switchgrass pollen
longevity decreased rapidly under sunny
atmospheric conditions, with a half-life of less
than 4.9 min and a complete loss of viability
in 20 min. Under cloudy atmospheric
conditions, the half-life of switchgrass pollen
was more than five-fold longer than under
sunny conditions, and it took approx. 150 min
to lose viability completely (Ge et al., 2011).
In both tall fescue and switchgrass, no
difference in pollen viability and longevity
was found between transgenic and non-
transgenic control plants. As wind-pollinated
grasses have a high potential to pass their
genes to adjacent plants, pollen flow is not
only a concern in transgenics; it has long been
a consideration for seed purity of conventional
cultivars.
Apomixis is known to exist in many warm-
season grasses, such as Poa and Paspalum
species. Apomictic reproduction mode is
characterized by embryo development, which

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is independent of fertilization of the egg cell,
but requires fertilization with compatible
pollen to produce the endosperm (Sandhu et
al., 2010). Transgenic Kentucky bluegrass was
used as a pollen donor to quantify intra- and
interspecific pollen mediated gene flow.
Twenty-five sexual and facultative apomictic
Poa species were used as pollen receptor and
placed at 0, 13 and 53 m distances from the
transgenic materials. Overall hybrid frequency
was 0.048% and hybrid frequency at the 0-m
distance was 0.53% (Johnson et al., 2006).
To quantify gene flow from apomictic
tetraploid bahiagrass (Paspalum notatum
Flugge) to tetraploid or diploid bahiagrass, the
glufosinate-resistant apomictic bahiagrass was
grown at close proximity (0.5–3.5 m) with
non-transgenic cultivars. Average gene
transfer between transgenic apomictic,
tetraploid and sexual diploid bahiagrass was
0.03%. Average gene transfer between
transgenic apomictic tetraploid and non-
transgenic, apomictic tetraploid bahiagrass
was 0.17% (Sandhu et al., 2010). While not
providing complete transgene containment,
gene transfer between apomictic species
occurs at low frequency and over short
distances (Johnson et al., 2006; Sandhu et al.,
2010). In a landscape-level of study for
‘Roundup Ready’ creeping bentgrass, it was
found that most of the gene flow occurred
within 2 km in the direction of prevailing
winds. The maximal gene flow distances
observed were 21 and 14 km in sentinel and
resident plants, respectively, that were located
in Alfalfa and white clover are predominantly
pollinated by insects. A large-scale field study
of ‘Roundup Ready’ alfalfa showed that
pollen-mediated gene flow diminished with
increasing distance from the source.
Gene flow is a natural event that happens all
the time, but the introduction of modern
biotechnology has brought new attention to
this natural process and raised ecological,
economic as well as intellectual property
issues for scientists and policymakers to
consider. A main focus in risk assessment
research should be placed on the consequences
of transgene flow. The phenotypes of
transgenic plants and their safety in the
environment, not the method used to produce
them, should be the main focus of risk
analyses and regulatory concern (Bradford et
al., 2005).
Deregulation of ‘roundup ready’ alfalfa
Although significant progress has been made
in the genetic engineering of forage, turf and
bioenergy species, to date, the only
deregulated crop is ‘Roundup Ready’ alfalfa.
The deregulation process is lengthy and
complicated. The trait was obtained by
transgenic expression of the 5-
enolpyruvylshikimate-3-phosphate synthase
(EPSPS) gene. The gene was derived from the
CP4 strain of Agrobacterium tumefaciens. The
transgenic alfalfa plants were obtained by
Agrobacterium-mediated transformation. The
non-selective herbicide glyphosate inhibits an
essential step in aromatic amine synthesis in
plants by blocking the action of the natural
EPSPS enzymes already present in the plant.
However, the CP4 EPSPS protein is not
inhibited by glyphosate; thus any plant
expressing sufficient levels of this protein is
tolerant to glyphosate application. The
‘Roundup Ready’ alfalfa is also called
glyphosate-tolerant (GT) alfalfa.
Herbicide-tolerant kentucky bluegrass
The Scotts Company produced new
glyphosate-tolerant transgenic Kentucky
bluegrass without using plant pest
components. Specifically, the transgenic
plants were produced by biolistic
transformation, without involving
Agrobacterium transformation or any other
plant pest regulated under the Plant Protection

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Act. The herbicide resistance gene EPSPS is
from Arabidopsis thaliana, the ubiquitin
promoter is from rice, the actin intron is from
rice and the alcohol dehydrogenase 3′
untranslated region is from maize. The Animal
and Plant Health Inspection Service (APHIS)
defines a ‘regulated article’ as: any organism
which has been altered or produced through
genetic engineering, if the donor organism,
recipient organism, or vector or vector agent
belongs to any genera or taxa designated in §
340.2 and meets the definition of plant pest, or
is an unclassified organism and/or an
organism whose classification is unknown, or
any product which contains such an organism,
or any other organism or product altered or
produced through genetic engineering which
the administrator determines is a plant pest or
has reason to believe is a plant pest.
Table.1
Crop
Trait Targeted
Reference
alfalfa, tall fescue and
perennial ryegrass
In-vitro dry matter
digestibility
Guo et al., 2001; Chen et al., 2003,
2004; Reddy et al., 2005; Tu et al.,
2010
alfalfa, white clover,
creeping bentgrass and
bahiagrass
Enhanced drought tolerance
J.-Y. Zhang et al., 2005, 2007; Fu et
al., 2007; Jiang et al., 2009, 2010;
Xiong et al., 2010
white clover and alfalfa
Increased phosphorus
acquisition
Ma et al., 2009, 2012
perennial ryegrass, tall
fescue and creeping
bentgrass
Enhanced salt tolerance, cold
tolerance or freezing
tolerance
Hisano et al., 2004; Hu et al., 2005;
Wu et al., 2005; Li et al., 2010
Tall fescue
Delay or inhibition of floral
development
Jensen et al., 2004
alfalfa
Enhanced aluminium
tolerance
Tesfaye et al., 2001; Barone et al.,
2008
alfalfa
Delay of leaf senescence
Calderini et al., 2007; C. Zhou et
al., 2011
perennial ryegrass and
white clover
Virus-resistance
Xu et al., 2001; Ludlow et al., 2009
tall fescue and creeping
bentgrass
Increased disease resistance
Fu et al., 2005; Dong et al., 2007,
2008; M. Zhou et al., 2011
bahiagrass
Improved turf quality
Agharkar et al., 2007; H. Zhang et
al., 2007
subterranean clover and tall
fescue
Accumulation of sulphur-rich
protein
Rafiqul et al., 1996; Wang et al.,
2001
alfalfa and switchgrass
increased sugar release
Chen and Dixon, 2007; Jackson et
al., 2008; Fu et al., 2011a, b;
Saathoff et al., 2011
switchgrass
improvement in bio ethanol
production
Fu et al., 2011

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Intragenesis and cisgenesis
One of the public concerns about transgenic
crops relates to the mingling of genetic
materials among distantly related organisms.
New molecular strategies have been designed
to address the issue. Intragenesis (Rommens
et al., 2004, 2007) or cisgenesis (Schouten et
al., 2006a, b) refers to the introduction of one
or more genes that are derived from the target
species itself or species that are sexually
compatible with the target species. Cisgenesis
is more restrictive in that it refers to the
transfer of a complete DNA copy of a natural
gene, including its promoter and terminator
(Schouten and Jacobsen, 2008). It is obvious
that intragenic or cisgenic plants are closer to
their natural counterparts than the above-
mentioned Kentucky blue grass.
Conventional breeding employs methods such
as introgression and mutagenesis to modify a
plant genome randomly and, as a result, create
genetic variation (Rommens et al., 2007). In
the case of intragenic or cisgenic plants, the
gene of interest, together with its regulatory
sequences, has been present in the species or
in a sexually compatible relative for centuries
(Schouten et al., 2006a). Therefore, the gene
pool exploited by intragenesis and cisgenesis
is identical to the gene pool available for
traditional breeding (Holme et al., 2012).
Furthermore, no changes in fitness occur that
would not happen through either conventional
breeding or natural gene flow. Intragenic or
cisgenic plants carry no additional risks –
such as effects on non-target organisms or soil
ecosystems, toxicity or a possible allergy risk
for GM food or feed –other than those that are
also incurred by conventional breeding
(Schouten et al., 2006a). By avoiding the
transfer of foreign or unknown DNA, crops
developed through intragenesis or cisgenesis
mimic the conventionally bred cultivars. In
fact, they have much less gene shuffling than
the conventional cultivars. By eliminating
various potential risk factors, the intragenic or
cisgenic method represents a relatively safe
approach to crop improvement (Rommens et
al., 2007). Therefore, it has been argued that
intragenic or cisgenic plants should be treated
as conventionally bred plants (Schouten et al.,
2006a; Rommens et al., 2007). Considering
gene flow and other biosafety issues in
forage, turf and bioenergy crops, the
intragenic or cisgenic approach may provide a
cost-effective way for genetic engineering of
these species.
Improving lysine and methionine levels in
forage crops
In forage crops the main consumed part is the
vegetative tissue, and therefore efforts to
increase the essential amino acid content in
vegetative tissues were mainly conducted by
constitutive expression of recombinant
constructs expressing seed vacuolar storage
proteins, fused to the 35S promoter. These
storage proteins which stably accumulate in
seeds vacuoles, failed to accumulate in the
protease-rich vegetative vacuoles, due to their
efficient degradation (Saalbach et al., 1994).
Preventing the trafficking of the seed storage
proteins from the endoplasmic reticulum (ER)
to the vegetative vacuole by engineering of an
ER retention signal (KDEL) into the C-
terminus of these proteins only partially
solved their stability problems (Khan et al.,
1996; Tabe et al., 1995; Wandelt et al., 1992).
More successful results were obtained by
using two methionine-rich seed storage
proteins of maize, namely, the 15-kDa β-zein
and the 10-kDa δ-zein, which naturally
accumulate in ER-derived protein bodies
(Shotwell and Larkins, 1989), Maize β-zein
and δ-zein genes, constitutively expressed
alone in transgenic tobacco plants,
accumulated in novel ER-derived protein
bodies and were moderately stable (Bagga et
al., 1995). Co-expression of the two proteins

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1235
together significantly increased their stability
(Bagga et al., 1997). Stability problems
associated with the expression of seed storage
proteins in vegetative tissues suggest that
expression of genes for other types of
nutritionally balanced proteins should also be
tried. Inasmuch as a number of plants also
naturally accumulate vegetative storage
proteins (VSPs) to high levels inside
vegetative vacuoles (Staswick, 1994), such
proteins may be better targets for nutritional
improvement of forage crops than seed
storage proteins. VSPs may also have
additional beneficial effects, such as
enhancement of shoot regrowth after cutting
of forage crops. Guenoune and co-workers,
1999 overexpressed the soybean VSPα gene,
fused to the Cauliflower mosaic virus
(CaMV) 35S promoter, in transgenic tobacco
plants. This protein was highly stable in
vacuoles of both vegetative and seed tissues.
The level of the soybean VSPα ranged
between 2 and 6% of the soluble proteins in
leaves of the transgenic plants, causing a
significant increase of total soluble lysine by
about 15%. This suggests that VSPs can serve
as excellent protein sources for improving the
nutritional quality of forage crops.
Plant improvement is needed to enhance our
ability to produce food, feed, fibre and fuel
and to ensure we have a safe, liveable
environment. Ideally, our plant improvement
efforts would be done in a way that is in
harmony with the environment (Brummer et
al., 2011). In addition to their main product or
function, forages, turf and bioenergy species
have positive effects on farming systems and
on the environment. For example, including
forage crops, such as alfalfa, into a crop
rotation with corn and soybean had both
environmental and economic benefits
(Olmstead and Brummer, 2008). The modern
plant improvement technologies developed
for genetic manipulation of various forage,
turf and bioenergy species have opened up
new opportunities for breeding these crops,
which may help make them more valuable to
cropping systems and hence more likely to
become a component of them, bringing along
their multifunctional benefits.
Transgenesis, including nuclear
transformation as well as intragenesis,
cisgenesis and chloroplast transformation,
provides a rapid means for plant
improvement, and should be among the
technologies being used as we attempt to
develop improved crops to be included into
sustainable cropping or landscape systems
(Ronald, 2011). The major challenge now is
how to apply the technology to generate new
genetic variability in a way that satisfies
regulatory requirements. The development of
an EIS for alfalfa and the deregulation of
herbicide-tolerant alfalfa paved the way for
future transgenic improvement of this
important forage legume crop. For grasses,
the development of intragenic or cisgenic
lines is likely to be the first practical step
toward deregulation.
Despite various concerns, major transgenic
crops have been widely cultivated and
intensively consumed in the last 16 years with
no documented cases of adverse effects on
health or the environment. A streamlined
regulatory system, designed to catch obvious
hazards but not prevent entry into the
marketplace by small companies and non-
profit organizations, needs to be developed.
By efficient incorporation of novel
germplasm into applied breeding
programmes, transgenic cultivars have the
potential to play a critical role in fulfilling the
increasing demand for animal products and
renewable fuels in the 21st century, and in
conjunction with ecologically driven farming
practices, leading to an economically and
environmentally sustainable agricultural
system.

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How to cite this article:
Rahul Kapoor, Tarvinder Pal Singh and Gaurav Khosla. 2018. Biotechnological Interventions
in Forage Crops-A Review. Int.J.Curr.Microbiol.App.Sci. 7(07): 1229-1240.
doi: https://doi.org/10.20546/ijcmas.2018.707.148