Download Transgenic Glyphosate Resistance Crops

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An overview: How to develop transgenic sugarcane for insects and glyphosate resistance?
Abstract
Sugarcane is an important sugar crop grown throughout the world. Sugarcane quality and
production is mostly affected by biotic and abiotic factors that caused low yield of sugarcane
products throughout the world. Using biotechnology and genetic engineering as a complement
for traditional breeding methods it is possible to introduce insect/pest, herbicide-tolerant traits
into various crop species. It has been found that the absence of insect/pest, abiotic and herbicide
tolerant genes in genetic pool of crop plant species makes traditional breeding programs
difficult. The varieties that are productive and at the same time has resistance against certain
pathogens and diseases, can improve yield. The development of biotic and abiotic resistant
sugarcane varieties through transgenic technology will be cost effective in controlling all type
of stresses which ultimately improve yield potential. The present review will provide its
readers the opportunity to understand the methods of Bt and glyphosate resistant genes in
sugarcane.
Keywords: Saccharum officinarum, gene transformation
Introduction
Sugarcane is tall perennial true grasses belongs to the grass family; Poaceae, of the
genus Saccharum. Sugarcane is an economically important crop and used for sugar production.
They have stout jointed fibrous stalks that are rich in sugar, and measure 6 to 19 feet tall. All
sugarcane species interbreed and the major commercial cultivars are complex hybrids. Agriculture
is central to economic growth and development in Pakistan. Sugarcane occupies an important
position in national economy in order to drive the large sugar industry. Agriculture contributes to
21.4% of GDP and employs 45% of the country’s labor force. During 2012-13, sugarcane was
cultivated on an area of 1124 thousand hectares. It adds 3.2% in agriculture and 0.7% in GDP. The
production of sugarcane during year 2012-13 was 62.5 million tonnes with 5.9% increase in yield
as compared to last year i.e. 58.4 million tonnes (Economic Survey of Pakistan, 2012-13).
Sugarcane is indigenous to tropical South and Southeast Asia. The genus Saccharum contains six
species; two wild species S. spontaneum and S. robstum; and 4 cultivated species S. edule, S.
officinarum, S. barberi and S. sinense (D’Hont et al., 1998; Peter Sharpe, 1998). Different species
originated in different locations e.g. S. barberi originated in India; S. edule and S.
officinarum in New Guinea. All commercial canes grown today are inter-specific hybrids
(Wrigley, 1982). Sugarcane originated in India about 300 B.C and subsequently spread through
trade routes to North Africa, the Mediterranean region, Spain and later on to Arabia, China and
Persia (Ullah et al., 2011). It was first domesticated as a crop in New Guinea around 6000 BC.
Approximately 70% of the sugar produced globally comes from S. officinarum. It is assumed that
multiple crossing between S. spontaneum, Erianthus asundinaceus and Miscanthus sinensis
resulted in S. officinarum (Daniels and Roach, 1987).
Sugarcane cultivars have a complex aneuploid, highly heterozygous and polyploid genome with
chromosome number varying from 80-120 (Joyce et al., 2010, D’Hont et al., 1998). Sugarcane
cultivars derive from recent inter-specific hybrids obtained by crossing S. officinarum
(2n=8x=80) and S. spontaneum (2n=5x=40 to 2n=16x=128) (D’Hont et al., 1996; Cuadrado et al.,
2004). The challenge in sugarcane sequencing is complexity of its genome structure that is highly
polyploidy and aneuploid with a complete set of homologous genes predicted to range from 8-10
homologous copies of most loci (Souza et al., 2011; Mudge et al., 2009). Multiple alleles are
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expressed although transgenes expressed from the corresponding promoters can undergo efficient
silencing (Moyle and Birch, 2013).
Sugarcane is largely grown in tropical and sub-tropical regions of the world and provides around
74 million tonnes of sugar annually and contributes to 2/3rd of world sugar production (Ullah et
al., 2011; Weng et al., 2010: Hillocks and Waller, 1997). Sugarcane propagation is through stem
cuttings of immature 8-12 months old canes. These are called "setts", "seed", "seed-cane" or "seedpieces". Cane is grown highest latitude at 34o N in northwest Pakistan and 37o N in southern Spain
(Sharpe, 1998). The best setts are taken from the upper 3rd part of the cane because the buds are
younger and less likely to dry out. It takes 12,500-20,000 setts to plant one hectare (Purseglove,
1979). The setts are lightly covered with soil until they sprout in 10-14 days and then the sides of
the furrow are turned inward (Sharpe, 1998). Sugarcane is a perennial crop which usually produces
crops for about 3-6 years before being replanted. The first crop is called the "plant crop" and takes
9-24 months to mature, depending on location (Purseglove, 1979).
Sugarcane Production in Pakistan
Pakistan is the 6th largest cane producing country in the world in term area and ranks 15th in sugar
production (FAO, 2011; Ullah et al., 2011; Qureshi, 1998). Sugarcane contains a major source of
edible sugars and many by-products are also produced by sugarcane.
Table 1: Area, Production and Yield of Sugarcane in Pakistan
Area
Production
Yield
Year
(000
%
(000
%
%
(Kg/Ha.)
Hectare) Change
Tonnes)
Change
Change
1029
50,045
48634
2008-09
943
-8.4
49,373
-1.3
52357
7.7
2009-10
988
4.8
55,309
12.0
55981
6.9
2010-11
1058
7.1
58,397
5.6
55196
-1.4
2011-12
1124
6.2
62,472
7.0
55580
0.7
2012-13
Source: Pakistan Bureau of Statistics, 2012-13.
Sugarcane Production in World
Cane is the world’s largest, important and major crop which is very sweet in taste. It is used as raw
material as 80% of sugar production is based upon it. It is cultivated in several countries. Brazil is
the biggest sugarcane producing country of world with a total production of 734,000,000 tonnes,
while Mauritius is the 2nd biggest producer after Brazil and India ranked 3rd (FAO, 2011).
Table 2: Ten Sugarcane Producers of World
Rank
Country
Production (000 Tonnes)
Brazil
734,000
1
Mauritius
645,600
2
India
342,382
3
China
115,124
4
Thailand
95,950
5
Pakistan
55,309
6
Mexico
49,735
7
Colombia
34,000
8
Philippines
26,656
9
Australia
25,182
10
Source: FAO, 2011
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Sugarcane Uses
Sugarcane is the main source of sucrose that is deposited in stalk internodes. Saccharum
officinarum L. is cultivated on a large scale as raw material for sugar and industrial products, such
as furfural, dextrans, alcohol, chip board, paper manufacturing, beverages, food processing,
confectionery, chemicals, plastics, paints, synthetics, fiber, insecticides and detergents. It accounts
for 75% of sugar production worldwide (World Sugar Statistics, 2009; Joyce et al., 2010;
Enríquez-Obregón et al., 1998; Patrau, 1989). Some natural pharmaceutical compounds are
derived from sugarcane (Nasir et al., 2014; Julian et al., 2005; Enríquez-Obregón et al., 1998;
MeneÂndez et al., 1993). It is also a valuable renewable source of biofuel/bio-energy for the
production of ethanol (Ricaud et al., 2012; Weng et al., 2011; Menossi et al., 2007). Due to this
reason, it has gained importance all over the world in the last 20 years. It is a prime candidate for
the future fuel crop due to its efficient biomass production (Bower and Birch, 1992; FAO, 1991).
Sugar Production
The sugar industry is the 2nd largest industry in Pakistan (Ullah et al., 2011). Global production of
table sugar is now exceeded up to 165 million tonnes a year. Approximately 70-80% is produced
from sugarcane. The remaining is produced from sugar beet, which is grown mostly in the
temperate zones of the northern hemisphere. Sugar is produced in 120 countries. 70 countries
produce sugar from sugarcane, 40 from sugar beet, and 10 from both (World Sugar Statistics,
2009). The 10 largest sugar producing nations represent roughly 75% of world sugar production
and 2/3rd of total world consumption. Brazil alone accounts for almost 25% of world production.
White sugar consumption in developed countries can be considered as saturated whereas
developing countries are considered as growing markets, particularly in Asia, and to a lesser extent
in the Middle-East and Africa. At the beginning of the 20th century, a world population of 1.6
billion people consumed roughly 8 million tonnes of sugar, i.e. 5.1 kg per capita. Today, a world
population of 7 billion people consumes roughly 165 million tonnes of sugar i.e. 23 kg per capita
on average (World Sugar Statistics, 2009).
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Fig 1: Major Sugar Consumers of World
Problems of Sugarcane Industry
Internal disputes between sugarcane growers and processors are big problem the industry.
Procurement practices such as buying and purchasing used by processors such as delaying the
crushing season, buying cane at less than the support price, and withholding payments hurt the
farmers’ profitability. On the other hand, sugar processors complaint that farmers grow
unapproved varieties that produce low sucrose content resulting in lower sugar production and
recovery rates. As a result of the fluctuations in quantity and quality of raw material, sugar mills
have been required to operate at 50% of their installed capacity. Furthermore, the lower sugarcane
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supplies have also forced most of the mills in cane producing areas to close 1-2 months earlier than
normal (www.thebioenergysite.com).
Low Yield of Sugarcane
Wide use of sugarcane and its relevant products have created a challenging situation for sugarcane
researchers and growers. In spite of extensive research the average yield of sugarcane in Pakistan
is very low as compared to other cane producing countries of the world (Imam, 2001). There are
many factors which are responsible for this low yield, among them severe attack of insect pests at
early and mature stages of crop are the most significant one. Sugarcane borers are the most
damaging among the pests attacking this crop (Ashraf and Fatima, 1980). These include top borer,
stem borer, root borer, and Gurdaspur borer. Borers may reduce the yield up to 80% (Kalra and
Sidhu, 1955). The damage caused by borers not only reduces the crop yield but also affects the
sucrose contents of cane.
Sugarcane Problems
Approximately, 100 diseases have been reported to hinder the growth of sugarcane all over the
world (Khurana and Singh, 1975). The most common are fungal diseases which occur as spots and
hinder the process of photosynthesis affecting the growth of plant and ultimately the yield (Patil
and Bodhe, 2011). A number of bacterial, fungal and viral pathogens attack the crop and cause
serious damage.
Bacterial Diseases
Some important bacterial diseases of sugarcane are gumming (Xanthomonas vaculorum pv.
vasculorum), leaf scald (Xanthomonas albilineans), mottled stripe (Pseudomonas
rubrisubalbicans), ratoon stunting disease (Clavibacter xyli) and red stripe or top rot
(Pseudomonas avenae, P. rubrilineans).
Fungal Diseases
Fungal diseases of sugarcane are red rot (Physalospora tucumanesis anamorph; Colletotrichum
falcatum), Pythium root rot (Pythium graminicola), Rhizoctonia sheath and shoot rot (Rhizoctonia
solani), pineapple diseases (Thielaviopsis paradoxa), downy mildew (Peronosclerospora
sacchari), wilt (Fusarium sacchari), rust (Puccinia melanocephala, P. kuehnii), smut (Ustilago
scitaminea) and seedling blight (Alternaria alternata) which are common and most prevalent in
cane growing areas. Smut is prevalent in Southeast Asia and Africa. The whip-like black organs
emerge from the center of the plant and damage the whole plant gradually (Sengar et al., 2011).
Viral Diseases
There are several known viral diseases the most damaging of which is Mosaic disease. It was first
recognized in Java in 1892. Currently, the sub-group SCMV of the genus Potyvirus is known to
have seven species out of which SCMV (Sugarcane Mosaic Virus) and SrMV (Sorghum Mosaic
Virus) are known to attack sugarcane plants (Alegria et al., 2003; Arencibia, 1997; Shukla et al.,
1989). Other viruses that infect the sugarcane are chlorotic streak virus, sugarcane dwarf virus
(SCDV) and sugarcane Fiji disease virus.
Insect Pests of Sugarcane
Insect pests are the major problem in agriculture which are responsible for the low yield of
sugarcane include termites, borers, pyrilla, white flies, mealy bugs and termites which cause heavy
losses to the crop in terms of quality and yield (Kleter et al., 2007; Qaim and Zilberman, 2003).
Globally, 1300 species of insects were recorded to be responsible for attacking sugarcane while
61 were recorded to be present in Pakistan (Hashmi, 1994). One of the major restriction factors
that affect sugarcane production is the damage caused by insects such as Lepidopterous stem
borers (Weng et al., 2010; Fauconnier, 1993). These sugarcane insects are often hard to control
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using chemical insecticides because of their ability to tunnel into and feed inside the sugarcane
stems. Moreover, the insect-resistant germplasm is not available in sugarcane varieties (Christy et
al., 2009). The annual loss caused by insects has been estimated to be more than 10% of the
sugarcane yield losses worldwide (Ricaud et al., 2012). In mainland China, sugarcane borer
Walker (Proceras venosatus) is one of the major pest which alone causes more than 7% of
sugarcane yield losses per year (Weng et al., 2010; Weng et al., 2006). According to Gupta &
Singh (1997), 25% reduction in weight was observed by the 3rd and 4th brood of the sugarcane
borers. Insecticides are extensively used to combat losses from insect pests. In 2011-12, 112,928
metric tons of pesticides were consumed which cost 11242 million rupees (Department of Plant
Protection, Ministry of Food and Agriculture, Pakistan).
Major Lepidopteran insect pests of sugarcane are stem borer (Diatraea saccharalis) (Rossato et
al., 2010), root borer (Emmalocera depressalis), sugarcane top borer (Chilo terrenellus) (Goebel
and Way, 2003), pink borer (Sesamia inferens) and Maxican rice borer (Eoreuma loftini) (Naidu,
2009). Borers may reduce the yield up to 80% (Kalra and Sidhu, 1955). The damage caused by
borers not only reduces the crop yield but also affects the sucrose contents of cane. Naqvi et al.
(1978) have reported a gross negative correlation between the intensity of infestation and sucrose
contents and recorded reduction of sucrose beyond 10% borer infestation on internodes basis. The
sugarcane beetles (Tomarus subtropicus), giant termite (Mastotermes darwiniensis) and ants can
affect sugarcane yield by damaging the roots and this damage can be minimized by using
appropriate insecticide like imidacloprid or chloropyrifos. The other pests that damage sugarcane
yield are moth or butterfly, sugarcane thrips (Fulmekiola serrate), cane aphid (Melanaphis
sacchari), grasshopper (Oxya chinensis), sugarcane whitefly (Aleurolobus barodensis) and
burrowing bug (Scaptocoris talpa) (Naidu, 2009). Sap-sucking insects include woolly aphid
(Ceratovacuna lanigera) is predominant in Asia (Srikanth, 2004). Overall, 10% of the yield
reduction is observed all over the sugarcane growing countries (Ricaud et al., 2012; Srikanth and
Kurup, 2011). Non- insect pests include rats which also damage sugarcane crop (Bashir et al.,
2005).
Weeds of Sugarcane
Weeds are considered undesirable or detrimental to other plants. Weeds are the plants that are not
part of the intended crop and compete for nutrients, water, light and other factors integral to crop
growth (Ross and Lembi, 1999). The presence of undesirable plants in sugarcane plantations
reduces crop yield. Weeds can cause major losses in terms of quality and quantity. They can reduce
cane yield by competing for moisture, nutrients, and light during the growing season (Rainbolt and
Dusky, 2006). Ahmad et al., (2012) reported that 15-30% reduction in yield of sugarcane is due to
weeds in Pakistan. Furthermore according to Netafim (2010), weeds are estimated to cause 12 to
72 % reduction in cane yield worldwide. In Pakistan, national average yield of sugarcane is about
58.0 tons/ha as compare to world average production of 65 tons/ha (Economic Survey of Pakistan,
2011-2012).
Reduction in the cane yield with the uncontrolled weeds was recorded up to 14-74%. However,
the intensity and the weed flora cause variation in the loss of yield (Malik et al., 1986; Singh and
Moolani, 1975). Weeds can be controlled by mechanical, chemical and biological methods. Now
a day, herbicides are used world widely to eradicate the weeds to minimize crop competition.
Weed control by traditional method in growing field of sugarcane requires lots of manpower,
moreover it tear the surface of soil resulting in uprooting of plants and cause injuries to the people
involved in mechanical uprooting of weeds. Also many of the weeds have deep and extensive roots
that cannot be uprooted by traditional method and majority of them can regenerate (Nasir et al.,
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2014). Using genetic engineering as a complement for traditional breeding methods it is possible
to introduce herbicide-resistant traits into Saccharum germplasm (Enríquez-Obregón et al., 1998).
Transgenic Sugarcane Crop
Sugarcane is an attractive crop for improvement aimed at sustainable biomaterial production,
because of high biomass productivity and an efficient gene transfer system (Birch, 2007). This has
been true even for input traits such as insect and herbicide resistance that need only coarse control
over the level of constitutive transgene expression (Mudge et al., 2009; Finnegan and McElroy,
1994). The required precision is expected to be higher in metabolic engineering that diverts
metabolic flows into novel and high-value products, because diversion of these flows in
inappropriate tissues will interfere with plant development (Mudge et al., 2009).
With the advent of genetic transformation techniques based on recombinant DNA technology, it
is possible to insert genes into genome of the plant that confers resistance to insects (Bennett, 1994;
Romeis et al,. 2006). Engineering crop plants for enhanced resistance to insect pests has been one
of the successes of transgenic technology. As a trait, insect resistance, either alone or combined
with herbicide resistance is currently ranked second in terms of the global area occupied by biotech
crops during 1996–2009 (James, 2009). Several sugarcane promoters have been isolated and
shown to drive transgene activity in callus or young plants but not in the expected pattern in mature
plants (Hansom et al., 1999; Wei et al., 2003). Several heterologous and synthetic promoters have
also been shown to drive strong expression in callus but little or no expression in mature plants,
with evidence for both transcriptional and post-transcriptional gene silencing (PTGS) effects. In
contrast, the maize Ubi-1 promoter has driven sustained reporter transgene expression in sugarcane
(Hansom et al., 1999).
Sugarcane Breeding
The progress in traditional breeding of sugarcane, a highly polyploid and frequently aneuploid
plant, is impeded by its narrow gene pool, complex genome, poor fertility and the long breeding
and selection cycle. These constraints make sugarcane a good candidate for molecular breeding.
In the past decade considerable progress has been made in understanding and manipulating the
sugarcane genome using various biotechnological and cell biological approaches. The creation of
transgenic plants with improved agronomic or other important traits; advances in genomics and
molecular markers and progress in understanding the molecular aspects of sucrose transport and
accumulation are notable aspects of transgenic crops. More recently, substantial effort has been
directed towards developing sugarcane as a bio-factory for high-value products (Lakshmanan et
al., 2005).
Traditional plant breeding techniques have been widely used to enhance important economic traits
in agronomic crops, but this approach is laborious and time-consuming, especially in vegetatively
propagated species like sugarcane (Liu et al., 2003). Moreover, various important traits such as
resistance to insects, viruses, and herbicides are often absent from the normal sugarcane
germplasm. The conventional breeding method of crossing between various germplasms has been
used successfully for maximizing the productivity of sugarcane (Weng et al., 2011; Fauconnier.
1993). However, due to the biological complexity of sugarcane crop and non-availability of
desirable germplasms, this valuable traditional approach has also had its limitations, in particular,
in breeding insect-resistant sugarcane varieties. Rapid progress of plant genetic engineering in the
last two decades suggests that sugarcane breeding could benefit a lot from the use of nonconventional methods. In particular, genetic engineering may not only be able to shorten the period
of breeding time and reduce costs for producing an improved sugarcane line. It can also introduce
new agronomic traits that are absent in the natural sugarcane germplasms e.g. transgenic sugarcane
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plants were shown to produce sorbitol, gentiobiose and gentiobitol (Chong et al., 2010; Jain et al.,
2007; Chong et al., 2007), and have resistance to herbicides or pest (Weng et al., 2006; Hernandez.,
2000). Since 1990s, genetic transformation using insecticidal Bt genes is regarded as an effective
method to develop insect-resistant transgenic plants (Kim et al., 2008; Valderrama et al., 2007).
Transformation Methods in Sugarcane
Transformation methods to introduce resistance genes into a plant genome have allowed the
generation of new sugarcane varieties resistant to herbicide spray (Enríquez-Obregón et al., 1998)
or insect attack (Arencibia et al., 1997; González-Cabrera et al., 1998). Genetic transformation
using insecticidal Bt genes is regarded as an effective method to develop insect-resistant transgenic
plants (Weng et al., 2011; Kim et al., 2008; Valderrama et al., 2007). Particle bombardment and
Agrobacterium co-cultivation are the two most common methods for delivery and expression of
transgenes, but a direct comparison of these systems have been limited in sugarcane and other
plant species (Liu et al., 2003). Since 1990s, several methods for generation of transgenic
sugarcane plant have been established, such as micro-projectile bombardment and electroporation
of embryogenic cells (Weng et al., 2011; Arencibia et al., 1995; Bower and Birch, 1992). The
progress in transformation methodology has facilitated incorporation of several useful genes into
sugarcane genomes, including a bacterial toxin degradation gene (Weng et al., 2011; Zhang et al.,
1999), three virus resistance genes (Gilbert et al., 2009; McQualter et al., 2004; Ingelbrecht et al.,
1999), a bovine pancreatic trypsin inhibitor gene (Christy et al., 2009), and several insect
resistance genes (Weng et al., 2006; Falco and Silva-Filho, 2003; Arencibia et al., 1997). However,
a common problem associated with these transgenic sugarcane plants is the low expression levels
of transgenes (Zhang et al., 2007), which might account for the less than satisfactory performance
of elite transgenic sugarcane lines in field trials (Weng et al., 2011; Arencibia et al., 1999).
Micro-Projectile Bombardment
The efficiency of gene transfer into embryogenic calli of sugarcane has been increased tenfold by
optimization of particle bombardment conditions and increase in stable transformation
frequencies. Genes co-precipitated on separate plasmids are co-transformed at high efficiency. The
development of a genetic transformation system opens the way for sugarcane molecular
improvement by introducing useful foreign genes, blocking the expression of undesired genes,
moving isolated genes between varieties, or tailoring the patterns of expression of key sugarcane
genes. Transgenic sugarcane plants were produced by bombardment of embryogenic callus with
high-velocity DNA-coated micro-projectiles, followed by a selection and regeneration procedure
designed for this target tissue. Optimal bombardment conditions for embryogenic callus required
higher micro-projectile velocities for sugarcane suspension culture cells (Bower and Birch, 1992).
Transgenic sugar-cane plants have been successfully recovered from cell suspensions and
embryogenic calli transformed by particle bombardment (González-Cabrera et al., 1998;
Arencibia et al., 1995; Bower and Birch, 1992).
Agrobacterium-mediated DNA Transformation
The most frequently used and most efficient technique to genetically transform dicot cells employs
the Gram-negative soil bacterium, Agrobacterium tumefaciens; without verified success in the
production of transgenic plants (Potrykus, 1991; Klee et al., 1987). However, successful
Agrobacterium-mediated uptake of DNA by monocot cells, especially the economically important
cereals, has been limited (Chan et al., 1992; Gould et al., 1991; Mooney et al., 1991; Raineri et
al., 1990). Optimization of the Agrobacterium-mediated DNA transfer to sugarcane meristems has
recently been reported (Enríquez-Obregón et al., 1998).
Electroporation
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The naked DNA molecules can be introduced into protoplast by electrically induced changes in
the membrane permeability. This has been used successfully for transformation of a wide range of
crop species (Christou et al., 1987; Fromm et al., 1986). Electroporation treatment of protoplasts
has allowed regeneration of transgenic rice and maize plants (Shimamoto et al., 1989; Rhodes et
al., 1988), but this approach is limited by severe difficulties with plant regeneration from
protoplasts for most cultivars. Transgenic sugarcane plants have been successfully recovered from
electroporated intact cells (González-Cabrera et al., 1998).
Polyethylene Glycol (PEG) Mediated Transformation
The development of monocot transformation systems has involved direct DNA transfer methods
such as PEG treatment of protoplasts (Mass and Werr, 1989; Meijer et al., 1991). This involved
use of chemicals like PEG and divalent cations designed to increase the membrane permeability
in freshly isolated protoplasts to DNA. PEG-mediated DNA uptake typically transforms 0.1-0.4%
of the total protoplast treated (Hayashimoto and Murai, 1990).
Microinjection
This involves immobilization of protoplast and microinjecting DNA directly into the nucleus.
Transformation efficiencies have ranged from 12-66% (Miki et al., 1987) with the highest
efficiency being obtained in tobacco. However, DNA-pollen mixtures (Ohta, 1986) or DNA
injection into young floral tillers, have not proved generally applicable (Bower and Birch, 1992;
de la Pena et al., 1987).
Insect Resistance in Plants
Biotechnology efforts have focused on developing GM crops that resist abiotic and biotic stress
and on increasing yields. Genetically modified (GM) crops were grown on 160 million ha globally
in 2011 (James, 2011). Among them, transgenic cotton expressing insecticidal proteins from
Bacillus thuringiensis (Bt) has been one of the most rapidly adopted GM crops in the world (Dong
and Li, 2007; Barwale et al., 2004). Transgenic insect-resistant crops carrying genes from Bacillus
thuringiensis were grown commercially for the first time in 1996 and increased quickly, with 14
million ha grown worldwide in 2002 (James, 2002). The insecticidal toxin gene of Bt, a known
insecticidal gene, is one of the most commonly used genes in the development of GM crops (Lee
et al., 2009). Bt cotton is considerably effective in controlling Lepidopteran pests, and is highly
beneficial to the grower and the environment by reducing chemical insecticide sprays and
preserving population of beneficial arthropods (Tabashnik et al., 2002; Gianessi and Carpenter,
1999). Crops contain Cry genes such as Cry1Ac, Cry1Ac + Cry2Ab or Cry1Ac + Cry1F. Larvae
from the resistant strain of European corn borer (Ostrinia nubilalis) did not survive on transgenic
corn that produces Cry1Ab or Cry1Ac (Huang et al., 2011; Tabashnik et al., 2003). EnríquezObregón et al., (1998) report with evidence of the first transgenic sugarcane lines resistant to stemborer attack.
Bacillus thuringiensis
Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming, soil-dwelling bacterium with
entomo-pathogenic properties. Bt produce insecticidal proteins during the sporulation phase as
parasporal crystals that produces crystalline protein inclusions known as -endotoxins (deltaendotoxin). The number of sequenced crystal proteins in Bt is more than 100, encoding Cry and
Cyt proteins (Schnepf et al., 1998). Crystal (Cry) and cytolitic (Cyt) protein families are a diverse
group of proteins with activity against insects of different orders e.g. Lepidopterans (Cohen et al.,
2000; Schnepf et al., 1998; Hofte and Whitely, 1989), Coleopterans (Herrnstadt et al., 1986; Krieg
et al., 1983), Dipteran insects (Andrews et al., 1987) and also against other invertebrates such as
nematodes. These genes are generally safe for human consumption (Bakhsh et al., 2010; Bently,
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2003; GMPP, 2000). These are being widely used to develop insect resistance in various crops.
The traditional breeding program could successfully accomplish pyramiding the foreign Bt genes
with native insect resistant trait, in a single genetic background (Sachs et al., 1996).
Mechanism of Bt Endotoxins
The mechanism of action of the Bt Cry proteins involves solubilization of the crystal in the insect
midgut, when ingested by larvae, toxin proteins bind to specific receptors in the midgut region and
toxin binding in susceptible insects disrupts midgut epithelium; thereby causing overall toxic
effects and ultimately resulting in death of the larvae (Kranthi et al., 2005). Their primary action
is lysis of midgut epithelial cells by inserting into the target membrane and forming pores in the
apical microvilli membrane of the cells (Bravo et al., 2005; Aronson and Shai, 2001; de Maagd et
al., 2001). Among this group of proteins, members of the 3-domain Cry family are used worldwide
for insect control. Like other PFTs that affect mammals, Cry toxins interact with specific receptors
located on the host cell surface and are activated by host proteases following receptor binding
resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast,
Cyt toxins directly interact with membrane lipids and insert into the membrane (Bravo et al., 2007).
Zhang et al. (2006) recently suggested that toxicity could be related to G-protein mediated
apoptosis following receptor binding. Cry proteins pass from crystal inclusion pro-toxins into
membrane-inserted oligomers that cause ion leakage and cell lysis. The crystal inclusions ingested
by susceptible larvae dissolve in the alkaline environment of the gut and the solubilized inactive
pro-toxins are cleaved by midgut proteases yielding 60–70 kDa protease resistant proteins (Bravo
et al., 2007; Bravo et al., 2005).
Bt Cry and Cyt toxins belong to a class of bacterial toxins known as pore-forming toxins (PFT)
that are secreted as water-soluble proteins undergoing conformational changes in order to insert or
translocate across cell membranes of their host. There are two main groups of PFT:
1. The α-helical toxins, in which α-helix regions form the trans-membrane pore
2. The β-barrel toxins, that insert into the membrane by forming a β-barrel composed of βsheet hairpins from each monomer (Bravo et al., 2007; Parker and Feil, 2005)
PFT-producing bacteria secrete their toxins and these toxins interact with specific receptors
located on the host cell surface. In most cases, PFT are activated by host proteases after receptor
binding inducing the formation of an oligomeric structure that is insertion competent. Finally,
membrane insertion is triggered by a decrease in pH that induces a molten globule state of the
protein (Parker and Feil, 2005).
Need to Develop Bt Transgenic Crops
The crystal protein “Cry endotoxins” produced by Bacillus thuringiensis are among the most
specific and potent insecticides known to man. Unfortunately, these toxins must be eaten by the
target insect and persist for only a few days after application sprays produced with Bt toxins have
had limited use. It was realized more than a decade ago that problem with persistence, complete
coverage of the plant which is impossible with sprays, especially for the control of internal feeders
like stem borers and therefore ingestion could be overcome by transferring the Cry genes to plants.
Plants able to defend themselves effectively even against high densities of insects were produced
by the early 1990s and finally commercialized in the USA and Australia in 1996. Bt-transgenic
varieties are referred to by the US-EPA as “plant pesticides”. Bt-transgenic crops can facilitate the
use of under-utilized management tactics such as crop rotation and pheromone disruption that are
not generally sufficiently effective alone at moderate-to-high pest densities (Roush, 1997).
Benefits of Bt Transgenic Crops
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One of the most successful applications of Bt has been the control of Lepidopteran defoliators.
The development of transgenic crops that produce Bt Cry proteins has been a major breakthrough
in the substitution of costly and laborious spraying of chemical insecticides by environmental
friendly alternatives (Bakhsh et al., 2010; Bravo et al., 2007; Qaim and Zilberman, 2003). In
transgenic plants the Cry toxin is produced continuously, protecting the toxin from degradation
and making it reachable to chewing and boring insects. Cry protein production in plants has been
improved by engineering Cry genes with a plant biased codon usage, by removal of putative
splicing signal sequences and deletion of the carboxyl-terminal region of the pro-toxin (Schuler et
al., 1998).
Glyphosate Tolerance in Plants
Glyphosate-resistant technology offers broad-based weed control with flexibility in application
timing. Herbicide-tolerant crops have been widely and rapidly adopted by farmers in several
countries due to enhanced weed control, lower labor and production costs, increased
environmental benefits and gains in profitability (Cao et al., 2010). The huge diversity of perennial
and annual weeds is found to occur in the crop fields. However, some particular weeds predominate different cropping systems and zones. Due to these reasons both broad spectrum
(selective and non-selective) herbicides are applied in weed rich fields. It is reported that
continuous application of some herbicides has induced resistant in weed plants and has provoked
weed problems, e.g. Phalaris minor developed resistance against isoproturon, a phenylurea
herbicide, in rice and wheat cropping system of Indian Punjab and Haryana, (Malik et al., 2004).
Herbicides using a mechanism of blocking specific metabolism pathways do not differentiate
between crop plants and weeds. These non-selective herbicides are commonly applied before
sowing of crop plants and their residual effects may affect crop seeds germination and growth of
seedlings. There is very limited flexibility in the timings of the herbicide use and their application
demand lot of precautions. However, few crop plants surprisingly possess gifted resistance to
specific herbicides. For example, 2,4-dichlorophenoxyacetic acid (2,4-D) causes death only to
broad-leaved weeds. Thus it can act as the best type of selective herbicide in monocot crops like
sugarcane, wheat and maize. In the same way, maize shows resistance to atrazine and simazine
(Bowler et al., 1992).
Glyphosate (N-phosphonomethyl glycine) is a well-known broad-spectrum systemic herbicide
used to kill weeds, especially annual broad-leaf weeds and grasses, which compete with
commercial crops. It is quite effective in killing a wide variety of weed plants, including broadleaf,
grasses and woody plants. On the contrary, it has a relatively small effect on some clover species.
It is one of the most widely used herbicides. It was initially patented and sold by Monsanto
Company in the 1970’s under the trade name Roundup, and its US patent expired in 2000. In 1983,
scientists of Monsanto and Washington University isolated the common soil bacteria,
Agrobacterium tumefaciens strain CP4, which is highly tolerant to glyphosate because its EPSPS
is less sensitive to inhibition by glyphosate than EPSPS found in plants (Watrud et al., 2004). By
1986, they had successfully inserted the CP4 EPSPS gene into the plant genome and obtained
glyphosate resistance (GR) plants. The initial GR crops were the most quickly adopted technology
in the history of agriculture. In 2007, 12 million growers in 23 countries planted 114.3 million ha
of biotech crops (Baum et al., 2007). Growers chose GR crops because glyphosate made weed
control easier and more effective, increased profit, required less tillage, and did not restrict crop
rotations. Before the introduction of GR crops, growers routinely used many different herbicides
with many different modes of action. Now, many growers rely only on glyphosate (Foresman and
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Glasgow, 2008; Gustafson, 2008). Applying glyphosate alone over wide areas on highly variable
and fertile weeds made the evolution of resistant weeds inevitable (Gower et al., 2003).
Weeds and their Control
Many types of weeds are present that crowd the sugarcane cultivated areas together with grasses,
broadleaf weeds and sedges. Grasses that present in cane growing districts are Bermuda grass
(Cynodon dactylon), wild sorghum (Sorghum spp.) and Guinea grass (Panicum maximum). Field
grasses in particular can be challenging when cane has to grow in territory consequently. Broadleaf
weeds are more regional and soil specific unlike sedges and present in all sugarcane growing areas
and soil types (McMahon et al., 2000). Some other weeds can also present in the field and can be
problematic such as hymenachne and giant sensitive plant. Hymenachne an aquatic grass and can
be 2.5 meters tall and declared as “weed of national significance” in Australia. It can block
irrigation and drainage channels in cane growing field and pollute the sugarcane crop. Spraying
with herbicide is used three months periodically to control hymenachne (Anonymous, 2003).
Glyphosate
Glyphosate (N-phosphonomethyl glycine) is a broad-spectrum, non-selective herbicide that has
little residual soil activity and is non-toxic to animals. Glyphosate inhibits aromatic amino acid
synthesis. It is a potent inhibitor of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS)
(Duke, 1987). Plant DNA encodes gene for glyphosate-tolerant enzyme 5-enolpyruvyl-shikimate3-phosphate (EPSP) synthase. The DNA encodes glyphosate-tolerant EPSPS modified by
substitution of an alanine residue for a glycine residue in a sequence found between positions 80
and 120, and a threonine residue for an alanine residue in a second conserved sequence found
between positions 170 and 210 in the mature wild type EPSPS (Eichholtz et al., 2001).
Mechanism of Glyphosate Action
Glyphosate is registered for use on more than fifty crops and has been used for more than 30 years
to manage annual, perennial, and biennial herbaceous grass, sedge and broadleaf weeds as well as
unwanted woody brush and trees. Many glyphosate formulations and salts are commercially
available; the most common salts are the mono-potassium and iso-propylamine. The type and
amount of adjuvant included in the various formulations differ greatly and strongly influence weed
control. This molecule is an acid, which dissociates in aqueous solution to form phyto-toxic anions.
Several anionic forms are known. The term “glyphosate” refers to the acid and its anions (Eichholtz
et al., 2001). Glyphosate strongly competes with the substrate phosphoenolpyruvate (PEP) at the
EPSPS enzyme-binding site in the chloroplast, resulting in the inhibition of the shikimate pathway.
Glyphosate inhibits the shikimic acid pathway which provides a precursor for the synthesis of
aromatic amino acids. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvate
(PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the
enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) which is the very important enzyme
of the pre-chorismate part of the shikimate pathway (Nasir et al., 2014; Eichholtz et al., 2001;
Steinrucken and Amrhein, 1980). Early kinetic characterization established that glyphosate is a
reversible inhibitor of EPSPS, acting by competing with PEP for binding to the active site. The
products of the shikimate pathway include the essential aromatic amino acids tryptophan, tyrosine,
and phenylalanine and other aromatic compounds and important plant metabolic products in
plants, fungi, bacteria, and apicomplexan parasites (Senseman and Armbrust, 2007). EPSPS
reaction proceeds through a tetrahedral intermediate formed between S3P and the carbocation state
of PEP, followed by elimination of inorganic phosphate (Eichholtz et al., 2001).
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Over-expression of EPSPS gene in cells renders glyphosate tolerance in transformed plants
(Pline-Srnic, 2006; Castle et al., 2004). Two mechanisms have been reported underlying the
glyphosate tolerance i.e.:
1. Over-expression of EPSPS gene
2. Herbicide-insensitive enzyme (Nasir et al., 2014; Ge et al., 2010)
Transgenic Glyphosate Resistance Crops
It has been shown that glyphosate-tolerant plants can be produced by inserting into the genome of
the plant the capacity to produce a higher level of EPSP synthase. In plants including weeds,
glyphosate inhibits chloroplast localized EPSP synthase (Castle et al., 2004; Pline et al., 2002).
The present invention provides a means of enhancing the effectiveness of glyphosate-tolerant
plants by producing variant EPSP synthase enzymes which exhibit a lower affinity for glyphosate
while maintaining catalytic activity (Eichholtz et al., 2001). The introduction of herbicide resistant
crops has dramatically changed weed management in crop production systems (Vencill et al.,
2012; Owen, 2008). Using genetic engineering as a complement for traditional breeding methods
it is possible to introduce herbicide-tolerant traits into Saccharum germplasm. Absence of
herbicide tolerant genes in genetic pool of wild relatives of sugarcane makes traditional breeding
programs difficult. The varieties that are productive and at the same time has resistance against
certain pathogens and diseases, can improve yield. The development of herbicide resistant
sugarcane varieties through transgenic technology will be cost effective in controlling weeds
which ultimately improve yield potential. Several crops have been genetically modified for
resistance against herbicide. The most significant was Roundup ready soybean which is the most
widely adopted biotech crop that contained a version of EPSPS gene (glyphosate) from the
Agrobacterium tumefaciens (Nasir et al., 2014).
Need for Glyphosate Resistance Crop
Herbicide-tolerant crop plants could reduce the need for tillage to control weeds, thereby
effectively reducing costs to the farmer. Currently, weeds in sugarcane crop are being controlled
by the application of certain organo-phosphorus herbicides like Roundup. The active ingredient of
roundup is glyphosate which is a non-selective, broad spectrum herbicide that translocated
symplastically to the meristems of the growing plants and is well known for its high effectiveness,
low toxicity, low residues in organisms and soil and overall limited environmental impact.
However, heavy use of glyphosate based herbicides increases the emergence of glyphosate
resistant weeds and also it is reported that once glyphosate travels to a plant's roots, it is released
into the rhizosphere where it is immobilized at the soil matrix (Tesfamariam et al., 2009) which
leads to disruption of soil and root microbial community (Busse et al., 2001). About 1-2% of
glyphosate runoff in rainfall after glyphosate is applied (Giesy et al., 2000). Paganelli et al., (2010)
indicated that low doses of glyphosate cause birth defects in frogs and chickens.
Promoters
Sugarcane is a crop of great interest for engineering of sustainable biomaterials and bio-fuel
production. Promoters isolated from sugarcane have generally not maintained the expected
patterns of reporter transgene expression. This could arise from defective promoters on redundant
alleles in the highly polyploid genome, or from efficient transgene silencing (Mudge et al. 2009).
In eukaryotic systems the expression of genes is directed by a region of the DNA sequence called
the promoter. In general, the promoter is considered to be that portion of the DNA, upstream from
the coding region that contains the binding site for RNA polymerase II and initiates transcription
of the DNA. The promoter region also comprises other elements that act as regulators of gene
expression. These include a TATA-box consensus sequence in the vicinity of about ~30bp and
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often a CAAT-box consensus sequence at about -75bp, 5'-end relative to the transcription start
site, or cap site (Messing, 1983; Breathnach and Chambon, 1981). In plants the CAAT-box may
be substituted by the AGGA-box (Messing, 1983; Christensen et al., 1996).
The promoter is a key DNA regulatory element that directs appropriate strength and patterns of
gene expression in a constitutive or specific manner, and therefore, plays a crucial role in
successful transformation studies. Moreover, the number and types of promoters that drive strong,
constitutive expression of transgenes are relatively few in sugarcane. For example, the viral
Cauliflower Mosaic Virus 35S (CaMV 35S) promoter has been widely used in the transformation
of many dicot and monocot crops, but activity in sugarcane has been low as demonstrated in
various studies (Liu et al., 2003; Elliott et al., 1998; Chowdhury et al., 1992). Nearly all transgenic
crops around the world utilize the CaMV 35S promoter (Odell et al., 1985), or similar promoters
from closely-related viruses to drive transgenes. It is only now becoming clear that this promoter
is not as robust as laboratory and glasshouse studies have suggested and its function is influenced
by as yet undefined physiological and perhaps environmental factors (Sunilkumar et al., 2002).
Foreign gene expresses in transgenic plants, it is necessary use efficient transcriptional promoters
that allow a constitutive expression of the transgene. Optimization of transgene expression has
been reported for other monocotyledonous plants such as maize and rice, obtaining the highest
expression levels when reporter gene was fused to an untranslated intron under the control of a
strong promoter (González-Cabrera et al., 1998; McElroy et al., 1991; Mascarenhas et al., 1990).
CaMV 35S Promoter
Cauliflower mosaic virus (CaMV) is a DNA virus that infects members of the Cruciferae. The
virus is approximately 8kb long and its complete nucleotide sequence has been determined (Hohn
et al., 1982). Transcript mapping experiments have identified two viral promoters, designated 19S
and 35S. During the virus life cycle, the 35S promoter is transcribed from the viral DNA minusstrand to produce an 8kb transcript referred to as the 35S RNA (Guilley et al., 1982). The 35S
promoter is at least 30 times stronger than the nopaline synthase (NOS) promoter. The strength of
35S promoter accounts for its widespread use for high level expression of desirable traits in
transgenic plants (Fang et al., 1989; Cuozzo et al., 1988; Hemenway et al., 1988). The CaMV 35S
promoter is the most commonly used promoter for driving transgene expression in plants. Though
it is presumed to be a constitutive promoter (Odell et al., 1985), some reports suggest that it is not
expressed in all cell types (Sunilkumar et al., 2002; Terada and Shimamoto, 1990; Yang and
Christou, 1990; Benfey and Chua, 1989; Williamson et al., 1989). In addition, the information
available on its expression profile in all possible cell and tissue types and during early stages of
development is incomplete. It is effective in driving transgene expression in both dicots and
monocots.
Ubiquitin Promoter
Ubiquitin, a 76 amino acid protein, is present in all eukaryotic cells and is one of the most
conserved proteins, differing in only three of 76 amino acids between higher plants and animals
(Ausubel, et al., 1994; Christensen et al., 1992). Ubiquitin is encoded by small multigenic families
containing two types of genes: polyubiquitin and ubiquitin extension-fusion genes. The best
characterized function of ubiquitin is its conjugation to target proteins as a recognition signal for
protein degradation (Callis et al., 1987). Ubiquitin is also involved in other cellular processes, such
as ribosome biosynthesis, chromatin structure and cell cycle control (Christensen and Quail, 1989;
Callis et al., 1988). The polyubiquitin genes were reported to be constitutively expressed in all
plant organs tested with an increased level in young tissues (Wang et al., 2000; Cornejo et al.,
1993; Kawalleck et al., 1993; Binet et al., 1991; Burke et al., 1988).
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The Ubi-1 promoter has been shown to be highly active in monocots; these constructs may be
useful for generating high-level gene expression of selectable markers to facilitate efficient
transformation of monocots, to drive expression of reference reporter genes in studies of gene
expression and to provide expression of biotechnologically important protein products in
transgenic plants. The general availability of strong promoters active in all or most cell types of
monocotyledonous plants would be useful in a variety of applications in gene transfer studies with
this plant group (Christensen et al., 1996; McElroy and Brettell, 1994).
Sivamani (2006) reported that polyubiquitin promoter has been isolated from several plants
including Arabidopsis (Callis et al., 1990; Norris et al., 1993), sunflower (Binet et al., 1991), maize
(Christensen et al., 1992), potato (Garbarino and Belknap, 1994), Nicotiana tabacum (Genschik
et al., 1994), rice (Wang et al., 2000; Wang and Oard, 2003), parsley (Kawalleck et al., 1993),
peas (Xia and Mahon, 1998), and sugarcane (Wei et al., 2003; Yang et al., 2003). The levels of
expression were higher for the polyubiquitin promoter than the CaMV 35S promoter in maize,
rice, and N. tabacum (Joung et al., 2006). The maize ubiquitin promoter Ubi-1 has been used to
drive constitutive transgene expression in sugarcane studies (Falco et al., 2000; Bower et al.,
1996). The control of gene expression appears to differ for the cereal and some of the floral and
non-cereal monocots. In sugarcane, the maize Ubi-1 promoter showed higher levels of transient
expression than the rice Act1 and CaMV 35S promoters (Joung et al., 2006; Gallo-Meagher and
Irvine, 1993). The maize Ubi-1 promoter has been used most frequently for developing transgenic
plants of cereal monocots that show high constitutive levels of expression, but this promoter is not
useful for high levels of expression in Gladiolus (Joung et al., 2006).
In both rice and sugarcane, these assays found that Ubi-1 produced more expression foci than other
tested promoters, including the rice Act1 promoter (McElroy et al., 1990, McElroy et al., 1991),
the pEmu promoter (Last et al., 1991; Chamberlain et al., 1994), and some configurations of the
CaMV 35S promoter. Ubi-1 has been used to produce stable transgenic rice (Cornejo et al., 1993)
and sugarcane (Gallo-Meagher and Irvine 1996, Ma et al. 2000). For these reasons, Ubi-1 is often
used as a standard of comparison when characterizing new monocot promoters (Schenk et al.,
1999; Wang et al., 2000; Schenk et al., 2001; Wei et al., 2003). The early commercial transgenic
traits in plants e.g. resistance against herbicides, insect pests or viruses were achieved through the
use of ‘constitutive’ promoters that drive expression across most plant tissues. Notable examples
are the virus-derived CaMV 35S promoter (Odell et al., 1985) and the maize Ubi-1 promoter
(Christensen et al., 1992), widely used in dicotyledonous and monocotyledenous plants,
respectively (Moyle and Birch, 2013).
GUS activity from constructs containing RUBQ1 and RUBQ2 promoters in rice suspension cells
was 10-15 fold greater than those using the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter,
and two to threefold greater than constructs with the maize polyubiquitin Ubi-1 promoter (Liu et
al., 2003; Wang et al., 2000). Although the widely-used CaMV 35S promoter is active in monocot
cells, its relative strength is substantially less than in dicot cells and it is inactive in some cell types,
e.g. pollen (McElroy and Brettell, 1994; Christensen et al., 1992; Bruce et al., 1989). Because of
their expected involvement in fundamental processes in all cell types, the genes for rice actin (Actl) (McElroy et al., 1990) and maize ubiquitin (Ubi-1) (Christensen et al., 1992) have been
investigated as potentially useful alternatives to the CaMV 35S and Adhl sequences. Both of these
monocot promoters have been shown to be significantly more active than the CaMV 35S promoter
in monocot cells (McElroy and Brettell, 1994; Cornejo et al., 1993; Gallo-Meagher and Irvine,
1993; Christensen et al., 1992; McElroy et al.,1990; Bruce et al., 1989) with the Ubi-1 promoter
being somewhat stronger than the Act-1 promoter where compared directly (Christensen et al.,
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1996; Wilmink et al., 1995; Schledzewski and Mendel, 1994; Cornejo et al., 1993; Gallo-Meagher
and Irvine, 1993).
Conclusion
It was concluded form all above discussions that the use of GMO (genetically modified organisms)
technology, the quality and production of sugarcane may be improved. The resistance against
insects/pests, diseases and glyphosate may be induced in sugarcane to increase sugar production.
It may be suggested that there is need to increase the use of GMO crops to fulfill world food
requirements.
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