Chapter 1 Introduction and Review of Literature

INTRODUCTION AND REVIEW OF LITERATURE

1.1 General introduction

Sugarcane (Saccharum officinarum L.) has worldwide importance for

food, feed and fiber. It ranks amongst the world’s top ten food crops and major
source of ethanol production . Globally, it contributes about 60-70% sugar
production, annually . Sugarcane is considered to have its origin in Asia and then
spread to other parts of the world for cultivation and sugar production . Presently,
it is mostly grown in tropical to subtropical zones of the world, under diverse
environmental conditions . Sugarcane belongs to genus Saccharum and family
poaceae. Saccharum is known as a complex genus with high ploidy level and
consists of six species viz- S.officinarum L., S.barberi L., S. sinensi L., S.
spontaneum L., S. robustum L. and S. eduLe. . Out of these six species, only
Saccharum officinarum L., (2n=80) was commercially cultivated for sucrose
extraction from its stalks till the end of 20 th century and then breeders made
hybridization of Saccharum officinarum L., with wild specie S. spontaneum that
is termed as nobilization. Currently, all commercial growing clones of sugarcane
are inter-specific hybrids . The hybrids are repeatedly backcrossed with
S.officinarum L. to restore the high sucrose contents, along with other desired
characters, such as resistance/tolerance to insect pests anddiseases, low in fiber
contents, thick stalk, rare flowering and limited tillering..

Commercially grown sugarcane (Saccharum officinarum L.) is a tall semi

perennial C4 grass, which make tillers to produce thick stem (3-6m length, 2.5-7.5 cm
diameter) , used for the extraction of sucrose . It is mainly grown in the tropical to sub
tropical countries of the world which extend in the latitudes about 37 0 N and 320 S of the
equator . Pakistan is located between the latitude range of 24 0 to 36N0 and longitude 620
to 780E , where coastal, subtropical and semi temperate climatic conditions are available
for sugarcane cultivation . In Pakistan, sugarcane is mainly grown along the coastal area,
plains of Indus River Sindh, Punjab and Peshawar valley of Khyber Pakhtunkhwa
(KPK).

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 Chapter 1 Introduction and Review of Literature

1.2 Sugarcane importance and uses

Sugarcane crop is grown globally in more than 105 countries . About 174 million
tons sugar is produced annually from sugar crops and 70-76 % of the total sugar
produce comes from the sugarcane and its share has increasing trend since last 50 years .
The most of the world agricultural produce are raw material source for agriculture
industry and the major one are the cereals and the two major sugar producing crops,
sugarcane (Saccharum officinarum L.) and sugar beet (Beta vuLgaris L.) . Generally, 70
% of the world sugar produce is consumed in the countries of its origin while the balance
is traded to the world markets to fill up the 24.30 kg per capita sugar consumption in the
world . In most of the countries, sugarcane is grown only for sugar (sweetener) purpose
but importance of sugarcane is increasing rapidly with huge biomass per unit area to
produce alcohol and bio-energy for the automobiles .

Sugarcane has attained a worldwide importance for industrial derivatives

due to which it is not only an important cash crop for the farmer but also has become
necessity for the sugar, alcohol and other agro based industries. While producing the
sugar from sugarcane in the industrial chemical process, there is generation of residues,
many of which are waste products of sugar extraction process, but these industrial
residues or waste products are used in many bi-products as the production of animal
food, feed and probiotic medium . The major bi-products and residues of sugarcane are
molasses and fiber . About 80% world’s molasses is used for alcohol production through
biochemical process and remaining for animal feed. Bagasse (fiber) is mainly used as a
source of renewable energy, paper, board and xylitol production purposes . Its raw juice
is used as a soft drink by the peoples of tropics and subtropics which contains medicinal
and antioxidant properties .

1.3 Economic importance of sugarcane in Pakistan

Pakistan has a prominent position in the sugar producing countries in the world.
It ranks 5th in sugarcane growing area, cane production and has 15 th global sugar
production position . Sugarcane is the second important industrial and profitable cash
crop and a good source of rural income in Pakistan . It is grown on an area of about one

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 Chapter 1 Introduction and Review of Literature

million hectares and provides raw material to 84 sugar mills to produce 7.0 million
tones of crystal sugar and related products annually . Sugar industry is the second largest
industry after textile and has an immense economic importance for the farmer prosperity
by generating the employment in the country. It is estimated that about 15,000 to 25,000
families earn their living in one sugarcane factory zone and 600 to 1000 skilled and
unskilled personnel are employed in one sugar mill . It contributes 3.2 and 0.7% in the
value added of agriculture and GDP of the country respectively . Sugarcane is very
important source of animal feed all over the world . In Pakistan, livestock rearing is a
vital integration with agriculture system and it requires sufficient quantities of fresh and
succulent fodder to feed the animals. Sugarcane green tops are used for animal fodder, as
a substitute or supplement with seasonal fodder for the conventional animal feeding .
Trash is used for energy and as mulch in the field for moisture conservation and on
incorporation the soil as source of organic fertilizer. Its bi- product, filter cake is used as
bio-compost (organic fertilizer) that is rich source of nutrients and organic matter and a
cheaper fertilizer source . Pakistan produces about 2.65 million tons molasses annually
resulting in production of about 0.123 million tons ethanol having worth earning of
$44.35 million from ethanol export per year . Ethanol is not only the energy source for
automobiles, the biogas produced from the fermentation process is another source of
cheaper energy for running the generators . One sugar factory produces about 30 %
bagasse of its total crushing in a season and as a whole sugar industry of Pakistan
produces about 30 million tons bagasse annually which is used for paper, wood
substitute and to run the boilers for energy generation. In Pakistan, there is potential to
generate 2000 MW electric power from bagasse .

1.4 Sugarcane as a bio-energy crop and diversity among Saccharum

complex for fiber characteristics

Due to increasing demand for bio-fuels in recent years, sugarcane is seen as a

promising source for ligno-cellulosic ethanol production because of its high biomass
production per unit area. However, ligno-cellulosic biomass is generally obstacle to
bioconversion because the lignin protective coat around cellulose impedes the efficient
hydrolysis of cellulose. For the easy conversion of ligno-cellulosic biomass to ethanol,

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the protective coat must be modified or removed so that efficient hydrolysis of cellulose
can occur. Reduction of lignin in sugarcane plants may be used to increase the sucrose
and ethanol yield with economical processing cost to fulfill the increasing energy
demands .

Different genera of Saccharum complex are known to differ widely in fiber

characteristics. Thus, the use of these diverse parents is considered imperative in
hybridization programs to maximize heterozygosity and maintain high levels of
variability in the progeny for selection of high sugar yielding and low lignin content
biomass sugarcane clones. Conventional breeding, molecular and biotechnological
approaches are being practiced as an attempt to alter the lignin composition of the plants
by down regulating some of the important enzymes in lignin biosynthetic pathway such
as cinnamoyl CoA reductase (CCR), cinnamoyl alcohol dehydrogenase (CAD), ferulate-
5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) .

Genetic diversity among the germplasm can be evaluated through different

molecular markers. Various molecular markers (RAPD, RFLP, SSR, AFLP and SRAP)
are being used for the study of genetic diversity in sugarcane germplasm . Along with
the natural variation in Saccharum complex, there is also possibility to create variability
and selection for desired characteristics through artificial means as the somaclonal
variation, a tissue culture tool, that have played role in varietal development for desired
characteristics . There is also need for the variability study in the micro propagated and
regenerated plantlets because it has been reported genetic variation in these plantlets as
compared to the parent clones. This variability may be useful for the selection of novel
characters as salt, drought and disease tolerance characters in crop plants . The
variability during tissue culture may be due to different reasons. Auxins concentration
and sub culturing may play important role for variability in the micro propagated
plantlets, derived from callus culture. This variation presence in the tissue culture may be
positive for selection of new agronomic desired characteristics, but in the genetic
transformation and micro -propagation such variation are not desired so these are
avoided . The possible genetic variability in the somaclones have been studied through
different molecular marker technique . It has been observed that to some extent the

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transgenic sugarcane plants also exhibit genotypic and phenotypic variability as

compared to plants of the parent cultivar and transgene show variable agronomic
response in the field . The variability might be due to somaclonal variations and foreign
gene expression. This genetic dissimilarity among the transgenic population and
untransformed control may be analyzed through some molecular marker techniques to
identify least genomic deviation from the parental clones that were used for
transformation . However, these tools sample the genomes randomly and may not
represent the true diversity for genes of interest. TRAP (target region amplification
polymorphism) marker technique is known to reflect the diversity in functional genes
(coding regions) and has been successfully applied on diversity in sucrose and drought
related genes and may be implied for diversity study among the Saccharum complex for
the desired genes .

1.5 Factors affecting sugarcane productivity

With the efforts of conventional, modern breeding and advance biotechnological

approaches, sugarcane has become the ideal crop for the future times that will full all the
requirements of rapidly growing population as food, feed, fiber and biofuel. To meet the
sugar need of the increasing population of the country and to make sugarcane a
profitable crop for the farmer it is much needed to improve the quantity and quality of
sugar per unit area. In spite of all management and varietal improvement efforts, sugar
and sugarcane production in Pakistan is much lower than most of advance cane growing
countries of the world .

There are multiple factors which affect the sugarcane yield and quality cane
production such as the genetic potential of the clone, pests including weeds, insect pest
borers’ infestation , low inputs availability, deficient irrigation (drought) or low quality
irrigation water availability , low and high temperature , salinity etc . In Pakistan, the low
cane and sugar yields are mainly attributed to salinity; drought, insect pest and diseases .

1.5.1 Insect pest problem

Among the other factors responsible for yield constraints in sugarcane, the insect
pest attack at the early and cane formation stage are very significant. In the insect pest

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population of sugarcane, top borrer Scirpophaga niveLLa (Lepidoptera: Pyralidae); stem

borer, ChiLo infuscateLLus (Lepidoptera: Crambidae); root borer, EmmaLocera
depresseLLa (Lepidoptera: pyralidae) and Gurdaspur borer, Acigona stenieLLa
(Lepidoptera: Crambidae) are the most harmful pests which cause significant loss in
sugarcane tonnage and sucrose contents . It has been reported that borers cause yield loss
up to 16-24 % in cane yield and 10 % in sugar recovery . Integrated approaches have
been implied for the control of borers in sugarcane as biological, chemical cultural
methods and breeding for resistant varieties. The most advance, economical and
environment friendly approach is the application of biotechnological techniques for the
development of insect resistant transgenic clones .

1.5.2 Drought conditions

Importance of water for yield of crop plants is very crucial, as it is very

essential structural element of plant tissues. Plants are constituted 85-95% of water and
essentially required for growth and development, any loss in water contents, causes
physiological and biochemical cellular changes in the plants . It is also essentially
required for food synthesis (Photosynthesis), nutrient uptake, movement, cooling agent
(Transpiration), respiration, support through turgor and a medium for intercellular
reactions . Numerous plant physiological processes are affected by even a mild water
stress (drought), the cell growth is most important one . Drought is the water deficit
condition for an extended period of time. Lack of precipitation and its spell vary in
different zones and in different years. It is normally the climatic feature and natural
disaster . Drastic effects of drought or water stress on agriculture crops are due to
shortage of soil moisture to meet the requirement of the crop .

In the drought stress conditions, a number of changes occur in plant

growth and metabolism however these changes vary with in the plant species . Drought
stress conditions normally causes reduction in biochemical process as protein synthesis,
photosynthesis, respiration and nucleic acid synthesis . About 17 % crop yield loss has
been estimated due to water stress conditions prevailing in the crop growing period .
Arid and semi arid climates accounts normally 30% of the world total area and Pakistan
is also situated in semi arid to arid climate of the world and receives low and variable
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rainfall in different months of the year mainly precipitation occurs in the moon soon
season (July -August) which is not sufficient for growing the crops . Ground water has
also declined due to insufficient recharge so in the region drought spell severely affect
the crop yield and some time complete failure in case of only artificial irrigation
arrangements as in barani areas of Pakistan.

Drought stress effects are variable for different crops and during different
growing periods, moderate deficit cause considerable morphological changes and severe
deficit may cause the death of the plant and it may be drastic for the crops like
sugarcane (Saccharum officinarum L.), a long growing period (9-12 months) and high
biomass producing crop per unit area, so it requires huge delta of water ( 60-80 acre
inches) till its maturity . Drought conditions in sugarcane hinder with sugar production
due to its glcophytic nature, which affects its growth rate, cane yield and sucrose quality
and contents . It has been estimated that drought stress may reduce the sugarcane crop
yield up to 50 % or even more depending upon the drought severity and growth period of
the crop and it may be more critical in Pakistan where the crop is grown only in irrigated
areas .

Irrigation and rainfall limitation are expected to amplify in the coming

decades , so the development of the genotypes with enhanced drought tolerance through
conventional breeding strategies, marker assisted selection and transgenic approaches
are too much desired and need of the time and future .

1.5.3 Salinity problem

Salinity is also among the most important factors, limiting the crop yield in
irrigated lands. It is difficult to access the precise losses due to salinity, however it is
estimated that the losses are significant and increasing with time . Salinity problem is a
crop production constraint, all over the world and it is estimated that 10-15 % of the total
land area of the world is affected by erosion, physical and chemical degradation
including soil salinity and sodicity . According to another estimate an area up to 955
million hectare is saline affected in the world, which makes approximately 25 % of the
geographical and 60 % of the cultivated lands of the world . The USDA salinity lab

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defines the soil a saline soil which has E.Ce 4 dSm-1 or more . Different crops show
different tolerance level to salinity and even some crops are affected at the E.Ce less than
4 dSm-1.

Pakistan is situated in arid to semiarid climate and thus evaporation

exceeds than precipitation which causes the development of salinity and sodicity in the
soils . Out of 20 million hectare cultivated land of Pakistan 6.7- 6.8 million ha is salt
affected . In the world large areas among the salt problem soils, exist under sodic soils
but in Pakistan saline sodic soils are more common than the pure saline or pure sodic
soils . The potential yield loss has been estimated 20 % due to salt affected soils in
Pakistan . In spite of the severe yield losses in problem soils, these are even taken into
some agriculture utilization and about 45% of the salt affected land is under cultivation
with better management practices for growing of different salt tolerant agricultural field
crops, forage grasses and shrubs etc .

Salinity affects the soil and plants both in different ways. Toxic effects of
salinity cause adverse effects on metabolism and growth of the plants and on soil
physical condition. Soil water osmotic potential is reduced under increased salt
concentration and subsequently the transpiration rate is also reduced . The toxic levels of
Na+1 and Cl-1 in the root system interfere with the uptake and transportation of water and
important nutrients. Sodium reduces the influx of Ca+2 by binding it to the plasma
membrane, inhibiting the influx and increasing the efflux of Ca +2 that causes depletion of
internal Ca+2 concentration . Similarly high level of Cl-1 would decrease nitrate (NO3-1)
and phosphate uptake under the saline conditions . Chloride induced NO3-1 deficiency is
responsible for growth retardation in plants exposed to high NaCl levels and there is
variable fertilization effect in saline conditions .

Different approaches are being employed to cope with the salinity problem,
as an extensive drainage system, conventional reclamation practices etc, but these
techniques are costly and time required, hence efforts are being employed on the
development of salt tolerant crops through conventional breeding, screening of the
germplasm for salt tolerance and adopting transgenic approaches to bring the salt

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affected lands under profitable cultivation and to feed the increasing population with self
sufficiency.

1.6 Need for the sugarcane crop yield improvement

Global food demand is increasing with the increasing population, about 80

million people increase per annum . It has been estimated that an increase of 38% global
food production will be needed by 2025 and 57% by 2050 to feed about 9 billion
population and in some reports it has been estimated that food production must be
doubled up to 2050 to feed this increasing population . Integrated efforts are being
implied through conventional breeding , Physio-biochemical molecular biology ,
biotechnology approaches and through better crop production and field management
practices on sustainable and precise basis to cope with the biotic and abiotic crop
limiting factors to maximize the yield, economic return and to fulfill the global
increasing demand for the food on sustainable basis .

Through the help of advance biotechnological approaches, a number of

transgenic crops having tolerance against herbicide, insect, salt and drought stress have
been developed and are being grown in many parts of the world commercially with
successful results to full fill the increasing demand of the world population .

1.7 Approaches for sugarcane improvement

1.7.1 Conventional breeding

Conventional breeding is an important tool to evolve varieties better than the

existing one, in terms of tons of cane and sugar per unit area. In the world, there has been
considerable achievement for the improvement of the sugarcane clones through
conventional breeding. Efforts have been implied to evolve the varieties with higher
yield and efficient utilizer of irrigation and fertilizers. A steady increase in yield is
required to meet the challenges of increasing costs of production inputs and to feed the
increasing population demand of the world. To evolve new cultivar through systematic
breeding programme is a lengthy process which takes normally 12-13 years for the
selection process, field evaluation and testing for genetic stability and some time at the
end of the selection cycle some varieties have to be abandon due to some disease
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susceptibility . Improvement of sugarcane through conventional breeding is only

possible in the countries where sugarcane plants flowers profusely and produces viable
cane fuzz .

In the climatic conditions of Pakistan, sugarcane plants does not flower

profusely and viable cane fuzz is not produced and the improvement or development of
new clones having tolerance against biotic and abiotic stresses is dependent only on
selection breeding of the imported cane fuzz . So there is dire need for the application of
modern biotechnological approaches for the improvement, development of sugarcane
clones having tolerance against biotic and a biotic stresses and promise for higher cane
and sugar yield.

1.7.2 Biotechnological approaches

Sugarcane has the most complex genome, than any crop plant , the
commercial sugarcane cultivars are the result of a limited series of crosses and
backcrosses derived from two Saccharum species and are poly-aneuploid hybrids with
chromosome numbers in excess of 100 . The genetic pool of sugarcane does not possess
resistance to many diseases and pests , high ploidy, low fertility, large genome and
complex environmental conditions make conventional breeding and genetic studies
difficult for this crop . In spite of all these complexes, although some important goals
have been achieved for cane improvement through plant breeding and genetics, however
it has been realized that exploitation of desired variation through conventional methods
has got limited success for sugarcane improvement against stress tolerance .
Furthermore, it has been observed that in most of the cases improvement of the
sugarcane through conventional breeding for some specific traits such as sugar contents
has reached at its plateau due to narrow gene pool and genomic complexity .

It is highly desirable to improve the sugarcane with in short time to fulfill

the increasing production demands according to the environmental conditions, through
the introduction of novel genes or manipulation of native genes . In the present scenario,
physio-biochemical, molecular and biotechnological approaches have assertion to meet
the most of the challenges of the sugarcane crop improvement .

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The currently available modern plant genetic resources are greatly

enhancing the ability to identify abiotic (salt, drought, cold) and biotic (Insect pests and
diseases) responsive genetic determinants . The identification, molecular cloning and
expression of these genes in the plant tissues, which enable plants to adapt or tolerate
such stresses, is very essential for the crop improvement programmes .

Different techniques are being applied for the improvement of the crops
plants through biotechnological approaches, as simple tissue culture to the advance
molecular and genetic engineering approaches for the desired gene integration.

1.7.2.1 Tissue culture (In vitro)

An established in vitro tissue culture system is a prerequisite for the most

sophisticated biotechnological studies. Tissue culture, is a simple biotechnology
technique used for micro-propagation, rapid in vitro multiplication as well as to make
improvements in the agronomic characteristics through somaclonal variation in
vegetatively grown plants . In sugarcane, a lot of work has been done for the
establishment of efficient in-vitro tissue culture system as rapid multiplication,
callogenesis and proficient shoot regeneration . Different tissue techniques are being
used for the crop improvement, as meristem tip culture for disease free nursery
production, rapid multiplication, somatic embryogenesis for high efficiency
regeneration, radiation exposure to induce somaclonal variation and anther, protoplast
culture for new varietal development . Sugarcane immature inflorescence segments have
been used for direct somatic embryogenesis without intervening callus phase to avoid
somaclonal variation. It may be used for the production of large number of plantlets in
short time and it may also be used as a target tissue for the genetic transformation with
great success . Meristem tip culture technique of tissue culture is being used extensively
in the world for healthy disease free and rapid seed multiplication of sugarcane
(Sugarcane mosaic free) and for the other vegetative grown crops .

Efficient protocols have also been developed for large scale rapid
multiplication through micro-propagation, of the local grown elite sugarcane commercial
clones, CP-77-400, BL-4, HSF-240 and CPF-237 in Pakistan . Screening could be done

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for the clones performing better against different deficiencies and diseases through the
regenerated soma clones . Through conventional breeding haploid cell production is
laborious, in the tissue culture technique anther culture has made it easy to produce
haploid embryos and to develop homozygous lines . The protoplast fusion technique of
tissue culture has make genetic transformation and integration successful for the desired
genes in the species which are incapable otherwise . Proficient and rapid in vitro protocol
have also been developed successfully for the direct somatic embryogenesis and
microprojectile bombardment with high transformation efficiency .

1.7.2.2 Molecular approaches

Integration of molecular and modern biotechnological approaches with the

conventional breeding tools, have facilitated to improve the plants for better qualitative
and quantitative traits . Molecular tools are being used as supplement tool in
conventional breeding of the crops in general and for sugarcane specifically . Molecular
approaches as marker techniques have made possible to identify the alleles or
quantitative trait alleles (QTA) at the quantitative trait loci (QTL) which contribute the
trait. The mapping in the population could be done using either SSR or SNP markers .
Molecular biology has also made possible the identification and characterization of
different causal organisms, which cause diseases in sugarcane and in other crops . The
precise characterization and identification of a problem, will ultimately lead towards the
effective solution within short period of time through modern available biotechnological
approaches.

There may be multiple goals for the improvement of a crop. In the breeding
of sugarcane (Saccharum officinarum L.), the major focus is given on variety
development with increased sugar yield. It may be improved either by increasing the
sugar contents (sugar recovery %) or the increase in stalk or cane yield, but the
correlation between the traits is very low . To make some significant improvements in
sucrose contents with other desirable traits it is very important to introduce novel or to
manipulate native genes to influence metabolism through molecular, biotechnological
techniques and transformation process .

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1.7.2.3 Genetic engineering

Development of transgenic crops has helped to meet the sustainable

development goals and has provided new opportunities to develop desirable agronomic
traits in crop , maximize the quality and quantity of food, feed and fiber . Genetic
manipulation of major crops has been possible by transfer of foreign DNA into the host
genome through direct DNA transfer procedures such as particle bombardment and
Agrobacterium mediated transformation methods . Many transgenic crops have been
developed successfully harboring the tolerance against biotic and abiotic stresses, the
key limiting factors for crop yield .

Protocols have been established for the gene transformation to improve

agronomic characteristics and tolerance against major biotic and a biotic stresses in
sugarcane and other important cereals such as rice, maize wheat, barley and sorghum .
Transgenic sugarcane having tolerance against a biotic and biotic stresses have been
developed successfully through microprojectile and Agrobacterium mediated
transformation of sugarcane callus and proficient protocol are available for regeneration
of the transformants to get maximum number of the putative transgenes . Both the
transformation methods (Agrobacterium and Microprojectile ) have their advantages and
disadvantages, however the transformation of sugarcane through Agrobacterium, faces
the problem of over growth of the bacterium and subsequent contamination in the sub
culturing of clumps of sugarcane cells . For the particle gene gun transformation of
sugarcane, standardization of different parameters i.e., DNA concentration, size of
explants (callus) to be transformed and firing distance between micro carrier launch
assembly and target tissue is much desirable for successful and high transformation
efficiency .

The plants with altered genome (transgenic plants) have been used
successfully for expression of recombinant proteins and this process may be an efficient
alternative for large scale proteins production. Sugarcane genome has been altered
successfully through genetic engineering for some specific purpose. It has been
demonstrated through many studies that cysteine, which are natural inhibitors of cysteine
protein-ases can be used successfully against the insect attacks. Sugarcane has also been
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used as a viable expression system for recombinant protein production under the control
of maize ubiquitin promoter . Different transgenic sugarcane lines with higher yield
potential have been developed successfully having resistance against, Fiji disease virus
(FDV), , ratoon stunting disease (RSD), sugarcane mosaic virus (SCMV), herbicide and
insect (sugarcane borers) especially for Diatraea saccharaLis (Lepidoptera: Crambidae)
through the transformation of the synthetic BaciLLus thuringiensis Cry1Ab,Cry1Ac and
Cry1b genes . Transgenic sugarcane clones have also been developed for altered
invertase isoform activity to increase sucrose accumulation , drought tolerant through the
transformation of trehalose synthase gene (TSase) and resveratrol synthase (RS) gene for
resveratrol biosynthesis to catalyze the coumaroyl CoA and malonyl CoA .

Studies have been conducted on the drought tolerant and sensitive clones
of sugarcane to make out the genes responsible for drought stress tolerance. It was
observed that few genes differentially expressed in tolerant plants, 94 % of these were up
regulated and 45% of the expresses genes in sensitive plants were down regulated during
the water stress situation . These studies have given insights for the identification of the
stress tolerant genes and development of stress tolerant sugarcane through the
transformation of these genes in the desired sugarcane clone (s).

The genetic transformation requires an efficient selection system to

reduce the escapes and to get maximum positive plantlets for the transformed gene.
Different selection agents i.e. neomycin phosphotransferase (npt11), hygromycin
phosphotransferase (hptII), phosphinothrincin (ppt) and phosphomannose isomerase have
been optimized with different concentrations by different researchers for different crops
and different cultivars of the same crop to get the stable transformants with maximum
transformation efficiency .

Sugarcane (Saccharum officinarum L.) is very complex poly-aneuploid

and consists of more than 100 chromosomes . Genetic complexity, low fertility and high
ploidy level 5X to 14 X (X= 5, 6, 8, 10, 12 or 14), make it difficult for the improvement
of some specific trait through conventional breeding . In Pakistan, its improvement
through genetic transformation is more attractive and needed, due to problem of viable
cane fuzz production and lack of conventional breeding. There is only selection breeding
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from exotic germplasm, and moreover it takes long time (10-12 years) to release a
variety . The alternative and sophisticated approach may be the genetic engineering,
which has make it possible to modify the plants to be able to grow under the biotic and
abiotic stresses without economic loss .

1.8 Rationale for the development of insect, drought and salt tolerant
transgenic sugarcane (Saccharum officinarum L.)

Sugarcane (Saccharum officinarum L.) is attacked by number of insect pest

species, sucking (wooly aphid, Jassid, whitefly, Pyrilla) and boring or chewing type (top,
stem, root borers). The insect pest attack at the economic threshold level, is one of the
important yield and quality limiting factor which cause considerable yield loss at farmer
field due to chlorophyll, yield reduction, secondary infection and deterioration of the
juice quality and ultimately the sugar recovery % loss at factory . There are considerable
losses (10-25%) in yield and quality of sugarcane due to insect pest (especially borers)
attack in sugarcane . The yield and quality losses may be variable in different varieties
and at different infestation level. It has been estimated that for every 1% bored and
rotting inter node there was 0.17- 0.23% sugar loss that makes 0.82 tons cane and 0.13
tons sugar yield loss per hectare .

To control the insect pest attack in crops chemical control is being used
intensively which kills the insect directly. However application of chemical pesticides
causes increase in cost of production and irreversible loss of natural environment,
ecosystem with the killing of non target insect and ill effects on human health . Through
conventional breeding it is not easy to develop pest resistant clones and some time at the
end of the selection period (10-15 years) the selected clone may become susceptible to
insect pests and diseases, that results in discarding the selected material . Modern
biotechnological approaches are ultimate solution for the development of insect resistant
crops with efficient and environment friendly mode. The biotechnological approaches
have scope to create effective resistance against lepidopterous pest complex of maize,
sugarcane and other crops . For the control of sugarcane borers, B. thuringiensis (Bt)
Cry1Ac/Cry1Ab genes have been integrated and expressed in sugarcane plants, resulted
in effective control of the borers of sugarcane under field growing conditions . The insect
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pathogen, BaciLLus thuringiensis (Bt), kills their hosts by the production of Cry and Cyt
toxins. The Cry toxins involves the sequential interaction with several insect midgut
proteins that causes the formation of oligomeric structure and insertion into the
membrane, which results in formation of pores in the membrane that kills the insect
midgut cells and leads to the death of the insect .

A biotic stresses are the great constraint in the crop production. About
70% loss of crop yield over worldwide is estimated due to the abiotic stresses . Water
deficiency, one of the major abiotic stress that limits the production of sugarcane
(Saccharum officinarum L.) and it is more critical in sugarcane growing areas where the
water deficient period prolongs or low rain fall period occurs during the grand growth
period of sugarcane . Different approaches and management practices are being applied
to overcome the water deficiency problem and one of the approach may be the breeding
and selection of the sugarcane families having resistant to drought or water stress
conditions but it is very difficult and lengthy process to select and then evaluate a variety
in the field having resistance to drought along with other good desired agronomic
characteristics then the ultimate precise and modern approach is the transgenic crop
plants development for abiotic stress resistance .

The molecular pathways and signals which are responsible for the biotic
and abiotic stress response, are not well understood in sugarcane . Different genes
responsive to stress tolerance have been isolated successfully from different plant
sources as Arabidopsis thaLiana (AtNHX1), ALDH (ZmALDH22A1) and AVP1 gene etc.
These stress tolerant genes, response against the abiotic stresses (Dehydration, high salt
exposure and absisic acid (ABA)), when present in plants naturally or transformed
through genetic engineering under some suitable promoter as CaMv 35- S promoter. The
tobacco transgenic plants over expressing the stress tolerant gene (ZmALDH22A1),
SodREF3 (a sugarcane cDNA that encodes a 201 –amino acid DNA –binding protein,
which acts a transcriptional regulator of ethylene responsive factor), have shown
elevated tolerance against stresses (salt, drought and osmotic) by the reduction of
malondialdehyde (MDA) derived from cellular lipid per oxidation and Sod REF3
induction by ethylene, ABA, salt stress and wounding .

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Salinity, sodicity (salt stress) are also amongst the major crop yield,
limiting factors over all the world . Toxic harmful effects of salinity cause growth and
yield loss due to osmotic effect or ion specific effects on plants . Plants productivity is
severely affected under the salt stress condition and changes in sodicity-salinity
parameters are one of the important characteristics changes in agro ecosystem . Different
field crops have variable tolerance for salinity and above the threshold level of salts, the
yield decreases as salinity increases . Considerable losses in yield are known, in
sugarcane crop due to the soil sodicity and salinity problem . In spite of the direct salt
affected soil, the brackish irrigation water also reduces the yield and quality of sugarcane
and other crops, however the losses may be variable in different cultivars .

To cope with the salinity problem, physical, chemical and biological

strategies are being practiced as development of salt tolerant species through
conventional breeding and exploitation of genetic variability, soil reclamation through
extensive drainage, development of genetically modified plants and use of
osmoprotectants etc . Application of novel approaches as the development of GM crops,
having tolerance against salt and other stresses has provided good opportunity to cope
with the problem in short time and in the precise manners .

1.9 Multiple gene transformation

Multiple gene transformation in the crop plants is necessary for

sophisticated genetic manipulation strategies, such as the stacking of transgenes
specifying different agronomic traits and the expression of different polypeptide subunits
making up a multimeric protein. The multiple gene stacking may be achieved by single
gene transformation followed by the crossing of plant lines carrying different transgenes,
but it is much quicker and convenient to introduce all the necessary genes
simultaneously . Multi gene transformation has been successfully accomplished by many
scientists, in different crop plants, through different techniques as co-transformation,
combined delivery of multiple transgenes and sexual crossing between the transgenic
having different transgenes . In sugarcane, transformation and integration up to six
plasmids had been reported through Agrobacterium mediated genetic transformation.
The integration was confirmed through PCR and Southern dot blot detection and
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bioassay confirmed the resistance of the transgenic sugarcane clones against the wooly
aphid (Ceratovacuna Lanigera) of sugarcane .

Sugarcane may be benefitted from multiple gene transformation.

Relatively stable transformed regenerate able calli and vegetative propagation in
sugarcane has advantage for development of transgenic sugarcane. Major technical
challenge facing plant transformation biotechnology is the development of methods and
constructs to produce transgene expression without collateral genetic and tissue damage .
Stable transformation and integration of genes for transgene expression can be confirmed
by detailed molecular analysis. To improve the complex agronomic traits and
biosynthetic pathways it is greatly needed to pool up the multiple genes into crops . The
technique of transformation depends upon the available facility and plant species to be
transformed. Among the different transformation techniques Agrobacterium and particle
gene gun (micro particle bombardment) transformation methods are being extensively
used for genetic modifications into plants .

Gene gun or particle transformation is a simple technique for foreign DNA

transformation into tissues. Multiple genes on a single plasmid or separate plasmids
could be transformed on a single plasmid or separate plasmids by co transformation
method . Hadi and colleagues have co-transformed 12 different plasmids successfully in
soybean through particle bombardment and have evaluated that all of the co-transformed
genes were present in most of the transgenic events and found no preferential uptake and
integration of any transformed plasmid . It was also evaluated in the transgenic events
with co transformation via particle bombardment that individual plasmids copy number
was approximately equal with in clones but there was found great variability between the
clones. Copies were confirmed through Southern analysis and it was also further found
that some clones contained up to three copies while others contained 10-15 copies of the
each plasmid transformed . Extensive research work has been undertaken to understand
the mechanisms of transformation and integration of foreign DNA into plant genome,
however little is known about equivalent mechanisms for foreign DNA integration into
plants generated through particle bombardment or other direct DNA transfer method
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and with the Agrobacterium tumefaciens . Increased transformation efficiencies are

requirement for the production of multiple transformants containing different constructs.
Through improved microprojectile mediated co-transformation and selection system,
now it has become possible to introduce and study the argonomically important genes
into commercial clones of sugarcane .

The present research work was conducted for the transformation and
integration of multiple genes (AtNHX1, AVP1 and Cry1Ac) into promosing and
commercial clones of sugarcane, for abiotic and biotic stress tolerance through particle
gene gun into with the following objective (s).

1.10 Objective of the study

The objective of the present research work was

 Optimization of tissue culture of different commercial and promising

clones of sugarcane.
 Genetic transformation of multiple genes (AtNHX1, AVP1 and Cry1Ac),
in the better tissue culture responding sugarcane clones through particle gene gun
method.

 Molecular, agronomic and physiological analysis of transgenic plants.

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