REVIEW ARTICLE

Biotechnological Developments in Sugarcane Improvement:An Overview

P. Suprasanna • V. Y. Patade • N. S. Desai • R. M. Devarumath •

P. G. Kawar • M. C. Pagariya • A. Ganapathi • M. Manickavasagam •

K. H. Babu

Received: 22 July 2011 / Accepted: 10 October 2011 / Published online: 2 November 2011! Society for Sugar Research & Promotion 2011

Abstract Sugarcane (Saccharum officinarum L.) is oneof the most important field crops grown in the tropics and

sub-tropics. More than half of the world’s sugar is derived

from sugar cane. Conventional methods have greatly con-tributed to crop improvement; however limitations such as

complex genome, narrow genetic base, poor fertility, sus-

ceptibility to biotic and abiotic stresses and long duration tobreed elite cultivars still impose a challenge. Sugarcane,

thus, is a suitable candidate for application of biotechnol-

ogy and genetic engineering tools. In this direction, in vitro

culture systems and related biotechnologies have beendeveloped as novel strategies for sugarcane improvement.

Studies have been conducted towards employing in vitro

culture combined with radiation/chemical induced muta-genesis for mutant isolation. Advancements in genomics

tools have paved the way for a detailed understanding of

the mechanism underlying biotic and abiotic stressresponses. The potential of the current genomics programs,

aimed at elucidating the structure, function, and interac-

tions of the sugarcane genes, will revolutionize the appli-cation of biotechnology to crop improvement. Genetically

modified sugarcane with increased resistance to agronomic

traits including biotic and abiotic stresses, yield and juicecould become useful in breeding for better varieties. This

review outlines some of the biotechnological developments

that are in place and tailored to address important issuesrelated to sugarcane improvement.

Keywords Sugarcane ! Biotechnology ! Genomics !Molecular markers ! Stress tolerance ! In vitro culture !Mutagenesis ! Transgenic plants

Introduction

Sugarcane (Saccharum spp.) is an important industrialcrop, ranking among the ten most planted crops in the

world. Besides being the major sugar contributor with more

than 70% of the world’s sugar, sugarcane is important asthe raw material for sugar producing and allied industries.

India is the largest single producer of sugar including tra-

ditional cane sugar sweeteners, khandsari and Gur equiv-alent to 26 million tonnes raw value followed by Brazil in

the second place (Indian Sugar 2008). The Saccharumcomplex includes the agronomically and industrially

P. Suprasanna (&)Functional Plant Biology Section, Nuclear Agriculture &Biotechnology Division, Bhabha Atomic Research Centre,Trombay, Mumbai 400085, Indiae-mail: penna888@yahoo.com

V. Y. PatadeMolecular Biology and Genetic Engineering Division, DefenceInstitute of Bio-Energy Research, Haldwani, Nainital 263 139,Uttarakhand, India

N. S. DesaiDepartment of Biotechnology & Bioinformatics, Padmashree Dr.D.Y. Patil University, Sect-15, C.B.D. Belapur, Navi Mumbai400614, India

R. M. Devarumath ! P. G. Kawar ! M. C. Pagariya ! K. H. BabuMolecular Biology & Genetic Engineering Division, VasantdadaSugar Institute, Manjari (Bk.), Pune 412307, Maharashtra, India

A. GanapathiDepartment of Biotechnology, School of Life Sciences,Bharathidasan University, Tiruchirapalli 620024, Tamil Nadu,India

M. ManickavasagamDepartment of Bioinformatics, School of Life Sciences,Bharathidasan University, Tiruchirapalli 620024, Tamil Nadu,India

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DOI 10.1007/s12355-011-0103-3

important sugarcane genotypes obtained from S. officina-rum, S. spontaneum and S. robustum crosses. Conventionalbreeding has greatly contributed to the development of

agronomically improved varieties, however limitations

such as narrow gene pool, complex genome, poor fertility,and the long breeding/selection cycle make it difficult to

undertake further improvement. In addition, modern vari-

eties have a variable chromosome number (2n = 100–120)and rarely flower. Sugarcane is a typical glycophyte and

hence exhibits stunted growth or no growth under salinity,with its yield falling to 50% or less than its true potential.

To sustain sugarcane production and to improve the pro-

ductivity, tolerance to biotic and abiotic stresses, nutrientmanagement, and improved sugar recovery are some of the

challenges. Both the conventional and biotechnological

methods need to be integrated in solving some of theseconstraints.

Agronomically improved sugarcane varieties endowed

with tolerance to biotic and abiotic stresses are highlybeneficial, as unfavourable environmental factors can

challenge cultivation and crop productivity. Although

crops tolerant to biotic and abiotic stresses have beenselected by traditional breeding programs, speeding up the

pace is essential in developing improved varieties. Salinity

in the root zones of sugarcane decreases the sucrose yield,through its effect on both biomass and juice quality. The

complexity and polygenic nature of salinity tolerance has

further limited the efforts to develop the tolerant cropvarieties through conventional breeding practices. In this

regard, biotechnological approaches including somaclonal

variation, in vitro mutagenesis and selection are beingapplied for the isolation of agronomically useful mutants

(Jain 2005). In vitro mutagenesis and selection has been

successfully used for improvement in agronomic traits likesalinity and drought tolerance in different crop plants

advocating that tissue culture selection is useful to select

stress tolerant clones. Many examples related to differentvegetatively propagated plants, show that the combination

of in vitro culture with selection is relatively inexpensive,

simple and efficient. Studies using in vitro cultures com-bined with radiation induced mutagenesis have been con-

ducted to isolate mutant clones in sugarcane (Patade and

Suprasanna 2008).There has been a tremendous success in gene transfer

from a wide variety of plant and non-plant sources to

sugarcane. With the availability of efficient transformationsystems, selectable marker genes and genetic engineering

tools, it should also be possible to clone and characterize

useful genes and improve commercially important traitsinto elite germplasm that subsequently can lead to develop

an ideal plant type of sugarcane (Lakshmanan et al. 2005).

Figure 1 presents areas of contemporary research interestand progress. The range of potential applications being

developed through transgenic plants in sugarcane include,

insect resistance, resistance to viruses, altered sucrosecontent, lignin modification, sucrose accumulation and

herbicide resistance. Sustained efforts are being made to

engineer sugarcane varieties that can produce high-valuecompounds such as pharmaceutically important proteins,

functional foods and nutraceuticals, biopolymers and pre-

cursors and enzymes and biopigments. This will go a longway in launching sugarcane as a biofactory (Brumbley

et al. 2008).

Sugarcane Fingerprinting and Marker AssistedSelection

DNA markers have contributed greatly, for fingerprintingof elite genetic stocks, assessing of genetic diversity,

increasing the efficiency of trait selection, and diagnostics.

Various molecular marker systems including restrictionfragment length polymorphism (Lu et al. 1994), ribosomal

RNA (Glaszmann et al. 1990), mitochondria and chloro-

plast genes (D’Hont et al. 1993), RAPDs (Nair et al. 1999;Kawar et al. 2009), and simple sequence repeats (SSR;

Cordeiro et al. 2003; Selvi et al. 2003) have shown use-

fulness for differentiating the genera and to assess germ-plasm diversity within the genus Saccharum.

Fig. 1 Sugarcane biotechnology: research areas of contemporaryprogress

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RAPD markers have been used to assess the genetic

diversity in elite and exotic sugarcane germplasm (Kawaret al. 2009; Srivastava and Gupta 2006; Nair et al. 1999,

2002) and construct genetic maps (Mudge et al. 1996). The

potential of ISSR markers for molecular profiling wasassessed in sugarcane using 42 varieties of subtropical

India (Srivastava and Gupta 2008). Virupakshi and Naik

(2008) used organellar genome inter-simple sequencerepeat markers (cplSSR and mtISSR) to analyze red rot

disease resistant/moderately resistant and susceptible elitesugarcane (Saccharum spp. hybrid) genotypes. The results

indicated that these markers may be used as a new tool for

the identification of the disease resistant varieties. In sug-arcane AFLP markers have been used to study diversity

existing among tropical and subtropical Indian sugarcane

cultivars (Selvi et al. 2005), and Saccharum complex andErianthus (Selvi et al. 2006; Besse et al. 1998).

One of the major ways sugarcane industries have

already benefited from molecular markers is the use ofSSRs for cultivar identification. SSR markers have been

used to fingerprint 180 sugarcane varieties and the data

stored in a database which could provide a source ofinformation to identify varieties of unknown or disputed

origin and as additional information in Plant Breeding

Rights applications and for quality assurance for deliveryof new cultivars to the industry (Piperidis et al. 2004).

Target region amplification polymorphism (TRAP) has

also been used to characterize the germplasm from thegenera Saccharum, Miscanthus, and Erianthus with the

help of six primers designed using sucrose- and cold tol-

erance-related expressed sequence tags (EST) sequences(Alwala et al. 2006). Development of new high-throughput

marker systems like single nucleotide polymorphisms

(SNPs) and diversity array technology (DArT) markers areexpected to have a major impact in the future for sugarcane

improvement.

Sugarcane Functional Genomics

In spite of immense economic importance, sugarcane

genetics has received relatively little attention as compared

to other crops, mainly due to its highly heterozygous,polyploid and frequently aneuploid nature, complex gen-

ome, poor fertility, and the long breeding/selection cycle

(Gupta et al. 2010). However advancement in moderntechnologies, including development of highly efficient

DNA sequencing techniques, identification of SNPs and

genome mapping, DNA microarray technologies for geneexpression analysis, RNAi (RNA interference) technology

and the rapid improvement in data mining tools, can have a

major influence on future sugarcane crop improvementprograms. At present, both micro- and macro arrays are

being used for the identification of genes expressed spe-

cifically in stems, disease resistance genes, and thoseinvolved in carbohydrate metabolism (Ulian 2000; Grivet

et al. 2001; Casu et al. 2005). Moreover the sequencing of

sugarcane ESTs greatly contributed to the gene discoveryprocess (Prabu et al. 2010). Prior to June 1996, the public

databases of DNA sequences had only 28 sequences from

sugarcane compared to the 270,000 sugarcane sequencesthat are deposited to date (P.G. Kawar, unpublished).

Another investigation using SSH approach has providedinsights into identification of salt induced genes in sugar-

cane leaves specifically to target rare transcripts, such as

those participating in cell signaling and the regulation ofgene expression (Patade et al. 2011b). The validated

expression data by real-time PCR can aid in assigning

function for the sugarcane genes and characterization ofregulatory sequences in sugarcane.

Genomics for Biotic Stress Tolerance

Sugarcane being a long standing crop is constantly chal-lenged by herbivorous insects, nematodes, fungi, bacteria,

and viruses. Plant defence responses to such perturbations

are largely mediated by phytohormones through triggeringconserved defence mechanisms, each with an intricate

signaling pathway leading to plant protection. It has been

shown that both the ethylene and jasmonic acid signalingpathways act synergistically in plant defence.

In a study to identify red-rot-related genes, Gupta et al.

(2010) used enriched subtractive cDNA library preparedfrom the C. falcatum challenged stem of sugarcane variety

(Co 1148) and reported at least 85 red-rot-specific clusters,

unique and not reported in the database previously. Inanother study cDNA-SSH library was constructed and

analyzed to identify the up-regulated genes in sugarcane

under SCGS infection condition. Subtracted library highlyrepresented genes potentially involved in cell rescue,

defence, ageing and apoptosis (13.1%). The forward SSH

approach implemented, allowed to explicate the transcrip-tional regulatory mechanisms of sugarcane in response to

SCGS infection and isolated the R2R3-MYB (SoMYB18)

gene, a potential candidate playing important roles in theregulation of secondary metabolism, signal transduction

during biotic, abiotic and other environmental stresses. In

an attempt towards studying the host–pathogen interactionand decipher the molecular basis of virulence of sugarcane

SCGS disease, Kawar et al. (2010a) isolated partial gen-

ome of first Asiatic strain of phytoplasma (SCGS) bygenomic-SSH. The library yielded 83 SCGS specific

fragments representing approximately 42% of the chro-

mosome of Sugarcane grassy shoot phytoplasma, com-prising approximately 85 predicted partial phytoplasmal

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CDS. Further, a species specific detection method was

developed for early detection of SCGS infection (Kawaret al. 2010b).

Genomics for Abiotic Stress Tolerance

Salinity and drought are important environmental factorsthat limit crop productivity. Sugarcane, being a typical

glycophyte, exhibits stunted growth or no growth undersalinity, with its yield falling to 50% or even more as

compared to its true potential (Akhtar et al. 2003; Wie-

denfeld 2008). High salt environment adversely affectsplant growth due to alterations in water relations, ionic and

metabolic perturbations, generation of reactive oxygen

species (ROS), and tissue damage (Patade et al. 2011a),enzymes involved in sugar metabolism (Gomathi and

Thandapani 2005) and respond with an altered expression

of stress responsive genes, which may ameliorate the det-rimental effects of salinity. Therefore construction of

cDNA libraries enriched for differentially expressed tran-

scripts is an important first step in attempting to studystress responsive genes. Patade et al. (2011b) constructed a

forward subtracted cDNA library from sugarcane plants

stressed with NaCl (200 mM) for 0.5–18 h to find mRNAspecies that are differentially expressed in sugarcane in

response to salinity stress. Sequencing the differentially

expressed few cDNAs clones led to the identification ofsalinity induced shaggy-like kinase (designated as sugar-

cane shaggy like protein kinase-SuSk). The expression was

induced by salt as well as PEG stress indicating that theinduction of this gene probably occurred in response to the

osmotic component of salt stress rather than the ionic

component.Gene expression profiling is an important tool to

investigate responses to environmental changes at the

transcriptional level. Transcriptomic study of short-term(up to 24 h) salt (NaCl, 200 mM) or iso-osmotic polyeth-

ylene glycol-PEG 8000 (20% w/v) stress has revealed

altered expression of representative stress responsive genesin sugarcane leaves (Patade et al. 2011c). Efficient

sequestration of Na? to vacuole, which reduces the cyto-

solic Na? concentration, is an important aspect of tissuetolerance to salinity. The authors reported down regulation

of a sugarcane homologue of NHX belonging to the family

of Na?/H? and K?/H? antiporters in response to the saltstress. Though the NHX transcript levels increased tran-

siently at 2 h PEG treatment, expression of NHX was

repressed in response to salt or PEG stress subsequently upto 24 h and in sugarcane plants stressed with salt or PEG

for 15 days and correlated to growth inhibition (Patade

2009). The transcript levels of P5CS, an important gene of

the proline biosynthesis pathway, did not alter significantly

upon exposure to salt stress, but were severely reduced inresponse to PEG stress. On the other hand, expression of

PDH, which plays a role in proline catabolism, was

inhibited in response to salt stress and induced under PEGstress treatments (Patade et al. 2011c). On long-term

exposure to salt or PEG stress the steady state levels of

both P5CS and PDH gene expression increased (Patade2009), which also correlated to proline accumulation under

these stress conditions (Patade et al. 2011a). The steadystate transcript levels of CAT2 were also lower in non-

primed stressed sugarcane plants in response to iso-osmotic

salt or PEG stress for 15 days however, the expressionlevel was maintained in priming induced stress tolerant

plants under the stressed conditions (Patade et al. 2009).

To understand the molecular basis of salt (NaCl 2%)stress response, Pagariya et al. (2010) carried out cDNA-

RAPD-based gene expression at early growth stage in

tolerant sugarcane variety Co 62175. Among 335 differ-entially expressed transcript-derived fragments (TDFs),

156 up- and 85 down-regulated were sequenced. The 17%

TDFs representing potential transcripts involved in stresstolerance and plant defense in sugarcane were reported.

Further, Pagariya et al. (unpublished) identified 137 salin-

ity tolerant candidate cDNAs from sugarcane, 20% ofwhich were novel sugarcane genes. These unique sequen-

ces that have so far not been reported to be stress related

might provide further understanding on the perception,response and adaptations mechanisms of non model plant

like sugarcane to salinity stress.

In the study of various tissue-specific EST librariessequence data of Indian subtropical sugarcane variety (CoS

767), 25 water-deficit stress-related clusters showed greater

than twofold relative expression during 9 h dehydrationstress (Gupta et al. 2010). Further, recently Prabu et al.

(2010) based on sqRT-PCR analysis showed higher tran-

script expression of WRKY, 22-kDa drought inducedprotein, MIPS and Ornithine-oxo-acid amino transferase at

initial stages of stress induction with a gradual decrease in

advanced stages. Analysis of the expression of these stress-responsive genes in sugarcane plants under water deficit

stress revealed a different transcriptional profile compared

with sucrose accumulation. Prabu et al. (2010) identifieddifferentially expressed transcripts in response to water

deficiency stress in sugarcane cv. Co740 using PCR-based

cDNA suppression subtractive hybridization technique. Ofthe sequenced 158 cDNA clones based on Dot blot, 62%

showed similarity with known functional genes, 12% with

hypothetical proteins of plant origin, while 26% repre-sented new unknown sequences. Annotation of these dif-

ferentially ESTs indicated their possible function in

cellular organization, protein metabolism, signal trans-duction, and transcription.

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Involvement of Sugarcane miRNA in Abiotic StressResponses

MicroRNAs (miRNAs) are small single stranded, non-

coding, naturally occurring, highly conserved families oftranscripts (18–25 nt in length). Several miRNAs are either

up regulated or down regulated by abiotic stresses, sug-

gesting that they may be involved in stress-responsive geneexpression and stress adaptation (Sunkar and Zhu 2004).

The involvement of miRNAs in abiotic stress has been

studied in plants in response to dehydration or NaCl byusing expression analysis, suggesting stress specific regu-

lation of expression of miRNA (Patade and Suprasanna

2010) in sugarcane. In response to long term (15 days) iso-osmotic (-0.7 MPa) NaCl or PEG stress, no change in

mature transcript level of miR159 over the control was

detected. However, under the short-term (up to 24 h) saltstress, transcript level of the mature miRNA increased to

112% of the control at 16 h treatment. The mature tran-

script level of miR159 was higher under all the PEGinduced osmotic stress treatments as compared to the

control, and it progressively increased with stress exposure

period (1.3 fold at 8 h treatment). This indicated thatexpression of miR159 gene was more responsive to osmotic

stress than ionic stress. The authors studied expression of

one of the predicted target MYB under the same stress(NaCl or PEG) conditions to study the changes in target

gene expression in response to over or under expression ofmiR159. The results on the expression of specific miR159and its targets could be useful in developing appropriate

markers for selection of tolerant cultivars in sugarcane.

In Vitro Culture Systems

In vitro culture methods in sugarcane have had a great

impact both on basic research and applied commercialinterest. These include micropropagation of elite clones,

production of disease-free planting material, generation of

agronomically superior somaclones, screening methods forbiotic and abiotic stress tolerance, and conservation of

novel and useful germplasm. In vitro techniques for the

mass propagation of healthy sugarcane plantlets via directand indirect regeneration pathways are well established and

are critical in numerous ongoing efforts to improve sug-

arcane germplasm through genetic engineering (Snymanet al. 2011). In direct morphogenesis, plants are regener-

ated directly from tissues such as immature leaf roll discs

and also from shoot tip culture, by which sugarcane ispropagated commercially (Hendre et al. 1983). Indirect

morphogenesis involves initial culturing of leaf roll sec-

tions or inflorescences on an auxin-containing medium toproduce an undifferentiated mass of cells, or callus.

Somatic embryogenesis has been useful for propagating

large number of uniform plants in less time, for obtainingvirus resistant plants through somaclonal variation, muta-

genesis and in vitro selection and developing transgenic

plants (Suprasanna and Bapat 2006; Suprasanna et al.2007a). The in vitro conservation methods can be useful in

order to obviate problems of viability during storage for

extended periods, manpower requirements and need forlarge growth facilities. These methods can also facilitate

maintenance of elite lines, transgenic material and mutantstill their field establishment and/or approval. In vitro

preservation of sugarcane germplasm has been explored

using slow growth (Chandran 2010) and cryopreservationtechniques (Gonzales-Arnao and Engelmann 2006).

Direct adventitious plant regeneration in sugarcane has

been achieved from immature inflorescence tissues (Desaiet al. 2004a), immature leaf thin cell layers (Lakshmanan

et al. 2006) and leaf segments (Manickavasagam and

Ganapathi 1998; Gill et al. 2006) and leaf midrib explants(Franklin et al. 2006). Table 1 presents successes achieved

in the area of in vitro plant regeneration from Indian sug-

arcane cultivars and Saccharum species. The direct somaticembryogenesis (DSEM) system is useful for cost-effective,

large-scale clonal propagation besides providing a

new target explant source for genetic transformation(Suprasanna et al. 2007a). For an efficient application, it is

also essential to ensure the rapid development of embryos

from cultured explants with subsequent regeneration intocomplete plants. The plants derived through DSEM have

been found to be uniform in growth pattern with more

vigour compared to plants derived through indirect somaticembryogenesis pathway and exhibited no variation at the

molecular level (Suprasanna et al. 2006b). This method

also yielded a large number of plants (7–8 per explant) in ashort span of 7 weeks. Assuming an average of 24 seg-

ments per inflorescence, the total number of plants that can

be generated could be around 185–200 (Desai et al. 2004a).Subsequently this technique of direct somatic embryogen-

esis has been extended to several other Indian sugarcane

cultivars suggesting wide adaptability.Studies have been made to refine and improve frequency

of callus induction, proliferation and plant regeneration by

using different media, growth regulators and other addi-tives (Snyman et al. 2011). Alternative strategies such as

partial desiccation (Desai et al. 2004b), silver nitrate and

copper sulphate have also been attempted for improvingregeneration response (Patel et al. 2007). Cefotaxime has

been found to be beneficial for improving frequency of

shoot multiplication and elongation (Kaur et al. 2008)Partial desiccation of the immature inflorescence derived-

embryogenic callus was found to enhance plant regenera-

tions frequency, faster regeneration response and vigorousgrowth of the plants (Fig. 2). Sugarcane propagation

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through mature sugarcane stems is expensive as it incurslabor costs and biologically the method has problems of

spreading viral, bacterial and fungal diseases. Synthetic

seed technology is emerging as a novel tool in plant biol-ogy (Suprasanna et al. 2006a) and synthetic seeds are

potential delivery systems and provide an alternative to

current high-cost vegetative propagation and for storage ofnovel and important germplasm. In sugarcane, there have

been very few studies demonstrating the synthetic seed

research. The somatic embryos derived through directsomatic embryogenesis of sugarcane (Desai et al. 2004a)

were encapsulated in sodium alginate and the beads

showed maximum percentage of germination (73%) onhalf strength MS media (Fig. 3). Synchronous somatic

embryo production combined with good plant regeneration

can be useful for synthetic seed technology in sugarcane.

Broadening Genetic Variability Through In VitroCulture and Mutagenesis

Somaclonal variation has emerged as an important in vitroculture tool for crop improvement. This system has been

adopted for improving the quality and production of

sugarcane and somaclones for yield, sugar recovery,disease resistance, drought tolerance, and maturity have

been isolated. Sugarcane was amongst the first plants

where somaclonal variation was reported (Heinz and Mee1969; Larkin and Scowcroft 1981). Physical and chemical

mutagens have been applied to in vitro cultures so as to

enhance the frequency of genetic variation and obtainbeneficial modifications in cultivars (Patade and Supra-

sanna 2008; Suprasanna et al. 2010a). Physical (gamma

rays, ion beams) and chemical (ethyl methanesulfonate(EMS), sodium azide and sodium nitrite) mutagens have

been used successfully and their optimum mutagenic

treatments have been devised (Patade et al. 2008; Ali et al.2008; Kenganal et al. 2008; Koch et al. 2010). For

example, the LD50 for gamma radiation was around 20 Gy(Saif-Ur-Rasheed et al. 2001; Patade et al. 2006), while the

ideal concentration and exposure time were reported as

40 mM for 2.5 h and 16 mM for 4 h for EMS (Kenganalet al. 2008 and Koch et al. 2010).

In vitro selection at the cellular level has been successful

in isolation of mutants for desirable traits by imposing invitro selection pressure either by incorporating fungal

pathotoxins or fungal culture filtrates for selecting disease

resistance (Rai et al. 2011) or incorporation of sodiumchloride, polyethylene glycol, mannitol for selecting salt or

drought tolerance (Suprasanna et al. 2008a). In sugarcane,

somaclonal variant lines resistant to eye spot diseasecaused by Helminthosporium sacchari were selected

(Larkin and Scowcroft 1983). Various researchers have

used mutagenesis and selection to isolate embryogeniccells and plants tolerant to the causal agent of red rot (Ali

et al. 2008; Singh et al. 2008; Sengar et al. 2009).

Mutation induction of in vitro cultures will require thatmeristematic cells or tissues and mitotically active cells are

cultured to prepare sufficient material for mutagenic

treatment. Intrasomatic competition discriminating muta-gen affected cells and causing loss of their cell progenies

may be controlled by modifying in vitro conditions

resulting in a better competitiveness of mutant cells. Insugarcane, partial desiccation has been used successfully to

stimulate and enhance somatic embryo differentiation

(Desai et al. 2004b) and enhance regeneration response ofgamma-irradiated embryogenic callus cultures (Suprasanna

et al. 2008b). Partial desiccation treatment can offer as a

simple and novel method in stimulating regenerationresponse of higher dose gamma- irradiated cultures.

Assessment of genetic fidelity among micropropagated

plants is important especially in polyploid plants likesugarcane. Besides morphological analysis, molecular

markers have been used for detecting genetic change in

tissue cultured raised plants (Jain et al. 2005; Lal et al.2008; Suprasanna et al. 2007a, b, 2010b), for detecting

Table 1 Development of in vitro regeneration systems in Indian cultivars of sugarcane

Cultivar(s) Explant Mode of regeneration Reference

Co94012, VSI 434 Shoot tips Direct regeneration Tawar et al. 2008

CoC-671 Shoot tips Direct regeneration Biradar et al. 2009

Co 91010, CoC 671 Inflorescence segments Indirect somatic embryogenesis Suprasanna and Bapat 2006

CoC 671 Immature Inflorescence Direct Somatic embryogenesis Desai et al. 2004a

Co Si 95071 Immature leaf roll Direct Somatic embryogenesis Geetha and Padmanabhan 2001

Co.J. 83, Co.J. 86 Inner leaf whorls Indirect somatic embryogenesis Gill et al. 2004

CoS96268 Young leaf rolls Direct adventitious regeneration Pandey et al. 2011

CoS 96268, CoS 95255 Shoot tips Direct regeneration Ramanand et al. 2007

Co J 64, Co J 83, Co J 86 Young leaf segments Direct regeneration Gill et al. 2006

Saccharum edule Immature Inflorescence Indirect somatic embryogenesis Chandran 2011

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genetic fidelity in meristem raised tissue culture plantlets

(Devarumath et al. 2007; Tawar et al. 2008; Doule et al.

2008) and for characterizing salt and drought tolerantradiation induced variants (Patade et al. 2006). Field testing

of tissue cultured progeny has been conducted by several

researchers and clones for improved traits have beenobtained (Geetha and Padmanabhan 2002; Sandhu et al.

2008; Suprasanna et al. 2008c).

Sugarcane Genetic Engineering

Sugarcane is the most suitable candidate for genetic engi-

neering because of its complex polyploidy nature, variable

fertility and genotype versus environment interactions. Theavailability of high frequency in vitro regeneration system

from various explants makes this crop as a suitable can-

didate for genetic manipulation. Several genes (for disease/pest resistance, salt and drought tolerance, and sugar

accumulation) targeted towards sugarcane improvement

have been introduced into sugarcane (Altpeter and Oraby2010; Hotta et al. 2011; Table 2). The success of transgenic

sugarcane plant production depends on the method used for

transformation, the target tissue/explants and tissue culture

regeneration system used. Various explant types (axillarybuds, apical meristems, immature inflorescences, leaf

segments)) have been used successfully to regenerate full

plants in sugarcane indicating that a wide range of totipo-tent target tissues are available for genetic transformation.

The improvements in microprojectile method and its sim-

plicity made this technology unavoidable in sugarcanegenetic engineering. Embryogenic callus can be used for

transformation via microprojectile method to develop

transgenic plants (Fig. 4). Despite the most useful, robustand routinely applied method, biolistic DNA method often

leads to complex transgene integration pattern which can

cause problems in subsequent analysis. On the contrary,Agrobacterium mediated transformation gained more usage

due to simplicity and efficiency to produce single copy

integration of transgene.First report by Arencibia et al. (1998) demonstrated

success in producing transgenic sugarcane plants and stable

integration of transgene and single copy of transgene notedin transgenic plants made the method useful (Enriquez-

Fig. 2 Desiccation ofembryogenic callus andimproved plant regeneration.a Embryogenic callus, b plantregeneration for embryogeniccallus, and c plant regenerationfrom desiccated callus

328 Sugar Tech (December 2011) 13(4):322–335

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Obregon et al. 1998; Manickavasagam et al. 2004).

Transgenic plant production requires selectable marker

genes that enable the selection of transformed cells, tissueand plants. The most routinely practiced are those that

exhibit resistance to antibiotics or herbicides. Since thereare perceived risks in the deployment of transgenic plants

containing these markers, alternate selection systems

referred to as positive selection and marker-free systemshave become useful (Suprasanna and Ganapathi 2010). In

sugarcane, Jain et al. (2007) used mannose for the selection

of embryogenic callus and found that increased mannoseimproved the overall transformation efficiency by reducing

the number of selection escapes.

Since the first report of successful transformation, sig-nificant progress (Fig. 5) has been made towards the

development of transgenic sugarcane endowed with resis-

tance to biotic stresses, particularly diseases and insectpests (Srikanth et al. 2011; Altpeter and Oraby 2010), viral

diseases, metabolic engineering and towards making sug-

arcane as a biofactory. Genes from bacteria such asBacillus thuringiensis (Bt) and Bacillus sphaericus, prote-

ase inhibitors, plant lectins, ribosome inactivating proteins,

secondary plant metabolites, and small RNA viruses havebeen used alone or in combination with conventional host

plant resistance to develop crop cultivars that suffer less

damage from insect pests (Srikanth et al. 2011). Transgenic

sugarcane for borer resistance was also reported using Cry

1Aa3 gene (Kalunke et al. 2009). Recently the efficacy of

native Cry1Aa, Cry1Ab and Cry1Ac against C. infusca-tellus in in vitro bioassays through diet-surface contami-

nation method and observed that the Cry1Ab as the mosttoxic among the three compounds (Arvinth et al. 2010).

Christy et al. (2009) transferred aprotinin genes to sugar-

cane cultivars. The in vivo bioassay studies showed thatlarvae of top borer Scirpophaga excerptalis Walker

(Lepidoptera: Pyralidae) fed on transgenics showed sig-

nificant reduction in weight and impairment of larvaldevelopment. Sucrose content is a highly desirable trait in

sugarcane. Sugarcane cultivars differ in their capacity to

accumulate sucrose and breeding programs routinely per-form crosses to identify genotypes able to produce more

sucrose. In this regard, transgenic approaches to manipu-

late native genes that influence metabolism may havesignificant application.

Most of the field trials of transgenic sugarcane are

related to the transgene expression for agronomic traits andare being undertaken in Brazil and Australia. The agro-

nomic traits like height, diameter and the number of stalks,

fibre content, disease resistance, and yield of transgenicclones were not significantly different from that of

untransformed sugarcane plants. However the field trials

of insect resistant transgenic sugarcane revealed some

Fig. 3 Synthetic seedsdeveloped using directsomatic embryos of sugarcanecv. CoC671. a Sodiumalginate beads with somaticembryos, b magnified view ofthe bead, and c germination ofembryos

Sugar Tech (December 2011) 13(4):322–335 329

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morphological, physiological and phytopathological vari-

ations (Arencibia et al. 1999). Sugarcane has a long historyof use as human food (as a source of sugar), molasses,

animal feed and for energy purpose. Possible exposure to

the GM sugarcane plants or pollen can be through workingwith the plants while conducting field trials or post harvest

analyses, and/or living near the area where the GM sug-

arcane plants are grown (Anonymous 2011). Commercialsugarcane cultivars rarely flower or produce seed in the

field, and exposure to non-GM sugarcane has not been

associated with any reports of allergic responses in Aus-tralia (Anonymous 2011). The report suggests that some

measures can be imposed to minimize exposure include

harvesting the GM sugarcane plants before flowering orremoving flower heads before anthesis.

Sugarcane (Saccharum hybrids) is an attractive candi-

date for metabolic engineering aimed at sustainable pro-duction of value-added biomaterials and feedstocks,

Table 2 Genetic engineering of sugarcane for different traits (modified from Hotta et al. 2011, and references therein)

Trait Gene Transformation method Reference

Reporter and Selection system

Neopmycin phosphotransferase npt-II Microprojectile Bower and Birch 1992

B-Glucuronidase uid-A Microprojectile Bower and Birch 1992

Hygromycin phosphotransferase hpt Agrobacterium Arencibia et al. 1998

Green fluorescent protein gfp Agrobacterium Elliott et al. 1998

Phosphinothricin acetyl transferase bar Agrobacterium Manickavasagam et al. 2004

Phosphomannose isomerase manA Microprojectile Jain et al. 2007

Herbicide resistance

Bialophos bar Microprojectile Gallo-Meagher and Irvin 1996

Phosphinothricine bar Agrobacterium Enriquez-Obregon et al. 1998

Glufosinate ammonium pat Microprojectile Leibbrandt and Snyman 2003

Disease resistance

SCMV SCMV-CP Microprojectile Joyce et al. 1998

Sugarcane leaf scald albD Microprojectile Zhang et al. 1999

SrMV SrMV-CP Microprojectile Ingelbrecht et al. 1999

Puccinia melanocephala Glucanase, chitanase & ap24 Agrobacterium Enriquez et al. 2000

SCYLV SCYLV-CP Microprojectile Gilbert et al. 2005

Fiji leaf gall FDVS9 ORF 1 Microprojectile McQualter et al. 2004

Pest resistance

Sugarcane stem borer cry1A Microprojectile Arencibia et al. 1999

Sugarcane stem borer cry1Ab Microprojectile Braga et al. 2003

Sugarcane stem borer cry1Ab Microprojectile Arvinth et al. 2010

Sugarcane stem borer cry1Aa3 Agrobacterium Kalunke et al. 2009

Proceras venosatus Modified cry1Ac Microprojectile Weng et al. 2010

Sugarcane canegrub gna Microprojectile Legaspi and Mirkov 2000

Mexican rice borer gna Microprojectile Setamou et al. 2002

Ceratovacuna lanigera gna Agrobacterium Zhangsun et al. 2007

Scirpophaga excerptalis Aprotinin Microprojectile Christy et al. 2009

Metabolic engineering/alternative products

Sucrose accumulation Antisense soluble acid invertase Microprojectile Ma et al. 2004

Fructo oligosaccharide IsdA Agrobacterium Enriquez et al. 2000

Polyphenol oxidase ppo Microprojectile Vickers et al. 2005

Polyhydroxybutyrate phaA, phaB, phaC Microprojectile Brumbley et al. 2007

p-Hydroxybenzoic acid hch1 and cp1 Microprojectile McQualter et al. 2004

Mannose manA Microprojectile Jain et al. 2007

Isomaltulose SI Microprojectile Wu and Birch 2007

Proline overproduction P5CS Microprojectile Molinari et al. 2008

330 Sugar Tech (December 2011) 13(4):322–335

123

particularly those derived from sucrose (a-D-glucopyrano-

syl-1,2-D-fructofuranose), the major storage product in

sugarcane (Birch 2007). Successful example of

establishing biorefineries at sugar mills to produce biofueland bioplastics and, engineering sugarcane to make new

bioplastics and alternative sugars could demonstrate that

this crop has the potential to contribute to BioEconomy(Brumbley et al. 2007).

Concluding Remarks

Sugarcane is a source of food and fuel, and biotechnologycan contribute to substantially increase the utility of this

crop. The successful application of biotechnological tools

will require reliable, high levels of transgene expressionand their stability over generations. The availability of

cellular and molecular toolbox has opened up a plethora of

prospects. Innovative in vitro culture systems have becomeavailable with potential for rapid propagation and gener-

ating novel germplasm with desirable traits. A greater

understanding of the crop using functional genomics andcellular methods will accelerate understanding responses to

biotic and abiotic stresses and their management. Profiling

of gene expression under conditions that affect crop yield

Fig. 4 Steps in genetic transformation using embryogenic callus in sugarcane. a Gus expression, b selection of callus on hygromycin medium,c regeneration on selection medium, d regeneration on selection medium, and e selection and regeneration

Fig. 5 Chronological events in the developments of GM sugarcane

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can aid in building up an ‘expression panel’ for sugarcane

cultivars which should become invaluable in target geneselection. Gene silencing is being used in transgenic

research aimed at down-regulation of endogenous genes in

sugarcane. Some of the important challenges include genediscovery, transgenics and controlled transgene expression,

sucrose metabolism and photosynthesis. The advances in

sugarcane biotechnology could become remarkable in thecoming years, both in terms of improving productivity as

well as substantially increasing the value and utility of thiscrop.

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