Biotechnology and Crop Improvement

Journal of Crop Improvement, 24:153–217, 2010Copyright © Taylor & Francis Group, LLCISSN: 1542-7528 print/1542-7535 onlineDOI: 10.1080/15427520903584555

Biotechnology and Crop Improvement

SATBIR S. GOSAL, SHABIR H. WANI, and MANJIT S. KANGPunjab Agricultural University, Ludhiana, India

Plant biotechnology, a major component of agricultural biotech-nology, deals with various aspects of plant tissue culture, genetictransformation, and molecular biology techniques. Tissue cul-ture methods offer a rich scope for creation, conservation, andutilization of genetic variability for the improvement of field,fruit, vegetable, and forest crops, and medicinal/aromatic plants.Micropropagation technology ensures true to type, rapid and massmultiplication of plants that possesses special significance in veg-etatively propagated plant species. This technology has witnesseda huge expansion globally, with an estimated global market of15 billion US$/annum for tissue-culture products. Some basictechniques of tissue culture, such as anther/microspore culture,somaclonal variation, embryo culture, and somatic hybridiza-tion, are being exploited to generate useful genetic variabilityfor obtaining incremental improvement in commercial cultivars.Production of secondary metabolites, such as food flavors, foodcolors, dyes, perfumes, drugs, and scented oils used in aromather-apy, through cell cultures and hairy root cultures, are leadingexamples of molecular farming. Cryopreservation of germplasmat the cell/tissue/organ levels, in liquid nitrogen at −196◦C, ishighly rewarding for establishing germplasm banks, especiallyfor vegetatively propagated crops and rare, endangered plantspecies. During the past 15 years, remarkable achievements havebeen made in the production, characterization, field evalua-tion, and release of transgenic varieties/hybrids in several crops.Transgenic varieties/hybrids of maize, cotton, soybean, potato,tomato, and papaya are now being commercially grown onabout 134 million hectares spread across 25 countries. Researchin genomics allows high-resolution genetic analysis for physicalmapping and positional gene cloning of useful genes for crop

Address correspondence to Dr. Satbir S. Gosal, School of Agricultural Biotechnology,Punjab Agricultural University, Ludhiana-141004, India. E-mail: [email protected]

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154 S. S. Gosal et al.

improvement. Molecular (DNA) markers help in precise character-ization of germplasm, construction of saturated linkage maps, andDNA fingerprinting of crop varieties. Molecular markers are nowincreasingly being used for marker-assisted gene pyramiding andalien gene introgression. Current research, involving large-scaleDNA sequencing, microarrays, and robotics, is heading towardsgene revolution and nanobiotechnology.

KEYWORDS biotechnology, tissue culture, micropropagation,haploids, genetic transformation, transgenic crops, gene-cloning,molecular markers, crop improvement

INTRODUCTION

Biotechnology in a broad sense has been practiced for centuries for curdmaking, food preservation, pickle making, and fermentation. However,biotechnology received a boost during the 1970s with the discovery ofrestriction enzymes, which led to the development of a variety of gene tech-nologies and is, thus, considered the greatest scientific revolution of the 20th

century. Biotechnology, in a true sense, deals with changing and improv-ing, more efficiently than traditional technologies can, the characteristics ofan organism at cellular and molecular levels for the benefit of mankind.Depending upon the biological system (organism) involved, the field ofbiotechnology may be divided into plant biotechnology, microbial biotech-nology, animal biotechnology, and human biotechnology. Plant biotechnol-ogy deals with cell and tissue culture, genetic transformation, gene cloning,DNA markers, and other molecular approaches. Unlike conventional plantbreeding, the biotechnological techniques for genetic modifications largelyoperate at organ, tissue, cell, protoplast, and molecular levels. These inno-vative techniques are considered an adjunct to the conventional methods forefficient and precision plant breeding (Kang et al. 2007).

TISSUE CULTURE

Plant tissue culture broadly refers to the in vitro cultivation of plants,seeds, and plant parts (tissues, organs, embryos, single cells, protoplasts) onnutrient media under closely controlled and aseptic conditions. It includesseveral specialized areas, such as induction of callus and plant regeneration,micropropagation, somatic embryogenesis, somaclonal variation, meristemculture, anther culture, embryo culture, protoplast culture, cryopreserva-tion, and production of secondary metabolites. Tissue-culture methods hold

Biotechnology & Crop Improvement 155

significant promise for creation, conservation, and utilization of genetic vari-ability for improvement of a wide variety of crop plants. Among these,micropropagation, somaclonal variation, and embryo, anther and protoplastculture have direct applications in crop improvement.

Microporpagation of Plants

Micropropagation of plants is one of the best and most successful exam-ples of the commercial application of tissue-culture technology. It entails invitro propagation of plants from very small plant parts (0.2-10 mm) in thelaboratory, followed by their establishment in soil under greenhouse condi-tions. An estimated more than 500 million plants belonging to different plantspecies are now being annually produced through micropropagation in theworld. Micropropagation industry is environment-friendly and requires verylittle raw material in the form of chemicals. This technology possesses thefollowing definite advantages:

1. True-to-type plants produced, i.e., identical to donor (in vitro cloning).2. Selected plant species can be multiplied anywhere in the world.3. Rapid and mass multiplication (1 to 10/cycle of 2 weeks each) of

elite clones/varieties otherwise difficult to multiply through conventionalmethods.

4. Independent of seasonal and raw material constraints.5. Production of disease-free plants.6. Elite plant material for national/international exchange of germplasm,

avoiding the risk of pathogens and insects.7. Micropropagated, field-grown plants give higher yield and exhibit better

quality.

Micropropagation involves four stages: 1) establishment of asepticcultures; 2) shoot bud/shoot multiplication; 3) induction of rooting andhardening; and 4) transfer of plantlets to soil. Micropropagation meth-ods for important field, ornamental, fruit, medicinal, aromatic, and forestplant species are now well established (Table 1). During genetic transfor-mation, this technique helps to increase the plant number of elite eventsfor precise characterization and efficient transfer of regenerated plants togreenhouse.

Likewise, during anther/pollen culture, micropropagation of regener-ants helps to minimize the risk of losing any genotype during hardeningand transfer to soil. Now scores of multimillion-dollar industries around theworld propagate a variety of plant species through tissue culture, whichallows environment-friendly industries to flourish. The clean planting mate-rial can certainly improve the yield potentials of vegetatively propagated


crops like sugarcane, potato, banana, strawberry, sweet potato, cassava, andornamental species. It is likely that automation of multiplication systems willbe commercially feasible within the next few years. Improvement of somaticembryogenesis (Gill, Gill, & Gosal 2004), coupled with embryo desiccationand encapsulation technology, may lead to the utilization of artificial seedsfor mass cloning of plants. Micropropagation possesses special significancein the following areas:

1. Production of high quality, disease-free, super elite planting material forfurther seed production, especially in the vegetatively propagated plantspecies.

2. Rapid spread of new varieties of vegetatively propagated crops like sug-arcane, potato, poplar, medicinal/aromatic plants (Gosal & Gill 2004) forcrop diversification.

3. Mass production of ornamental plants otherwise difficult to multiplythrough conventional methods for domestic and international markets.

4. Rejuvenation of old varieties/clones of vegetatively propagated crops forimproving their yield and quality.

5. Mass cloning of rootstocks in horticultural plants like citrus, peach, andapple.

6. Mass cloning of genetically superior (plus trees), cross-pollinated, andseed-propagated trees like eucalyptus.

7. Multiplication of male-sterile lines for hybrid seed production or themultiplication of F1 hybrids and transgenic clones/varieties.

8. Interstate/international exchange of germplasm without the risk ofpathogens and insects.

9. This technology possesses tremendous potential for making environmentclean and green.

Somaclonal Variation

Success of a plant-breeding program largely depends upon the nature andextent of genetic variability available in the germplasm. In this regard,somaclonal variation, i.e., the variation among callus-derived plants, is apotent force for broadening the genetic base, more particularly in the vege-tatively propagated species (Evans & Sharp 1986). Using in vitro selection,many million cells/protoplasts can be screened against various biotic andabiotic stress factors in a single petri dish, which is more efficient as com-pared with field screening that requires more time and space (Kaur et al.2001). Several interesting and potentially useful traits have been recoveredusing this method in sugarcane, potato, tomato, maize, rice, alfalfa, rape-seed, and mustard (Table 1). Recovery of novel variants that either do notexist or are rare in the natural gene pool—for example, atrazine resistancein maize, glyphosate resistance in tobacco, improved lysine and methionine


contents in cereals, increased seedling vigor in lettuce, jointless pedicelsin tomato, and Fusarium resistance in alfalfa—is of much significance.Genetic, cytogenetic, and molecular evidences for increased recombinationfrequency through cell cuture have now been provided (Larkin et al. 1993).Tissue culturing of wide-hybrids also helps in breaking undesirable link-ages and achieving introgression from alien sources. Several new varietieshave been developed through somaclonal variation in tomato, sugarcane,potato, celery, brassica, and sorghum. This simple and cost-effective tech-nique possesses a huge potential for the improvement of apomictic andvegetatively propagated plant species and, of course, seed-propagated cropplants with narrow genetic base (Table 1). In India, a somaclonal variantof a medicinal plant, Citronella java, has been released as a commercialvariety, ‘B-3,’ which gives higher yield and oil content than the originalvariety. Likewise, ‘Pusa Jai Kishan’ is a variety of B. juncea released as asomaclonal variant of ‘Varuna’ variety. However, under several situations,low plant-regeneration ability and the lack of correspondence in expres-sion of the trait in field-grown plants are the major problems (Karp 1995).The occurrence of somaclonal variation has been described in a wide vari-ety of crop plants for various traits (Table 1), including potato (Das et al.2000), sugarcane (Leal et al. 1994; Doule 2006; Jalaja et al. 2006; Singh et al.2008; Sengar et al. 2009), banana (Hwang & Ko 2004; James et al. 2007;Oh et al. 2007b), Prunus persica (Hammerschlag & Ognjanov 1990), applerootstocks (Rosati & Predieri 1990), and Heliconia bihai (Rodrigues 2008).Several somaclones have been released as improved cultivars.

Anther/Pollen Culture for Production of Haploids/Doubled Haploids

In self-pollinated crops, an inordinately long period is required to assem-ble desirable gene combinations from different sources in homozygousform. Generally, it takes 8-10 years to develop stable, homozygous andready-to-use materials from a fresh cross of two or more parental lines. Incross-pollinated crops, because of inbreeding depression, it becomes dif-ficult to develop vigorous inbreds for hybrid seed production programs.In this regard, haploids possessing a gametic chromosome number arevery useful for producing instant homozygous true-breeding lines. In addi-tion, haploids constitute an important material for induction and selectionof mutants, particularly for recessive genes. In conventional breeding, theearly segregating-generation populations involve variation attributable toboth additive and non-additive genetic effects (Khush & Virk 2002), whereasdoubled haploid (DH) lines exhibit variation only of additive genetic nature,including additive x additive type of epistatis, which can be easily fixedthrough a single cycle of selection. The elimination of dominance effectsleads to high narrow-sense heritability, and availability of sufficient seed ofeach DH line allows for replicated testing. Thus, in contrast to relatively


large segregating populations in conventional genetic studies, fewer DHlines are required for the purpose of selection of desired recombinants. Forinstance, in rice about 150 DH lines derived from F1, instead of 4,000-5,000F2 plants, are sufficient for selecting desirable genotypes. Production of hap-loids has also been exploited during wide hybridization for the developmentof addition and substitution lines.

Anther/pollen culture is an attractive alternative for developing hap-loids (sporophytes with gametophytic chromosome number). One of thevery popular methods for production of haploids is anther or microsporeculture. Incubation of cultures under optimum conditions leads to growthof microspores into sporophytes. The following parameters have been rec-ognized as particularly important for successful anther/microspore culture:(i) growth conditions of donor plant, (ii) genotype of donor plant, (iii)pretreatment of anthers, (iv) developmental stage of anthers/microspores,(v) composition of culture medium, and (vi) physical conditions during cul-ture growth. Anthers are cultured in liquid or on semisolid agar medium (Gillet al. 2003), where they may directly give rise to embryoids or may lead tocallus formation before differentiation. The embryoids develop into haploidplantlets or doubled haploids in some crops (because of spontaneous dou-bling of chromosomes during callus proliferation). Haploids may be treatedwith colchicine to obtain fertile, doubled haploid homozygous plants forfield testing and selection. Production of haploids/doubled haploids throughanther culture from F1 rice plants results in true-breeding plants in lessthan one year, which otherwise takes 7 to 8 generations through conven-tional methods (Gosal et al. 1996). Several cultivars are either in test orhave been released in rice, wheat, maize, rapeseed, and mustard in China,Canada, Denmark, the United States and France (Guzman & Zapata-Arias2000; see Table 1). But in several instances, poor androgenesis, occurrenceof mixoploids, and albino plants have been the recurring problems.

Attempts have also been made to produce haploids in grain-legumeslike pigeon pea (Bajaj, Singh, & Gosal 1980) and vegetable crops, includingtomato (Segui-Simarro & Nuez 2007). In the case of tomato, both game-tophytic and sporophytic calli were produced from cultured anthers. Invitro-induced disturbance of cytokinesis and subsequent fusion of daugh-ter nuclei may be the reason for mixoploidy and genome doubling duringtetrad compartmentalization and callus proliferation (Segui-Simarro & Nuez2007). After the first report on androgenesis (Guha & Maheshwari 1964) fol-lowing this approach, haploids have been produced in more than 247 plantspecies and hybrids belonging to 38 genera and 34 families of dicots andmonocots (http://www.biotechnology4u.com/). Hundreds of varieties havebeen developed through anther culture in rice, brassica, barley, and wheat.The doubled-haploid approach is also being used for the rapid developmentof QTL-mapping populations, construction of genetic linkage maps for traitsof interest, and rapid fixing of transgenes.


TABLE 1 Plant Tissue and Protoplast Culture Techniques in Relation to Crop Improvement

Technique Plant material Remarks Reference

Micropropagation Banana Musa spp.(‘Philippine,’ ‘Lacatan,’‘Grande Naine’)Plantain(‘Pelipita’ and ‘Saba’).

Rapidly multiplying culturesfrom excised shoot tips.

Cronauer &Krikorian,1984; Damascoet al. 1997

Strawberry Fragariaananassa

Efficient method for massproduction of plantingmaterial.

Chopra, Dhaliwal& Gosal 1993

Eucalyptus tereticornis In vitro clonal propagationthrough nodal segments.

Gill, Gill, & Gosal1994

Rice Oryza sativa L. var.Jaya

Clonal propagation of indicarice through proliferationof axillary shoots.

Sandhu et al.1995

Dalbergia sissoo Roxb. Rapid in vitro propagationfrom mature trees.

Gill, Gill, & Gosal1997

Banana Musa spp cv.Grande Naine

Sunlight for micropropagationsystems as a way ofreducing tissue culturecosts.

Kodym & Zapata1999

Potato Solanum tuberosum In vitro propagation throughmicrotubers.

Gopal et al. 1998

Dianthus caryophyllusL.(carnation) cv. scania

In vitro propagation throughaxillary shoot proliferation.

Sooch et al. 1998

Sugarcane Saccharumofficinarum L.

Micropropagation protocolfor mass plant production.

Gosal et al.1998;Gill, Malhotra,& Gosal 2006;Sood et al.2006; Jalajaet al. 2006;Jalaja,Neelamathi, &Sreenivasan2008

Poplar P. deltoides Protocol for mass productionof two important clonesviz., G3 and G48 throughinduced shootdifferentiation of leaf, stemand root explants collectedfrom adult trees.

Chaturvedi et al.2004

Brahmi Bacopa monnieri Efficient protocol formicropropagation throughaxillary shoot proliferation.

Gill, Gupta, &Gosal 2004

Rice Oryza sativa L.var.‘Mocoi FCA‘ItapéP.A.‘Fortuna INTA,‘EMBRAPA-7- Taim,’ and‘CT 6919,’ ‘BR IRGA409’)

Clonal propagation protocolusing shoot tip cultures,and the genetic stability ofthe micropropagated plantswas verified by isozymeanalysis.

Medina et al.2004

(Continued)


TABLE 1 (Continued)


Potato Solanumtuberosum L.

A protocol for production ofpre-basic potato materialby micro cuttings, obtainedfrom plants with a shortperiod of acclimatization.

Pereira & Fortes2004

Neem Azadirachta indica Macro and micro propagationprotocols.

Gill, Chauhan, &Gosal 2006

Potato (Solanumtuberosum L.) Nif,Clone 122, Agria andResy

The highest microtubernumber (2.8), microtuberyield (278.1 mg) and singlemicrotuber weight (92.2mg) were obtained in theMS medium containing 2.0mg BAP/l and 60 g sucrose.

Ozturk & Yldrm2006

Potato (Solanumtuberosum L.) cv.Marfona

Use of continuous and semicontinuous bioreactors andtheir functions at shootmultiplication and microtuberization of potato.

Ebadi,Iranbakhsh, &Khaniki 2007

Banana Protocol formicropropagation of Musasapientum using shootmeristems.

Kalimuthu,Saravanakumar,& Senthikumar2007

Grapevine (Vitis. vinifera) Best shoot development forthe initial culture ofrootstock VR043-43 in vitrousing nodal segments andbest micro cuttingsmultiplication using e QLmedium.

Machado et al.2007

Strawberry Fragariaananassa

Mass propagation viameristem tip culture.

Mohamed 2007

Sugarcane Saccharumofficinarum L.cv. CoS99259

Spacing of 90cm × 60 cmwas the most suitable fortransplantingtissue-cultured plantlets.

Ramanand et al.2007

Banana Musa spp. Inflorescence apices werefound more suitable forrapid in vitro propagation.

Resmi & Nair2007

Grapevine (Vitis vinifera)cv. ‘Bidaneh Sefid’

Micropropagation protocolfor quick multiplication.

Salami et al. 2007

Sugarcane Saccharumofficinarum L.cv. Co86032

Sets obtained frommicropropagated plantletsresulted in higher seedyields.

Salokhe 2007

Aloe vera Micropropagated plantsexhibited elevated levels ofbioactive compounds.

Thind, Jain, &Gosal 2007

(Continued)


TABLE 1 (Continued)


Chrysanthemumcinerariifolium (Trev.)

Rapid propagationtechnology was establishedand optimized in vitro.

Liu & Gao 2007

Banana Musa spp. cv.‘Cavendish Dwarf’ and‘Valery’

Efficient medium for clonalmass propagation (MS + 30gl-1 of sucrose,N-phenyl-N- 1, 2,3-thidiazol 5-yl Urea(0.5 mgl-1) and IAA(2 mgl-1).

Farahani et al.2008

Grape vine Vitis viniferaL. cv. Perlette

Clonal propagation of grapesfor increasing plantmaterial for cultivation.

Jaskani et al.2008

Sugarcane Saccharumofficinarum L

No variation was detectedamong the regeneratedplants of a particularvariety on the basis ofRAPD markers and theprofiles of micropropagated clones werecomparable to those of therespective donor plants.

Lal et al. 2008

Potato Successful micropropagationwas achieved using nodalsegments as explants.

Badoni &Chauhan 2009

Scoparia dulcis A suitable protocol wasestablished throughmultiple shoot inductionfrom nodal segment andshoot tip explants of thisimportant medicinal herb.

Rashid et al. 2009

Somaclonalvariation

Prunus persica cvs.Sunhigh, Red haven

Somaclones S156 and S122resistant to leaf spot,moderately resistant tocanker.

Hammerschlag &Ognjanov 1990

Apple rootstocks (M26,MM106

S-2 (M26) performed betteragainst Phytophthoracactorum.

Rosati & Predieri1990

Banana Giant Cavendisha 10 somaclones. GCTCV215-1released for commercialplanting.

Hwang & Ko,1992; 2004

Apple cv. Greensleeves 16 somaclones. 21% lesssymptoms thanGreensleeves against fireblight strain T.

Donovan et al.1994

(Continued)


TABLE 1 (Continued)



Results confirmed thesuperiority of twosomaclones, one resistantand one tolerant to eyespotdisease.

Leal et al. 1994

Potato Solanumtuberosum L.

Somaclones for heat tolerance Das et al. 2000

Potato Solanum tuberosumL cv. Desiree

Somaclones IBP-10, IBP-27and IBP-30, infected withAlternaria solani andStreptomyces scabiei,exhibited higher resistanceto the pathogen, comparedto the susceptible cultivarDesiree.

Veitia-Rodriguezet al. 2002

Actinidia deliciosa cv.Tamuri

5 somaclones tolerant to NaCl(85.5 mM)

Caboni et al.2003

Potato cv. Desiree, Tomatocv. Amalia leaves,Soyabean cv. William 82Coffee cv. Robusta

Monomorphism was detectedin tomato, coffee, andsoyabean, indicating thegenetic stability of thecrops. Potato callusesshowed variations in theelectrophoretic patterns ofperoxidase and esteraseisoenzymes, indicatingsomaclonal variation in thecrop.

Lara et al. 2003

Oryza sativa L. cv. CICA-8 Four somaclones showedsignificantly higher degreeof partial resistance whencompared with the parentcultivar CICA-8.

Araujo & Prabhu2004

Durum wheat (Triticumdurum Desf.) cv.(Selbera, Sebou, andKyperounda)

Somaclonal variation thusappears to induce a widerange of modificationsamong individualcomponents ofdrought-resistancemechanisms.

Bajji et al. 2004

Sugarcane Saccharumofficinarum L.cv.CP-43/33

The somaclones performedbetter than the sourceplant.

Khan et al. 2004

Maize (Zea mays) Somaclones thus derivedwere tolerant to NaCl.

Zheng et al. 2004

(Continued)


TABLE 1 (Continued)


Bread wheat (Triticumaestivum) cv. (Sakha 8,Sakha 69, Giza 157, Giza160, Lerma Rojo 64, andTobari 66)

Somaclones were superior totheir original cultivars.

Ahmed &Abdelkareem2005

Wheat (Triticum aestivum)cv. Sakha 61

Twenty-one out of the 23somaclones outperformedthe original cultivar Sakha61 in terms of leaf rustresistance and grain yield.

Sabry et al. 2005

Oryza sativa L. Somaclones were obtainedfrom anther culture ofhybrid combinations INCALP-10/C4 153,Amistad-82/C4 153, INCALP-10, as well as fromAmistad-82.

Cristo, Gonzalez,& Perez 2006


Six tissue culture-derivedsugarcane somaclonesTC-434, TC-435, TC-436,TC-237, and TC-045 fromCoC671 and somaclonenumber TC-338 fromCo7219 were evaluated.Somaclone TC-435 gavehigher cane yield at 12months crop age over CoC671. Somaclone TC-435 hadsignificantly higher millablecane height and number ofinternodes than that ofdonor parent CoC671.

Doule 2006


A new sugarcane variety, Co94012, was released in thename of Phule Savitri forcultivation in Maharashtra,India, for Pre and Suruseasons. It is an early,sugar-rich, high-yieldingvariety with high CCS yield.Co 94012 is a somaclonalvariant of CoC 671, withbetter sucrose content andmoderate resistance to redrot (Glomerellatucumanensis) and smutdiseases (Ustilagoscitaminea). This is the first

Jalaja et al. 2006

(Continued)


TABLE 1 (Continued)


sugarcane variety to bereleased in India throughthe use of somaclonalvariation.

Alfalfa Medicago sativa An increase of variability wasnoted in importantquantitative and qualitativetraits compared with theinitial cultivars, includingproductivity of the aboveground mass and seeds,resistance to fungaldiseases, and winterhardiness.

Rozhanskaya2006

Oryza sativa L. cv.‘Pokkali’

The grain yield wassignificantly high insomaclones ‘BTS 11-1,’‘BTS 28,’ ‘BTS 24,’ ‘BTS9-2(S),’ ‘BTS-17(S),’ ‘BTS10-2,’ and ‘BTS 11-7,’ whichexhibited higher flag leafarea and moderate leafarea index compared toother somaclones.

Elanchezhian &Mandal 2007

Olive cv. ‘Frangivento’ Somaclonal variation couldbe found in olive plantsregenerated throughsomatic embryogenesis;this appears in matureplants in the field.

Leva &Petruccelli2007

Banana In banana cultivars (Musa xacuminata, Musa xbalbisiana), somaclonalvariation can be useful inselecting for clones withimproved agronomiccharacteristics.

James et al. 2007;Oh et al. 2007b

Dieffenbachia cvCamouflage, Camille,Star Bright.

Potential for new cultivardevelopment by selectingcallus-derived somaclonalvariants of dieffenbachiawas demonstrated.

Shen et al. 2007


Development of somaclonesresistant to red rot diseaseusing in vitro and fieldselection.

Singh et al.2008,Sengaret al. 2009

(Continued)


TABLE 1 (Continued)


Anther culture/

HaploidProduction

Wheat Doubled haploid wheatvariety ‘Florin’ wasdeveloped.

De Buyser et al.1986

Wheat A single 2,4-D treatmentgiven to spikes one dayafter pollination withmaize, enabled embryos tobe recovered from all 19varieties.

Laurie &Reymondie1991

Rice Revised medium was used forincreasing anther cultureefficacy and improvedfeasibility of using doubledhaploids in genetic andbreeding research withindica rice.

Raina & Zapata1997

Rice ‘Bicol’ first F1 antherculture-derived line froman indica/indica cross insaline-prone areas.

Senadhira et al.2002

Rice An improved method forpollen culture in rice.

Sarao et al. 2003;Grewal, Gill, &Gosal 2006b

Durum Wheat Dicamba and 2,4-D, was bestfor improving the yield ofhaploid plants of durumwheat through crosses withmaize.

García-llamas,Martín, &Ballesteros2004

Maize Zea mays L. Embryogenic induction ofmicrospores within anthersin in vitro conditions wasthe best when combinationof cold treatment, TIBA(0.1 mg l-1) in media andcolchicine (0.02% duringfirst 3 days of culture) wasapplied.

Obert &Barnabas 2004

Citrus (Citrus clementina) Anther culture as a rapid andattractive method ofobtaining new triploidvarieties in clementine.

Germanà et al.2005

Citrus (Citrus clementina) Influence of light quality onanther culture of Citrusclementina Hort. ex Tan.,cultivar Nules wasreported.

Antonietta et al.2005

(Continued)


TABLE 1 (Continued)


Maize Zea mays L. 15, 10, 10, and 3 fertiledoubled-haploid plantswere obtained in culturestreated with paraquat,t-BHP, methioninecombined with riboflavin,and menadione,respectively.

Ambrus et al.2006

Wheat Simplified wheat x maizehaploid productionprotocol that is 100%effective across all breadwheat cultivars, generatingdata means of 25% forembryo excision, 90% to95% for plantletregeneration, and between95% to 100% for doubledhaploid (2n = 6x = 42,AABBDD) outputs.

Mujeeb-Kaziet al. 2006

Maize Zea mays L. Maize haploid plants by invitro culture of pollinatedovaries.

Tang et al. 2006

Durum Wheat Triticumdurum

Novel pretreatmentcombining mannitol 0.3Mand cold for seven dayshad a strong effect on thenumber of embryosproduced and regeneratedgreen plants; 11.55 greenplants were produced per100,000 microspores.

Labbani, deBuyser, &Picard 2007

Tomato Lycopersiconesculentum L.

Embryogenesis and plantregeneration by in vitroculture of isolatedmicrospores and wholeanthers of tomato.

Segui-Simarro &Nuez 2007

Rice The 9 DH lines could providethe basic materials forbreeding on Dian-typehybrid rice with both goodquality and high blastresistance in the future.

Zhahg-Yi et al.2008

Maize Doubled haploids should beinduced from F2 plantsrather than from F1 plants.

Bernardo 2009

(Continued)


TABLE 1 (Continued)


Cryopreservation Rice Oryza sativa L. Rice plants (cv. Taipei 309)were regenerated fromdifferent cryopreservedcalli, and cryopreservationwas declared to be areliable way to storetransformation competentrice lines.

Moukadiri &Cornejo 1996

Potato Solanumtuberosum L

Shoot tips of in vitro grownpotato (Solanumtuberosum L.) plants werecryopreserved by amodified vitrificationmethod.

Halmagyi et al.2004

Potato Solanumtuberosum L

In vitro plants of potatocultivars ‘Superior’ and‘Atlantic’ were coldacclimated, and axillarybuds were precultured,osmoprotected, exposed toPVS-2 solution, plungedinto liquid nitrogen,thawed, and finally plantedin the regenerationmedium. Aftercryopreservation, vitrifiedshoot tips resumed growthwithin a week.

Zhao et al. 2005b

Rubus spp. (blackberry andraspberry)

Encapsulation-dehydrationand PVS2-vitrificationcryopreservation protocolswere found successful forpreserving diverse Rubusgermplasm (Shoot tips)

Gupta & Reed2006

Arabidopsis Arabidopsis can besuccessfully cryopreservedusing either plantvitrification solution 2(PVS2) or plant vitrificationsolution 3 (PVS3) ascryoprotectants prior torapidly cooling shoot tipsin liquid nitrogen (LN).

Volk et al. 2007

Potato Solanum tuberosumL. cv. Dejima and(STN13)

Protocol was tested with 12selected cultivated varietiesand wild species and thesurvival percentagesobtained ranged between64.0% and 94.4%.

Yoon-Ju et al.2007

(Continued)


TABLE 1 (Continued)


Citrus Up to 98.1% of the plantsobtained bycryopreservation were freefrom HLB bacterium, ascompared with a sanitationrate of 25.3% yielded byconventional meristem tipculture.

Ding et al. 2008

Protoplast fusion Potato S. brevidens xS. tuberosum

Somatic hybrids wereproduced by electrofusion.

Fish, Karp, &Jones 1988

Brassica B. juncea, B.nigra and B. carinata XB. napus.

Resistance to Phoma lingamwas expressed in allsymmetric hybrids, and in19 of 24 toxin-selectedasymmetric hybrids.

Sjödin et al. 1989

Potato S. brevidens xS. tuberosum

Twenty hybrids testedexpressed a high level ofresistance to PVY.

Rokka et al. 1994

Brassica Brassica napus xBrassica oleracea

Inoculations Xanthomonascampestris pv campestrisidentified four somatichybrids with highresistance.

Hansen et al.1995

Brassica Brassica oleraceax Brassica rapa

Disease assays showed thatmost somatic hybrids hadlower disease severityratings against bacterial softrot.

Ren, Dickson, &Earle 2000

Citrus Citrus sinensis L.Osbeck x C.volkameriana Pasquale,C. reticulata Blanco

Somatic hybrids combinedcharacteristics from bothsources and have potentialfor tolerance to blight andcitrus tristeza virus (CTV).

Mendes et al.2001

Banana ‘Maçã’(Musa AABgroup) x Lidi’ Musa sp.AA group.

Somatic hybrids wereidentified by using RAPDmarkers.

Matsumoto,Vilarinhos, &Oka 2002

Citrus Mandarin xpummelo, Sweet orangex pummelo

Somatic hybrids wereconfirmed by leafmorphology, ploidyanalysis via flow cytometry,and RAPD analysis. Somatichybrids are beingpropagated by tissueculture for furtherevaluation of diseaseresistance and horticulturalperformance in field trials.

Ananthakrishnanet al. 2006

(Continued)


TABLE 1 (Continued)


Potato Aminca-Cardinal xCardinal-Nicola

Complete resistance to PVYwas noted for one somatichybrid line (CN2). All otherhybrids also showedimproved tolerance toPythium aphanidermatuminfection during tuberstorage or after plantinoculation.

Nouri-Ellouzet al. 2006

Potato Solanum tuberosumx Solanum tuberosum

Tetraploid intraspecificsomatic hybrids between16 different diploidbreeding lines of Solanumtuberosum L. wereproduced by PEG-inducedfusion.

Przetakiewiczet al. 2007

Brassica Brassica oleraceax Brassica rapa

Calli were screened by RAPDanalysis for their hybridcharacter. This is the firstreport about a hybridformation between twohaploid protoplasts.

Liu et al. 2007a

Brassica napus(2n = 38) XOrychophragmusviolaceus (2n = 24)Brassicanapus(2n = 38) xOrychophragmusviolaceus (2n = 24)

Symmetric fusions ofmesophyll protoplasts andsubsequent development ofB. napus- O. violaceouschromosome addition lines.

Zhao et al. 2008

Embryo/ Ovule Culture

During wide hybridization, when parents are genetically diverse, endospermdegeneration leads to embryo abortion and the failure of the cross. Undersuch situations, embryo culture/rescue is a practical approach to obtaininterspecific and intergeneric hybrids (Gosal & Bajaj 1983). Embryo cul-ture is also used to produce haploids from wide crosses (Bains et al. 1995;Verma et al. 1999). Embryo culture has been successfully used to transferdesirable genes from wild relatives into cultivated varieties of several fieldand vegetable crops (Kaur, Satija, & Gosal 2002). Ovule culture in grapes(Singh, Brar, & Gosal 1991) aids in developing hybrids, even in seedlessgrapes.


Protoplast Culture and Somatic Hybridization

Protoplast culture and somatic cell hybridization involving fusion of proto-plasts from different plant species are important approaches for combiningcharacteristics from otherwise sexually incompatible species. Further, cybrids(cytoplasmic hybrids) and organelle recombinants, not possible through con-ventional methods, can also be developed (Hinnisdaels et al. 1988). Sincethe production of the first somatic hybrid between Nicotiana glauca andN. langsdorfii in 1970, numerous intraspecific, interspecific, and intergenericsomatic hybrids have been produced. Somatic hybrids can be classified intotwo categories, viz., symmetric and asymmetric. Symmetric hybrids carry thecomplete sets of chromosomes from both the parents, whereas asymmetrichybrids possess the full chromosome complement of only one parent andpartial genome of the second parent.

Earlier efforts were to combine full genomes from both the parents andto develop symmetric hybrids among closely related and cross compatiblespecies, where somatic hybrids resembled sexual hybrids. For instance, thesomatic hybrids between Brassica campestris and B. oleracea resembled theB. napus. Later, researchers thought to produce novel hybrids by fusingprotoplasts from genetically distant species. However, the somatic hybridsthus produced, particularly among the phylogenetically distant species, haveexhibited somatic incompatibility, genetic instability, and sterility. It is obvi-ous that such problematic somatic hybrids cannot be incorporated intobreeding programs. Therefore, the interest has moved from creation of novelhybrids to the production of cybrids, chromosome transfer, and gene intro-gression. It is well known that alloplasmic association leads to male sterilityas a consequence of interactions between nuclear and mitochondrial ele-ments. Male sterility has been developed by fusing protoplasts of Nicotianatabacum with X-irradiated protoplasts of N. africana. Likewise, male sterilityhas been transferred from Raphanus sativus into Brassica napus. Moreover,resistance to herbicide triazine has been combined with male sterility byfusing Brassica napus protoplasts from a male-sterile line with B. napusprotoplasts from a triazine-resistant parent. Cytoplasmic genetic male sterilityhas also been successfully transferred into rice.

Resistance to some diseases like potato leaf roll virus, PVX, and PVYhave been incorporated into Solanum tuberosum from Solanum brevidensand Solanum phureja through protoplast fusion (Rokka et al. 1994, Nouri-Ellouz et al. 2006). Likewise, resistance against Phoma lingam disease inBrassica species (Sjödin & Glimelius 1989) and tristeza virus (CTV) in cit-rus (Mendes et al. 2001) was developed through protoplast fusion. Hybridsproduced following protoplast fusion among hybrids of Iris fulva (4x) xIris laevigata; because Iris fulva has unique brown flowers, this trait couldbe very useful for flower color improvement in Iris laevigata, which lacksthis color (Inoue et al. 2006). The protoplast-to-plant system developed in


basmati rice (Jain et al. 1995; Kaur et al. 1999) can be exploited for single-step transfer of male sterility from one line to another for production ofhybrid rice. Pollen protoplasts have also been fused using pollen proto-plasts of Brassica oleracea var. italica and haploid mesophyll protoplasts ofBrassica rapa (Liu et al. 2007 a). Calli were screened by random amplifiedpolymorphic DNA analysis for their hybrid character.

Cryopreservation and In Vitro Germplasm Storage

The aim of germplasm conservation is to ensure the ready availability ofuseful germplasm. In seed-propagated crops, seed is extensively used forconservation of germplasm using conventional methods. However, in vege-tatively propagated species where conventional storage techniques are used,it is very difficult to store germplasm for long durations. The conservation ofplant parts in vitro has a number of advantages over in vivo conservation,e.g., in vitro techniques allow conservation of plant species in danger ofbeing extinct. In vitro storage of vegetativly propagated plants can result ingreat savings in storage space and time, and sterile plants that cannot bereproduced generatively can be maintained in vitro. Complete plants havebeen successfully regenerated from tissues cryopreserved at −196◦C in liquidN2 in several crops for several months to years (Ford, Jones, & Van Staden2000; Panis, Swennen, &. Engelmann 2001; Halmagyi et al. 2004; Gupta &Reed 2006; Ding et al. 2008). This method is now being practically used atseveral national and international germplasm banks.

Successful cryopreservation of plant shoot tips is dependent uponeffective desiccation through osmotic or physical processes. Cryoprotectivetreatments, which favor survival of small, meristematic cells and young leafcells, are most likely to produce high survival rates after liquid-nitrogenexposure. Further, microscopy techniques have been used to determinethe extent of cellular damage and plasmolysis that occurs in pepper-mint (Mentha piperita) shoot tips during the process of cryopreservation,using cryoprotectant plant vitrification solution 2 (PVS2) (30% glycerol,15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) prior toliquid-nitrogen exposure (Volk & Casperson, 2007). Arabidopsis, which isincreasingly being used in genomic studies, can be successfully cryopre-served using either PVS2 or PVS3 as cryoprotectants prior to rapidly coolingshoot tips in liquid nitrogen (LN). PVS3 contains 50% glycerol as comparedto PVS2 that contains 30% glycerol. PVS3 was less injurious than PVS2. Allof the shoot tips regrew after LN exposure when cryoprotected with PVS3for 60 min at 22◦C (Towill, Bonnart, & Volk 2006). The high levels of shootformation after LN exposure of Arabidopsis shoot tips make this a desirablesystem in which molecular tools can be used to examine how alterations inbiochemical, metabolic, and developmental processes affect regrowth aftercryoprotective treatments.


In Vitro Production of Secondary Metabolites

Several plant species are known to produce secondary metabolites in vivoor in vitro. These metabolites do not perform vital physiological functions,but some act as potential predators and attract pollinators. Further, thesemetabolites act as a valuable source of a vast array of chemical compounds,including fragrances, flavors, natural sweeteners, and industrial feed stocks.Cultured cells/organs produce a wide range of secondary products. Mainlythree approaches have been followed: (i) the rapid growth of cell sus-pension cultures in large volumes, (ii) immobilization of plant cells, and(iii) growing hairy root cultures in vitro. There are several advantages ofthe cell-culture systems over the conventional cultivation for production ofsecondary metabolites, e.g., 1) independence from various environmentalfactors; 2) any cell of the plant could be multiplied to yield specific metabo-lite; and 3) a culture of cells may prove suitable in cases where plants aredifficult or expensive to grow in the field because of their long life cycles.Since the 1970s when the possibility of producing useful secondary productsin plant cell cultures was first recognized, considerable progress has beenmade, and a number of plant species have been found to produce secondaryproducts, such as shikonin, diosgenin, caffeine, glutathione, capsaicin, andanthraquinone (Havkin-Frenkel, Dorn, & Leustek 1997; Varindra et al. 1997;Fischer et al. 1999). Large-scale production of such compounds (molecularfarming) is becoming increasingly popular with the industry where somephysical and chemical conditions for growth and product formation havebeen optimized.

GENETIC TRANSFORMATION AND MOLECULAR APPROACHES:PLANT GENETIC ENGINEERING

During the past 15 years, the combined use of recombinant DNA technology,gene transfer methods, and tissue-culture techniques has led to the effi-cient transformation and production of transgenics in a wide variety of cropplants (Chahal & Gosal 2002). In fact, transgenesis has emerged as an addi-tional tool to carry out single-gene breeding or transgenic breeding of crops.Unlike conventional breeding, only the cloned gene(s) of agronomic impor-tance is/are being introduced without co-transfer of other undesirable genesfrom the donor. The recipient genotype is least disturbed, which eliminatesthe need for repeated backcrosses. Above all, the transformation methodprovides access to a greater gene pool as the gene(s) may come fromviruses, bacteria, fungi, insects, animals, human beings, unrelated plants,and even from chemical synthesis in the laboratory. Various gene-transfermethods (Christou 1994; Sudhakar et al. 1998; Maqbool & Christou 1999;Gosal & Gosal 2000; Ahmed et al. 2002) have been developed for genetic


transformation of plants. Rapid and remarkable achievements have beenmade in the production, characterization, and field evaluation of transgenicplants in several field crop, and fruit and forest plant species. However, themajor interest has been in the introduction of cloned gene(s) of agronomicimportance into commercial cultivars for their incremental improvement.Using different gene-transfer methods and strategies, transgenics carryinguseful agronomic traits have been developed and released in several crops.

Engineering for Herbicide Resistance

There have been two approaches to develop herbicide-resistant transgenicplants.

TRANSFER OF GENES WHOSE ENZYME PRODUCTS DETOXIFY THE HERBICIDE

(DETOXIFICATION)

In this approach, the introduced gene produces an enzyme that degrades theherbicide sprayed on the plant. For instance, introduction of the bar gene,cloned from bacteria Streptomyces hygroscopicus into plants, makes themresistant to herbicides. The bar gene produces an enzyme, phosphinothricinacetyl transferase (PAT), which degrades phosphinothricin into a non-toxicacetylated form. Plants engineered with the bar gene were found to growin phosphinothricin at levels 4-10 times higher than those found in normalfield applications (Gordon-Kamm et al. 1990; Choi et al. 2007; Shizukawa& Mii 2008). Likewise, bxn gene of Klebsiella ozaenae, which producesnitrilase enzyme, imparts resistance to plants against herbicide bromoxynil.Other genes, including tfdA for 2, 4-D tolerance and GST gene for atrazinetolerance, have also been used (Table 2).

TRANSFER OF GENES WHOSE ENZYME PRODUCT BECOMES INSENSITIVE TO

HERBICIDE (TARGET MODIFICATION)

Under this approach, a mutated gene is introduced into the plant, whichproduces a modified enzyme in the transgenic plant that is not recognizedby the herbicide. Hence, the herbicide cannot kill the plant. For instance,a mutated aroA gene from Salmonella typhimurium has been used fordeveloping tolerance to glyphosate herbicide. The target site of glyphosateis a chloroplast enzyme 5-enol pyruvylshikimic acid 3-phosphate synthase(EPSPS). Introduction of the mutated aroA gene produces modified EPSPS,not recognizable to glyphosate. Likewise, sulphonylurea and imidazolinoneherbicides inhibit acetolactate synthase (ALS) chloroplast protein. Toleranceto these herbicides has been achieved by engineering the expression of themutated ALS gene derived from plants (Table 2).


TABLE 2 Important Transgenes/Transgene Products being Used for Engineering Crop PlantsPossessing Herbicide Resistance

Trnasgene (s) Source Plant species Target herbicide Reference

Bar Streptomyceshygroscopicus

Nicotianatabaccum,Solanumtuberosum,Lycopersiconesculentum

Phosphinothricin andbialaphos

De Block et al.1987


Zea mays Glufosinate, bialaphos Gordon-Kammet al. 1990

Bxn Klebsiellaozaenae

Nicotianatabaccum

Bromoxynil(3,5-dibromo-4-hydroxybenzonitrile)

Stalker,Mcbride, &Malyj 1998

Bxn Klebsiellaozaenae

Clover Bromoxynil(3,5-dibromo-4-hydroxybenzonitrile)

Dear et al. 2003

GAT – Arabidopsis,tobacco, maize

Glyphosate Castle et al.2004


Sweet potato Glufosinate Choi et al. 2007

CYP1A1,CYP2B6,CYP2C19

Homo sapiens Oryza sativa Metolachlor Kawahigashiet al. 2008

MxPPO Myxococcusxanthus

Tall fescue Oxyfluorfen, acifluorfen Lee et al. 2008


Nierembergiarepens

Bialaphos Shizukawa et al.2008

Engineering for Insect Resistance

There have been two approaches to develop insect-resistant transgenicplants by transferring insect-control-protein genes.

INTRODUCTION OF BACTERIAL GENES

Bacillus thuringiensis synthesizes an insecticidal crystal (cry) protein, whichresides in the inclusion bodies produced by the Bacillus during sporula-tion. Upon ingestion by insect larvae, this crystal protein is solublized in thealkaline environment of the midgut of the insect and processed by midgutproteases to produce a protease-resistant polypeptide toxic to the insect.Lepidopteran-specific Bt gene from Bacillus thuringiensis subsp. kurstakihas been widely and successfully used in tobacco, tomato, potato, cotton,rice, and maize to develop resistance against several lepidopteran insectpests (Table 3) (DeMaagd, Bosch, & Stiekema 1999; Gosal et al. 2003; Bashiret al. 2005; Wei et al. 2006; Cao, Shelton, & Earle 2008; Jafari et al. 2009).The use of redesigned synthetic Bt gene has also been made in some of


these crops and, in several instances, the synthetic versions have exhibitedup to 500-fold increases in the expression (Brandy et al. 2001). Insect-resistant transgenic varieties/hybrids of several crops have been releasedfor commercial cultivation in many countries (James 2009).

INTRODUCTION OF PLANT GENE(S)

Several insecticidal proteins of plant origin, such as lectins, amylaseinhibitors, and protease inhibitors, can retard insect growth and develop-ment when ingested by insects at high doses (Jongsma & Boulter 1997;Tamayo et al. 2000). Some genes like CpTi, PIN-1, PIN 11, αA-1, and GNAhave been cloned from different plants and are being used in the genetic-transformation programs aimed at developing insect resistance in plants (Buet al. 2007; Maheswaran et al. 2007; McCafferty, Moore, & Zhu 2008). A listof transgenes used for developing insect-resistant transgenic plants is givenin Table 3.

TABLE 3 Important Transgenes/Transgene Products being Used for Engineering Crop PlantsPossessing Insect Resistance

Transgene (s) Source Plant species Target pest Reference

Cry genesCry9C Bacillus

thuringiensisZea mays Ostrinia nubilalis

(Hübner)Jansens et al.

1997Cry1Ab Bacillus

thuringiensisOryza sativa Chilo suppressalis,

Scirpophaga incertulas,Cnaphalocrocismedinalis,Herpitogrammalicarisalis, Sesamiainferens, Narangaanescens, Mycalesisgotama, Hesperiidae,Parnara guttata

Shu et al. 2000

Cry1Ac Bacillusthuringiensis

Oryza sativa Yellow stem borer Gosal et al.2003;Grewal,Gill, & Gosal2006a

Cry1Ac Bacillusthuringiensis

Pinus taeda Dendrolimuspunctatus Walker,CrypyotheleaformosicolaStaud

Tang & Tian2003

Cry1Ac +CpT1

Bacillusthuringiensis,Vignaunguiculata

Oryza sativa Chilo suppressalis Zhang et al.2004

Cry1Ac +cry2A

Bacillusthuringiensis

Oryza sativa Lepidoptera Bashir et al.2005

(Continued)


TABLE 3 (Continued)

Transgene (s) Source Plant species Target pest Reference

Cry2A Bacillusthuringiensis

Oryza sativa Lepidoptera Chen et al. 2005

Cry1AbCry1Acfusion


Oryza sativa Lepidoptera Huang et al.2005

Cry1c Bacillusthuringiensis

Oryza sativa Lepidoptera Wei et al. 2006

Cry1Ac +Cry1c


Brassicajuncea

Plutella xylostella Cao, Shelton &Earle 2008

Cry1Ac,Cry1Ie


Nicotianatabacum

Helicoverpa armigera Lian et al. 2008

Cry1Ab Bacillusthuringiensis

Beta vulgaris Spodoptera littoralis Jafari et al. 2009

Cry1Ac +Cry2Ab


Gossypiumhirsutum L.

Helicoverpa armigera L. Gao et al. 2009

GNA genesGNA Galanthus

nivalis.Oryza sativa Nilaparvata lugens Xiaofen et al.

2001GNA Galanthus

nivalis.Zea mays Rhopalosiphum maidis

FitchWang et al.,

2005GNA Galanthus

nivalis.Carica papaya Tetranychus

cinnabarinusMcCafferty,

Moore, & Zhu2008

GNA Galanthusnivalis.

Saccharumofficinarum

Ceratovacuna lanigeraZehnther

Zhangsun et al.2008

CpTI genesCpTI Vigna

unguiculataOryza sativa Chilo. Spp. Xu et al. 1996

CpTI Vignaunguiculata

Triticumaestivum

Sitotroga cerealellaOlivier

Bi et al. 2006

OthersICP Bacillus

thuringiensisOryza sativa Scirpophaga incertulas Nayak et al.

1997PINII-2x Solanum

tuberosumOryza sativa Chilo suppressalis Walker Bu et al. 2007

PI Nicotianatabaccum

Malus Spp. Epiphyas postvittiana Maheswaranet al. 2007

OCII Oryza sativa Medicagosativa

Phytodecta fornicataBrüggemann

Ninkovic et al.2007

Aprotinin Bos Taurus Saccharumofficinarum

Scirpophaga excerptalis Christy et al.2009

Engineering for Disease Resistance

VIRUS RESISTANCE

The genetic engineering of virus-resistant plants has exploited new genesderived from viruses themselves in a concept referred to as ‘pathogen-derived resistance’ (PDR). Some such genes are discussed below.


Coat-protein-mediated resistance (CP-MR). Introduction of viral coat-protein gene into plants makes the plants resistant to the virus from whichthe gene for the CP was derived. This was first demonstrated for TMV intobacco (Powel-Abel et al. 1986). Subsequently, virus-resistant transgenicshave been developed in tomato, melon, rice, papaya, potato, and sug-arbeet (Table 4) (Fuchs & Gonsalves 2007). A variety of yellow squashcalled ‘Freedom 11’ has been released in the United States. Several vari-eties of potato, cucumber, and tomato, in which CP-MR has been used, areunder field evaluation. Transgenic papaya resistant to papaya ring-spot virus(PRSV) has been developed and is being commercially grown in the UnitedStates (Ferreira et al. 2002).

TABLE 4 Genetic Engineering for Disease Resistance in Plants

Transgene Source Target species Pathogen Reference

BacterialDiseases

T4 Lysozyme Human Tobacco ErysipheCichoracearum,Pseudomonassyringae pv.tabaci

Nakajima,Muranaka, &Ishige 1994

NPR1 Arabidopsisthaliana

Arabidopsisthaliana

Pseudomonassyringae andPeronosporaparasitica

Cao, Li, & Dong1998

Pto Lycopersiconpimpinellifolium

Lycopersiconesculentum

Pseudomonassyringae pv. tomato

Tang et al. 1999

chly Chicken Potato Erwinia carotovorasubsp, atroseptica

Serrano et al.2000

Synthetic D4E1 Cecropia (insect) Populus tremula,Populus alba

Agrobacteriumtumefaciens,Xanthomonaspopuli pv. Populi.,Hypoxylonmammatum

Mentag et al.2003

NH1 (NPR1) Oryza sativa Oryza sativa Xanthomonas oryzaepv. oryzae

Chern et al.2005

Rxo1 (NBS-LRR) Zea mays Oryza sativa Xanthomonas oryzaepv. oryzicola

Zhao et al.2005a

Xa21 (NBS-LRR) Oryza sativa Oryza sativa Xanthomonas oryzaepv. oryzae

Wang et al.2007; Zhaiet al. 2002

Fungal DiseasesPR-3(I) French Bean Tobacco

RapeseedRhizoctonia solani Broglie et al.

1991Vst1 (Stilbene

(resveratrol)synthase)

Vitis vinifera Nicotianatabacum

Botrytis cinerea Hain et al. 1993

(Continued)


TABLE 4 (Continued)


Aglul, RCH10 Alfalfa, Rice Nicotianatabacum

Cercospora nicotianae Zhu et al. 1994

PR-3(I) Rice Rice Rhizoctonia solani Lin, Anuratha, &Datta 1995

Aglul, RCH10 Alfalfa, Rice Alfalfa Phytophthoramegasperma f. sp.medicaginis (Pmm)

Masoud,Mojtaba, &Behzad 1996

PR-3(I) Rice Cucumber Botrytis cinerea Tabei, Kitade, &Nishizawa1998

Vst1 (Stilbene(resveratrol)synthase) pss(pinosylvinsynthase)

Vitis vinifera,Pinus sylvestris

Hordeumvulgare,Triticumaestivum

Botrytis cinerea;Puccinia reconditaf.sp. tritici &Stagonospora(Septoria) nodorum

Leckband &Lorz 1998;Serebriakova,Oldach, &Lorz 2005

RCC2 Rice Grape vine Uncinula necator,Elisinoe ampelina

Yamamoto,Iketani, &Ieki 2000

Synthetic D4E1 Cecropia (insect) Nicotianatabacum

Colletotrichumdestructivum

Cary et al. 2000

AiiA Bacillus Solanumtuberosum

Pectobacterium(Erwinia)carotovora

Dong et al. 2001

RC7 chitinasePR-3

Oryza sativa Oryza sativa Rhizoctonia solani. Datta et al. 2001

gf-2.8 (oxala-teoxidase)

Triticum aestivum Glycine max Sclerotiniasclerotiorum

Cober et al.2003;

Cry1Ab (Bttoxin)


Zea mays Fusarium spp Clements et al.2003;Hammondet al. 2004

Rpi-blb2(NB-LRR)

Solanumbulbocastanum

Solanumtuberosum

Phytophthorainfestans

Van derVossenet al.2003, 2005

Vf (Cf) Malus floribunda Malus domestica Venturia inaequalis Belfanti et al.2004

gf-2.8 (oxalateoxidase)

Triticum aestivum Populuseuramericana

Septoria musiva Liang et al. 2004

Rchit Rice Pigeon pea Kumar et al.2004

9f-2.8 (oxalateoxidase)&TaPERO(peroxidase)

Triticum aestivum Triticumaestivum

Blumeria graminisf.sp. tritici

Altpeter et al.2005

Chit French Bean Cotton Verticillium dahliae Masoud et al.2005

Chi11(chitinase)Tlp (PR-4)

Oryza sativa Oryza sativa Rhizoctonia solani Kalpana et al.2006

(Continued)


TABLE 4 (Continued)


NPR1 Arabidopsisthaliana

Triticumaestivum

Fusariumgraminearum

Makandar et al.2006

KP4 Virus infectingUstilago maydis

Triticumaestivum

Tilletia caries Schlaich et al.2006;

Gfzhd101 Clonostachys rosea Zea mays Fusariumgraminearum

Igawa et al.2007

Synthetic D4E1 Cecropia (insect) Gossypiumhirsutum

Thielaviopsis basicola Rajasekaranet al. 2007

PFLP Pepper Tomato R. Solanacearum Huang et al.2007

Chi 18(chitinase)

Solanumtuberosum

Raphanus sativusLinn

Rhizoctonia solani Yang et al. 2009

Viral DiseasesREP (TYLCV) Tomato yellow leaf

curl virus(TYLCV)


TYLCV Yang et al. 2004

SyntheticsequencegeneratingmiRNA basedon P69TYMV) andHC-Pro(TuMV)

Turnip yellowmosaic virus(TYMV) & turnipmosaic Virus(TuMV)

Arabidopsisthaliana

TYMV & TuMV Niu et al. 2006

PSRV coatprotein gene

Papaya ringspotvirus (PRSV)

Carica papaya PRSV Fuchs &Gonsalves2007

C1 (TYLCV) Tomato YellowLeaf Curl Virus(TYLCV)


TYLCV Fuentes et al.2006

ORF2b (PLRV) Potato leaf-rollvirus (PLRV)

Solanumtuberosum

PLRV Vazquez,Asurmend, &Hopp 2001

Suppressor 2b(CMV)

Cucumber mosaicvirus (CMV)

Nicotianatabacum

CMV Qu, Ye, & Fang2007

RNAi Technology. Antiviral RNAi technology has been successfullyused for viral disease management in human cell lines (Bitko & Barik2001; Gitlin, Karelsky, & Andino 2002; Jacque, Triques, & Stevenson 2002;Novina et al. 2002). The effectiveness of the technology in generating virus-resistant plants was first reported for PVY in potato harboring vectors forsimultaneous expression of both sense and antisense transcripts of thehelper-component proteinase gene. Many examples of extreme virus resis-tance and post transcriptional gene silencing of endogenous or reportergenes have been described in transgenic plants containing sense or antisensetransgenes. In these cases of either cosuppression or antisense suppression,there appears to be induction of a surveillance system within the plant


that specifically degrades both the transgene and target RNAs. Transformingplants with virus or reporter gene constructs that produce RNAs capable ofduplex formation confered virus immunity or gene silencing on the plants.It has been accomplished by using transcripts from one sense gene and oneantisense gene colocated in the plant genome, a single transcript that hasself-complementarity, or sense and antisense transcripts from genes broughttogether by crossing (Waterhouse, Graham, & Wang 1998). Likewise, RNAi-mediated silencing of African cassava mosaic virus (ACMV) resulted in a 99%reduction in Rep transcripts and a 66% reduction in viral DNA (Vanitharani,Chellappan, & Fauquet 2003). The siRNA was able to silence a closelyrelated strain of ACMV but not a more distantly related virus. More than40 viral suppressors have been identified in plant viruses (Ruiz-Ferrer &Voinnet 2007).

Qu, Ye, and Fang (2007) used a different amiRNA vector to target the2 b viral suppressor of the Cucumber mosaic virus (CMV)—a suppressorthat interacted with and blocked the slicer activity of AGO1. This suppres-sor has been shown to confer resistance to CMV infection in transgenictobacco. A strong correlation between virus resistance and the expressionlevel of the 2 b-specific amiRNA was shown in individual plant lines. Itis evident from the above-mentioned reports that the RNA components,such as single-strand template RNA, dsRNA, and/or siRNA of the silenc-ing pathways, are the preferred targets of most viral suppressors. However,plant viruses are known to have evolved a counter-silencing mechanism byencoding proteins that can overcome such resistance (Li & Ding 2006; Díaz-Pendón & Ding 2008). These suppressors of gene silencing, often involvedin viral pathogenicity, mediate synergism among plant viruses and resultin the induction of more severe disease than that caused by individualviruses. Simultaneous silencing of such diverse plant viruses can be achievedby designing hairpin structures that can target a distinct virus in a singleconstruct (Díaz-Pendón & Ding 2008).

Fungal Resistance

Genetic engineering for fungal resistance has been limited. But several newadvances in this area now present an optimistic outlook. Many reports(Makandar et al., 2006; Huang et al. 2007; Yang et al. 2009) show positiveresults relative to transgenic plants expressing genes for disease resistance(Table 4).

ANTIFUNGAL PROTEIN-MEDIATED RESISTANCE

Introduction of chitinase gene in tobacco and rice has been shown toenhance fungal resistance in plants (Lee & Raikel 1995; Nishizawa et al.


1999). Chitinase enzyme degrades the major constituents of the fungal cellwall (chitin and a-1,3 glucan). Coexpression of chitinase and glucanasegenes in tobacco and tomato plants confers a higher level of resistance thanthat imparted by either gene alone. Use of genes for ribosome-inactivatingproteins (RIP), along with chitinase, has also shown synergistic effects.A radish gene encoding antifungal protein 2 (Rs-AFP2) was expressed intransgenic tobacco, and resistance to Alternaria longipes was observed(Broekaert et al. 1995). Other pathogenesis-related proteins/peptides includeosmotin, thionins, and lectins (Florack & Stiekema 1994).

ANTIFUNGAL COMPOUND-MEDIATED RESISTANCE

The low molecular weight compounds, such as phytoalexins, possess antimi-crobial properties and have been implicated in imparting plant resistance tofungal and bacterial pathogens (Leckband & Lorz 1998). Expression of a stil-bene synthase gene from grapevine in tobacco resulted in the production ofa new phytoalexin (resveratrol), which enhanced resistance to infection byBotrytis cinerea. Active oxygen species (AOS), including hydrogen peroxide,also play an important role in plant defense responses to pathogen infection(Wu et al. 1995). Transgenic potato plants expressing an H2O2-generatingfungal gene for glucose oxidase were found to have elevated levels of H2O2

and enhanced levels of resistance both to fungal and bacterial pathogens—particularly to the Verticillium wilt pathogen. Further, overexpression ofdefense-response genes in transgenic plants has provided enhanced resis-tance to a variety of fungal pathogens (Muehlbauer & Bushnell 2003). Forexample, transgenic wheat lines carrying a barley-seed class II chitinaseexhibited enhanced resistance to powdery mildew (Bliffeld et al. 1999;Oldach, Becker, and Lorz 2001). Varying levels of resistance toward powderymildew were observed in transgenic wheat lines carrying a barley chitinaseor a barley β-1,3-glucanase (Bieri, Potrykus, & Futterer 2003). With respectto fusarium head blight (FHB), a transgenic wheat line carrying a rice tlpand a line carrying a combination of a wheat β-1, 3-glucanase and chiti-nase exhibited delayed symptoms of FHB in greenhouse trials (Chen et al.1999; Anand et al. 2003). In addition, transgenic Arabidopsis plants carryingan overexpressed Arabidopsis thionin have exhibited increased resistance toF. oxysporum (Epple, Apel, & Bohlmann 1997). Transgenic wheat express-ing the Arabidopsis NPR1 gene, a gene that regulates defense responses, wasshown to exhibit a high level of resistance to FHB in greenhouse evaluations(Makandar et al. 2006).

RNA-mediated gene silencing (RNA silencing) is being tried as a reversetool for gene targeting in fungi. Homology-based gene silencing induced bytransgenes (co-suppression), antisense RNA, or dsRNA has been demon-strated in many plant pathogenic fungi, including Cladosporium fulvum(Hamada & Spanu 1998), Magnaporthae oryzae (Kadotani et al. 2003),


Venturia inaequalis (Fitzgerald, Kha, & Plummer 2004), Neurospora crassa(Goldoni et al. 2004), Aspergillus nidulans (Hammond & Keller 2005), andFusarium graminearum (Nakayashiki et al. 2005). Fitzgerald, Kha, andPlummer (2004), using hairpin-vector technology, have been able to trig-ger simultaneous high-frequency silencing of a green fluorescent protein(GFP) transgene and an endogenous trihydroxynaphthalene reductase gene(THN ) in V. inaequalis. The GFP transgene acted as an easily detectable vis-ible marker, while the trihydroxynaphthalene reductase gene (THN ) playeda role in melanin biosynthesis.

Nakayashiki and colleagues (2005) developed a protocol for silenc-ing the mpg1 and polyketide synthase-like genes. The mpg1 gene is ahydrophobin gene essential for pathogenicity, as it acts as a cellular relayfor adhesion and trigger for the development of appressorium (Talbot et al.1996). Nakayashiki and colleagues (2005) were successful in silencing theabove-mentioned genes to varying degrees by pSilent-1-based vectors in70%–90% of the transformants. Ten to fifteen percent of the silenced trans-formants exhibited almost “null phenotype.” This vector was also efficientlyable to silence a GFP reporter in another ascomycete fungus, Colletotrichumlagenarium.

Bacterial Resistance

Genetic engineering for bacterial resistance has been met with relatively lit-tle success. The expression of a bacteriophage T4 lysozyme in transgenicpotato tubers led to increased resistance to Erwinia carotovora. In addition,the expression of barley a-thionin gene significantly enhanced the resistanceof transgenic tobacco to Pseudomonas syringae. Advances in the cloningof several new bacterial resistance genes (Tables 4, 6) should provide bet-ter understanding of plant-bacterial interactions. Further, RNAi technologyhas been tried to control bacterial diseases. Escobar and colleagues (2001)developed a crown gall disease-management strategy that targets the pro-cess of tumorigensis (gall formation) by initiating RNAi of the iaaM andipt oncogenes. Expression of these genes is a prerequisite for wild-typetumor formation. Transgenic Arabidopsis thaliana and Lycopersicon escu-lentum transformed with RNAi constructs that targeted iaaM and ipt gene(s)showed resistance to the crown gall disease. Transgenic plants generatedthrough this technology contained a modified version of these two bacte-rial gene(s); this was the first report on managing a major bacterial diseasethrough RNAi. The extra genes recognize the bacterial gene and effectivelyshut down the expression of the corresponding bacterial gene during infec-tion, thus preventing the spread of infection. The invading bacteria could notmake the hormones needed to cause tumors; plants deficient in the silenc-ing mechanism were hyper-susceptible to A. tumefaciens (Dunoyer et al.


2007). Successful infection relies on a potent anti-silencing state establishedin tumors, whereby siRNA synthesis is specifically inhibited. The proce-dure can be exploited to develop broad-spectrum resistance in ornamentaland horticultural plants susceptible to crown gall. This approach can beadvocated for the effective management of those pathogens that multiplyvery rapidly and cause tumor formation, e.g., Albugo candida, Synchytriumendobioticum, and Erwinia amylovora. The natsiRNA (nat-siRNAATGB2)was strongly induced in Arabidopsis upon infection by Pseudomonassyringae pv. Tomato; it down-regulated a PPRL gene that encodes a neg-ative regulator of the RPS2 disease-resistance pathway. As a result, theinduction of nat-siRNAATGB2 increased the RPS2-mediated, race-specificresistance against P. syringae pv. tomato in Arabidopsis (Katiyar-Agarwalet al. 2006b).

Recently, the accumulation of a new class of sRNA, 30 to 40 nucleotidesin length, designated as long-siRNAs (lsiRNAs), was found to be associatedwith P. syringae infection. One of these lsiRNAs, AtlsiRNA-1, contributed toplant bacterial resistance by silencing AtRAP, a negative regulator of plantdefense (Katiyar-Agarwal et al. 2007). A Pseudomonas bacterial flagellin-derived peptide was found to induce the accumulation of miR393 inArabidopsis. The miR393 negatively regulated mRNAs of F-box auxin recep-tors, causing increased resistance to P. syringae, and the overexpression ofmiR393 was shown to cause a five-fold reduction in plant’s bacterial titer(Navarro et al. 2006).

Engineering for Male Sterility

The exploitation of heterosis (hybrid vigor) through the use of hybrid vari-eties is one of the major achievements of conventional plant breeding.However, in many crops, an efficient and economical method of producinghybrid seed is not available. To overcome these difficulties, genetic engi-neering of male sterility (Williams 1995) and its restoration have emergedas tangible options for the development of male sterile and restorer linesfor hybrid seed production. There are several biotechnological approachesto develop male sterile lines, but the barnase-barstar genes have beenused with greater success. The barnase gene, from the bacterium Bacillusamyloliquefaciens, encodes the enzyme barnase (ribonuclease), which isproduced in the transgenic plant/line during the development of the anthers.Barnase destroys the tapetal tissues, preventing pollen production and con-ferring male sterility. The introduction and expression of the barstar gene,also from Bacillus amyloliquefaciens, into another plant/line result in thedevelopment of a restorer line. The hybrid plants derived from crosses ofmale sterile and restorer lines are fully fertile. This system has been com-mercially exploited in maize and oilseed rape (Denis et al. 1993; Bisht et al.


2004, Ray et al. 2007). This method can be extended to other crops for theproduction of hybrid seeds.

Engineering for Improved Nutritional Quality

There is now a growing interest in improving the nutritional quality offood crops. Genes cloned from plants and microbes are being introducedinto crop plants to enhance their nutrional value. For instance, introduc-tion of provitamin A and β carotene genes have resulted in the productionof ‘golden rice’ (Burkhardt et al. 1997; Ye et al. 2000; Beyer et al. 2002).Likewise, high protein ‘phaseolin’ and AmA1 genes have been introducedto hetereologous systems such as tobacco and potato. Intoduction of AmA1gene into potato (Chakraborty, Chakraborty, & Datta 2000; Randhawa, Singh,& Sharma 2009) has improved its yield, protein content, and quality. Vitamin-producing transgenic plants have also been developed (Herbers 2003)and emphasis is being laid on multigene engineering (Daniell & Dhingra,2002). The main objective of these crops is to add value to agri-foods(Newell-McGloughlin 2008).

Engineering for Molecular Farming/Pharming

An additional goal for the development of transgenic plants is the useof living systems for the production of metabolites at the industrial scale,e.g., specialty chemicals, antibodies, pharmaceuticals, edible vaccines, etc.The cell’s metabolic networks that evolved in nature are not optimizedfor industrial production of these metabolites. So, the performance ofmetabolic pathways is manipulated so that metabolites are over-produced.The introduction of heterologous genes and regulatory elements in theliving systems is commonly called ‘metabolic engineering,’ There are anumber of reports dealing with the production of specialty chemicals, bio-pharmaceuticals, and edible vaccines that can be stored and distributed asseeds, tubers, or fruits (Fischer & Emans 2000; Gidding et al. 2000, Daniell,Streatfield, & Wycoff 2001; Daniell & Dhingra 2002; Bonetta 2002; Maliga2003). Solulin is a recombinant, soluble derivative of human thrombomod-ulin (Weisel et al. 1996). To evaluate the production of the pharmaceuticalprotein in plants, expression vectors were generated using four differentN-terminal signal peptides. Immunoblot analysis of transiently transformedtobacco leaves showed that intact ‘solulin’ could be detected using threeof these signal peptides (Schinkel et al. 2005). Furthermore, transgenictobacco plants and BY2 cells producing solulin were generated. It hasbeen shown that plants and plant cell cultures can be used as alterna-tive systems for the production of an active recombinant thrombomodulinderivative.


Engineering for Abiotic Stress Tolerance

The transfer of cloned genes has resulted in transgenics tolerant to someabiotic stresses (Gosal, Wani, & Kang 2009; Table 5). For instance, for frostprotection, an antifreeze protein gene from fish has been transferred intotomato and tobacco. Likewise, a gene coding for glycerol-3-phosphate acyl-transferase from Arabidopsis has been transferred to tobacco to enhancecold tolerance in tobacco. Hal2 gene is being tried for developing salt tol-erance. P5CS from Vigna aconitifolia was introduced through the ‘particlegun’ method of gene transfer into Saccharam officinarum under the action

TABLE 5 Plant Genetic Engineering for Abiotic Stress Tolerance

Gene and source Plant species Remarks Reference

P5CS (Pyrroline-5-carboxylatesynthetase) fromVigna aconitifolia

N. tabacum Transgenic plants produced 10-18fold more proline than controlplants. Overproduction ofpraline also enhanced rootbiomass and flowerdevelopment in transgenicplants.

Kishore et al.1995

codA fromA. globiformis

Oryza sativa Transgenic plants had high levelsof glycinebetaine and grewfaster as compared to wild typeson removal of stress.

Sakamoto,Murata, &Murata 1998

bet A and bet B fromE. coli

N. tabacum Transgenic plants showedincreased tolerance to salt stressas measured by biomassproduction of green housegrown plants.

Holmstromet al. 2000

HVA1 from Hordeumvulgare

Triticumaestivum

Transgenic plants showedimproved growth characteristicsin response to soil-water deficits.Field trials showed that HVA1gene had the potential to conferdrought-stress protection ontransgenic spring wheat.

Sivamani et al.2000;Bahieldinet al. 2005

CBF1 from A. thaliana Lycopersiconesculentum

Transgenic tomato plants weremore resistant to water-deficitstress than the wild-type plants.

Hsieh et al.2002

mtlD from E. coli Triticumaestivum

Ectopic expression of the mtlDgene for the biosynthesis ofmannitol in wheat improvestolerance to water stress andsalinity

Abebe et al.2003

DREB 1A fromA. thaliana

Triticumaestivum

Transgenic plants expressingDREB1A gene demonstratedsubstantial resistance to waterstress under greenhouseconditions.

Pellegrineschiet al. 2004

(Continued)


TABLE 5 (Continued)


CBF3/ DREB 1A fromA. thaliana

Oryza sativa Tolerance to drought and highsalinity without growthretardation or any phenotypicalteration.

Oh et al. 2005

DREB1A fromA. thaliana

Solanumtuberosum

Transgenic plants showedsignificant tolerance againstsalinity stress (1M NaCl).

Behnam et al.2007

GmTP55 from Glycinemax

A. thalianaandN. tabacum

Ectopic expression of GmTP55 inboth Arabidopsis and tobaccoconferred tolerance to salinityduring germination and to waterdeficit during plant growth.Antiquitin may be involved inadaptive responses mediated bya physiologically relevantdetoxification pathway in plants.

Rodrigues et al.2006

CMO(cholinemonooxygenase)from Spinaciaolearceae

Oryza sativa Transgenic plants were tolerant tosalt and temperature stress atseedling stage. CMO expressingrice plants were not effective foraccumulation of glycinebetaineand improvement ofproductivity.

Shirasawa,Takabe, &Kishitani 2006

P5CS from Vignaaconitifolia

Saccharumofficinarum

Stress-inducible prolineaccumulation in transgenicsugarcane plants underwater-deficit stress acts as acomponent of antioxidativedefense system rather than as anosmotic adjustment mediator.

Molinari et al.2007

TPS1-TPS-2 fromE. coli

A. thaliana No morphological growthalterations were observed inlines over-expressing theTPS1-TPS2 construct, while theplants over expressing the TPS1alone under the control of 35Spromoter had abnormal growth,color, and shape.

Miranda et al.2007a


Oryza sativa Transgenic rice resulted in anincrease in tolerance to drought,high salinity, andlow-temperature stresses withoutstunting growth.

Oh et al. 2007a


N.tabaccum Expression was up-regulated byAtDREB1A in transgenic plantsunder normal conditions andfurther enhanced by high salt,

Cong et al. 2008

(Continued)


TABLE 5 (Continued)


which at least partiallycontributed to the high salttolerance inAtDREB1A-expressing plants.

AtMYB44 fromArabidopsis

Arabidopsis Transgenic plants exhibitedreduced rate of water loss, asmeasured by the fresh-weightloss of detached shoots, andremarkably enhanced toleranceto drought and salt stresscompared to wild-type plants.

Jung et al. 2008

of AIPC promoter. Results indicated that stress-inducible proline accumu-lation in transgenic sugarcane plants under water-deficit stress acted as acomponent of antioxidative defense system rather than as an osmotic adjust-ment mediator (Molinari et al. 2007). TPS1-TPS2 fusion gene construct wasintroduced into Arabidopsis through Agrobacterium-mediated gene trans-fer under the control of CaMV 35S or stress-regulated Cd 29A promoter.No morphological growth alterations were observed in lines overexpress-ing the TPS1-TPS2 construct, whereas the plants overexpressing the TPS1alone under the control of 35S promoter showed abnormal growth, color,and shape (Miranda et al. 2007a). Thus, it can be concluded that engineer-ing trehalose metabolism in plants can substantially increase their capacityto tolerate abiotic stresses. Late embryogenesis abundant (LEA) proteins aremainly low molecular weight (10–30 kDa) proteins involved in protectinghigher plants from damage caused by environmental stresses, especiallydrought (dehydration) (Hong, Zong-Suo, & Ming-An 2005). OsLEA 3-1 hasbeen transformed into rice through Agrobacterium-mediated gene transfermethod under the control of different promoters. Constitutive and stress-inducible expression of Os LEA 3-1 under the control of CaMV 35S andHVA1-like promoter, respectively, resulted in transgenic rice plants showingincreased tolerance to drought under field conditions (Xiao et al. 2007).

The gene regulating protein factors that regulate gene expressionand signal transduction and function under stress responses may be use-ful for improving the abiotic stress tolerance in plants. These genescomprise regulatory proteins, i.e., transcription factors (bZip, MYC, MYB,DREB, NACs, NAM, ATAF and CUC), protein kinases (MAP kinase, CDPkinase, receptor protein kinase, ribosomal protein kinase, and transcriptionregulation protein kinase), and proteinases (phosphoesterases and phos-pholipase) (Katiyar-Agarwal et al. 2006a). Transgenic overexpression ofHvCBF4 from barley in rice resulted in increased tolerance to drought,


high salinity, and low-temperature stresses without stunting plant growth.Using the 60 K Rice Whole Genome Microarrays, 15 rice genes wereidentified that were activated by HvCBF4. When compared with 12 targetrice genes of CBF3/DREB1A, five genes were common to both HvCBF4and CBF3/DREB1A, and 10 and 7 genes were specific to HvCBF4 andCBF3/DREB1A, respectively (Oh et al. 2007a). Overexpression of stress-responsive gene SNAC1 (stress-responsive NAC 1) significantly enhanceddrought resistance in transgenic rice (22%–34% higher seed-set than control)in the field under severe drought stress conditions at the reproductive stagewhile showing no phenotypic changes or yield penalty. The transgenic riceexhibited significant improvement in drought resistance and salt toleranceat the vegetative stage. Compared with wild type, the transgenic rice wasmore sensitive to abscisic acid and lost water more slowly by closing morestomatal pores, yet displayed no significant difference in the rate of photo-synthesis. DNA chip analysis revealed that a large number of stress-relatedgenes were up-regulated in the SNAC1-overexpressing rice plants. SNAC1holds promise in improving drought and salinity tolerance in rice (Hu et al.2006). Several transcription factors have been used to develop transgenicplants tolerant to abiotic stresses (Pellegrineshci et al. 2004; Oh et al. 2005;Cong et al. 2008; Jung et al. 2008).

Engineering for Improved Post-Harvest Preservation

The ripening of fleshy fruits involves changes in color, texture, and fla-vor. This process is mediated through the action of enzymes. The activitiesof many of the key enzymes are regulated by the controlled expressionof ‘ripening genes.’ In climacteric fruits, such as tomato, the expressionof ripening genes is stimulated by ethylene, which functions as a ripen-ing hormone. Many ripening genes have now been cloned, includingpolyglacturonase and pectinesterase involved in cell wall softening; phy-totene synthase, required for carotenoid synthesis; and ACC synthase andACC oxidase that catalyse the production of ethylene. A number of tech-niques are now available for overexpression of particular genes in fruits tochange their physiological and biochemical properties. The techniques ofgene silencing either using antisense genes or sense-supression have beenused successfully to reduce or inactivate the expression of specific genesand determine their function. Ripening of fruits and senescence in flow-ers can be delayed by antisense expression of genes involved in pectinmetabolism or ethylene biosysnthesis. Genetically engineered tomatoes pos-sess lower activity of the ripening enzyme, polygalacturonase, which delaysripening. Transgenic tomato variety ‘Flavr Savr’ possessing longer shelf lifewas released in United States. Reduction in ethylene synthesis has beenshown to improve quality and storage life of tomato and melon. Fruit ripen-ing was delayed by controlling ethylene production in apple (Yao et al.


1995; Hrazdina et al. 2000), grapes (Mehlonbacher 1995), and citrus (Wonget al. 2001). Further, these approaches are now being used to improve flow-ering plants by changing plant architecture, flower color, and shape, andto improve vase-life of cut flowers to meet consumer demand. Transgeniccarnations and geraniums that produce less ethylene exhibit delayed petalsenescence. Attempts are now being made to develop marker gene-freetransgenic plants carrying only the gene(s) of agronomic importance toaddress biosafety issues (Puchta 2003).

GENE CLONING

With the availability of a variety of cloning vectors, useful genes can now beisolated, cloned, and characterized. The discovery of the reverse transcrip-tase enzyme made it possible to isolate genes for specific proteins or geneshaving tissue-specific expression. Nowadays, PCR cloning has become rou-tine especially for those genes whose base sequences are known. Severalgenes, particularly for disease resistance, have been isolated and cloned(Table 6) from genomic or cDNA libraries using heterologus or synthetricprobes. Using such genes, suitable gene constructs carrying marker gene(s),promoter, and terminator sequences are developed for plant transformation.

MOLECULAR MARKERS

Development and utilization of molecular markers to detect differencesin the DNA of individual plants has many applications of value to cropimprovement. These differences are known as molecular markers becausethey are often associated with specific genes and act as ‘signposts’ to thosegenes. Several types of molecular markers that have been developed andare being used in plants include: restriction fragment-length polymorphism(RFLP), amplified fragment-length polymorphism (AFLP), randomly ampli-fied polymorphic DNA (RAPD), sequence-tagged sites (STS), expressedsequence tags (ESTs), simple sequence repeats (SSRs) or microsatellites,sequence-characterized amplified regions (SCARs), and single nucleotidepolymorphisms (SNPs)(Paterson, Tanksley, & Sorrels 1991; Hoisington,Listman, & Morris 1998; Joshi, Ranjekar, & Gupta 1999). Such markers,closely linked to genes of interest, can be used to select indirectly for thedesirable allele, which represents the simplest form of marker-assisted selec-tion (MAS) and is now being exploited to accelerate backcross breedingand pyramid several desirable alleles (Singh et al. 2001). Using molecularmarkers, genotypes can be distinguished at plant, tissue, or even cellularlevel. These molecular markers are not stage-specific. Selection of a markerflanking a gene of interest allows selection for the presence (or absence) of


TABLE 6 List of Cloned/Fine Mapped Important QTL for Disease Resistance

Gene Host Pathogen Reference

Xa1 Rice Xanthomonas oryzae pv.Oryzae

Yoshimura et al.1998

RPM1 Arabidopsis Pseudomonas syringae Boyes, Nam, &Dangl 1998

Pm24 Wheat Blumeria graminis f. sp. Tritici Huang et al. 2000Ve Tomato Verticillium dahliae Kawchuk et al. 2001Lr10 Wheat Puccinia triticina Feuillet et al. 2003Pm5e Wheat Blumeria graminis f. sp. tritici Huang et al. 2003Pm3 Wheat Blumeria graminis f. sp. tritici Huang et al. 2004PmPS5A,

PmPS5BTriticum

carthlicumBlumeria graminis f. sp. tritici Zhu et al. 2005

Pi-d2 Rice Magnaporthae oryzae Chen et al. 2006Pm34 Aegilops

tauschii cossBlumeria graminis f. sp. tritici Miranda et al. 2006

PmY201 andPmY212

Aegilopstauschii Coss

Blumeria graminis f. sp. tritici Sun et al. 2006

Pi9 Rice Magnaporthae oryzae Qu et al. 2006NCA6 Pm T. monococcum Blumeria graminis f. sp. tritici Miranda et al. 2007cPm35 Aegilops tauschii Blumeria graminis f. sp. tritici Miranda et al. 2007bPi40(t) Rice Magnaporthae oryzae Jeung et al. 2007Pi36 Rice Magnaporthae oryzae Liu et al. 2007bPi-ta Rice Magnaporthae oryzae Huang et al. 2008Snn1 Wheat Stagonospora nodorum Reddy et al. 2008Lr 60(LrW2) Wheat Puccinia triticina Hiebert et al. 2008Pi20(t) Rice Magnaporthae oryzae Li et al. 2008Pik-h Rice Magnaporthae oryzae Xu et al. 2008Pm36 Triticum turgidum

var. dicoccoidesBlumeria graminis f. sp. tritici Blanco et al. 2008

Pm37 Triticumtimopheevii

Blumeria graminis f. sp. tritici Perugini et al. 2008

Pm23 Wheat Blumeria graminis f. sp. tritici Hao et al. 2008Lr34/Yr18 Wheat (Puccinia triticina) and

(P. striiformis f. sp. tritici)Spielmeyer et al.

2008YrC591 Wheat Puccinia striiformis f. sp.

TriticiLi et al. 2009

Pm43 Thinopyrumintermedium

Blumeria graminis f. sp. Tritici He et al. 2009

a gene in new progeny. Thus, molecular markers can be used to follow anynumber of genes during the breeding program (Paran & Michelmore 1993).DNA markers are now extensively being used for gene mapping/tagging(Vikal et al. 2004; Gill et al. 2007; Kuraparthy et al. 2007a, b; Singh et al.2007; Gill et al. 2008; Lagudah et al. 2009; Li, Niu, & Chen 2009; Tsilo,Chao, & Jin 2009). Further, these can also be used for dissecting poly-genic traits into their Mendelian components or quantitative trait loci (Luo &Kearsey 1992; Yamasaki et al. 1999; Table 7). Likewise, molecular markersare also extensively used to probe the level of genetic diversity among dif-ferent varieties and related species. The applications of such evaluations aremany, including DNA profiling, fingerprinting for patenting and IPR issues,


TABLE 7 QTLs in Relation to Drought Tolerance in Different Plant Species

Species Cross Environment Main traits Reference

Maize Lo964 × Lo1016 Field Root traits and yield Tuberosa et al.2002

Rice CT9993-5-10-1-M ×IR62266-42-6-2

Field Root morphology,plant height, grainyield

Babu et al.2003

Rice IR62266 x IR60080. Greenhouse Osmotic adjustment Robin et al.2003

Maize F2 × F252 Field Silking date, grainyield, yield stability

Moreau,Charcosset,& Gallais2004

Rice Zhenshan 97 ×Wuyujing 2

Field Stay-green Jiang et al.2004

Rice Bala × Azucena Field Grain yield, yieldcomponents

Lafitte, Price, &Courtois2004

Rice CT933 × IR62266 Field Grain yield Lanceras et al.2004

Rice Teqing × Lemont Field Yield components,phenology

Xu et al. 2005

Cotton G. hirsutum ×G. barbadense

Field 12C/13C, osmoticpotential, canopytemperature, drymatter, seed yield

Saranga et al.2004

Wheat Beaver × Soissons Field Flag leaf senescence Verma et al.2004

Arabidopsis Landsberg × CapeVerde

Greenhouse 12C/13C, floweringtime, stomatalconductance,transpirationefficiency

Juenger et al.2005

Barley Tadmor × Er/Apm Field 12C/13C, osmoticadjustment, leafrelatives watercontent, grain yield

Diab et al.2004

Wheat SQ1 × ChineseSpring

Field Water-use efficiency,grain yield

Quarrie et al.2005

Rice Kalinga III ×Azucena

Field Root length Steele et al.2006

Rice Vandana × WayRarem

Field Grain yield, underreproductive stage

Bernier et al.2007

Rice Akihikari × IRAT109

Screeningfacility

Specific water use,(SWU), Water useefficiency (WUE)

Kato et al. 2008

efficiently managing genetic resources, and facilitating introgression of chro-mosomal segments from alien species. In addition, markers and comparativemapping of various species have been very valuable in enhancing our under-standing of genome structure and function, and have allowed the isolation ofgenes of interest via map-based cloning. Molecular maps (Singh et al. 2007)


have been developed for several crop plants such as tomato, wheat, andrice. Molecular markers have facilitated the dissection of quantitative traitsinto their simple genetic components (Tanksley 1993; Pelleschi et al. 2006;Bernier et al. 2007; Kato et al. 2008). Further, these markers assist selectionand pyramiding of QTL alleles through MAS (Ribaut et al. 2004; Chhunejaet al. 2007; Neeraja et al. 2007; Ribaut & Ragot 2007). A novel upland ricevariety, Birsa Vikas Dhan 111 (PY 84), has recently been released in theIndian state of Jharkhand (Steele 2009). It was developed using marker-assisted backcrossing, with selection for multiple quantitative trait loci (QTL)for improved root growth to improve its performance under drought condi-tions. It is an early-maturing, drought-tolerant and high-yielding variety withgood grain quality suitable for direct-seeding in uplands and transplantingin medium lands of eastern India. It outyields its recurrent parent by 10%under rainfed conditions.

CONCLUSIONS

Innovative approaches of cellular and molecular biotechnology haveemerged as a valuable adjunct to supplement and complement the con-ventional methods for precise and efficient breeding of a wide varietyof crop plants. Biotechnological approaches are now increasingly beingused for creation, conservation, characterization, and utilization of geneticvariability for germplasm enhancement. Micropropagation ensures true-to-type and rapid multiplication for bulk production of super-elite plantingmaterial, especially of the vegetatively propagated plant species. This tech-nology has witnessed a huge expansion globally, with an estimated globalmarket of US$15 billion/annum for tissue-culture products. Techniquesof anther/microspore culture, somaclonal variation, embryo culture, andsomatic hybridization are being exploited to obtain incremental improve-ments in plants. Production of secondary metabolites, such as foodflavors, food colors, dyes, perfumes, drugs, and scented oils used in aro-matherapy, through cell culture/hairy root culture are leading examplesof molecular farming/pharming. Cryopreservation of germplasm at thecellular/tissue/organ level in liquid nitrogen at −196◦C is highly rewardingfor establishing germplasm banks, especially in the case of vegetatively prop-agated and rare, endangered plant species. Several genes, particularly forinsect and disease resistance, have been isolated and cloned from differentorganisms using genomic or cDNA libraries, heterologus or synthetic probes,transposon tagging, promoter- enhancer traps, subtractive hybridization, andmap-based cloning.

Several vector and vectorless methods have been developed for genetictransformation of plants. Agrobacterium and ‘particle gun’ methods havebecome routine and are now being widely used for efficient production of


transgenic plants. In fact, transgenesis has emerged as an additional toolto carry out single-gene breeding or transgenic breeding of crop plants.Unlike conventional breeding, only the cloned genes of agronomic impor-tance are introduced without co-transfer of other, undesirable genes fromthe donor. The transformation method provides access to a larger genepool as the gene(s) can be acquired from viruses, bacteria, fungi, insects,animals, human beings, unrelated plants, and even from chemical synthe-sis in the laboratory. Remarkable achievements have been made in theproduction, characterization, and field evaluation of transgenic plants in sev-eral field, fruit, vegetable, ornamental and forest crops. Transgenic varietiesbelonging to 10 crops are now being commercially grown across an area ofabout 134 million hectares spread across 25 countries. Genetically modifiedfoods, such as tomato containing high-lycopene flavonols as antioxidants,cavity-fighting apples, golden rice with enhanced vitamin A, golden brassicawith pro-vitamin A, canola rich in vitamin E, proteinaceous potatoes, ediblevaccines, decaffeinated tea and coffee, and nicotine-free tobacco are lead-ing examples of genetically engineered crops. Several types of molecularmarkers, such as RFLPs, AFLPs, RAPDs, STS, ESTs, SSRs or microsatellites,SCARs, SNPs, and insertions/deletions (INDELS) are being used in allelemining, developing saturated linkage maps, marker-assissted breeding, andgene pyramiding. Biotechnolgy has made tremendous a contribution towardincreasing crop yields; improving nutritional quality; enabling crops to beraised under adverse conditions; increasing shelf life of fruits, vegetablesand flowers; and developing resistance to pests and diseases.

REFERENCES

Abebe, T., A.C. Guenzi, B. Martin, and J.C. Cushman. 2003. Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity. Plant Physiol.131:748–1755.

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