Compendium of Transgenic Crop Plants

Volume 1: Transgenic Cereals & Forage Grasses

1. Rice2. Maize3. Wheat4. Barley5. Oat6. Sorghum7. Pearl millet8. Finger Millet9. Cool Season Forage Grasses10. Bahia Grass

Volume 2: Transgenic Oilseed Crops

1. Soybean2. Oilseed Brassicas3. Sunflower4. Peanut5. Flax6. Sesame7. Safflower

Volume 3: Transgenic Legume Grains and Forages

1. Common bean2. Cowpea3. Pea4. Faba Bean5. Lentil6. Tepary Bean7. Asiatic Beans8. Pigeonpea9. Vetch Pea10. Chickpea11. Lupin12. Alfalfa 13. Clovers

Volume 4: Transgenic Temperate Fruits and Nuts

1. Apple2. Pears3. Peach4. Plum5. Berries6. Cherry7. Grapes8. Almond9. Persian Walnut

Volume 5: Transgenic Tropical and Subtropical Fruits and Nuts

1. Citrus Fruits2. Grapefruit3. Banana4. Pineapple5. Papaya6. Mango7. Avocado8. Kiwifruit9. Passion Fruit10. Persimmon

Volume 6: Transgenic Vegetable Crops

1. Tomato2. Egg plant 3. Capsicums4. Vegetable Brassicas5. Radish6. Carrot 7. Cucurbits 8. Alliums9. Asparagus 10. Leafy Vegetables

Volume 7: Transgenic Sugar, Tuber and Fiber Crops

1. Sugarcane2. Sugar beet3. Stevia4. Potato5. Sweet potato6. Cassava7. Cotton

Volume 8: Transgenic Plantation Crops, Ornamentals and Turf Grasses

1. Tobacco2. Coffee3. Cocoa4. Tea5. Rubber 6. Medicinal Plants7. Ornamentals8. Turfgrasses

Volume 9: Transgenic Forest Tree Species

1. Poplars 2. Eucalypts 3. Pines 4. Spruces 5. Chestnuts 6. Birches 7. Douglas-fir 8. American Elm 9. Black Walnut 10. Casuarinaceae 11. Black Locust 12. Sandalwood 13. Teak

Volume 10: Master Index

Compendium of Transgenic Crop Plants: 9 Volume Set + Index This series offers a comprehensive review of the commercially relevant transgenic plants developed and presently utilized. Volumes 1-9 cover around 100 plant species, including all economic plants from crops to forest trees. Volume 10 is the master index volume.

Each chapter covers one particular species (or sometimes group of closely related species) and the transgenic versions developed for that particular species.

Key features:

Offers a complete description of the successfully developed transgenic plants for around 100 commercially relevant crops from fruits and vegetables to grains, industrial crops and forest tree species Covers the transgenic techniques used in the molecular tailoring of traits relevant from an agricultural, medicinal and environmental point of view Explores future developments, from desired traits to expected new technologies Examines risks and concerns around the use of GM crops Discusses public perception, industrial perspectives and political and economic consequences Contributions from over 300 leading scientists from around the world

Provides a valuable and inclusive reference for students, researchers, scientists and industry professionals, studying and working on transgenic plants.

ISBN: 978-1-4051-6924-0 Hardcover 2800 pages October 2008

*Pre-publication prices: 695.00* 965.00* $1390.00* Valid until 31st December 2008, thereafter 825.00*

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1

RiceChandrakanth Emani1, Yiming Jiang1, Berta Miro2,

Timothy C. Hall1 and Ajay Kohli21Department of Biology, Texas A&M University, College Station, TX, USA

2Division of Biology, Newcastle University, Newcastle upon Tyne, UK

Rice is LifeTheme of UN International Year of Rice, 2004

Cutting stalks at noon time Perspiration drips to the earthKnow you that your bowl of rice Each grain from hardship comes?

Cheng Chan-Pao, Chinese Philosopher

1. INTRODUCTION

Rice serves as the principal source of nourishmentfor over half of the global population and vieswith wheat andmaize as themost important cerealcrop. As a symbol of life, prosperity, fertility,and self-sufciency, it is deeply embedded in thecultural heritage of many societies. The globalimportance of this crop reached its pinnacle onthe occasion of the 57th session of the UnitedNations General Assembly, when the Food andAgricultural Organization declared the year 2004as the International year of Rice (FAO, 2004). Thisunprecedented step in United Nations history ofdevoting a year to a commodity was based onthe need to heighten awareness of the role ofrice in alleviating global poverty andmalnutrition.The declaration was also aimed at the need tofocus world attention on the role that rice canplay in providing food security and eradicatingpoverty in the attainment of the internationallyagreed development goals (UNDeclaration, 57thSession, 2004).

Rice accounts for more than one-fth of thecalories consumed by human beings in theirglobal diets (Smith, 1998). In Asia, rice andits derivatives account for 6070% of energyintake for over 2 billion people (FAO, 2004). Asan important commercial crop, it is the mostrapidly growing food source in Africa and isof signicant importance to food security inan increasing number of low-income food-decitcountries. Rice-based production systems andtheir associated postharvest operations employnearly 1 billion people in rural areas of developingcountries, and about four-fths of the worlds riceis grown by small-scale farmers in low-incomecountries (FAO, 2004). The production of 700million metric tons (FAO data for 2005) makesrice the worlds largest seed crop, closely followedby maize and wheat.

1.1 History, Origin, and Distribution

The word rice is believed to be of IndoIranianorigin, the term being derived from the Tamil word

Compendium of Transgenic Crop Plants: Transgenic Cereals and Forage Grasses. Edited by Chittaranjan Kole and Timothy C. HallC 2008 Blackwell Publishing Ltd. ISBN 978-1-405-16924-0

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2 TRANSGENIC CEREALS AND FORAGE GRASSES

Arisi and the Arabic Ar-ruzz. The English originsare attributed to Greek Oryza, via Latin Oriza,Italian Riso, and the Old French Ris (Wikipedia,2007).

Cultivation of rice is thought to have originatedalong the Yangtze Delta, one of the cradles ofEast Asian civilization in China. Paleobotanicalhypothesis credits the Chinese of the LatePleistocene era with collecting wild rice, leading toits eventual domestication about 6400 BC (Zhao,1998). Early Neolithic groups are also known tohave cultivated rice, possibly as early as 9000 BCwith radiocarbon dating evidence going back toat least 7000 BC (Crawford and Shen, 1998;Diamond, 1999; Zohary and Hopf, 2000).

The oldest scriptures in India mention rice (theterm used was Dhanya), rice dishes, and aspectsof rice cultivation (Nene, 2005). Farmers practicedmixed farming techniques planting the rice cropin integrated elds during the Indus ValleyCivilization (Kahn, 2005). Wild rice appeared asearly as 5440 BC in the Belan and the Gangesvalleys of northern India. Along with barley, meat,dairy products, and sh, rice was a dietary stapleof ancient Dravidian society (Taylor, 2004).

Dryland ricewas introduced to Japan andKoreabetween 3500 and 1200 BC (Crawford and Lee,2003). Wetland cultivation techniques migrated toIndonesia around 1500 BC and then to Japanby 100 BC. The Niger River delta extending toSenegal was the rice farming region in Africaduring 1500800 BC. The Moor invasion of theIberian Peninsula in 700 AD introduced rice toSpain, from whence it spread during the 15thcentury through Italy and France to all continentsduring the great age of European exploration(Wikipedia, 2007). Rice arrived in North Americain 1694, from Madagascar to South Carolina.Plantation owners in Georgetown, Charlestown,and Savannah learnt techniques of rice culturesuch as dyking of marshes and periodical oodingof elds from African slaves, who also broughtwith them rice mills made of wooden paddles andwinnows made of sweetgrass baskets. Subsequentimprovements in rice production can be attributedto the invention of rice mill and the additionof waterpower to these mills. The predominantstrains of rice in Carolina were christenedCarolina Gold (Wikipedia, 2007).

Plants of the genus Oryza are known to thrivein desert, hot, humid, ooded, dry, and cool

conditions, and grow in saline, alkaline, and acidicsoils. There are 24 species of Oryza. Domesticatedor cultivated rice comprise two main species, theAsian rice Oryza sativa, and the African riceOryza glaberrima. O. sativa was domesticatedfrom wild Asian rice, Oryza rupogon, originatingin the foothills of the Himalayan mountainranges, with O. sativa var. Indica of India andO. sativa var. Japonica from China and Japan(Londo et al., 2006). O. sativa cultivars consistof three groups: the short-grained Japonicaor Sinica (e.g., Japanese rice); long-grainedIndica varieties (e.g., Basmati rice); and thebroad-grained Javanica rice (e.g., Jefferson rice)(Zohary and Hopf, 2000).

Originating from itsAsian homeland, rice showsa diverse distribution and is cultivated in 113countries and 6 continents (none in Antarctica!).Rice is grown under a wide range of soil moistureregimes, from deep ood to dry land, and invarious soil conditions. Rice-based productionsystems span from 53 N, in the Heilongjiang.Province of China, to 35 S in New SouthWales ofAustralia; from the tropical rain forest climate ofthe Congo to the continental temperate climate inKrasnodar of Russia; from the arid desert climatefound inEgyptsNileDelta, to the sea-level regionsin Guinea-Bissau, to 2700m above sea level asin the Himalayan mountain chains in Nepal andIndia (FAO, 2004).

1.2 Botanical Description

1.2.1 Scientific classification

Kingdom: PlantaeDivision: MagnoliophytaClass: LiliopsidaOrder: PoalesFamily: PoaceaeGenus: OryzaSpecies: O. glaberrima and O. sativa

1.2.2 The plant

Rice is a semiaquatic, monocarpic annual grassplant, its height varying from 0.6 to 1.8m (26 ft.)tall. The plants tiller, i.e., develop multiple shoots,depending on the variety, spacing, and soil fertility.

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RICE 3

The grass has long, slender leaves 50100 cm longand 22.5 cm broad. The small wind-pollinatedowers are produced on a branched archingto pendulous inorescence 3050 cm long. Theinorescence is an open panicle. Flowers aredistinct in having six anthers as opposed to thecommonly seen three anthers in other grasses.Spikelets have a single oret, lemma, and paleaenclosing a grain (caryopsis) 512mm long and23mm thick that can be yellow, red, brown,or black. The lemmas may be awnless, partly orfully awned. The rice kernel has four primarycomponents: the hull or the husk, the seed coator bran, the embryo or germ, and the endosperm.Rice milling procedures yield a variety of productsdepending on the extent of outer layer removal.If just the inedible husk is removed, it results inbrown rice where, the nutritious high-ber branis a source of 8% protein, iron, calcium, and Bvitamins. Removal of the bran and germ results inwhite or polished rice, which is greatly diminishedin nutrients.

1.2.3 Habit and habitat

The ideal climate for rice growth is 75 F (24 C).Its growth cycle is 36 months. In nonindustrial-ized nations, rice elds are typically prepared byploughing (by cattle-drawn ploughs or a tractor),fertilizing (traditionally with dung or sewage), andsmoothing (a process of dragging a log acrossthe eld). Seedlings are prepared in seedling beds,and after a month, are transplanted manuallyto the elds, which have been ooded by rainor river water. Dike-controlled canals or manualwatering maintains irrigation during the entiregrowth period. Before harvesting the crop, theelds are allowed to drain.

1.2.4 Genome

Evolutionary lineages of major owering plants,monocots, and dicots, as illustrated by rice andArabidopsis, were shown to have diverged about200 million years ago. The genomes of thesetwo plants do not share extensive synteny, butsimilarities exist amongst many encoded proteins.Rice genes diverge from Arabidopsis in guanine-

cytosine (GC) content, codon usage, and aminoacid usage. There is also a gradient in the GCcontent of rice genes that is not seen inArabidopsisgenes, with the 5 end being up to 25% richer inGCcontent than the 3 end.

The draft genome sequences of both Indicarice (Yu et al., 2002) and Japonica rice (Goffet al., 2002) were published in the same year.These analyses gave an average gene size of 4.5 kbwith some 513 ribosomal RNA gene repeats and688 centromeric repeats. The average rice genomeis a compact 430Mb, one-sixth the size of thehuman and maize genomes (Leach et al., 2002).It is the smallest of all genomes amongst grasses,commonly grown as crops. The smaller genome isreected in a higher gene density relative to othercereals, with an average of one gene every 15 kb(Goff, 1999). However, the overall organizationof genes is preserved in such a way that ricegenomic information can be used as a useful guidein deciphering the larger genomes of maize andbarley. The genomic similarity makes it possible toapproximate the genomes of these grain species ina concentric circle, and use the smaller rice genomeas a guiding point to nd related genes of interestin the larger genomes (Figure 1).

The characteristics of rice genome are summa-rized in Table 1 (Goff et al., 2002; Yu et al., 2002).

Barley

Figure 1 Genome colinearity among cereals [Reproducedfrom Rice Genome Poster (2002)]

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Table 1 Salient features of the rice genome(a)

Rice strain Indica Japonica

Genome size (Mb) 466 420Number of genes 4656K 3250KDuplicated genes (%) 74 77Number of transposable elements >24.9% 4220Number of single sequence repeats 1.7% 46 666

(a)Reproduced from the Rice Genome Poster (2002)

1.2.5 Cytological features

O. sativa L. has a chromosome number of 2n =24 (Kuwada, 1910). The DNA content wasdetermined to be 0.870.96 pg/2C (Martinez et al.,1993). In contrast to the rapid progress inmolecular analysis, progress in cytological char-acterization of the rice genome has had limitedsuccess. Early studies using uorescent in situhybridization techniques to map DNA sequenceson chromosomes (Fukui et al., 1994; Jiang et al.,1995; Ohmido et al., 1998) involved mapping onmitotic metaphase chromosomes. This approachprovided limited details due to low mappingresolution. Cheng et al. (2001a) successfullyused pachytene chromosome karyotyping byhybridizing 24 bacterial articial chromosome(BAC) clones and a rice centromere DNA-specic probe with all 24 chromosomal arms.The arm-specic BAC clones unambiguouslyidentied rice chromosomes in both mitotic andmeiotic cells at 2n = 24. The longest waschromosome 1 (61.12m) and the shortest waschromosome 10 (24.74m), the descending orderof chromosome length being 1, 3, 2, 6, 4, 5,7, 8, 11, 9 (not including the ribosomal DNA(rDNA)), 12, and 10. Heterochromatic regionsthat have a highATcontent relative to euchromaticregions were preferentially stained by DAPI(4, 6-diamidino-2-phenylindole). Chromosomes 4and 10 have the most distinct heterochromaticpatterns, with one-third of the chromosome,including the entire short arm and part ofthe long arm, being highly heterochromatinized.Other heterochromatic regionswere the pericentricregion of chromosome 5, the distal region ofthe long arm of chromosome 11, and the shortarm and pericentric region on the long arm ofchromosome 10. The three longest chromosomes(1, 2, and 3) are more euchromatic than the others.

1.3 Economic Importance

1.3.1 Production and trade

Rice has a key role in the food security of theworld. In 2005, world rice production reacheda record level of 621 million tons (FAO, 2005).Much of the expansion was concentrated in Asia,with mainland China boosting production by 6million tons compared to the previous year. Largeincreases were also reported from Bangladesh,India, Myanmar, Pakistan, Sri Lanka, andThailand. Developing countries account for 95%of the total world rice production, 83% of exports,and 85% of imports, with China and Indiaaccounting for more than 50%. According to FAOfact sheets, global trade in rice grew at 7% a yearthroughout the 1990s.

The top 10 rice-producing countries in terms ofmetric tons are as in Table 2 (FAO, 2004).

1.3.2 Nutrition

Rice provides 20% of the worlds dietary energysupply, and is followed by wheat (19%) and maize(5%) (FAO, 2004). Rice is a good source ofthiamine, riboavin and niacin. Brown rice is agood source of dietary ber. The overall aminoacid prole for rice seed shows high values forglutamic and aspartic acid, with lysine as thelimiting acid. Rice has a rich genetic diversity withover 1000 varieties and its natural colors includebrown, red, purple, and black. Nutrient contentsof these varieties are summarized in Table 3.

Rice is an integral part to the culinary traditionsof many cultures with personal preferencesregarding texture, taste, color, and stickiness. Dry

Table 2 Top 10 rice producers(a)

Country Metric tons

1. China 166.0 1062. India 133.5 1063. Indonesia 51.8 1064. Bangladesh 38.0 1065. Viet Nam 34.6 1066. Thailand 27.0 1067. Myanmar 21.9 1068. Philippines 13.2 1069. Brazil 10.2 106

(a)Reproduced from FAO (2004)

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RICE 5

Table 3 Nutrient contents of rice varieties(a)

Type of rice Protein (g/100 g) Iron (mg/100 g) Zinc (mg/100 g) Fiber (g/100 g)

White 6.8 1.2 0.5 0.6Brown 7.9 2.2 0.5 2.8Red 7.0 5.5 3.3 2.0Purple 8.3 3.9 2.2 1.4Black 8.5 3.5 4.9

(a)Reproduced from FAO Rice Fact Sheet: Rice and Human Nutrition (2004)

aky rice is preferred in SouthAsia and theMiddleEast; moist sticky rice in Japan, Taiwan, Korea,Egypt, and northern China; red rice and long-grained scented rice in India. Many countrieshave signature recipes such as sushi (Japan), friedrice, pulav, and biryani (India), paella, risotto, andpancit (Italy).

1.4 Traditional Breeding: BreedingObjectives, Tools and Strategies, andAchievements

The science of plant breeding has been developedmainly in the 20th century.However, as a practice itgoes back thousands of years when art and scienceas we know them now, were a unied learningprocess for enhanced survival. Archaeologicalnds in India and China indicate the beginningof rice cultivation from earlier than 10 000 yearsago (Chang, 2000). Starting from India andChina,the dispersal and selection of ancient varieties ofO. sativa in South-East Asia, Austronesia, andAfrica date back to 5000 years. At present, riceis the staple food of nearly half of the worldpopulation. Nearly 90% of all rice is grown inAsia with China and India together accountingfor nearly one-third of the total global productionof 618 million tons in 2005 (International RiceResearch Institute, 2006). Only a little more than5% of the global rice production is traded ininternational markets indicating its importance infeeding the domestic population. Since after theSecond World War, there has been an exponentialgrowth in population and in the number of peoplebelow poverty line in Asia and Africa. Hence,through major international research initiatives,rice breeding efforts have targeted the increase ofrice yield. TheRiceDevelopment Program of FAOrecommends increases in rice production in all

ecologiesfrom high altitude to coastal areas andfrom temperate to tropical climates. Various ricebreeding efforts globally have directly or indirectlyaimed at increasing the rice yield to support theunprecedented growth in population.

1.4.1 Breeding objectives

1.4.1.1 Abiotic stress resistance

This includes drought, salinity, waterlogging, coldand frost, soil pH, mineral deciency, and toxicity.

1.4.1.2 Biotic stress resistance

This includes the following:

(a) Major pests such as yellow stem borer(Scirpophaga incertula), gall-midge (Orseoliaoryzae), brown planthopper (Nilaparvatalugens), white-backed planthopper (Sogatellafurcifera), green leafhopper (Nephotettix),striped stem borer (Chilo suppressalis), riceleaf folder (Cnaphalocrocis medinalis and/orMarasmia exigua).

(b) Major pathogens such as bacterial blight(Xanthomonas oryzae), sheath blight (Rhi-zoctonia solani), fungal blast (Magnaporthegrisea), fungal stem rot (Sclerotium oryzae),root-knot nematode (Meloidogyne), ricedwarf virus, rice grassy stunt virus (RGSV),rice hoja blanca virus (RHBV), rice yellowmottle virus (RYMV), and rice tungro virus(RTD).

1.4.1.3 Other agronomic traits

Other major agronomic traits include: lifecycletime, weed competitiveness, nitrogen (and other

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macronutrient) uptake and utilization, plantheight, number of tillers and panicles, photoperiodsensitivity, owering time, wide compatibility,male sterility and/or self-incompatibility, fertilityrestoration, grain number, weight, size, shape,fragrance, composition, and nutritional qualityincluding starch, proteins, macro- and micronu-trients.

1.4.2 Breeding tools and strategies

According to Khush and Brar (2002) the tools andstrategies used to achieve the above objectives canbe divided into two phases, the evolution phaseor broadening the gene pool of rice cultivars,whereby rice populations are created that can serveas selection stocks and the evaluation phase orincreasing selection efciency, whereby superiorgenotypes are selected.

1.4.2.1 Evolution phase

The following approaches have been used toexpand the genetic pool of rice:

(a) Wide hybridization refers to creating hybridsbetween the cultivated species of O. sativacontaining the AA genome and any one ofthe wild variety represented by 24 speciescontaining any one of the 10 possible genomecompositions (AA, BB, CC, BBCC, CCDD,EE, FF, GG, HHJJ and HHKK). The strat-egy of embryo rescue has been successfullyused to transfer a number of genes forresistance to insect pests and pathogens fromthe wild species into the cultivated speciescreating nuclear genome diversity (Jena andKhush, 1990; Khush et al., 1990; Brar andKhush, 1997). Additionally, Lin and Yuan(1980) created cytoplasmic diversity whenthey successfully used the nucleus of thecultivatedO. sativa and cytoplasm of the wildO. sativa L. f. spontanea. This hybrid hasbeen the source of cytoplasmic male sterilityin the highly successful Chinese hybrid riceprograms.

(b) Somaclonal variation refers to genomicchanges created by dedifferentiation and

regeneration cycle of the tissue cultureprotocols. It serves, as amethod for natural se-lection of mutations since in vitro-regeneratedplants must retain genome functionality togive rise to a largely normal plant. Thetechnique was successfully used to create avariety with superior cooking qualities andincreased resistance to rice blast (Araujo andPrabhu, 2002).

1.4.2.2 Evaluation phase

The following methods are used for efcientlyselecting for desired characteristics:

(a) Field-based evaluation on hybrids or variantsgenerated through embryo rescue or tissueculture, respectively was the main route ofselection of desired phenotype in the con-ventional breeding methods. However, theprocess of evaluating and selecting the desiredphenotype also now starts in the laboratory,for example, anther culture can be used togenerate doubled haploid (DH) plants thatserve as true breeding lines thus shorteningthe time toward generating a new variety(Khush and Brar, 2002). Selection efciencyof DH lines is higher if dominance variationas evidenced through a number of newvarieties developed using DH lines (Khushand Brar, 2002).

(b) Molecular genetics based evaluation is nowthe norm since DNA markers have highlyfacilitated the tracking of characters throughcrosses and generations. Although the meth-ods have not been extensively used on widehybrids or somaclonal variants, they havebeen very useful in tagging and trackingrice fungal blast and bacterial blight resis-tance quantitative trait loci (QTLs) throughvarious crosses (Young, 1996). Variousmolecular markers are now available foruse such as restriction fragment lengthpolymorphism, amplied fragment lengthpolymorphism, random amplied polymor-phic DNA, simple sequence repeats, singlenucleotide polymorphisms, etc. Semagn et al.(2006) provide a good review of the differentmolecularmarkers used in plant breeding, the

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RICE 7

principles on which these are based, limitedinformation on laboratory protocols and theadvantages of each. Additionally, Khush andBrar (2002) provide a detailed account andexamples of how rice breeding efforts havebeen positively affected by efcient selectionusing molecular marker technology, molecu-lar maps, synteny relationships, marker-aidedselection, QTL mapping, gene tagging andgene pyramiding, map-based cloning, andfunctional genomics.

Laboratory based techniques, such as the widehybridization and embryo rescue, anther culture,somaclonal variation, and molecular markers mayhave indeed hastened the process of selection andreduced the time to generating a new variety, yetsubstantial advances and achievements were madeusing classical breeding systems of pedigree andbulk methods, three-way remote crosses, recurrentparent, and rapid generation advancement. Thechromosomal deletion lines, recombinant inbredlines, near isogenic lines, and pure lines were madeavailable through sheer diligence and perseverancein the elds. These methods were facilitated thenby ethylmethane sulfonate (EMS)- and radiation-mediated mutation. Creation of semidwarf vari-eties, resistance to certain pests and pathogens, andmaintaining cytoplasmic and genic male sterilitywere all achieved through classical breeding. Bymid 1980s the rice production was double that ofmid 1950s. Nearly half that improvement was dueto additional land being cultivated but the otherhalf was due to high-yielding varieties developedthrough conventional breeding methods. Theachievements of conventional plant breeding havebeen reviewed at regular intervals (Bingham, 1981;Duvick, 1986; Reeves and Cassaday, 2002).

1.5 Limitations of Conventional Breedingand Rationale for Transgenic Breeding

A major constraint of conventional breedingapproaches is the nonavailability of desirable geneswithin the gene pool, for example in rice, thereis limited variability for resistance to diseasessuch as sheath blight, stem borer, and otherinsect pests, bacteria and fungi contain genes,

such as the Bacillus thuringiensis crystal proteins(Bt Cry) and chitinase, respectively, which can beuseful against pests and pathogens. Transferringgenes from one kingdom to another invokes fortransgenic approaches. A variety of Bt genes havebeen transferred to rice using electroporation, bi-olistic or Agrobacterium-mediated transformationmethods (Fujimoto et al., 1993; Wunn et al., 1996;Ghareyazie et al., 1997; Nayak et al., 1997; Chenget al., 1998; Maqbool et al., 1998, 2001; Tu et al.,2000). In cases when genes are available withinthe gene pool of rice for transfer to cultivatedvarieties, conventional breeding methods sufferfrom drawbacks such as hybridization barriers,low selection efciency, and long breeding cyclesthat take a long time to generate a new variety.Transgenic approaches along with methods suchas anther culture and marker-assisted breedingcan overcome these limitations. Additionally,the genes available within the gene pool maynot be expressed at desirable levels or maynot have the desirable expression patterns, thusneeding modication of regulatory elements. Suchmodications and the downstream incorporationof the gene into the genome are possible onlythrough transgenic approaches. For example, theendogenous rice chitinase gene was modied toconstitutively express at higher than endogenouslevels under the CaMV 35S promoter, conferringenhanced resistance to sheath blight (Lin et al.,1995).

Introducing designer characteristics in modify-ing the nutritional quality and quantity of ricegrains or having genes expressed at specic timeand place of the plant life cycle also calls fortransgenic approaches. For example, in the caseof Golden Rice, enriching the rice grains with-carotene was possible by expressing three genesresponsible for provitamin A synthesis (Ye et al.,2000). Rice anther specic expression of agene isolated from anther complementary DNA(cDNA) was conrmed through transgenic plants.The gene is responsible formale fertility and hence,important in hybrid rice programs (Luo et al.,2006).

Molecular markers have fast-tracked plantbreeding. Similarly, molecular approaches tocreating mutant lines using transgenic approacheswith transposons or transfer DNA (T-DNA) havefast-tracked mutation detection. Either a known

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gene of interest is examined for function and/orexpression patterns or novel genes are identiedthrough this approach. The added advantageof these approaches is that a genome saturatedinsertion-mutation library can be created andstored. Such a library represents a population ofseeds with insertion mutation in every single gene,ideally one gene mutation per seed, so that anychanges in the plant arising from such a seedcan be tracked to changes in that one mutatedgene. Such libraries have been created for riceusing an endogenous retrotransposon (Hirochika,2001; Miyao et al., 2007), heterologous maizeAc or Ac/Ds transposon (Enoki et al., 1999;Kohli et al., 2001, 2004; Kolesnik et al., 2004;Upadhyaya et al., 2006), and the T-DNA (Jeonet al., 2000; Chen et al., 2003; Sallaud et al.,2003; An et al., 2005a, b; Fu et al., 2006; Lianget al., 2006). A number of novel genes have beenidentied using insertional mutations. Recently,a dwarf mutant gene was identied from theT-DNA tagged population (Zhou et al., 2006).In a comprehensive review on advances in ricebiotechnology, Kathuria et al. (2007) list othergenes identied and isolated through moleculartagging approaches. These include genes involvedin gibberellic acid biosynthesis (Margis-Pinheiroet al., 2005), blast resistance (Kim et al., 2003),tapetum development (Jung et al., 2005), andpollen development (Han et al., 2006).

2. DEVELOPMENT OF TRANSGENIC RICE

In the 1960s, doubling of the worlds populationto more than 6.4 billion, together with a drasticdecrease in cultivated land led to a global foodcrisis. However, a Nobel Peace Prize winningeffort by Norman E. Borlaug, now christened thegreen revolution, averted a large-scale famine inthe semi-arid tropics of South Asia, specicallyin India, Pakistan, the Philippines, and Mexico.The introductions of high-yielding semidwarfvarieties of wheat and rice, in combinationwith applications of large amounts of nitrogenfertilizer to increase grain yields and promoteleaf and stem elongation, was crucial for thesuccess of the rst green revolution (Sakamotoand Matsuoka, 2004). The situation now comesfull circle as the world population, estimated to

touch 8 billion by 2025, again looks out forstrategies to increase grain production. The secondgreen revolution calls for genetic engineering offood crops (Sakamoto and Matsuoka, 2004), andadvances in transgenic rice production would beextremely benecial toward achieving the neededgoals. Parameters identied to be associated withincreases in yield potential are plant height, tillernumber, photosynthesis, photoperiod insensitivity,and disease and insect resistance (Khush, 1999).The green revolution genes, namely the wheatgreen revolution gene Reduced height1 (Rht1) andthe rice green revolution gene semidwarf1 (sd1)have been identied and have been found toencode mutant gibberellin response modulators(Peng et al., 1999). Genetic manipulations ofgibberellin (GA) levels thus would prove to bea promising rst step to generate semidwarfvarieties that, in combinationwith articial controlof tillering, would lead to a dramatic increasein the yield potential of rice (Sakamoto andMatsuoka, 2004). Commercial use of transgenicrice is far less than that of transgenic maizethat is now planted on more than 15 millionhectares around the world (James, 2003). Theambitious 16-year-long programof theRockefellerFoundation for rice biotechnology was a keyfactor in positioning rice biotechnology to beadvanced to the domains of public and federalfunding (OToole et al., 2001). Sequencing of therice genome provides added impetus to advancesin transgenic rice technology, and also cementsthe model monocot status of rice. The rapidadvances in rice biotechnology, starting nearlytwo decades ago with the production of therst transgenic rice plants (Toriyama et al.,1988; Zhang and Wu, 1988) have resulted inthe development of high-throughput, reproducibletransformation protocols in the crop. Transgenicrice plants with improved tolerance to bi-otic/abiotic stresses, improved insect/pest/diseasetolerance, and increased nutritional values havebeen developed (Upadhyaya et al., 2000a; Bajajand Mohanty, 2005; Kathuria et al., 2007). Ricehas also served as a model to understand theprocess of plant transformation in general, bothin terms of transgene integration and modulationof gene expression (Kohli et al., 1998, 1999a, b;Iyer et al., 2000; Cheng et al., 2001b; Kloti et al.,2002; Vain et al., 2002; Kathuria et al., 2007).

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2.1 Donor Genes

The need for transgenic technologies to improvethe qualitative and quantitative yields and otheragronomic characteristics of rice cannot beoverstated. Lack of desirable genes within therice gene pool and a consequent search for thesegenes in other organisms pushed the frontiersin locating, isolating and cloning heterologousgenes, and designing transgene constructs for ricetransformation.

The donor genes for rice transformation over theyears have been isolated from different kingdoms.Examples abound for multiple genes from eachkingdom that have been introduced in rice. Specicexamples of a gene or a class of genes introduced inrice fromeachkingdom, highlighting an importantagronomic or technical advance are mentioned inTable 4. The cited references for each of these genespoint either to recent research or review papers orto one of the rst reports in literature. Additionalgenes and regulatory sequences introduced inrice and their source organisms are mentionedin different sections of this chapter as directlyrelated to the aim of generating the transgenicplants.

The donor gene sources ranging from viruses tohumans, and the aims of transforming rice withthese genes can be categorized as in Table 4.

2.1.1 Plant transformation

Increasingly efcient transformation, selection,and screening tools techniques and genes are aprerequisite for generating transgenic rice with im-proved agronomic characteristics. New selectablemarker and reporter genes are continually beingexplored for their use in efcient selection oftransformed cells/tissues or reporting transgeneexpression status. Increasingly these genes arechosen and designed to be neutral in termsof their substrates and products affecting theplant per se or the consumer. In most caseshowever, the effort now is to generate marker-freetransgenic plants. The hpt and the pat genes havebeen the most frequently used selectable markersfor rice transformations while the gus and thegfp genes have been the most frequently usedreporter genes. Recently Lee et al. (2007) have

used theMyxococcus xanthusprotoporphyrinogenoxidase (MxPPO) as an efcient selectable markerusing the peroxidising herbicide butafenacil.Although the MxPPO is only an addition to theherbicide resistance class of selectable markersfor rice transformation, it supplies an exampleof another experimentally tested system in rice.Latest comprehensive reviews on transgenic riceby Bajaj and Mohanty (2005) and Kathuriaet al. (2007) illustrate the novel selectable markersand reporter genes used. For example, somenovel and neutral selectable markers include thephosphomannose isomerase gene (Lucca et al.,2001b), the -subunit of anthranilate synthase(Yamada et al., 2004); conditional negativeselection with cytosine deaminase (Dai et al.,2001a), indole acetic acid hydrolase (IAAH/tms2;Upadhyaya et al., 2000b), aminoglycoside 3-adenyltransferase (aadA; Oreig et al., 2004), andD-amino acid oxidase from yeast (dao1; Eriksonet al., 2004). Modied gus, gfp, luc, and syntheticsialidase and xylanase in combination with gusused as the latest reporter genes have also beendescribed (reviewed in Bajaj and Mohanty, 2005).

Generating marker-free transgenic rice plantshas been accomplished through different strate-gies. For example, simple molecular analysesof the segregating population for the lack ofselectable marker when it integrates separatefrom the gene of interest. With Agrobacterium-mediated transformation, the use of independentconstructs to carry the marker gene and the geneof interest increases the frequency of independentintegration and hence facilitates segregation in thefollowing generations. Cleverly designed twin T-DNA vectors also accomplish the same (Komariet al., 1996; Lu et al., 2001), as does the dualbinary vector system of pGreen/pSoup (Vainet al., 2003). Alternatively, molecular excisionof the selectable marker was achieved throughsite-specic recombination using cre/loxP (Gleaveet al., 1999; Moore and Srivastava, 2006), ipaserecombination enzyme (FLP)/ipase recombina-tion target (FRT) sites (Srivastava and Ow, 2004)and/or Ac/Ds transposon system (Cotsaftis et al.,2002). Endo et al. (2002) used the multiautotransformation (MAT) vector containing theAgrobacterium isopentyl transferase (ipt) geneas a marker in combination with the R/RSsite-specic recombination system to generate

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Table 4 Donor genes for generating transgenic rice

Kingdom Source Gene Functional category Reference

Prokaryota Viruses:Rice stripeRice tungroRice yellow mottle

Class of viral genes Viral disease resistance Hayakawa et al. (1992);Sivamani et al. (1999);Pinto et al. (1999); Kouassiet al. (2006)

Baculoviruses andEntomopoxviruses

EPV-fusolin Insect disease resistance Hukuhara et al. (1999)

Eubacteria:Bacillus thuringiensis

Class of Bt Cry genes Insect disease resistance Khush and Brar (2002); Bajajand Mohanty (2005); Rohet al. (2007)

Escherichia coli -glucuronidase Screenable marker Kathuria et al. (2007); Teradaand Shimamoto (1990)

Trehalose biosynthetic genes Abiotic stress resistance Garg et al. (2002)Phosphomannose isomerase Selectable marker Lucca et al. (2001b)

Erwinia uredovora Phytoene desaturase -Carotene synthesis Ye et al. (2000)Protista

Fungi Streptomyces Phosphinothrycin N-acetyltransferase

Selectable marker andherbicide tolerance

Christou et al. (1991)

Hygromycinphosphotransferase

Selectable marker Meijer et al. (1991)

Chitinase Fungal disease resistance Itoh et al. (2003)Aspergillus niger Phytase Nutritional enhancement Drakakaki et al. (2006)

Plantae Rice Class of Xa21 genes Bacterial blight resistance Song et al. (1995)Oryzacystatin Nematode resistance Vain et al. (1998)Ca2+ dependent protein

kinaseCold/salt/drought tolerance Saijo et al. (2000)

Pyruvate decarboxylase Submergence tolerance Quimio et al. (2000)NAC transcription factors Drought tolerance Hu et al. (2006)Gibberellic acid 2-oxidase Dwarsm and yield Sakamoto et al. (2003)

Cowpea Cowpea trypsin inhibitor Insect resistance Xu et al. (1996)Soybean Soybean trypsin inhibitor Insect resistance Lee et al. (1999)Barley Barley trypsin inhibitor Insect resistance Alfonso-Rubi et al. (2003)Snowdrop Galanthus nivalis

AgglutininInsect resistance Rao et al. (1998); Sudhakar

et al. (1998)Daffodil Phytoene synthase and

Lycopene B cyclase-Carotene (provitamin A)

synthesisYe et al. (2000)

Maize Phytoene desaturase Higher -carotene synthesis Paine et al. (2005)Anthocyanin genes Fungal blast resistance Gandikota et al. (2001)Ribosome inactivating protein Sheath blight resistance Kim et al. (2003)

Arabidopsis Phytochrome A Grain Yield Garg et al. (2006)LEAFY Plant architecture and early

oweringHe et al. (2000)

CBF3/DREB1A/ABF3 Abiotic stress tolerance Oh et al. (2005)Glycerol-3-phosphate

acetyltransferaseChilling tolerance Yokoi et al. (1998)

Heat shock protein 101 Heat tolerance Agarwal et al. (2003)Gibberellin insensitive Dwarsm Peng et al. (1999)H+/Ca2+ antiporter Seed calcium content Kim et al. (2005a)

Phaseolus vulgaris Ferritin Increase seed iron content Lucca et al. (2001b)Wheat DREB/CBF Abiotic stress tolerance Shen et al. (2003)

Animalia Human Cytochrome P450 monooxygenase genes

Herbicide tolerance Inui et al. (2001)

Jellysh Lactoferrin Iron binding supplement Nandi et al. (2002)Lysozyme Valuable industrial enzyme Hennegan et al. (2005)Human granulocyte colony

stimulating factorValuable industrial factor Hong et al. (2006)

Green uorescent protein Selecting/screening marker Vain et al. (2003)

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marker-free plants. All of the strategies mentionedhave been reviewed and extensively referenced byBajaj and Mohanty (2005) and Kathuria et al.(2007). A further advancement and simplicationon the use of cre/loxP system was achievedby circumventing the need to express cre underspatiotemporal control. A fusion of themembranetranslocation sequence from the Kaposi broblastgrowth factor (FGF-4) with CRE was used ascell permeable recombinase to deliver the latterinto plant cells and effect marker excision (Caoet al., 2006). An additional strategy to generatetransgenic plants devoid of undesirable sequencessuch as the vector DNA sequences and markergenes is to biolistically co-transform plants withminimal cassettes of the marker gene and the geneof interest (Fu et al., 2000a; Breitler et al., 2002;Loc et al., 2002;Agrawal et al., 2005). This strategyled to higher frequency of single copy integrationevents as also a higher frequency of independentintegration of the marker and the gene of interest,which can be analyzed for segregation in the nextgeneration.

2.1.2 Agronomic trait enhancement

The desirable characteristics in any crop plantare a healthy life cycle leading to copious yields.In almost 20 years since the rst transgenic riceplants were generated (Toriyama et al., 1988;Zhang and Wu, 1988), the state of the art nowallows facile examination and assessment of therole of heterologous and endogenous genes foragronomic trait improvement. For improvementof rice through transgenic approaches these traitstranslate into several agronomically desirablecharacters as detailed below.

2.1.2.1 Virus resistance

Viruses affecting rice crop include the RYMV,rice tungro bacilliform or spherical virus (RTBV/RTSV), rice stripe virus (RSV), rice ragged stuntvirus (RRSV), RGSV, and RHBV. Transgenicapproach to generate virus-resistant rice was rstreported by Hayakawa et al. (1992) against RSVusing the coat protein (CP)-mediated resistance.Other examples include CP against RHBV(Lentini et al., 2003),RTSV (Sivamani et al., 1999),

and RYMV (Kouassi et al., 2006). Additionalstrategies used expression of ribozyme againstdwarf virus (Han et al., 2000), spike proteinagainst RRSV (Shao et al., 2003), and replicaseagainst RTSV (Huet et al., 1999). Silencing ofthe viral RNA dependent RNA polymerase wasused to generate resistance to RYMV (Pinto et al.,1999). Modication of host factors involved invirus replication against RTBV (Dai et al., 2006)or even translation initiation factor 4G againstRYMV (Albar et al., 2006) has also been used.Partial to complete resistance to viral infectionwas demonstrated but no eld studies have beencarried out.

2.1.2.2 Bacterial resistance

Bacterial blight in rice is caused by X. oryzaepv. oryzae (Xoo). Resistance to bacterial blightis conferred to a limited extent by a 30-memberfamily of rice endogenous genes called Xa1 toXa29 (Xiang et al., 2006). However, the expressionpatterns of these do not restrict the natural varia-tion and evolution of resistance in the bacterialstrains thus necessitating transgenic approachesusing the endogenous genes with heterologouspromoters for a powerful and enduring resistanceresponse. Using Xa21, the most promising geneof the family (Song et al., 1995) and Xa26,transgenic rice resistant to blight in eld testswas created (Tu et al., 1998; Sun et al., 2004;Zhai et al., 2004). Additionally, overexpressionof endogenous transcription factor OSWRKY71implicated in defense signaling pathway was usedto obtain resistance to blight (Liu et al., 2006).Recently overexpression of the OSWRKY13 ledto resistance against bacterial blight and fungalblast with evidence for links through activation ofsalicylic acid (SA)-dependent and suppression ofjasmonic acid (JA)-dependent signaling pathways(Qiu et al., 2007). Other endogenous rice genesused against bacterial diseases are the OSYK1against bacterial stripe (Hayashi et al., 2005), theOSRAC1 (Ono et al., 2001), and OSRACB (Junget al., 2006) against bacterial blight. Heterologousgenes used against bacterial diseases in rice arethe antibacterial peptide cecropin from Bombyxmori (Sharma et al., 2000), the oat ASTH1 gene(Iwai et al., 2002), the sweet pepper ferredoxingene (Tang et al., 2001) and the Arabidopsis

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NPR1 gene (Chern et al., 2001, 2005). Yuanet al. (2007) conrmed that the rice NPR1 isan ortholog of the Arabidopsis NPR1 and itsoverexpression also confers resistance againstbacterial blight and fungal blast perhaps throughmediating antagonistic crosstalk between the SA-and the JA-dependent pathways was shown toconfer resistance to bacterial blight.

2.1.2.3 Fungal resistance

M. grisea is the causal organism for rice blast andR. solani is the causal organism for rice sheathblight disease. Transgenic rice overexpressing theendogenous resistance genes (R, Pi-ta, Nbs2-Pi9, Pid2) against fungal blast under alteredexpression regimes was shown to confer resistanceto M. grisea (Bryan et al., 2000; Chen et al.,2004; Qu et al., 2006). Chitinase genes fromrice or other fungi have been used to generatetransgenic rice resistant against Magnaportheand Rhizoctonia (Lin et al., 1995; Datta et al.,2001; Sridevi et al., 2003). Pathogenesis-relatedprotein PR2 was used by Nishizawa et al. (2003)and a defense-related gene RIR1b was used bySchaffrath et al. (2000) for rice resistance againstMagnaporthe. Wang et al. (2006) demonstratedthat the thiamine biosynthesis gene OSDR8 thatacts in the signal transduction pathway couldconfer blast resistance, while Uchimiya et al.(2002) demonstrated the similar use of OSKY1,a maize HM1 homolog. The OSWRKY13 gene,which confers blight resistance, also confers blastresistance through the involvement of salicylate-and jasmonate-dependent signaling (Qiu et al.,2007). TheB.mori antibacterial peptides cecropinswere also used as antifungal peptides (Coca et al.,2006). Recently, Zhu et al. (2007) introduced fourantifungal genes, two basic chitinases RCH10and RAC22, and the -glucanase and B-RIP(ribosome inactivating protein) in super hybridrice and demonstrated high resistance againstnot just blast but also against false smut andkernel smut caused by Ustilaginoidea virens andTilletia barclayana, respectively. Manipulating theendogenous levels of JA in transgenic rice throughpathogen inducible regulation of allene oxidesynthasea key enzyme in JA biosynthesisledto enhanced activation of pathogenesis related(PR) genes and blast resistance (Mei et al., 2006).

Examples of glucanases, ribozyme inactivatingproteins (RIPs), and other genes used to developblast-resistant rice are illustrated in Kathuria et al.(2007).

2.1.2.4 Nematode resistance

The endogenous gene oryzacystatin was alteredand used to generate transgenic rice (Vain et al.,1998), which resulted in 55% reduction in eggproduction by Meloidogyne incognita or the root-knot nematode.

2.1.2.5 Insect resistance

The major insect pests affecting rice cropinclude Nephotettix virescens (green leafhopper),N. lugens (brown planthopper), O. oryzae (gall-midge) S. incertulas (yellow/white stem borer), C.suppressalis (striped stem borer). Insects act asprimary pest and as vectors for viral infectionsof rice. Transgenic strategies using baculovirusthrough the insects enhanced susceptibility byexpressing the virus-enhancing factor (Hukuharaet al., 1999) did not become popular due to theslow mode of action of the virus. Expressingprotease inhibitors targeted against the insectproteases has been a popular strategy. Proteaseinhibitors such as the SBTI, CPTI, PINII, andITR1 have been used with limited success (Duanet al., 1996; Xu et al., 1996; Lee et al., 1999;Alfonso-Rubi et al., 2003). However, by far themost popular approach has been the use of BtCry genes. Since the rst transgenic rice expressingthe cry1Ab generated by Fujimoto et al. (1993),various rice varieties have been transformed withdifferent cry proteins in their original, modied,or fully synthetic versions (extensively reviewedin Tyagi et al., 1999; Khush and Brar, 2002;Bajaj and Mohanty, 2005; Kathuria et al., 2007).Countering the capacity of target insects todevelop concomitant resistance to cry proteins hasbeen the subject of extensive research in deployingthe transgenic Bt cry protein strategy, which holdspromise due to its initial desirable effects oncontrolling the insect infestation and crop damage.Gene pyramiding of different cry proteins isseen as one viable strategy (Maqbool et al., 2001;

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Loc et al., 2002; Bashir et al., 2005). Fusionbetween cry proteins and carbohydrate-bindingmoieties of lectins is another strategy that wasshown to increase the range of target insects(Mehlo et al., 2005). Field tests of rice expressingcry proteins have shown limited success (Tuet al., 2000; Ye et al., 2001, 2003 Wu et al.,2002; Bashir et al., 2004; Breitler et al., 2004).The fusion proteins, gene pyramiding not justwith cry proteins but also with other insecticidalproteins, and agronomic deployment strategiesas part of the integrated pest management hasbeen suggested as the route to creating insect-resistant plants (Christou et al., 2006; Ferry et al.,2006). The cry proteins are not effective againstthe hemipteran insects. Plant lectins such as thegalanthus nivalis agglutinin (GNA) have beenshown to be effective against insects such as brownplanthopper (BPH), green leafhopper (GLH),and white-backed planthopper (Rao et al., 1998;Sudhakar et al., 1998; Sun et al., 2002; Nagadharaet al., 2003, 2004). The Allium sativum leaf lectinwas shown by Saha et al. (2006) to be effectiveagainst BPH and GLH. The GNA in combinationwith the cry proteins (Maqbool et al., 2001;Ramesh et al., 2004) or in combination with SBTI(Li et al., 2005) extended the range of target insectsto which the transgenic plants were tolerant. Yetother molecules such as the avidin (Yoza et al.,2005) and the spider insecticidal protein (Qiu et al.,2001) have been tested in transgenic rice.

2.1.2.6 Drought, salt, submergence, andtemperature stress tolerance

Although rice is a sturdy grass crop that grows invaried climates and weather conditions, the gap inyield potential and yield harvest as affected by abi-otic stresses cannot be afforded under the presentdemand regimes. However, transgenic approachesto address a particular stress often favorablyimpact the other due to amelioration of a commonstress response pathway. One approach that simul-taneously affects favorably more than one abioticstress is overproduction of cell solutes such asglycine betaine, trehalose, proline, polyamines andlate embryogenesis abundant proteins. The genesused to achieve solute overproduction in trans-genic rice are reviewed in Kathuria et al. (2007).A recent example of using osmolyte accumulation

against salt and drought tolerance is the overpro-duction of mannitol in rice using the Escherichiacoli mannitol-1-phosphodehydrogenase (Pujniet al., 2007). Ion channel and water channel pro-teins that facilitate modication of the cells andthe vacuoles osmotic balance have been used todevelop drought-tolerant rice. For example, over-expression of the aquaporinRWC3 from rice (Lianet al., 2004) and that of the Na+/H+ antiporterfrom rice, Suaeda salsa, Artiplex or E. coli (Ohtaet al., 2002; Fukuda et al., 2004; Zhao et al., 2006)increased salinity tolerance in transgenic rice. Theusefulness of the strategy to increase salt tolerancein transgenic rice through a rice Na+/H+ an-tiporter gene (OSNHX1) was reinforced recentlyby Chen et al. (2007). Production of free radicalsand reactive oxygen species mostly accompaniesdifferent abiotic stresses (Mittler et al., 2004) andthe common signaling pathways for the perceptionand response to oxidative stress indicate thatstrategies targeting oxidative stressmay amelioratemultiple stresses. The presence of oxidative stressresponse cis elements in abiotic stress responsegenes such as OSISAP1 (Mukhopadhyay et al.,2004; Tsukamoto et al., 2005) supports this strat-egy. Hoshida et al. (2000) obtained salt-toleranttransgenic rice by overexpressing glutathionesynthase, a reactive oxygen species metabolismenzyme. Similarly, constitutive overproduction ofpea superoxide dismutase (Wang et al., 2005)and protoporphyrinogen oxidase (Jung and Back,2005) led to salt-tolerant rice. An additionalcommon reaction to stress is the Ca2+-mediatedsignaling to activate response pathways (Knightand Knight, 2001). Proteins involved in Ca2+-mediated signaling such as calcineurin A (Maet al., 2005), calcium-dependent protein kinases(OSCDPK7; Saijo et al., 2000), and MAP kinases(OSMAPK5; Xiong and Yang, 2003) were shownto generate salt-, drought-, and cold-tolerantrice.

Recently the DREB/CBF regulatory geneshave also been shown to be common to cold,dehydration, and salt stress (Dubouzet et al.,2003). Overexpressing endogenous and heterol-ogous (wheat and Arabidopsis) DREB/CBFclass of genes in rice has led to a betterunderstanding of the signaling pathways andgenerated multiple-stress-tolerant rice (Oh et al.,2005). Overexpression of other regulatory genessuch as the transcription factor SNAC1 has also

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been shown to generate drought- and salt-tolerantrice (Hu et al., 2006).

As opposed to salt and drought tolerance,submergence tolerance is important in certainareas. Quimio et al. (2000) generated the rstrice transgenics exhibiting submergence toleranceby overexpressing the pyruvate decarboxylasegene (PDC1). Xu et al. (2006) demonstratedsubmergence tolerance in transgenic rice byexpressing an ethylene responsive-factor-like gene(SUB1A), which affects the expression levels ofother genes of the same family (SUB1C) and thealcohol dehydrogenase (ADH1) gene expression.

Temperature stress tolerance has been engi-neered through the use of heat shock proteinsfor high temperature from rice itself (SPL7;Yamanouchi et al., 2002) or from Arabidopsis(HSP101; Agarwal et al., 2003; SHSP17.7;Murakami et al., 2004). Low-temperature stresstolerance was engineered through overexpressionof genes affecting the level of unsaturation of thefatty acids in the chloroplast phosphatidylglycerol(Ariizumi et al., 2002; Takesawa et al., 2002).

2.1.2.7 Mineral stress tolerance

Plant stress associated with excess or lack ofmineral nutrients can adversely affect yields toa large degree. Soil pH dictates the availabilityof nutrients such as iron, phosphorus, aluminum,etc. High pH soils limit the availability ofiron, which was ameliorated by overexpressingnicotianamine synthase, because it is functionalin the biosynthetic pathway of iron chelatingphytosiderophores. Barley phytosiderophore syn-thesizing genes (NAAT-A and NAAT-B) werealso used to show enhanced survival in highpH, low iron availability soils (Takahashi et al.,2001). Phosphate starvation in pH imbalancedsoils was relieved through the expression of a ricetranscription factor OSPTF1 (Yi et al., 2005).Begum et al. (2005) demonstrated that transgenicrice expressing the maize PEPC led to exudationof oxalate thus enhancing the plants capacity toadapt to high pH, low phosphorus soil conditions.Alternately, low pH soils pose the problem oftoxic aluminum ions that inhibit root elongation.Aluminum ion complexing organic acids such ascitrate and malate led to transforming rice with a

wheat malate transporter (ALMT-1; Sasaki et al.,2002) to generate aluminum-resistant plants butthe approach was not very successful.

2.1.2.8 Herbicide tolerance

The trait of herbicide tolerance was one of therst to be introduced in rice, tested in the elds,and used commercially. Transgenic rice has beendeveloped expressing the herbicide tolerance genebar from streptomyces (Christou et al., 1991;Dattaet al., 1992), Cytochrome P450 monooxygenasesfrom humans (Inui et al., 2001; Kawahigashi et al.,2005, 2006) or pigs (Kawahigashi et al., 2005) andthe rice glutathione S-transferases (Deng et al.,2003). Tolerance to a number of herbicides wasdemonstrated through these studies. Lee et al.(2000) and Jung and Back (2005) also generatedherbicide tolerance in rice through expressing theBacillus subtilis protox gene.

2.1.2.9 Increased grain yield

With the increasing population, increasing cropyield through contribution of the transgenictechnologies in the breeding programs is almosta prerequisite now. Although addressing bioticand abiotic stress resistance is an indirect routeto yield increase, a more direct route is toincrease grain number, size, and weight. Enhancedactivity of adenosine dephosphate-glucose py-rophosphorylase led to increase in seed weightper plant (Smidansky et al., 2003). Manipulatingcomplex traits such as light signal transductionand photosynthetic efciencies through effortsto convert the rice C3 plant into C4 plant isalso underway. Kong et al. (2004) demonstratedan increase in seed yield in transgenic riceexpressing the Arabidopsis phytochrome A gene.Plant hormones inuence grain yield. Sakamotoet al. (2003) overexpressed the gibberellin 2-oxidase, generating a semidwarf, high-yieldingtransgenic after a connection was establishedbetween dwarsm and gibberellin response (Penget al., 1999; Sasaki et al., 2002). Lack of C-22hydrolase, a brassinosteroid biosynthetic enzymeleads to erect leaves and increased grain yield(Sakamoto et al., 2006). Transgenic rice expressing

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the brassinosteroid receptor gene OSBR1 andexhibiting brassinosteroid insensitivity showedsimilar changes inmorphology and increased yield(Morinaka et al., 2006). Similarly, expressing thedefective allele of OSCKX2a cytokinin oxi-dase/dehydrogenase showed enhanced cytokininand increased grain yield (Ashikari et al.,2005). Flower development is directly relatedto grain yield capacities. Manipulation of thetransgenic expression of different rice MADSbox transcription factors was shown to affectower morphology indicating genes necessary fornormal ower development (Prasad et al., 2001,2005; Kyozuka and Shimamoto, 2002; Sentokuet al., 2005; Chen et al., 2006a; Yamaguchi et al.,2006). These studies helped to map the riceower development on the known ABC modelof Arabidopsis ower development. Genes otherthan the MADS box transcription factors thataffect the ower morphology have also beenelucidated (Jang et al., 2003, 2004). Other ricegenes studied through transgenic rice for theirrole in owering time, shoot-to-ower transitiontime, panicle number, and density and othergrowth and development patterns have beenreviewed in Kathuria et al. (2007) including thegenes concerned with phytohormone biosynthesismentioned above and the additional ethylenebiosynthesis and response genes that affect plantmorphology (Mao et al., 2006). Regulatory genesapart from the transcription factors, such as thoseconcerned with giving rise to further regulatorymoieties of micro-RNA also provide an insightinto plant architecture (Liu et al., 2005). Flowerdevelopment and viability also feeds into theimportant aspect of generating hybrid rice throughmale sterility. Generating cytoplasmic or genicmale sterility or restoration of the same hasbeen demonstrated through transgenic approaches(Komori et al., 2004; Wang et al., 2006). Thesestudies help in understanding factors that canbe used to develop an optimal plant architectureand life cycle that further helps increase grainyields. Interestingly, roots that sustain the entireplant and whose normal growth and developmentdictates that genetic growth patterns of the aerialparts are adhered to through supply of water andnutrients, have not been investigated to the sameextent as perhaps shoot branching patterns andower development. Recently, transgenic studies

have elaborated the role of some rice genes in rootdevelopment showing relationships to auxin levels(Ge et al., 2004; Nakamura et al., 2006).

2.1.2.10 Nutritionally enhanced seeds

Since rice is the staple food of a large populationin economically underdeveloped, and developingcountries, which cannot afford expensive fruitsand vegetables, enhancement of the nutritionalquality of the rice seeds can directly contributeto the health of this population. The rst targetin this regard was increasing the content ofcarotenoids to result in enhancement of vitaminA in the seeds. Over the last decade, rice hasbeen engineered with carotenoid biosynthesisgenes from rice, maize, daffodil, and bacteriasequentially improving the expression levels byapproaches such as codon optimization and tissue-specic expression (Burkhardt et al., 1997; Yeet al., 2000; Datta et al., 2003, 2006 Hoa et al.,2003; Paine et al., 2005). The second target is toimprove the rice seeds for essential amino acidcontent. To increase the content of tryptophanthe biosynthetic enzyme anthranilate synthasegene was used (Morino et al., 2005; Wakasaet al., 2006). Similarly, the seed methionine andcysteine content has been improved by expressingthe sesame albumin protein (Lee et al., 2003).Increasing the levels of polyunsaturated fatty acid-linolenic acid in rice bran has also been achieved(Anai et al., 2003). Seed iron content was increasedby transforming rice with human lactoferrin gene(Nandi et al., 2002), the Phaseolus vulgaris ferritingene (Lucca et al., 2001a), the soybean ferritingene (Goto et al., 1999; Vasconcelos et al., 2003)or the Aspergillus fumigatus phytase gene (Luccaet al., 2001a). In another breakthrough, the seedcalcium content was manipulated to lower theincidence of osteoporosis, an incidental problemoccurring through rice consumption amongst theeconomically poorer communities. A H+/Ca+transporter gene was integrated to achieve highercalcium content (Kim et al., 2005a). However,any nutritional improvement of rice seed canmost easily be affected by changes in starchcontent. Overexpression of one allele of the starchsynthase gene resulted in the increase of seed starch(Hirano et al., 1998). A change in the amylose to

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amylopectin ratio of the rice seed was targetedby transforming rice either with isoamylase 1silencing construct or with the E. coli glycogen-branching enzyme (Fujita et al., 2003; Kim et al.,2005b) to change the physiological properties ofstarch. A postharvest value-added modicationfor the use of rice starch was engineered byintroducing the wheat puroindoline genes PINAand PINB that rendered the grains much softerthan the control. The Soft-Rice has numerousapplications in the food industry.

2.1.2.11 Variation in transgene constructs

Most transgenic plants are primarily developedusing strong constitutive promoters. For rice,the most commonly used promoters are theCaMV 35S, rice ACT1 and the maize UBI1.The CaMV 35S promoter was shown to containa recombination hotspot (Kohli et al., 1999a),readily leading to transgene rearrangements.Otherviral promoters have been used in rice such asthe one from cestrum yellow leaf curling virus(CYLF) (Stavolone et al., 2003) and milk vetchdwarf virus (MVDV) (Shirasawa-Seo et al., 2005).Among the nonviral constitutive promoters thathave been used, the rice cytochrome c genepromoter was highly active in different parts ofthe plant including the embryo and the calli(Jang et al., 2002). Using introns further enhancedexpression of the constitutive promoters. Tanakaet al. (1990) used the rst intron of castor beancatalase to enhance gus expression driven bythe CaMV 35S promoter in rice. The maizeADH1 rst intron became quite popular in planttransformation constructs and was rst used inrice by Kyozuka et al. (1991). Simultaneously,McElroy et al. (1991) used the rst intron ofthe ACT1 gene in combination with the ACT1promoter. However, the most popular intronwas the rst intron of the maize UBI1 gene incombination with the UBI1 gene promoter itself(Toki et al., 1992; Cornejo et al., 1993). TheADH1 intron 1 was, however, used downstreamof the CaMV 35S promoter (Taylor et al.,1993). Other DNA sequences tested for theirfunctionality in regulating the level of expressionof a transgene were the matrix attachment region(MAR) sequences (Vain et al., 2002). Xue et al.

(2005) used the tobacco TM2 MARs to enhanceexpression from both constitutive and induciblepromoters. Novel regulatory sequences such asthe REB activator fused to the GLB promoterincreased the transgene activity (Yang et al.,2001) while the SRS element fused to the ACTpromoter also achieved enhanced expression levels(Lu et al., 1998). Nguyen et al. (2004) increasedconstitutive expression up to vefold, by using theT7RNA polymerase-directed expression systemin rice. However, constitutive promoters are notalways desirable since transgene expression levelsmay need to be spatiotemporally controlled andcoordinated. This can be achieved either throughtissue/stage-specic or inducible promoters per se(Ito et al., 2006) or through coupling regulatory,inducible/responsive cis elements to workhorseconstitutive promoters (Su et al., 1998). Furtherexamples of both categories have been extensivelyreviewed in Bajaj and Mohanty (2005) andKathuria et al. (2007). Promoters from diversesources have been tested in rice suggesting itssuitability for heterologous functional analysis orcomplementation of promoter elements of otherorganisms (Kathuria et al., 2007).

2.2 Methods of Genetic Transformation

2.2.1 Protoplast technology

The rst transgenic rice plants were obtained withrice protoplasts using electroporation (Toriyamaet al., 1988) and polyethylene glycol (PEG)-mediated methods (Zhang and Wu, 1988).Dividing rice protoplasts from which fertileplants can be regenerated are usually generatedfrom embryogenic cell cultures normally derivedfrom immature embryos (Vasil, 1988). Toriyamaet al. (1988) used protoplasts isolated fromanther-derived cell suspension cultures of O.sativa L. cv. Yamahoushi to electroporate twoplasmids containing CaMV 35S promoter-drivenaminoglycoside phosphotransferase II and -glucuronidase genes. Shimamoto et al. (1989) werethe rst to recover fertile transgenic Japonicarice using the electroporation method. Dattaet al. (1990) were the rst to recover fertiletransgenic Indica rice (O. sativa var. ChinsurahBoro II) from protoplasts derived from immature

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pollen grains (microspores) with a hygromycinphosphotransferase (hph) gene under control ofthe CaMV 35S promoter using the PEG-mediatedtechnique.

Protoplast-mediated transformation has manylimitations. These include difculties in regen-eration of elite Japonica and Indica varieties(Ayres and Park, 1994), the production of sterileand phenotypically abnormal regenerated plants(Datta et al., 1992), the integration of multiplecopies of genes into genomes (Tada et al.,1990), combined with the fragmentation andrearrangement of transgenes (Xu et al., 1995) andtheir occasional non-Mendelian inheritance (Penget al., 1995).

2.2.2 Particle bombardment

Continuous and directed research efforts touse Agrobacterium-mediated transformation forcereals have recently made the technology routinein many laboratories around the world. However,earlier difculties in transforming cereals withAgrobacterium led to a search for alternativestrategies. Wang et al. (1998) rst used thebiolistics technology of bombarding DNA-coatedgold particles accelerated under vacuum to achievetransient expression of transgenes in rice. Christouet al. (1991) reported the rst stable transformationusing a modication of the same technologywhereby the particles were accelerated by anelectric arc. This method of transformationbecame the method of choice for nearly a decadesince it is genotype independent and less laborintensive. A number of genes were introduced inrice using the biolistics technology (reviewed inGiri and Laxmi, 2000; Bajaj and Mohanty, 2005;Kathuria et al., 2007). An Indica rice populationwas created with Ac/Ds transposon for saturationmutagenesis and functional genomics using thebiolistics method (Kohli et al., 2001). Initialobservations of transgene rearrangements andmulticopy insertion events in transgenic rice lines(Kohli et al., 1998, 1999a) were ascribed as reasonsfor transgene silencing (Kumpatla andHall, 1998a,b; Dai et al., 2001b). Transgene rearrangementswere indeed shown to be responsible for lack oftransgene expression but integration of multicopytransgenes does not always lead to silencing

(Gahakwa et al., 2000; Kohli et al., 1999b).In fact, fragmentation and rearrangement oftransgene(s) were shown to be responsible forsilencing (Yang et al., 2005), especially whenmultiple copies are present. However, reducingthe chances of rearrangements by removing thevector backbone sequences from the transformingconstructs led to independent integration of intactmulticopies, which exhibited high and stableexpression patterns (Fu et al., 2000a; Agrawalet al., 2005). This approach also allayed theconcerns on antibiotic selectable marker andother vector backbone sequence integration inthe transgenic plants. This approach of cleanDNA transformation using the biolistics methodis gaining popularity for transformation of rice(Breitler et al., 2002; Loc et al., 2002), other cerealssuch as wheat (Yao et al., 2006), and yet otherplants such as potato (Romano et al., 2003) andgrapevine (Vidal et al., 2006).

2.2.3 Agrobacterium-mediatedtransformation

Agrobacterium tumefaciens is a phytopathogenicsoil bacterium, sometimes named as a naturalgenetic engineer due to its ability to transformplant cells with a part of its tumor-inducingplasmid (Ti plasmid) known as the T-DNAsegment (Chilton, 2001). Wounding of plantsallows entry of bacteria and provides phenoliccompounds that activate the DNA transfermachinery of A. tumefaciens in which a seriesof molecular events results in transfer of T-DNA from the bacterial cell to a plant cell andsubsequently into the plant genome (Gelvin, 2000,2003; Zhu et al., 2000; Zupan et al., 2000). Diversearrays of sophisticated plant transformationvectors have been derived to exploit this naturallyoccurring gene transfer mechanism in the geneticengineering of plants (Bevan, 1984; Tzira andCitovsky, 2006). Thismode of gene delivery is bothconvenient and efcient. Among its list of majoradvantages is the transfer of a small number ofintact DNA fragments (Hamilton et al., 1996; Daiet al., 2001b) that exhibit a normal Mendeliangene transmission to progeny (Budar et al.,1986).

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2.2.3.1 Transformation of dicots

The earliest reports of successful Agrobacterium-mediated transformation were for several dicotyle-donous plants. Murai et al. (1983) demonstratedthat a part of a bean phaseolin seed proteingene was transcribed in transformed sunowercells. Horsch et al. (1985) combined gene transfer,plant regeneration, and an effective kanamycin-based selection for transformants to generatetransgenic petunia, tobacco, and tomato bymeansof a novel leaf disk transformationregenerationmethod. Subsequently, Agrobacterium-mediatedtransformation was extended to other dicots suchas soybean (Hinchee et al., 1988) and walnut(McGranahan et al., 1988) culminating in the inplanta Agrobacterium-mediated gene transfer byinltration of adult Arabidopsis thaliana plants(Bechtold et al., 1993).

2.2.3.2 Transformation of monocots

De Cleene and De Ley (1976) showed thatmembers of the Liliales and Arales are susceptibleto Agrobacterium, and early reports exist forAgrobacterium-mediated transformation of bothAsparagus ofcinalis (Hernalsteens et al., 1984)and Dioscorea bulbifera (Schafer et al., 1987).Grimsley et al. (1986, 1987) demonstrated transferof T-DNA in maize seedlings infected with A.tumefaciens containing the maize streak virus(MSV) in the T-DNA (a process known as agroin-fection). Nevertheless, monocots, especially thecereals were generally considered outside the hostrange of A. tumefaciens (Potrykus, 1990; Smithand Hood, 1995). As a consequence, protoplastelectroporation and particle bombardment tech-niques were extensively used for transformation ofcereals until Hiei et al. (1994) provided rigorousevidence for the production of many fertilerice plants using a well-dened Agrobacterium-mediated transformation procedure.

This built upon the rst successful demonstra-tion ofAgrobacterium-mediated transformation ofrice by Raineri et al. (1990) who used matureembryos from wounded germinated seeds ofJaponica cultivars Nipponbare and Fujisaka5to deliver Agrobacterium strain LBA4404 withnos-nptII and CaMV 35S-gus. They obtainedkanamycin-resistant calli, and showed T-DNA

integration by Southern analysis, but failed toregenerate transgenic plants.

Chan et al. (1992) obtained G418-resistant callifrom root explants of the Indica variety Taichungnative I and showed T-DNA integration by South-ern analysis. Later (Chan et al., 1993) successfullyregenerated four transgenic rice plants, only oneof which produced progeny. To achieve successfulinfection, they added extracts from a potatosuspension culture to the co-cultivation medium,as these are rich in phenolic compounds that aidin susceptibility to Agrobacterium.

In the landmark paper of Hiei et al. (1994),Agrobacterium strains LBA 4404 and EHA101harboring pTOK233, a superbinary vector,were employed. In these strains, the plasmidcontaining the T-DNA bears a fragment of theS-Vir region that includes additional virB and virGgenes derived from pTiBo542. Transgenic plantswere successfully obtained for Japonica varietiesTsukinohikari, Asanohikari, and Koshihikari. T-DNA regions in the transformed plants weredetected by Southern analysis; their boundariessequenced, and Mendelian transmission of trans-genes to the R2 generation was demonstrated.This achievement was followed by the rst reportsof transgenic Indica rice plants, namely, Basmati370 and Basmati 385 (Rashid et al., 1996),and of Javanica rice, Gulfmont and Jefferson(Dong et al., 1996). All these studies usedcalli derived from scutellar tissue, and includedacetosyringone, a phenolic compound, in theco-cultivation media. Dong et al. (1996) alsoconducted sequence analysis of right borderfragments of one of their transgenic lines,conrming that insertion was into a coding regionof rice nuclear DNA. This analysis also revealedthe presence of relatively short regions of permutedT-DNA border sequences, similar to those foundafter Agrobacterium-mediated transformation ofdicots. Figure 2 details the production offertile transgenic rice plants by Agrobacterium-mediated transformation of scutellum-derivedcalli.

Broadening the range of tissues competentfor Agrobacterium-mediated transformation wasanother signicant step in rice biotechnology.Immature embryos from both Japonica (Radon)and Indica (TCS10 and IR72) varieties (Aldemitaand Hodges, 1996) and isolated shoot apices fromtropical Japonica (Maybelle) varieties (Park et al.,

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RICE 19

(7)

(1)(5)

(6)(2) (3) (4)

Figure 2 Production of fertile transgenic rice plants byAgrobacterium-mediated transformation of scutellum-derived calli. (1) Callion selection medium (50mg l1 hygromycin); (2, 3, 4) GUS expression on different hygromycin-resistant calli; (5) plantlets after 34weeks on regeneration medium; (6) fertile plant; (7) detail of panicle

1996) were used successfully. Dong et al. (2001)used inorescence explants from the Japonicavariety Taipei 309 and regenerated transgenicrice plants at frequencies as high as 80% frominorescences when the individual oral organswere 12mm long. This provided a simpler andmore rapid alternative to the scutellar-based ap-proach, by minimizing the tissue culture steps andreducing somaclonal variation, and also enhancedthe potential for transformation of genotypesrecalcitrant to tissue culture and regeneration.Another in planta method by Supartana et al.(2005) employed 2 days presoaked seeds ofJaponica variety Koshihikari to obtain transgenicplants by piercing a site of the husk overlyingthe embryonic apical meristem with a needle thathad been dipped in an Agrobacterium inoculum.The inoculated seeds were then grown to maturityand allowed to pollinate naturally to set seeds.Transformation efciency was estimated to be40% by polymerase chain reaction (PCR) and43% by -glucuronidase histochemical assay. Inother high-throughput methods, Terada et al.(2004) employed seed-derived calli to obtain 1000stable transformants from as few as 150 explantsin Japonica rice; Toki et al. (2006) successfullyregenerated transgenic rice plants within a month(as opposed to the standard three months inall related techniques), from scutella of 1-dayprecultured mature seeds, thus reducing the risk

of somaclonal variation attributable to prolongedtissue culture.

A more recent method of efcient Agrobac-terium-mediated transformation in rice usedmicrospore-derived haploid callus cells (Y. Jiangand T.C. Hall, personal communication). Com-pared to methods using diploid cells or tissuesas starting materials for Agrobacterium-mediatedtransformation, rice transformation of haploidcells such as microspore-derived callus hasseveral advantages. Chromosome doubling of atransgenic haploid cell produces DH transgenichomozygous cells that provide a method forrapid genetic xation of the gene of interest thatis transferred. Homozygous plants are valuablefor plant breeding because almost all of thenal products or cultivars released from plantbreeding are homozygous. Traditional methodsto obtain homozygous breeding lines followinghybridization or mutagenesis often require yearsto succeed. Another advantage of haploids forgenetic analysis is that dominance and recessivityare less likely to obscure gene expression, andthe phenotype is a direct manifestation of thegenotype since there is only one chromosomeset in the haploid. Although Agrobacterium-mediated transformation of microspore-derivedembryos was reported some time ago for Daturainnoxia and Nicotiana tabacum (Sangwan et al.,1993), limited success for rice was reported only

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recently (Chen et al., 2006a, b). The protocol fortransgenic rice extends the methods for a highlyefcient production of transgenic haploids andDH procedure using microspore-derived haploidcallus as a target for Agrobacterium-mediatedtransformation.

2.2.4 Miscellaneous methods

Reports of several novel methods for successfultransformation exist in the literature. Luo andWu (1989) described a method to transform ricethrough a pollen tube pathway method. Guo et al.(1995) reported an effective system for introducingexogenous DNA into cells of embryonic calliof Japonica rice by rst dehydrating the cellsin a hypertonic buffer, then placing the cellsin a medium of less negative osmotic potentialcontaining the exogenous DNA, and employinga laser beam to puncture holes in cell wall andmembrane to enable uptake of DNA expressingthe uorescent calcein. Yoo and Jung (1995)reported the uptake of gusA and hpt genes byimbibition of dry and viable rice embryos froma DNA solution. Matsushita et al. (1999) usedscutellar tissues of rice embryos obtained frommature seeds and vortexed them in liquid mediumcontaining plasmid DNA and silicon carbidewhiskers to obtain transgenic plants expressinggusA and bialaphos resistance.

2.3 Selection Methods for Transgenic Plants

There are numerous critical factors that are ofparamount importance in obtaining transgenicrice plants, and the relative efciency, economy,experimental time, and reproducibility must betaken into consideration to adopt a particulartechnology whether it is particle bombardmentor Agrobacterium-mediated transformation. Theultimate aim is to select an easy, efcient, andinexpensive method suited to the introduction of asingle gene or multiple genes or a study, which re-quires functional genomics to identify the functionof numerous genes in a high-throughput fashion.

2.3.1 Bacterial strains and vectors

Successful Agrobacterium-mediated rice transfor-mation procedures are mostly based on the use

of supervirulent strains and superbinary vectorscarrying the virulence region of pTiBo542 (Hoodet al., 1986; Komari et al., 1986). Strain A281(Hood et al., 1987; Komari, 1989) with a widehost range and higher transformation efciencywas used by both Raineri et al. (1990) and Chanet al. (1993). The strain EHA101 (Hood et al.,1986) was developed with two vector versions, oneharboring a disarmed version of pTiBo542 andanother with the superbinary vector, where aDNA fragment with the virB, C, and G geneswas introduced into a small T-DNA-carryingplasmid that is used in a binary vector system(Komari, 1990).Hiei et al. (1994) tested the efcacyof both the supervirulent EHA101 and anotherordinary strain LBA4404 in combination withpIG121Hm, a derivative of a normal binaryvector pBIN19 (Bevan, 1984), and pTOK233,a derivative of superbinary vector pTOK162(Komari, 1990). The LBA4404 (pTOK233) com-bination was slightlymore effective than LBA4404(pIG121Hm) and EHA101 (pIG121Hm), whileEHA101 (pTOK233) alone was not very effective.Dong et al. (1996) demonstrated that LBA4404(pTOK233) was most effective in Javanica rice. Ingeneral, an ordinary vector/strain combinationproved effective in transforming cultivars thatare easy to grow in tissue culture, and in caseof cultivars difcult or recalcitrant to tissueculture, the choice of vectors and strains is morecrucial. More recently, Jeon et al. (2000) showedthat a combination of LBA4404 with commonbinary vectors resulted in callus transformationefciencies of up to 40% from which transgenicplants were regenerated at a frequency of 85%.

2.3.2 Induction of vir genes

Agrobacterium was found to be attracted towounded plant cells in response to severalphenolic signal compounds, including 4-acetyl-2, 6-dimethoxy phenol (commonly known asacetosyringone) and 4-(2-hydroxy acetyl)-2,6-dimethoxy phenol (-hydroxy acetosyringone)that activate the vir genes on Ti plasmids (Stachelet al., 1985). Seven phenolic compounds werefound to induce the expression of vir genes (Boltonet al., 1986) but monocots appear not to produce,or produce at insufcient levels, these compoundsto act as signals (Smith andHood, 1995). Rice cells

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may be capable of producing sufcient levels ofinducing agent, as the rst report of transgenic calli(Raineri et al., 1990), did not require any phenoliccompound addition in the co-cultivation media.The transfer of T-DNA is enhanced at an earlystage of co-cultivation, and the efciency of genetransfer is also increased when both the bacteriaand the tissues are pretreated with acetosyringone(Aldemita and Hodges, 1996). Certain otherfactors, such as an acidic pH (Turk et al., 1991),culture temperatures below 28 C (Alt-Moerbeet al., 1988), high osmotic pressure (Usami et al.,1988), the presence of opines (Veluthambi et al.,1989), and aldoses likeD-glucose (Cangelosi et al.,1990) also increase vir gene induction.

2.3.3 Tissues amenable to transformation

Binns and Thomashaw (1988) pointed out adistinct correlation between cell wounding andcompetency of such cells for transformation, andproposed that processes related to DNA synthesisand cell division are critical for the integrationof delivered DNA into the host genome. Thewound responses of monocots differ signicantlyfrom those of dicots, with the cells at the woundsites of monocots becoming lignied or scleriedwith no apparent cell division (Kahl, 1982). Thisrenders the choice of dicot explants such asleaf discs and hypocotyls useless vis-a`-vis theirmonocot counterparts; hence, actively growingtissues need to be supplemented with vir-inducingcompounds to ensure successful transformation.For generating transgenic rice, the favored choicehas been mature or immature embryo (scutellum)-derived callus cultures (Hiei et al., 1994; Donget al., 1996; Rashid et al., 1996; Toki, 1997).Shoot apices (Park et al., 1996) and immatureembryos (Aldemita and Hodges, 1996) have alsobeen employed with limited success.

The choice of tissue for transformation is alsodependent on the rice genotype. Japonica andJavanica varieties are usually highly responsive totissue culture and actively dividing callus culturesare the tissues of choice for these varieties (Hieiet al., 1994; Dong et al., 1996). A majority ofIndica varieties falling under a different group,designated group I based on isozyme analysis(Glaszmann, 1987) are recalcitrant to tissueculture and transformation procedures. Aldemita

and Hodges (1996) used immature embryos toachieve transformation of two group I Indicacultivars, namely TSC10 and IR72, a methodextended by Hiei et al. (1997) to many other groupI varieties.

2.3.4 Co-cultivation and culture mediaconditions

The composition and incubation conditions ofthe co-cultivation and subsequent callusing mediaduring the transformation process play a criticalrole in the initial transient transformation efcien-cies and subsequent stable transgene integration.A modied N6 medium with B5 vitamins, 2,4-dichlorophenoxy acetic acid (2,4-D) supplementedwith acetosyringone during co-cultivation wasthe medium of choice for both Japonica andIndica varieties (Hiei et al., 1994; Rashid et al.,1996; Toki, 1997), while MS-based media wasused for Javanica varieties (Dong et al., 1996).Media solidied with a gelling agent was preferredover liquid media, where -glucuronidase (GUS)expression was low (Hiei et al., 1997). Theoptimization ofmedia (e.g., choice ofN6 overMS)can be monitored based on transient expressionof reporter genes like gus, as conditions allowingfor a high level of transient expression aregenerally associatedwith ahigh frequencyof stablytransformed calli and subsequent transgenic plants(Hiei et al., 1997). The gusA gene containing anintron in the coding region has been a very usefulreporter as the GUS expression is limited to plantcells (Ohta et al., 1990; Vancanneyt et al., 1990).

2.3.5 Reporter genes and selectable markergenes

The most commonly used reporter gene in ricetransformation was initially the -glucuronidasegene (gusA) (Jefferson et al., 1987), which servedas a valuable tool to monitor transient expressionearly after transformation, providing an indicationof the expected frequency of stable transgeniclines. Histochemical GUS activity was primarilylocalized at or around the vascular tissue in leaf,root, and ower organs and was also detectedin the embryo and endosperm of dormant andgerminating seeds (Terada and Shimamoto, 1990).Battraw and Hall (1990) also observed GUS

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activity in transgenic rice in the leaf epidermis,mesophyll, vascular bundles, in the cortex andvascular cylinder of the root, andmarginal activityin the root epidermis. Fluorometric assay ofvarious organs showed that GUS activity intransgenic rice plants was comparable to thatseen in transgenic tobacco plants. A more recentinnovation in the GUS reporter system involvesthe development of GUS plusTM (CAMBIA),which is a new reporter gene isolated fromStaphylococcus sp. with superior properties to E.coli gusA. A version with the rice glycine-richprotein signal peptide for extracellular secretionproviding rapid, in vivoGUSassays has been testedextensively in transgenic rice, and the gene has beencodon optimized for high expression in plants.Vickers et al. (2003) developed a synthetic, codon-optimized xylan

Documents

Compendium of Transgenic Crop Plants