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Peppers

Wing-Yee Liu1, Chee-Hark Harn2, Molly Jahn3, Byung-Dong Kim1

and Byoung-Cheorl Kang1

1Department of Plant Sciences, Seoul National University, Seoul, Republic of Korea,2Biotechnology Institute, Nong Woo Bio Co. Ltd., Jeongdan, Republic of Korea, 3University of

Wisconsin-Madison, Madison, WI, USA

1. INTRODUCTION

The genus Capsicum is one of the most importantmembers of Solanaceae crops that include tomato,potato, eggplant, tobacco, petunia, and others.Almost five billion people in the world utilizepeppers not only for food and spices but also forcolorants and medicines. Peppers are cultivated insix continents ranking seventh in the harvestedarea under vegetable crops in the world and theclassical breeding program for pepper cultivationhas been well established in Asia and Europe withincreasing number of elite F1 hybrid varieties.Traditional pepper breeding has been focusingon traits such as yield, environment adaptation,fruit color and shape, and disease resistance.However, limited genetic resources for breedingand increasing demand for better pepper varietiesrequire new tools for pepper breeding.

Two most important biotechnological toolsfor crop improvement are plant transformationand molecular marker-assisted breeding. For thelast 10 years, farmers have constantly increasedtheir cultivation area under genetically modified(GM) crops every year since GM crops werefirst commercialized in 1996. However, no GMpepper varieties have been commercialized; noGM peppers have been under field test due to thedifficulties in transformation of peppers. One ofthe most important steps in plant transformation

is plant regeneration (Valera-Montero and Ochoa-Alejo, 1992; Ebida and Hu-C 1993; Harini andSita, 1993). Despite continuous efforts, successfulreports of pepper transformation are very limited.Even among the successful groups in peppertransformation, the transformation efficiencieshave been very low and the transformation wasneither consistent nor reproducible. Therefore,commercial GM varieties in peppers are still a longway to go. In this review, we will present a generaloverview of the genus Capsicum and current statusof pepper transformation. In addition, we willintroduce one of the successful protocols forpepper transformation at the end of this review.

1.1 History, Origin, and Distribution

The origin of Capsicum species, commonly knownas peppers, is believed to be in South America,either central Bolivia along Rıo Grande (Andrews,1995) or Brazil along the Amazon (DeWitt andBosland, 1993). These areas concentrated withlarge wild species of peppers are also known asthe nuclear areas (DeWitt and Bosland, 1993;Andrews, 1995). There are at least two centersof domestication, one in central America and theother in the Andean region of South America.Five species, among approximately 30 in the genus,were independently domesticated and have been

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

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cultivated primarily for use as spice and vegetablefor thousands of years (Andrews, 1995). Columbusintroduced Capsicum into Europe in the 15thcentury, subsequently it was distributed rapidlyaround the world. Following its arrival in westernEurope, both pungent and nonpungent forms ofthe species C. annuum came into wide cultivation,assuming a particularly important role in thecuisines of some parts of Europe, e.g., Hungary,West Africa, and many regions of Asia.

In western Europe and North America,relatively large, nonpungent bell type (C. annuum)became dominant in the 18th and early 19thcentury (Boswell, 1937). The pungent types remainfavored in Latin America, Asia, and Africa.Today Capsicum is grown in tropical, subtropical,and temperate regions worldwide and Capsicumvarieties are utilized for a diverse range of foodproducts as vegetable and spice, fresh, dehydratedor processed, in medicine, in pest and animalcontrol, and even in law enforcement, whichmake this crop of immense cultural and economicimportance (Bosland and Votava, 2000).

1.2 Botanical Features

1.2.1 Taxonomy

The genus Capsicum belongs to the tribe Solanae,a family member of the Solanaceae (Hunziker,2001). Capsicum is closely related to Solanum,to which two other important vegetable crops,tomato (S. lycopersicum) and potato (S. tubero-sum), belong (Olmstead et al., 1999; Martins andBarkman, 2005). About 30 species are currentlyrecognized under the genus of Capsicum (Baraland Bosland, 2002), although the actual number ofspecies is still debatable. In fact, taxonomy of thegenus of Capsicum has long been confusing andarguable. Botanists traditionally define nomencla-ture and classification of Capsicum species basedon morphological and anatomical characteristics.Phylogenetic relationship can also be determinedby investigating hybridization performance be-tween the Capsicum species (Tong and Bosland,1999). Recently, molecular phylogenetic study hasbeen carried out with 11 species of Capsicum withthe aim of clarifying the phylogenetic ambiguityrelated to Capsicum (Walsh and Hoot, 2001).

C. annuum, C. chinense, and C. frutescens arethe three main cultivated Capsicum species inagriculture worldwide. Types of cultivated C.annuum include bell pepper, jalapeno, New Mexicochile, ancho, Anaheim, and banana pepper, toname just a few. Many local varieties andlandraces are also grown widely mostly in LatinAmerica. In addition to these three species,two other domesticated species, C. pubescensand C. baccatum, are grown primarily in LatinAmerica. C. pubescens has distinctive purpleflowers and black seeds and was domesticated inthe Peruvian and Bolivian Andes. Wild forms of alldomesticated species have been identified, exceptof C. pubescens (Pickersgill, 1997).

1.2.2 Germplasm

Germplasm collection is extremely important inpepper breeding. Diversity of genetic resourcesis utilized for bringing new traits, particularlyfor resistance to new diseases and alternativeresistance sources for existing diseases. Besidescrop improvements, germplasm also plays animportant role in various scientific researches,such as elucidation of evolution and classificationof Capsicum species, understanding biology andbiochemistry in peppers. Agricultural ResearchService of United States Department of Agri-culture (USDA-ARS) in Griffin (Georgia, USA)and Asian Vegetable Research and DevelopmentCenter (AVRDC), the world’s vegetable centerbased in Taiwan, have the world’s largestcollections of Capsicum germplasm and are activein characterization, evaluation and conservation.AVRDC has a total of 7726 accessions of Capsicumspp. as of September 2006 (htt://www.avrdc.org);USDA-ARS has more than 4700 accessions of 16Capsicum taxa in its active collection program inGriffin (Stoner, 2004). These two organizationsalso maintain databases for their collections anddistribute seeds to pepper researchers and breedersall over the world. Other institutions, universities,government or nongovernment organizations,and private seed companies have various scalesof Capsicum germplasm collections (Berke andEngle, 1997). Germplasm of Capsicum of at least15 countries have been reported in Capsicum andEggplant Newsletter (CENL) in the past 20 years(1983–2002).

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1.2.3 Habitat and morphology

Wild Capsicum plants are perennial shrubs;commercial pepper cultivars are usually grownas annual crops. As a perennial, the plant turnsfrom herbaceous to woody with age. Capsicumspecies has perfect and complete flowers. Asa member of Solanaceae, flowers of Capsicumtypically have five sepals, petals, stamens, andpistil. Color of petals (or corolla), stamens,and pistil can be white, greenish white, greenishyellow, or purple, combination varies dependingon species and varieties. Cultivated Capsicumspecies are primarily self-pollinated and do notdisplay inbreeding depression. Peppers can alsobe cross-pollinated by insects, outcrossing ratevaries depending on the cultivars. Temperature,particularly night-time temperature is criticalfor fruit set (Andrews, 1995). The optimal daytemperature for fruit setting of cultivated pepper isbetween 20–30 ◦C in chili pepper and 21–24 ◦C insweet pepper, but fruits fail or have difficulty to setwhen night temperature exceeds 24 ◦C; growth ofthe plants is also reduced when air temperature isless than 15 ◦C or more than 32 ◦C for chili pepperand less than 18 ◦C or more than 27 ◦C for sweetpepper (http://www.avrdc.org).

Pepper fruit is the commodity of the pepperplant; therefore, fruit morphology, flavor, andpungency are the most economically impor-tant characteristics in Capsicum. A tremendousamount of genetic variation is known with respectto fruit traits such as size, shape, color, and flavor,resulting in more than 50 commercially recognizedpod types. Bosland (1992) and Andrews (1995)described the major fruit types.

1.2.4 Genome size, karyotype,and ploidy level

The genome size of pepper was estimatedto be 7.65 pg/nucleus for C. annuum and9.72 pg/nucleus for C. pubescens (Belletti et al.,1998). Genome size in nucleotides is estimated tobe about 3000 Mbp (mega base pairs) (Arumu-ganathan and Earle, 1991). Most Capsicum specieshas 12 pairs of chromosomes; however, C. ciliatumhas 13 pairs of chromosomes. Polyploidy is veryrarely observed in Capsicum (Lippert et al., 1966).The development of doubled haploid (DH) lines by

anther culture is common to obtain homozygousgenotypes of interest and to develop populationsfor inheritance studies and the construction ofgenetic maps (Pochard et al., 1983a; Lefebvre et al.,1995). A set of trisomic lines were produced byPochard (1977) and used for the chromosomalassignment of several mutations. Various types ofchromosomal rearrangements are prevalent in thegenus both within and between species (Pochard,1977; Onus and Pickersgill, 2004).

1.3 Economic Importance andConsumption of Pepper

Peppers are the third-most important vegetablecrops worldwide, grown in most countries inthe world with production more than 22 milliontons annually (FAO, 2002). The world’s largestproducer of peppers is China (more than 8million tons in 2001) followed by Mexico andTurkey, which produced 1 900 000 and 1 400 000tons, respectively in 2001. Spain, Italy, and theNetherlands are the main growers in Europe.Peppers are important source of income forfarmers in most parts of the world.

Consumption of pepper is closely associatedwith cultures and diets. People in differentcountries or different regions of a country havedifferent demands for fruit type, color, shape,maturity, taste, and pungency of peppers. Forfresh- and dried-vegetable market, C. annuum isthe dominated species of cultivated Capsicum,although C. chinense and C. frutescens are alsocommonly grown in some regions (Andrews,1995). C. pubescens and C. baccatum have minormarket in Latin American culture. Pepper fruit ismainly used as fresh and dried vegetable; leaf ofpepper plants is also consumed as leafy vegetablein some cultures. Fresh and dried vegetablepeppers can also be used in making sauces; thefamous “Salsa” sauce is made with serrano andjalapaneos (Andrews, 1995).

Peppers are also important crops for processedfood industry. Three species of Capsicum are usedfor processed pepper: C. annuum, C. chinense,and C. frutescens. The major uses of peppersin processing industries are pickle, sauce andpowder. Thompson (1995) collected and describedmore than 350 hot sauces from all over theworld, including the best-known pepper sauce,

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Tabasco (C. frutescens). Pepper fruits are driedor dehydrated and processed to produce pepperpowder, also known as paprika in Europe. Pepperpowder is an important spice particularly inHungary and India.

In addition to its importance as a food andspice, peppers have been used and continue tobe used widely for diverse medicinal applications(Bosland and Votava, 2000). Because capsaici-noids interact specifically with the mammalianpain receptor, VR1, they have been widely usedas topical analgesics (Caterina et al., 2000). Whilepain control is the most familiar application,hundreds of publications each year report variouspharmacological applications of these moleculesfrom weight loss to cancer.

1.4 Chemical Composition and Nutrition

1.4.1 Capsaicinoids

Capsicum may be best known for its biosynthesisof capsaicinoids that account for the pungent or“hot” sensation when consumed. Capsaicinoidshave only been found in Capsicum species, butnot all Capsicum species are pungent, for instance,nonpungent form can be found in C. annuum,C. ciliatum, and C. chacoense. Capsaicinoids arethought to play an important role in the evolutionof Capsicum species. Tewksbury et al. (2006)studied the geographic variation of pungencywithin three species of ancestral Capsicum inBolivia and found that production of capsaicinoidsshifts across elevations. It was suggested thatcapsaicinoids entails both costs and benefitsin response to selection pressure (Tewksburyet al., 2006). It has been hypothesized thatthis trait evolved to deter mammalian herbivory,where crushing molars and acidic digestion aredetrimental to seed survival (seed predator). Thefavored agents of dispersal are various speciesof birds. Avian species do not perceive pain inresponse to capsaicin and are attracted by thebrightly colored fruit of Capsicum (Tewksbury andNabhan, 2001).

Capsaicinoids include a family of up to 25related alkaloid analogs produced in epidermalcells of the placenta or dissepiments of thefruit. The two major capsaicinoid compounds arecapsaicin and dihydrocapsaicin. The gene AT3

(acyltranferase 3) at Pun1 locus was identified forthe biosynthesis of capsaicin (Stewart et al., 2005).Nevertheless, the genetic base of biosynthesisof other quantitatively inherited capsaicinoidsis still poorly known (Blum et al., 2003).Biosynthesis of capsaicinoids is complicated andaccumulation of capsaicinoids varies dramaticallywithin and between Capsicum species. A numberof analytical techniques have been reported forthe determination of pungency in pepper andsummarized by Pruthi (2003). Scoville indexand high-performance liquid chromatographyanalysis represent the two generations of the mostwidely used techniques in pungency measurement(DeWitt, 1999); spectrophotometeric method isused as ISO Reference method (Pruthi, 2003).

1.4.2 Pigments and vitamins

The most common colors of pepper fruits aregreen, red, yellow and orange, and chocolateor purple. Green peppers are usually notmature; the mature color ranges from lemon oryellow through orange, peach, and red. Growingand storage conditions alter color of pepperfruits (Gomez et al., 2003). Pepper color isassociated with its pigment content. Carotenoidand anthocyanin pigments are responsible forfruit color and for nutritional value of Capsicumfruit. Red color is resulted from the accumulationof different carotenoids in fruit chromoplastsduring fruit ripening. The predominant redpigments are capsanthin and capsorubin, theyellow and orange pigments are lutein, β-carotene(provitamin A), zeaxanthin, violaxanthin, andantheraxanthin (Buckenhuskes, 2003). Carotenoidbiosynthetic pathway in pepper fruit and putativecorrespondences with quantitative and qualitativeorgan-color loci identified in the Solanaceae wasproposed by Thorup et al. (2000).

Peppers are well known as nutritious crop withvery low calories, particularly rich in vitamin Aand C. Peppers are ranked first in antioxidant con-tent among vegetables with very high levels of vita-min C (Palevitch and Craker, 1995). Consumptionof a single pepper fruit is enough to meet an adultRecommended Dietary Allowances for this vita-min. A list of nutrients of different peppers is pro-vided at www.chilepepperinstitute.org. Vitamin Cconcentration is high in green fresh fruits, but is

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lower in mature fruits and it breakdowns in driedor dehydrated pepper products. As for vitamin A,its accumulation increases as fruits turn maturedto red or orange. Unlike vitamin C, high levelof vitamin A is retained in dried fruits (DeWitt,1999).

1.5 Breeding Objectives

1.5.1 Breeding for yield

Yield, like all other crops, is always a majorobjective in pepper breeding. Yield in pepperoften associates with plant vigor, number, andconcentration of fruit set, fruit weight andsize, harvesting period. In addition, floweringtime, fruit set at extreme temperature, growthhabit (adaptation to open field or greenhouseproduction, mechanical or hand harvesting)are also considered. Besides actual increase inyield, stability of pepper production is equallyimportant.

1.5.2 Breeding for disease resistance

Disease is often the major constraint of pepperproduction worldwide; therefore, disease resis-tance is one of the major objectives in pepperbreeding and genetic studies (Paran et al., 2004).Pernezny et al. (2003) has an overview ofmost of the known pepper diseases. Resistancesources in wild species or domesticated peppershave been reported for tobacco mosaic virus(TMV) (Boukema, 1980), cucumber mosaic virus(CMV) (Pochard, 1982; Shifriss and Cohen, 1990),potato virus Y (PVY) (Pochard et al., 1983b),pepper vental mosaic virus (PVMV) (Hobbset al., 1998), tomato spotted wilt virus (TSWV)(Rosello et al., 1996), bacterial spot (Xanthomonascampestris pv. vesicatoria) (Hibberd et al., 1983),bacterial wilt (Ralstonia solanacearum) (Pereraet al., 1992), Verticillium wilt (Gil Ortega et al.,1990), Fusarium wilt (Jones and Black, 1992),Anthracnose (Colletotrichum spp.) (Voorrips et al.,2004), Phytophthora root rot, stem rot, and foliarblight caused by Phytophthora capsici (Pochardand Daubeze, 1982), and nematodes, etc.

Disease resistance breeding and genetics havebeen studied extensively in the past years.

Introgression of disease resistance genes fromwild germplasm to elite backgrounds contributessignificantly in crop improvement, particularlyin terms of yield and quality enhancementand stability in pepper production. Successfulexamples are as follows: resistance to TMVfrom C. chinense (L3) and C. chacoense (L4)(Boukema, 1980; Berzal-Herranz et al., 1995;De la Cruz et al., 1997), resistance to TSWVfrom C. chinense (Boiteux et al., 1993), andresistance to bacterial leaf spot disease (Bs2)from C. chacoense (Cook and Guevara, 1984;Kim and Hartmann, 1985; Hibberd et al., 1987)have been introduced to commercial Capsicumannuum cultivars. Additional genes for resistanceto potyviruses, CMV, nematodes, P. capsici, andpowdery mildew have been identified in severalCapsicum species and are being utilized.

However, introgression of disease resistancegenes into elite cultivars is particularly difficultwhen those resistance traits are inherited bycomplex quantitative mechanism and linked toundesirable horticultural and economic traits,such as low yield and small fruit size. Incase of CMV and many other virus diseases,due to recombination and wide diversity ofthe virus, breeding for sustainable resistanceremains a difficult task. With these challenges,breeding for disease resistance remains a pri-oritized breeding objective in pepper breedingin the future. Identification of new resistancesources, breeding for multiple disease resistances,and pyramiding different sources of resistancewill be important in breeding for sustainableagriculture.

1.5.3 Breeding for quality

There are many types of peppers that are beingutilized for different purposes, each with differentquality requirements and traits required forsuccessful production. A unique aspect of pepperbreeding is of course the degree of pungency.Understanding people’s diet and favors in terms ofpungency level is particularly important in pepperbreeding. Pungency level is particularly importantfor breeding for processed food industry. Theindustry also emphasizes on color, stability anduniformity of color, and color intensity (Bosland,1993). For quality control reason, analytical

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techniques for both color and pungency are oftenapplied to assist breeding (Pruthi, 2003). For freshvegetable pepper, major traits under selection arealways related to fruit quality and taste: fruit colorand color intensity, size, shape, pericarp thickness,taste, and degree of pungency. Furthermore, giventhe important role of long-distance transportationof vegetable produces now a days, shelf lifealso becomes an important breeding objectivefor fresh vegetable pepper. The major markettypes and their important fruit quality traits weresummarized by Poulos (1994). These include freshconsumption; fresh for processing into sauce,paste, canned or pickled product; dried for spice(whole fruits and powder); oleoresin extraction;and ornamental types.

1.6 Tools and Strategies of Pepper Breeding

1.6.1 Classical genetics and conventionalbreeding

Classical inheritance studies and quantitativegenetics have been used to study pepper genetics;conventional breeding methods and strategieshave been used for crop improvement in pepper,for instance, single plant selection by pedigreemethod, population improvement by recurrentselection, backcrossing, single-seed descent (SSD),etc. A set of recombinant inbred lines derivedfrom PSP-11 × PI201234, produced by SSD hasbeen generated and utilized for breeding andgenetic studies of P. capsici resistance in pepper(Ogundiwin et al., 2005). For commercial peppers,both open-pollinated and F1 hybrid varieties areavailable.

1.6.2 Hybrids

F1 hybrids (or single-cross hybrids), which areobtained from crossing between two homozygousinbred lines, were firstly used in corn. Now adays, F1 hybrid cultivars are produced in manyother field and vegetable crops. Hybrid cultivarsare exploited on the basis of heterosis or hybridvigor. F1 hybrid peppers generally show significantimprovements in yield, plant vigor, number of fruitset, disease resistance, etc. and have been producedby seed companies worldwide. The drawback of F1

hybrids is the cost of seed production, as it involveshand emasculation of each flower. The use of male-sterility can, therefore, dramatically reduce the costof production of F1 hybrid peppers by eliminatingemasculation in hybridization. Cytoplasmic-genicmale-sterility, also known as cytoplasmic male-sterility (CMS) was discovered in pepper (C.annuum L.) by Peterson in 1958. Pepper CMSsystem is similar to that found in rice, petunia, andradish. Generally, in CMS system, male-sterility iscaused by a cytoplasmic factor (S) and inheritedmaternally; normal cytoplasm is termed as (N).Recently, an additional abnormal mitochondrialgene orf456 and a dysfunctioning mitochondrialgene atp6 have been found in association withmale-sterility in pepper (Kim and Kim, 2005,2006); a dominant nuclear gene, Rf , carriesout male-fertility restoration. In hybrid seedproduction, a CMS line (A line), (S) rfrf , serves asfemale and pollinated with a male-fertile restorerline (R line), (N) RfRf . A CMS line is maintainedby an isogenic male-fertile maintainer line (B line)(N) rfrf . CMS system can give rise to 100% male-sterility and has been utilized particularly in hybridseed production. However, this system tends tobe more stable in certain types of hot pepper, forinstance, hot dry-type pepper in Korea (Shifriss,1997; Lee, 2001), but shows instability in otherhot/sweet types, especially under low temperatures(Shifriss and Guri, 1979; Shifriss, 1997). R linesare quite common in hot peppers, but rare insweet peppers (Zhang et al., 2000; Kumar et al.,2001; Yazawa et al., 2002). Pepper types, therefore,restrict the utilization of CMS in hybrid seedproduction. An alternative male-sterility system,genic male-sterility (GMS), is available and hasbeen used for sweet pepper production. In GMSsystem, male-sterility is conferred by a pair ofhomozygous recessive gene (ms/ms), homozygousdominant or heterozygous (Ms/Ms or Ms/ms)plants are male-fertile. To maintain and producea male-sterile line, two isogenic lines with onlydifference at the Ms locus (Ms/Ms and ms/ms)are crossed, that gives rise to a progeny mixtureof 50% male-fertile (Ms/ms) and 50% male-sterile(ms/ms) plants. In hybrid seed production field,the male-fertile plants are manually identifiedand removed, the remaining male-sterile (ms/ms)plants are used as female to pollinate with thesecond parental line (Ms/Ms) for hybrid seedproduction.

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1.6.3 Tissue culture in pepper breeding

In vitro haploid plant production is exploitedby plant breeders to facilitate the developmentof inbred lines particularly for hybrid breedingand widely adaptation in various field andvegetable crops. Nitsch and Nitsch producedhaploid plants from pollen grains in 1969. Acomprehensive protocol for anther culture inpepper was published by Dumas de Valuix et al.(1981). Various modifications or improvements ofthe original protocol have been reported (Qin andRotino, 1993; Mityko et al., 1995; Mityko andJuhasz, 2006). Other approaches for generatinghaploid plants have also been studied, includingmicrospore culture and shed-microspore culture(Mityko and Fari, 1994; Supena et al., 2006). Inprinciple of haploid culture, plants are generateddirectly from haploid gamete (n = x = 12 inC. annuum), chromosome number of haploidplants is doubled by treating with colchicine andhomozygous DH lines are produced (Sopory andMunshi, 1996). Once a protocol is established,robust tissue culture can be carried out to generatea number of DH lines. In conventional breeding,it takes 6–8 generations to produce homozygousinbred lines. In comparison, with haploid andDH technology, number of homozygous DH lineswith different recombinant traits can be generatedby culturing recombined haploid gametes fromhybrid plants. The resulting DH lines are readyfor testcross trials for hybrid breeding (Khush andVirmani, 1996). Tremendous time and cost couldbe saved by this strategy. Furthermore, DH canalso be used in other aspects of genetic research,such as in genetic mapping and evaluationof complex quantitative traits (Thabuis et al.,2003, 2004b).

Embryo culture is an important in vitro culturetechnique for plant breeding. One of the mostsignificant applications is to overcome hybridiza-tion barrier between distant crosses (Brown andThorpe, 1995). In the genus of Capsicum, only afew species are utilized for cultivation. However,tremendous genetic resources, for instance diseaseresistance genes, are hidden in other wild species.Most of these species belong to the secondary ortertiary gene pools, where crosses between differentspecies may fail or may give rise to weak or sterileF1 or backcross plants because of hybridizationbarriers. Even within the primary gene pool

barriers may occur between C. annuum, C.chinense, and C. frutescens. Hybridization barrierscan occur in different stages. Embryo culture isparticularly useful to overcome postzygotic orpoor endosperm development, where endospermfails to properly nourish the embryo and seeddevelopment is aborted (Brown and Thorpe, 1995).This technique allows researchers and breedersto transfer desirable genes/traits from a wild-related species. In practice, immature embryos arecollected after hybridization and transferred togrow on basal growth medium and plants areregenerated from the rescued embryos. In addition,embryo culture can also be utilized in overcomingseed dormancy, slow seed germination, andimmaturity in seeds. Besides embryo culture, othermethods used to overcome hybridization barrierinclude using bridge cross through related species,mixing of pollens, and protoplast fusion, etc.

1.6.4 Molecular approaches in breeding

The use of molecular markers and genetic mapsin pepper breeding is currently in developmentalstage. The aim of molecular breeding is tosupplement conventional breeding, to achievefaster and more efficient breeding through marker-assisted selection (MAS) and/or marker-assistedbackcrossing (MAB). Molecular markers thatare closely linked to the trait of interest are tobe identified and applied in gene pyramiding,facilitating introgression of desirable traits intocultivars, early selection, etc. For more complextraits conferred by polygenes, quantitative traitloci (QTL) analysis is carried out. Furthermore,molecular markers and genetic maps also havesignificant contributions in other plant scienceresearch, particularly in map-based cloning.

Molecular markers and genetic maps areimportant resources for breeding and geneticstudies, molecular resources have been buildingup in the past 20 years. The first generation ofmolecular markers are protein based isozymes.The first study in which isozymes were used forlinkage mapping in Capsicum was reported byTanksley (1984) who mapped 14 isozyme markersin an interspecific cross of C. annuum and C.chinense, of which nine were arranged in fourlinkage groups. From the 1990s, different typesof DNA markers: AFLP (amplified fragment

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length polymorphism), RAPD (random amplifiedpolymorphic DNA), and RFLP (restrictionfragment length polymorphism) were applied inpepper. Livingstone et al. (1999) used RFLPmarkers derived from tomato complementary-DNA (cDNA) and genomic DNA of pepper,which also allowed comparative genetic studiesbetween tomato and pepper. Kang et al. (2001)developed sets of pepper-specific DNA probes forRFLP system. Toward the 21st century, molecularmarker development has preceded to user-friendlypolymerase chain reaction (PCR)-based DNAmarkers. Simple sequence repeat (SSR) markersin pepper are available in both private and publicsectors (Lee et al., 2004a; SOL Genomic Networks,http://www.sgn.cornell.edu). The future develop-ment is expected to move toward development ofsingle-nucleotide polymorphism (SNP) and single-feature polymorphism (SFP) markers. SNP/SFPmarkers are believed to have the most potential inapplication of markers in crop improvement.

Several pepper genetic maps have been pub-lished in the past, most of these maps were pro-duced in F2 interspecific crosses: AC99, (Capsicumannuum × C. chinense) (SOL Genomic Networks);FA03, (C. frutescens × C. chinense) (Sol GenomicNetworks); SNU, (C. annuum × C. chinense)(Kang et al., 2001); and SNU2, (C. annuum ×C. chinense) (Lee et al., 2004a). SNU2, FA03,and AC99 are SSR-based genetic maps containing46, 489, and 359 SSR markers, respectively.An integrated map has been developed recentlyby pooling data of six genetic maps, includingtwo interspecific genetic maps (C. annuum ×C. chinense) and four intraspecific genetic maps(C. annuum × C. annuum) (Paran et al., 2004).The integrated map covering 1832 cM (centi-Morgans) contains 2262 markers including 1528AFLP, 440 RFLP, 228 RAPD, 3 isozyme, and3 morphological markers. This integrated mapsignificantly reduced the length of gaps betweenmarkers to 0.8 cM and is expected to serve asan important platform for locating markers andaligning with other pepper genetic maps.

A large number of dominant and recessivegenes have been identified and mapped in pepperthrough both classical and molecular approaches,including other potyviruses (Pvr4, pvr6) (Carantaet al., 1996; Caranta et al., 1999; Arnedo-Andreset al., 2002) and bacterial spot resistance (Bs3)(Pierre et al., 2000); resistance to TSWV (Tsw)

(Jahn et al., 2000; Moury et al., 2000), nematodes(Me3, Me4) (Djian-Caporalino et al., 2001)and TMV (L) (Lefebvre et al., 1995). As formore complex disease resistance traits, QTLstudies have been carried out for resistance toCMV (Caranta et al., 1997; Ben Chaim et al.,2001; Caranta et al., 2002), potyvirus (Carantaet al., 1996), powdery mildew (Lefebvre et al.,2003), anthracnose (Voorrips et al., 2004) andPhytophthora (Nahm, 2001; Thabuis et al., 2003;Thabuis et al., 2004a; Ogundiwin et al., 2005).

To date, two genes have been cloned in pepperconferring disease resistance by candidate geneand map-based cloning approaches respectively:pvr1 (=pvr2), a recessive gene for potyvirusresistance (Ruffel et al., 2002; Kang et al., 2005);Bs2, a dominant, disease resistance gene (R gene)for bacterial spot resistance against Xanthomonascampestris pv. vesicatoria (Tai et al., 1999; Taiand Staskawicz, 2000). Although the progress ofgene cloning is relatively slow, with successful genecloning, PCR-based molecular markers can bedeveloped and applied in plant breeding program(Yeam et al., 2005).

1.7 Limitations of Genetic and MolecularResources

The current status of genetic and molecularresources limits their use in applied pepperbreeding. Genetic resource is extremely important,however, the progress of characterization ofcollected germplasm is relatively low, for instance,800 accessions have been characterized for USDA-ARS’s Capsicum germplasm collection. Further-more, not many valuable genetic populations, suchas nearly isogenic lines, are available in pepper.

As for molecular resources, although the recentpublication of the integrated map (Paran et al.,2004) has improved the situation, there is stilllack of correspondence between different peppermaps and QTL analyses in pepper (Varshneyet al., 2005). Most of these QTL studies reliedon AFLP markers, which have relatively lowreproducibility and do not include overlappingsets of markers. These studies also suffer tovarying degrees from common limitations of QTLanalyses: QTL from individual studies may beeither overestimated or underestimated becauseof environmental factors and QTL intervals are

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too often very large (Yano and Sasaki, 1997;Carlborg and Haley, 2004). The differences intypes of populations, inoculation and scoringmethods, and QTL assessment between differentstudies of P. capsici resistance in pepper, may affectestimation of QTL. In addition, resolution of QTLwas further constrained by limited polymorphismoften due to lack of polymorphism in intraspecificpopulations. Considering the limitations of QTLanalysis and genomic resources in pepper, thediversity of the pathogens, and the complexityof genetic resources for resistance, molecularmarkers have important potential for MAS inpepper. Particularly, breeding for complex diseaseresistance traits in pepper remains a challenge forplant breeders.

With the limitations of genetic and molecularresources, genetic transformation provides analternative approach for crop improvement inCapsicum. Transformation further overcomes thebarriers between different species and allows usto introduce “useful” genes or novel traits intopepper.

2. DEVELOPMENT OF TRANSGENICPEPPERS

2.1 Donor Genes: Sources, Isolation,Cloning and Designing Transgenes

The first pepper transformation was reportedin 1990 (Liu et al., 1990). However, Liu andhis colleagues failed to regenerate the transgenicpepper plants. In fact, whole plant regenerationof pepper has been known as difficult with poorreproducibility that has been a key limiting factorfor pepper transformation. Several studies onestablishing and optimizing protocols for reliableregeneration have been reported in the past15 years (Lee et al., 1993; Manoharan et al.,1998; Mihalka et al., 2000; Kim et al., 2002;Li et al., 2003). Recently, Lee et al. (2004b)have reported an improved protocol by callus-mediated shoot formation. Despite these efforts,pepper transformation and regeneration efficiencyare relatively poor comparing with that of othersolanaceous crops, such as tomato and tobacco,where transformation protocols are well developedand have been routinely used for both biologicalstudies and crop improvement.

Summaries of pepper transformation, regen-eration methods and medium used for peppertransformation are presented in Tables 1 and 2,respectively. Besides establishing transformationand regeneration protocol, researchers are mostlyinterested in pepper transformation for diseaseresistance, particularly, virus resistance (Table1). It has been found that by introducing viralgene(s) to the host plant, transgenic plantsdisplayed resistance to the target and relatedvirus. This disease resistance mechanism was firstdemonstrated by Abel et al. (1986) by transformingcoat protein (CP) encoding gene of TMV intotobacco; the transgenic plants showed delay ofdisease development when infected with TMV andrelated tobamoviruses (Abel et al., 1986; Beachy,1999). Based on this resistance mechanism, peppertransformation has been studied by differentresearch groups for resistance to TMV byintroducing TMV-CP gene (Lee et al., 2004b),pepper mild mottle virus (PMMV) by Pepper-PMMV interaction 1 transcription gene (Lee et al.,2004b), tomato mosaic virus (ToMV) by ToMV-CP gene (Shin et al., 2002b), CMV by CMV-CPgene (Shin et al., 2002a) and CMV-satellite RNA(Kim et al., 1997). Such transgenic virus resistancemechanism was later known as RNA silencing(Voinnet, 2001). Production of transgenic peppersby RNA silencing approach is considered to bespecific and effective against targeted virus and/orviral groups.

Other donor genes might be used for developingbroad-spectrum resistance in pepper (Table 1).Shin et al. (2002b) demonstrated transformationinto and overexpression in pepper of Tsi1, atobacco pathogenesis-related (PR) gene and theresulting transgenic plants displayed resistanceor improved resistance to a broad spectrumof pathogen types including PMMV, CMV, abacterial pathogen Xanthomonas campestris pv.vesicatoria and an oomycete pathogen P. capsici.

Besides transformation for disease resistance,studies on other aspects of transformation inpepper are limited. In other plants like Arabidopsis,tobacco, and tomato, transformation technology isalso widely applied for functional studies of genes.Due to preliminary transformation technology inpepper, study on gene expression in transgenicpepper is extremely rare. Characterization ofisolated pepper genes has not been carriedout by transformation in foreign plants. With

JWBK232-Kole k0603 July 21, 2008 20:8

82 TRANSGENIC VEGETABLE CROPS

Tabl

e1

Sum

mar

yof

pepp

ertr

ansf

orm

atio

n

Pep

per

cult

ivar

s/A

grob

acte

rium

Pep

per

tiss

uefo

rSe

lect

ion

mar

ker/

Gen

e-of

-int

eres

t/ge

noty

pes

stra

ins

tran

sfor

mat

ion

resi

stan

cepr

omot

erP

lasm

id/v

ecto

rR

efer

ence

Yol

oW

onde

r,E

arly

Cal

iforn

iaW

onde

r,N

VH

3051

,Ju

pite

r,L

iber

tyB

ell,

Gua

tem

alan

wild

acce

ssio

n

A28

1,C

58(G

V38

50)

You

ngtr

uele

aves

,co

tyle

dons

,hyp

ocot

yls

nptI

I/ka

nam

ycin

GU

S/C

aMV

35S

p3-1

-GU

SL

iuet

al.,

1990

Gol

den

tow

erL

BA

4404

Cot

yled

ons

Kan

amyc

in,c

arbe

nici

llin

Cuc

umbe

rm

osai

cvi

rus

I 17N

-sat

ellit

eR

NA

/CaM

V35

S

pRok

1/10

5L

eeet

al.,

1993

Zho

ngH

uaN

o.2

GV

3111

-SE

You

ngtr

uele

aves

,co

tyle

dons

,hyp

ocot

yls

Kan

amyc

inC

MV

-CP

/CaM

V35

SpH

CM

40Z

huet

al.,

1996

Pus

ajw

ala

EH

A10

5C

otyl

edon

snp

tII/

kana

myc

inG

US/

CaM

V35

SpB

I12

1M

anoh

aran

etal

.,19

98N

o.40

017

C58

C1R

ifR

,L

BA

4404

,E

HA

101,

A28

1

Cot

yled

ons

nptI

I/ka

nam

ycin

and

gene

tici

n,hp

t/hy

grom

ycin

,dh

fr/m

etho

rexa

te,

bar/

phos

phin

othr

icin

GU

S/C

aMV

35S

pRG

Gpl

asm

idse

t(c

ontr

acte

din

this

stud

y)

Mih

alka

etal

.,20

00

Noc

kkw

ang

LB

A44

04H

ypoc

otyl

snp

tII/

kana

myc

inO

sMA

DS

1/C

aMV

35S

pGA

1209

Kim

etal

.,20

01V

S300

-1L

BA

4404

You

ngem

bryo

nic

tiss

ue,

coty

ledo

nssu

rB/c

hlor

sulf

uron

CaC

el1/

CaM

V35

SpW

TT

2132

Har

pste

ret

al.,

2002

Gol

den

Tow

erL

BA

4404

Cot

yled

ons,

hypo

coty

lsnp

tII/

kana

myc

inC

MV

-CP

,ToM

V-C

P/

CaM

V35

SpM

BP

2Sh

inet

al.,

2002

a

Noc

kkw

ang

–C

otyl

edon

s,hy

poco

tyls

nptI

I/ka

nam

ycin

Tsi/

CaM

V35

SpM

BP

2Sh

inet

al.,

2002

b

F1

Xia

ngya

n10

,Z

hong

jiao,

Zho

ngjia

o5,

Zho

ngjia

o6

LB

A44

04C

otyl

edon

snp

tII/

kana

myc

inG

US/

CaM

V35

SpB

I121

Lie

tal.,

2003

Feh

eroz

onSh

oote

rGR

ifR

GV

3170

Rif

RC

otyl

edon

shp

t/hy

grom

ycin

ornp

tII/

kana

myc

inG

US/

CaM

V35

SpR

GG

hpt,

pRG

Gne

oM

ihal

kaet

al.,

2003

P91

5,P

4090

,P41

0,P

101

EH

A10

5,L

BA

4404

Cot

yled

ons,

hypo

coty

lsnp

tII/

kana

myc

inT

MV

-CP

,PP

I1/C

aMV

35S

pCA

MB

IAL

eeet

al.,

2004

b

JWBK232-Kole k0603 July 21, 2008 20:8

PEPPERS 83

Tabl

e2

Sum

mar

yof

rege

nera

tion

met

hods

and

med

ium

used

for

pepp

ertr

ansf

orm

atio

n

Reg

ener

atio

nm

etho

d

Shoo

tin

duci

ngm

ediu

m/s

elec

tion

med

ium

Elo

ngat

ion

med

ium

Roo

tin

duci

ngm

ediu

mR

egen

erat

ion

rate

Tra

nsfo

rmat

ion

rate

Ref

eren

ce

Dir

ect

rege

nera

tion

MSL

2m

ediu

m:M

Ssa

lts,

L2

med

ium

,BA

(10

mg

l–1),

IAA

(1m

gl–1

)(w

hole

plan

tre

gene

rati

onfa

iled)

––

––

Liu

etal

.,19

90

Dir

ect

rege

nera

tion

MS

salt

s,B

5vi

tam

ins,

2%su

cros

e,ka

nam

ycin

sulf

ate

(100

mg

l–1),

carb

enic

illin

(250

mg

l–1),

NA

A(0

.05

mg

l–1),

zeat

in(2

.0m

gl–1

)

MS

salt

s,B

5vi

tam

ins,

2%su

cros

e,ka

nam

ycin

sulf

ate

(100

mg

l),c

arbe

nici

llin

(250

mg

l),N

AA

(0.0

1m

gl–1

),ze

atin

(2.0

mg

l–1)

MS

salt

san

dvi

tam

ins,

3%su

cros

e,ka

nam

ycin

sulf

ate

(50

mg

l–1)

57.5

%4%

Lee

etal

.,19

93

Dir

ect

rege

nera

tion

MS

basa

lmed

ium

,ka

nam

ycin

(50

mg

l–1),

carb

enic

illin

(30

mg

l–1),

sucr

ose

(30

gl–1

),B

A(8

mg

l–1),

IAA

(2m

gl–1

)

EM

1:M

Sba

salm

ediu

m,

BA

(2m

gl–1

),G

A3

(2m

gl–1

),A

BA

(2m

gl–1

);E

M2:

MS

basa

lmed

ium

,B

A(2

mg

l–1),

GA

3(2

mg

l–1),

AB

A(0

.5m

gl–1

)

MS

basa

lmed

ium

wit

hka

nam

ycin

(50

mg

l–1),

carb

enic

illin

(30

mg

l–1),

NA

A(0

.1m

gl–1

)

––

Zhu

etal

.,19

96

Dir

ect

rege

nera

tion

MS

med

ium

,2%

sucr

ose,

kana

myc

in(5

0m

gl–1

),ce

fota

xim

e(4

00m

gl–1

),th

idia

zuro

n(T

DZ

)(0

.5m

gl–1

)

SER

med

ium

:Hal

fst

reng

thM

Sm

ediu

m,k

anam

ycin

(25

mg

l–1),

cefo

taxi

me

(200

mg

l–1),

IAA

(0.5

mg

l–1)

21.2

–84.

3%(i

ndep

ende

ntfo

rmtr

ansf

orm

atio

n)–

–M

anoh

aran

etal

.,19

98

Dir

ect

rege

nera

tion

MSB

5gl:

MS

salt

s,B

5vi

tam

ins,

2%gl

ucos

e,ce

fota

xim

e(5

00m

gl–1

)or

augm

enti

n(4

00m

gl–1

),be

nzyl

aden

ine

(BA

P)

(4m

gl–1

),in

dol-

3-ac

etic

acid

(IA

A)

(0.5

mg

l–1)

(opt

imiz

edre

gene

rati

onpr

otoc

ol)

––

––

Mih

alka

etal

.,20

00

(con

tinu

ed)

JWBK232-Kole k0603 July 21, 2008 20:8

84 TRANSGENIC VEGETABLE CROPS

Tabl

e2

Sum

mar

yof

rege

nera

tion

met

hods

and

med

ium

used

for

pepp

ertr

ansf

orm

atio

n(c

onti

nued

)

Reg

ener

atio

nm

etho

d

Shoo

tin

duci

ngm

ediu

m/s

elec

tion

med

ium

Elo

ngat

ion

med

ium

Roo

tin

duci

ngm

ediu

mR

egen

erat

ion

rate

Tra

nsfo

rmat

ion

rate

Ref

eren

ce

Dir

ect

rege

nera

tion

MS

med

ium

,cef

otax

ime

(500

mg

l–1),

kana

myc

in(1

50m

gl–1

),ze

atin

(3m

gl–1

),IA

A(0

.3m

gl–1

)

MS

med

ium

,kan

amyc

in(5

0m

gl–1

),N

AA

(0.3

mg

l–1)

–0.

8%–

Kim

etal

.,20

01

Dir

ect

rege

nera

tion

MS

basa

lmed

ium

,ka

nam

ycin

(100

mg/

l–1),

sucr

ose

(30

gl–1

),ze

atin

(2m

gl–1

),N

AA

(0.1

mg

l–1)

––

––

Shin

etal

.,20

02a

Dir

ect

rege

nera

tion

MS

basa

lmed

ium

,ka

nam

ycin

(200

mg

l–1),

sucr

ose

(30

gl–1

),ze

atin

(2m

gl–1

),N

AA

(0.1

mg

l–1)

––

––

Shin

etal

.,20

02b

Dir

ect

rege

nera

tion

MS

salt

s,B

5vi

tam

ins,

2%su

cros

e,ka

nam

ycin

(50

mg

l–1),

carb

enic

illin

(500

mg

l–1),

IAA

(1.0

mg

l–1),

BA

(5.0

mg

l–1),

AgN

O3

(10

mg

l–1),

DJ

nutr

ient

s(5

000

mg

l–1;p

repa

red

inth

isst

udy)

MS

salt

s,B

5vi

tam

ins,

2%su

cros

e,ka

nam

ycin

(50

mg

l–1),

carb

enic

illin

(500

mg

l–1),

IAA

(1.0

mg

l–1),

BA

(3.0

mg

l–1),

AgN

O3

(10

mg

l–1),

2m

gl–1

GA

3,D

Jnu

trie

nts

(500

0m

gl–1

;pre

pare

din

this

stud

y)

MS

salt

s,B

5vi

tam

ins,

2%su

cros

e,N

AA

(0.2

mg

l–1),

IAA

(0.1

mg

l–1)

81.3

%(d

iffe

rent

iati

on);

61.5

%(e

long

atio

n);

89.5

%(r

ooti

ng)

40.8

%of

rege

nera

ted

plan

ts

Lie

tal.,

2003

Dir

ect

rege

nera

tion

MSB

5glm

ediu

m,

cefo

taxi

me

(500

mg

l–1)

MSB

5glm

ediu

m,c

efot

axim

e(3

00m

gl–1

)M

Sba

salm

ediu

m,g

luco

se(1

5m

gl–1

),m

alto

se(1

5m

gl–1

),IA

A(0

.5m

gl–1

);M

Sba

sal

med

ium

,glu

cose

(15

mg

l–1),

mal

tose

(15

mg

l–1),

kana

myc

in(1

50m

gl–1

)

–2.

4%M

ihal

kaet

al.,

2003

Indi

rect

callu

s-m

edia

ted

shoo

tre

gene

rati

on

MS

med

ium

,kan

amyc

in(8

0–10

0m

gl–1

),ce

fota

xim

e(3

00m

gl–1

)or

lilac

illin

e,ze

atin

(2.0

mg

l–1)+

NA

A(0

.5m

gl–1

)or

zeat

in(2

.0m

gl–1

)+

IAA

(0.1

mg

l–1)

MS

med

ium

,kan

amyc

in(6

0–10

0m

gl–1

),ce

fota

xim

e(3

00m

gl–1

),ze

atin

(2.0

mg

l–1)+

NA

A(0

.01

mg

l–1)

orze

atin

(2.0

mg

l–1)+

IAA

(0.0

1m

gl–1

)

MS

med

ium

,kan

amyc

in(2

0–30

mg

l–1),

cefo

taxi

me

(200

mg

l–1),

28/3

750

0(n

umbe

rof

shoo

tsw

ith

root

s/nu

mbe

rof

expl

ants

)

0–0.

19%

Lee

etal

.,20

04b

JWBK232-Kole k0603 July 21, 2008 20:8

PEPPERS 85

an exception, Harpster et al. (2002), whodemonstrated suppression of a ripening-relatedendo-1,4-β-glucanase in transgenic pepper. Onthe other hand, transformation of foreign genesidentified in other plant species into pepper is alsouncommon. Nonetheless, by transformation andconstitutive expression of a rice gene, OsMADS1, adwarf transgenic pepper was successfully produced(Kim et al., 2001).

Classical transgene designs have been used inpepper transformation (Table 1). The transgeneconstruct, which contains β-glucuronidase (GUS)gene as reporter gene, driven by cauliflower mosaicvirus (CaMV) 35S promoter and with nopalinesynthase (NOS) as terminator, is dominated inpepper transformation studies, particularly forthe studies aiming at establishing and optimizingpepper transformation protocol (Liu et al., 1990;Manoharan et al., 1998; Li et al., 2003; Mihalkaet al., 2003). CaMV 35S promoter is commonlyused for overexpression of genes of interest (Zhuet al., 1996; Kim et al., 2002; Shin et al., 2002b; Leeet al., 2004b).

2.2 Methods Employed

2.2.1 Agrobacterium-mediatedtransformation

Agrobacterium-mediated transformation system iscommonly employed for pepper transformation.Other transformation systems, for instance genegun, have not been reported. Wild type A.tumefaciens generally is not considered as apathogen of Capsicum spp. under field conditions,although certain strains of Agrobacterium showedvirulence to pepper (De Cleene and De Lay,1976). Capability of Agrobacterium strains toinduce gall formation under laboratory conditionsis required for successful genetic transformation.Use of strains also determines the efficacyof transformation (Liu et al., 1990; Mihalkaet al., 2000). Liu et al. (1990) compared theeffect of two different Agrobacterium strains inpepper transformation The strain C58 consistentlyshowed distinctively higher average percentage ofexplants forming tumors than the strain A281.

Cotyledons and/or hypocotyls are the mostcommonly used explants (Manoharan et al., 1998;Pozueta-Romero et al., 2001; Kim et al., 2002;

Lee et al., 2004b). In addition, use of youngleaf for pepper transformation has also beendemonstrated (Liu et al., 1990; Zhu et al., 1996).For transformation, Agrobacterium is co-culturedwith the pepper explants in Murashige and Skoog(MS) medium (Pozueta-Romero et al., 2001; Leeet al., 2004b) or MSB5gl medium (MS saltswith B5 vitamins and glucose) (Mihalka et al.,2000, 2003).

Different pepper cultivars/genotypes also showvariation in response to Agrobacterium-mediatedtransformation (Liu et al., 1990; Lee et al., 2004b).Transformation has been carried out mainly inC. annuum, both chili pepper and sweet pepper(Zhu et al., 1996; Manoharan et al., 1998);transformation of other Capsicum spp. is rarelytested except for C. frutescens.

2.2.2 Selection of transformed tissue

Antibiotic resistance has been the most commonlyused method for the selection of transformedpepper tissue (Table 1). To screen for positive trans-formation, explants are transferred to antibiotic-containing medium after transformation or gallformation for selection, positive transformantswith antibiotic resistance would survive. Similarto other protocols for selection of transformantsin other plants, kanamycin resistance is commonlyused for selection in pepper transformation.Kanamycin resistance is contributed by theneomycin phosphotransferase II gene (nptII) inthe vector construct (Liu et al., 1990; Kim et al.,1997; Manoharan et al., 1998; Mihalka et al.,2000; Pozueta-Romero et al., 2001; Shinet al., 2002a, b; Li et al., 2003). NptII is usuallydriven by a NOS gene promoter (Manoharanet al. 1998; Kim et al., 2002; Li et al., 2003).NptII confers resistance to kanamycin as wellas geneticin. Alternative selection markers forpepper transformation have been demonstratedto be effective by using hygromycin resistancegene (hpt) for hygromycin resistance, methorexateresistance gene (dhfr) for methorexate resistanceand phosphinothricin acetyltransferase gene (bar)for phosphinothricin resistance (Mihalka et al.,2000).

However, use of antibiotic or herbicide resis-tance genes as selection markers for transforma-tion has risen environmental and health concerns.

JWBK232-Kole k0603 July 21, 2008 20:8

86 TRANSGENIC VEGETABLE CROPS

Alternative selection methods have been investi-gated. Shin et al. (2002a) discussed the potentialuse of ToMV-CP gene as a selection marker. Inthis screening system, TMV is inoculated to thetransgenic plants and positive transformants areidentified by a distinctive hypersensitive response.However, further experiment is necessary toevaluate the efficiency and effectiveness of thisselection system.

Another alternative selection system wasdemonstrated by transformation and expression ofisopentenyl transferase gene (ipt) identified from“shooter” mutants of Agrobacterium (Mihalkaet al., 2003). With the presences of ipt, positivetransformants were shown to produce directshooting on growth regulator-free medium. Thesignificant morphological characteristic allowsvisual selection of positive transgenic plants.This system has also been demonstrated intransformation of other crops like tomato,tobacco, and muskmelon. The use of binary vectorin pepper transformation has also been reported(Mihalka et al., 2003). Selection markers, whichmight carry undesirable genes, and the genes ofinterest are integrated in different chromosomalpositions in the transgenic pepper under the binaryvector system. Therefore, selection marker genescould easily be removed subsequently.

2.2.3 Regeneration of whole plants

Regeneration of pepper plants has long beena challenging task for pepper transformationstudies. Several studies attempted to improve,modify, and optimize protocols for regenerationof transgenic pepper plants. However, theseexperiments are often hardly reproducible.

Direct regeneration from shoot is the mostcommon practice for transgenic pepper regen-eration. In this system, explants transformedwith Agrobacterium are grown on shoot inducingmedium or selection medium for 3–4 weeks,shoots or multishoots are formed directly at thesites of wounded explants. Then induced shootbuds are transferred to the elongation mediumand grown for 20 days to 8 weeks. The shootsformed are excised and grown on root-inductingmedium. Recently, an alternative regenerationmethod, named callus-mediated shoot formation,has been proposed (Lee et al., 2004b). In this study,

six different types of callus have been identifiedfrom transformed tissue and two of which wereable to form shoots. Desirable type of calluswere isolated and propagated (instead of directshooting); propagated transformed callus was thensubcultured for shoot formation. Although thetransformation rate remains low (<0.19%), callus-mediated shoot formation method is believed tobe more reproducible and reliable. More detailedprotocol of this approach is presented in the nextsection.

MS medium is used as basal medium fortransgenic plant regeneration, depending on theresearch group; different concentrations and com-positions of supplements were applied (Table 2).Particularly, effects of hormones have been studiedby several research groups with the aim ofoptimizing regeneration conditions. Nevertheless,there was lack of consensus between differentresearch groups. Different compositions and ratiosof cytokinins (benzyladenine, BAP), thidiazuron(TDZ), naphthalene acetic acid (NAA), zeatin,(2-isopentyl adenine (2IP)) to auxin (indol-3-aceticacid, IAA) have been tested (Lee et al., 1993; Zhuet al., 1996; Manoharan et al. 1998; Mihalka et al.,2000; Li et al., 2003). Furthermore, kanamycin inmedia is commonly used for selection of positivetransformants. Concentration and effects of otherantibiotics (geneticin, hygromycin, methorexate,phosphinothricin) in the regeneration media havealso been studied (Mihalka et al., 2000). Inaddition, effects of other important factors onregeneration have been studied, for instance, effecton explant tissue for shoot bud initiation (Zhuet al., 1996), genotypes/cultivars used for planttransformation and regeneration (Liu et al., 1990;Kim et al., 2002; Lee et al., 2004b).

2.3 Case Study: Transformation UsingCallus-Mediated Indirect RegenerationMethod

Most of the previously described transformationprocedures for pepper transformation are notvery useful for routine transformation due to thelack of reproducibility. The low rate of peppertransformation indicates that DNA transfervia Agrobacterium infection into cut-injuredcotyledon or hypocotyl tissues hardly occurs.This also suggests that Agrobacterium may not

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penetrate and/or infect cotyledon or hypocotyltissues of pepper. Lee et al. (2004b) demonstratedthat selection of the right callus type is mostcritical for successful pepper transformation.Indeed, the transformation via callus-mediatedshoot formation proved to be reproducible andthis selection method provided a reliable systemfor pepper transformation. The protocol forsuccessful pepper transformation and the resultsobtained using the protocol are described below.

2.3.1 Transformation protocol

Seeds from commercial inbred lines (C. annuuminbred line P915, P409, P410, P101: NongwooBio Co. proprietary) are commonly used for theexperiment. These lines showed a very high rateof regeneration (90%) among the 30 inbred linestested (Kim et al., 2002).

2.3.1.1 Germination and explant

Seeds are surface-disinfested in 95% ethanol for30 s, 50% bleach for 10 min and rinsed threetimes with sterilized water. Sterilized seeds arethen placed in 1/2 MS medium (Murashige andSkoog, 1962) and allowed to germinate in light ordark at 25 ◦C. Germination under light or darkconditions does not influence the transformationrate. Cotyledons and hypocotyls from 8- to 10-day-old plants are excised and used as explants.

2.3.1.2 Preculture and Agrobacteriuminoculation

Explants are transferred to a pre-culture mediumconsisting of MS medium supplemented withzeatin 2 mg l–1 and NAA 0.05 mg l–1 or IAA0.1 mg l–1, and placed in light room at 25 ◦C for2–36 h and this preculture step is necessary. Fortransformation, Agrobacterium strain EHA105 isthe best choice for the pepper transformation.Agrobacterium is grown in YEP (2% bactopeptone; 1% yeast extract) media supplementedwith kanamycin 50 mg l–1, rifampicin 50 mg l–1

and 100 µM acetosyringone. The Agrobacteriumculture is centrifuged and then diluted with MS tooptical density 0.3–0.8. This bacterial suspension

is then mixed with MS liquid containing 100 µMacetosyringone and inoculated to explants for10–20 min, co-cultured in dark for 38–96 h andthen washed with cefotaxime 500–800 mg l–1 orlilacilline 500–800 mg l–1 three times.

2.3.1.3 Callus and shoot formation

In order to obtain callus formation and devel-opment, explants are transferred to a selectionmedium consisting of 80–100 mg l–1 kanamycin,300 mg l–1 cefotaxime or lilacilline with zeatin2.0 mg l–1 + NAA 0.05 mg l–1 or IAA 0.1 mg l–1 for6–8 weeks. For shoot formation and elongation,the calli are transferred to a regenerationmedium consisting of 60–100 mg l–1 kanamycin,300 mg l–1 cefotaxime with zeatin 2.0 mg l–1 +NAA 0.01 mg l–1 or IAA 0.01 mg l–1 for 7–10weeks.

2.3.2 Transformation experiment usingthe protocol

Two general patterns of pepper shoot formationwere identified. Firstly, a shoot or multishootsformed directly from the wounding or cut regionof explants (direct regeneration) (Figure 1).This pattern was frequently observed in manycases of pepper transformation and the stagesof shoot development observed were directshoot formation, multishoot elongation, shootelongation, and root formation. Secondly, shootswere selected from callus tissues that had formedaround cut after 4–5 weeks after culture on theshoot selection medium (callus-mediated indirectregeneration) (Figure 2) These were unusualbecause calli are not easily formed from thewounded region of cotyledons. Five stages of shootdevelopment were distinct: callus formation, callusdevelopment, shoot formation, shoot elongation,and root formation. Six different types of callusdeveloped from the explants have been identified.Shoot regeneration capability was dependent onthe callus types. Those that were able to formshoots were designated type A (white hard surfaceand green tissue inside) and type B (dark greencallus with hard surface, but a little bit moist)(Figure 3). The regeneration rate of type A wasmuch higher than that of type B (ca. 90%, data

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88 TRANSGENIC VEGETABLE CROPS

(a) (b) (c)

(d) (e)

Figure 1 Development of direct shoot formation after co-culture: (a) shoot formation (5 weeks old on selection medium); (b)multishoot formation (7 weeks old); (c) multishoot elongation (9 weeks old); (d) a single shoot elongation from multishoot; (e) rootformation (14 weeks old) (the red dot line indicates a cut to move to the next culture)

(a) (b) (c)

(d) (e)

Figure 2 Development of indirect shoot formation after co-culture: (a) callus formation (5 weeks old on selection medium); (b)callus development (7 weeks old); (c) shoot formation (9 weeks old); (d) shoot elongation (11 weeks old); (e) root formation (14 weeksold) (the red dot line indicates a cut to move to the next culture)

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(a) (c)(b)

(d) (f)(e)

Figure 3 Callus types for shoot formation (a and b) and for nonshoot formation (c, d, e, f): (a) white hard surface and green tissueinside; (b) dark green callus with hard surface, but a little bit moist; (c) yellow and easily brittle; (d) yellow and hard surface; (e) moistand a little bit transparent; (f) green, moist, and easily brittle

not shown). The callus types that were not able toproduce shoots were designated type C (yellow andbrittle) and also included type D (yellow and hardsurface), type E (moist and a little transparent),and type F (green, moist, and brittle).

In order to investigate the direct shoot formationrate after transformation, 151 700 explants fromfour different lines were transformed with theTMV-CP and PPI1 genes. The rate of developinga direct shoot in the shoot medium after co-culture was 5.3% (8089/151 700) (Table 3). Therate of shoot survival in the rooting medium was0.93% (1407/151 700). Among four lines, P915 line

showed the highest rate of shoot development.There was no significant shoot formation ratebetween transformation experiments of two genes.To find the indirect shoot formation rate, 37 500explants from four different lines were transformedwith the TMV-CP and PPI1 genes. The frequencyof generating calli from explants was 1.2%(459/37 500) (Table 4). However, all calli didnot produce the shoot. The frequency of shootdevelopment from callus was 11.6% (53/459).The frequency of root formation from thecallus-mediated shoots was 52.8% (28/53). Outof four lines, 68% (19/28) of the total shoots

Table 3 Frequency of direct shoot formation

Number of explant Number of shoot Number of shoot with root

Gene P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P401 P101

TMV-CP 30 312 26 039 21 983 21 983 2106 1017 1697 628 392 156 311 49PPI1 14 413 14 080 7 488 13 078 1024 587 720 310 186 103 176 34Subtotal 44 925 40 110 31 595 35 060 3130 1604 2417 938 578 259 487 83Total 151 700 8089 1407

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Table 4 Frequency of callus development and shoot formation from the callus

Number of shoot Number of shoot callusNumber of shoot from

callus Number of shoot with root

Gene P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101

TMV-CP 5 188 6917 5 1 7 5491 7 2 10 81 30 25 94 14 12 3 0 2PPI1 2 903 3056 4 2 5 3798 2 9 37 107 46 17 59 15 7 2 1 1Subtotal 8 091 9973 9 3 12 9289 10 147 188 76 42 153 29 19 5 1 3Total 37 500 459 53 28

in the rooting medium were obtained from P915line, suggesting a genotype preference of shootformation.

A total of 1407 direct shoots grown in therooting media were tested by PCR to identifytransformed pepper plants and none of theshoots contained inserts (Tables 3 and 5).This indicates that the direct shoots grownfrom explants are not transformed. To test thetransformation rate of indirect callus-mediatedshoots, the final 28 shoots in the rooting mediumwere analyzed by PCR (Tables 4 and 5; Figure 4).The transformation rate was 0.19% (15/37, 500)for P915 line and 0.03% (3/37, 500) for P409 line.However, the transformation rate as determined bythe number of PCR positive versus the number ofcallus-mediated shoot was 34% (18/53), indicatingthat shoots grown from the callus could betransformed with high probability. Therefore, theselection of a callus-mediated shoots from amonga large number of shoots growing on selectionmedium tended to discriminate transformed plantsfrom nontransformed plants.

DNA samples from transformed peppers (T0),randomly chosen from the PCR positive peppers,were isolated and 30 µg DNA was digested tocompletion with XbaI for TMV-CP and BglIIfor the PPI1 gene. Figure 5a shows a Southern

blot analysis of TMV-CP transformed peppersdigested by XbaI. Transformed peppers showeddifferent TMV-CP insertion sites and those hadone copy of CP gene inserted. The PPI1 insertdigested with BglII was also localized at differentsites in PPI1 transformed peppers (Figure 5b).The 5-kb band on the Southern blot was presentin all T0 plants, and represented the pepperPPI1 gene internally embedded in the genome,whereas the other inserts were newly transformed.Interestingly, all of the transformed peppers hadonly one copy of the PPI1 gene inserted.

In summary, the protocol described hereprovides a more reliable pepper transformationplatform. However, several key points should betaken into account for successful transformation.First, plant material for pepper transformation iscritical. For maximum transformation efficiency,callus-mediated indirect shoot should be used.Most shoots seem to grow well directly fromthe wounded surface of explants on selectionmedium, however, no transformants have beenobtained from shoots grown directly. In contrast,callus-mediated indirect shoots showed very high-transformation efficiency. Another importantfactor is to select a right callus type, which canproduce transgenic shoot. Among the six differentcallus types, the type A callus generated most

Table 5 Transformation efficiency of direct and indirect shoot formation

Direct shoot Indirect shoot

Gene P915 P409 P410 P101 P915 P409 P410 P101

TMV-CP 0 0 0 0 10 5491 7210 81PPI1 0 0 0 0 5 3798 2937 107Total 0 0 0 0 15 3 0 0Transformation rate(a) (%) 0 0 0 0 0.19 0.03 0 0%

(a)Percentages were obtained by dividing the number of PCR positive with the number of explant used

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1 2 3 4 5 6 7 8 9 10 11 12 N1 N2 N3 P1 P2

TMV-CP718 bp

5´ 3´

35S promoter TMV-CP

9087837669M

PPI 1683 bp

92 N1 N2 P1 P2

5´ 3´

35S promoter PPI1

(a)

(b)

Figure 4 (a) PCR analysis of transformed peppers with TMV-CP gene (T0): 1–12,: transformed; N1–N3, nontransformed; P1–P2,positive control (cloned bacterial cell and pTMV-CP, respectively) (b) PCR analysis of transformed peppers with PPI1 gene (T0): 69,76, 83, 87, 90, and 92, transformed; N1–N2, nontransformed; P1–P2, positive control (cloned bacterial cell and pPPI1, respectively)

XbaI

5 63 N1 N2

Bgl II

69 76 N1 83 87 N2

5.0 kb

3.0 kb

4.0 kb

2.0 kb

3.0 kb

(a) (b)

Figure 5 (a) Southern blot analysis of transformed peppers with TMV-CP gene (T0)′ ′ 5 and 63, transformed; N1–N2,nontransformed (b) Southern blot analysis of transformed peppers with PPI1 gene (T0): 69, 76, 83, and 87, transformed; N1–N2,nontransformed (the 5.0-kb band belongs to the endogenous gene of PPI1)

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transgenic shoots. Therefore, the best way toscreen putative transformed shoots during thepepper transformation procedure is to identifyshoots regenerated from type A callus. Thirdly,plant genotypes and Agrobacterium strains forthe transformation are very important factors.Therefore, regeneration rate of each pepper lineshould be tested. The higher regeneration rate pro-duces the better transformation rate. Accordingto our experimental results Agrobacterium strain,EHA105, is best strain for pepper transformation.

3. FUTURE ROAD MAP

Capsicum transformation is still at its infantstage comparing with that of other well-studiedsolanaceous crops, such as tomato and tobacco.Therefore, no transgenic pepper lines are underfield test; no GM pepper varieties have beencommercialized. In spite of continuous effortsworldwide, successful reports of pepper transfor-mation are very rare. Even among the successfulgroups, the transformation efficiencies have beenextremely low and inconsistent. We presenteda callus-mediated indirect shoot transformationmethod and expect that this will provide a betterplatform for pepper transformation.

Nevertheless, further improvement in trans-formation efficiency is required. Since bothtransformation efficiency and plant regenerationis genotype dependant. In order to obtainmaximum transformation rate and for rou-tine transformation, the most virulent/effectiveAgrobacterium strain and the most susceptiblepepper genotypes with the highest regenerationrate should be identified. Genotypes with thehighest transformation rate do not necessarilyregenerate well (Liu et al., 1990). However,screening large numbers of pepper inbred lines mayrequire joint efforts of laboratories worldwide.

Since pepper transformation efficiency is ex-tremely low at present, introducing transgenesto elite lines can also be very slow even if anyuseful transgenic lines may be available. In orderto expedite transgene introgression, conventionalbackcrossing and molecular marker techniqueshould be utilized. Molecular linkage maps andvarious types of molecular markers are currentlyavailable for genome scanning and marker-assistedselection. Hence, as was shown in soybean and

other well-studied field crops, useful transgenes aretransferred to the genotype, which can be easilytransformed, and introgressed by backcrossing toelite lines those are recalcitrant to be transformed.

Virus resistant peppers are expected to be thefirst possible commercialized transgenic peppersbecause these traits have been most extensivelystudied and are relatively simple to manipulate. Forvirus resistance, virus CPs are easiest target genesto be transformed into pepper. For instance, CMVresistant transgenic peppers harboring CMV-CP gene are very promising future product. Inaddition to viral genes, engineering host factorcan be a target for virus resistance. Since diseaseresistance breeding by conventional method relieson the availability of disease resistance gene(s)in existing Capsicum germplasm and confinedby sexual barriers between domesticated andwild Capsicum species. Transformation techniqueis, therefore, extremely useful against potentialpathogens when there is lack of genetic resourcesamong available Capsicum species.

Although insect resistance has not been adominated research target in the past peppertransformation studies, Bacillus thuringiensis (Bt)-transgenic pepper also has potential to be com-mercialized (if it is available), particularly whenpolitical and regulatory issues are considered.Transformation of Bt-gene, mainly Cry1Ac, forinsect resistance has been demonstrated in manyother crops and the related products have beentested extensively and released for commercialproduction for long time; health and environmen-tal assessments and regulations; administrativeand political processes for the release of Bt-transformed products are well established. Infact, release of Bt-transgenic eggplant (anotherclosely related relative) in several targeted Asiancountries is under progress. These experiences inBt-transformed products and Bt-GM eggplant willserve as an example for future release of Bt-pepper.

Since for breeding pungency level is particularlyimportant and difficult object, development oftransgenic plant with different pungency levelwill be very useful approach. The gene, whichcontrols presence and absence of capsaicinoid, wasrecently identified as acyltranferase 3 (AT3) andexpression level of this gene controls pungencylevel. Therefore, it is theoretically feasible tocontrol pungency level by controlling expressionlevel of AT3. Other possibility of transgenic pepper

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development is biofortification for micronutrientenhancement. Although peppers are rich invitamin A and C, transformation approach forfurther increase or stable vitamins’ production willbenefit consumers and growers/manufacturers aswell particularly in the areas with malnutritionproblems.

Finally, once transgenic pepper plants aredeveloped, potential risks should be addressed.For example, damage to human health and envi-ronment, disruption of current cropping schemesand food production in developed countries,and of traditional practices and economies inless developed countries, the regulatory measuresadopted by Institutional Biosafety Committee(IBC), Animal and Plant Health InspectionService (APHIS), Food and Drug Administration(FDA), Environmental Protection Agency (EPA),and international agreements for new transgenicproducts are also to be considered.

ACKNOWLEDGMENTS

This work was supported by a grant fromSeoul National University Foundation for newlyappointed professors and by a grant from theCenter for Plant Molecular Genetics and BreedingResearch via the Korea Science and EngineeringFoundation (KOSEF) and Korea Ministry ofScience and Technology (MOST).

REFERENCES

Abel, P.P., Nelson, R.S., De, B., Hoffmann, N., Rogers, S.G.,Fraley, R.T. and Beachy, R.N. (1986) Delay of diseasedevelopment in transgenic plants that express the tobaccomosaic virus coat protein gene. Science 232, 738–743.

Andrews, J. (1995) The Domesticated Capsicums. New Edition.University of Texas Press, Austin.

Arnedo-Andres, S., Gil-Ortega, R., Luis-Arteaga, M. andHormaza, I. (2002) Development of RAPD and SCARmarkers linked to the Pvr4 locus for resistance to PVYin pepper (Capsicum annuum L.). Theoretical and AppliedGenetics 105, 1067–1074.

Arumuganathan, K. and Earle, E.D. (1991) Nuclear DNAcontent of some important plant species. Plant MolecularBiology Reporter 9, 208–219.

Baral, J.B. and Bosland, P.W. (2002) An updated synthesis ofthe Capsicum genus. Capsicum and Eggplant Newsletter 21,11–21.

Beachy, R.N. (1999) Coat-protein-mediated resistance totobacco mosaic virus: discovery mechanisms and exploita-

tion. Philosophical Transactions of the Royal Society forBiological Sciences 354, 659–664.

Belletti, P., Marzachi, C. and Lanteri, S. (1998) Flow cytometricmeasurement of nuclear DNA content in Capsicum(Solanaceae). Plant Systematics and Evolution 209, 85–91.

Berke, T. and Engle, L. (1997) Current status of major Capsicumgermplasm collections worldwide. Capsicum and EggplantNewsletter 16, 80–82.

Berzal-Herranz, A., De La Cruz, A., Tenllado, F., Diaz-Ruiz,J.R., Lopez, L., Sanz, A.I., Vaquero, C., Serra, M.T. andGarcia-Luque, I. (1995) The Capsicum L3 gene-mediatedresistance against the tobamoviruses is elicited by the coatprotein. Virology 209, 498–505.

Blum, E., Mazourek, M., O’Connell, M., Curry, J., Thorup, T.,Liu, K., Jahn, M. and Paran, I. (2003) Molecular mappingof capsaicinoid biosynthesis genes and quantitative trait locianalysis for capsaicinoid content in Capsicum. Theoreticaland Applied Genetics 108, 79–86.

Boiteux, L.S., Cupertino, F.P. and Reifschneider, F.J.B.(1993) Capsicum chinense PI159236: a source of resistanceto Phytophthora capsici and tomato spotted wilt virus.Capsicum and Eggplant Newsletter 12, 76.

Bosland, P.W. (1992) Chiles: a diverse crop. HorticultureTechnology 2 (1), 7–10.

Bosland, P.W. (1993) Breeding for quality in Capsicum.Capsicum and Eggplant Newsletter 12, 25–31.

Bosland, P.W. and Votava, E.J. (2000) Peppers: Vegetable andSpice Capsicums. CAB International, New York.

Boswell, V.R. (1937) Improvement and genetics of tomatoes,peppers, and eggplant. In: Agriculture, U.S.D.O. (ed.)Year Book of Agriculture. Government Printing Office,Washington DC, pp. 176–206.

Boukema, I.W. (1980) Allelism of genes controlling resistanceto TMV in Capsicum L. Euphytica 29, 433–439.

Brown, D.C.W. and Thorpe, T.A. (1995) Crop improvementthrough tissue culture. World Journal of Microbiology andBiotechnology 11, 409–415.

BuckenhusKes, H.J. (2003) Current requirements on paprikapowder for food industry. In: De, A.K. (ed.) Capsicum:The Genus Capsicum. Taylor & Francis, New York,pp. 223–230.

Caranta, C., Palloix, A., Gebre-Selassie, K., Lefebvre, V.,Moury, B. and Daubeze, A.M. (1996) A complementationof two genes originating from susceptible Capsicum annuumlines confers a new and complete resistance to pepper vienalmottle virus. Phytopathology 86, 739–743.

Caranta, C., Palloix, A., Lefebvre, V. and Daubeze, A.M. (1997)QTLs for a component of partial resistance to cucumbermosaic virus in pepper: restriction of virus installation inhost-cells. Theoretical and Applied Genetics 94, 431–438.

Caranta, C., Pflieger, S., Lefebvre, V., Daubeze, A.M.,Thabuis, A. and Palloix, A. (2002) QTLs involved in therestriction of Cucumber mosaic virus (CMV) long-distancemovement in pepper. Theoretical and Applied Genetics 104,586–591.

Caranta, C., Thabuis, A. and Palloix, A. (1999) Developmentof a CAPS marker for the Pvr4 locus: a tool for pyramidingpotyvirus resistance genes in pepper. Genome 42, 1111–1116.

Carlborg, O. and Haley, C.S. (2004) Epistasis: too oftenneglected in complex trait studies? Nature Review of Genetics5, 618–625.

JWBK232-Kole k0603 July 21, 2008 20:8

94 TRANSGENIC VEGETABLE CROPS

Caterina, M.J., Leffler, A., Malmberg, A.B., Martin, W.J.,Trafton, J., Petersen-Zeitz, K.R., Koltzenburg, M., Bas-baum, A.I. and Julius, D. (2000) Impaired nociceptionand pain sensation in mice lacking the capsaicin receptor.Science 288, 306–313.

Chaim, A., Grube, R.C., Lapidot, M., Jahn, M. and Paran,I. (2001) Identification of quantitative trait loci associatedwith tolerance to cucumber mosaic virus in Capsicumannuum. Theoretical and Applied Genetics 102, 1213–1220.

Cook, A.A. and Guevara, Y.G. (1984) Hypersensitivity inCapsicum chacoense to race 1 of the bacterial spot pathogenof pepper. Plant Disease 68, 329–330.

De Cleene, M. and De Lay, J. (1976) The host range of crowngall. Botanical Review 42, 389–466.

De La Cruz, A., Lopez, L., Tenllado, F., Diaz-Ruiz, J.R.,Sanz, A.I., Vaquero, C., Serra, M.T. and Garcia-Luque,I. (1997) The coat protein is required for the elicitationof the Capsicum L2 gene-mediated resistance against thetobamoviruses. Molecular Plant–Microbe Interactions 10,107–113.

DeWitt, D. (1999) The Chile Pepper Encyclopedia. WilliamMorrow, New York, pp. 56–57, 118–129, 181–183, 211–214,219–224.

DeWitt, D. and Bosland, P.W. (1993) The Pepper Garden. TenSpeed Press, Berkeley, pp. 1–209.

Djian-Caporalino, C., Pijarowski, L., Fazari, A., Samson,M., Gaveau, L., O’byrne, C., Lefebvre, V., Caranta, C.,Palloix, A. and Abad, P. (2001) High-resolution geneticmapping of the pepper (Capsicum annuum L.) resistanceloci Me3 and Me4 conferring heat-stable resistance to root-knot nematodes (Meloiogyna spp.). Theoretical and AppliedGenetics 103, 592–600.

Dumas De Valuix, R., Chambonnet, D. and Pochard, E.(1981) Culture in vitro d’antheres du piment (Capsicumannuum L.): amelioration des taux d’obtention de planteschez differents genotypes par des traitements a +35 ◦C.Agronomie 1, 859–864.

Ebida, A. and Hu-C, Y. (1993) In vitro morphogeneticresponses and plant regeneration from pepper (Capsicumannuum L. cv. Early California Wonder) seedling explants.Plant Cell Reports 13, 107–110.

FAO (2002) Production database, FAOSTAT, Food andAgriculture Organization of the United Nations.http://faostat.fao.org.

Gil Ortega, R., Palazon Espafiol, C. and Barriuso Vargas, J.(1990) Search for Verticillium dahliae resistance in Capsicumspp. Capsicum and Eggplant Newsletter 8–9, 60–61.

Gomez, R., Pardo, J.E., Alvarruiz, A., Gonzalez, M. andVaron, R. (2003) Color differences in peppers and paprikas.In: De, A.K. (ed.) Capsicum: the genus Capsicum. Taylor &Francis Inc., New York, pp. 236–247.

Harini, I. and Sita, G.L. (1993) Direct somatic embryogenesisand plant regeneration from immature embryos of chili(Capsicum annuum L.). Plant Science 89, 107–112.

Harpster, M.H., Brummell, D.A. and Dunsmuir, P. (2002)Suppression of a ripening-related endo-1,4-β-glucanase intransgenic pepper fruit does not prevent depolymerizationof cell wall polysaccharides during ripening. PlantMolecular Biology 58, 345–355.

Hibberd, A.M., Bassett, M.J. and Stall, R.E. (1987)Allelism tests of three dominant genes for hypersensitive

resistance to bacterial spot of pepper. Phytopathology 77,1304–1307.

Hibberd, A.M., Stall, R.E. and Subtamanya, R. (1983)Hypersensitive resistance in a Capsicum annuum genotypeto Xanthomonas campestris pv. vesicatoria races 1 and 2.Capsicum and Eggplant Newsletter 2, 121–122.

Hobbs, H.A., Valerde, R.A., Black, L.L., Pufresne, D.J.and Ariyaratne, I. (1998) Resistance of Capsicum spp.genotypes to pepper mottle potyvirus isolates from thewestern hemisphere. Capsicum and Eggplant Newsletter 17,46–49.

Hunziker, A.T. (2001) Genera Solanacearum: The Genera ofSolanaceae Illustrated, Arranged According to a New System.A.R.G. Gantner Verlog, Ruggell.

Jahn, M., Paran, I., Hoffmann, K., Radwanski, E.R.,Livingstone, K.D., Grube, R.C., Aftergoot, E., Lapidot,M. and Moyer, J. (2000) Genetic mapping of the Tsw locusfor resistance to the Tospovirus tomato spotted wilt virusin Capsicum spp. and its relationship to the Sw-5 genefor resistance to the same pathogen in tomato. MolecularPlant–Microbe Interactions 13, 673–682.

Jones, M.M. and Black, L.L. (1992) Sources of resistanceamong Capsicum spp. to Fusarium wilt of pepper. Capsicumand Eggplant Newsletter 11, 33–34.

Kang, B.C., Nahm, S.H., Huh, J.H., Yoo, H.S., Yu, J.W., Lee,M.H. and Kim, B.D. (2001) An interspecific (Capsicumannuum × C. chinense) F2 linkage map in pepper usingRFLP and AFLP markers. Theoretical and Applied Genetics102, 531–539.

Kang, B.C., Yeam, I., Frantz, J.D., Murphy, J.F. andJahn, M.M. (2005) The pvr1 locus in Capsicum en-codes a translation initiation factor eIF4E that inter-acts with Tobacco etch virus VPg. Plant Journal 42,392–405.

Khush, G.S. and Virmani, S.S. (1996) Haploids in plantbreeding. In: Jain, M., Sopory, S.K. and Veilleus, R.E. (eds.)In vitro Haploid Production in Higher Plants. Volume 1:Fundamental Aspects and Methods. Kluwer , Dordrecht, pp.11–34.

Kim, B.S. and Hartmann, R.W. (1985) Inheritance of a gene(Bs3) conferring hypersensitive resistance to Xanthomonascampestris pv. vesicatoria in pepper (Capsicum annuum).Plant Disease 69, 233–235.

Kim, D.H. and Kim, B.D. (2005) Development of SCARmarkers for early identification of cytoplasmic male sterilitygenotype in chili pepper (Capsicum annuum L.). Moleculesand Cells 20, 416–22.

Kim, D.H. and Kim, B.D. (2006) The organization ofmitochondrial atp6 gene region in male fertile and CMSlines of pepper (Capsicum annuum L.). Current Genetics 49,59–67.

Kim, J.Y., Jung, M., Kim, H.S., Lee, Y.H., Choi, S.H., Lim,Y.P., Min, B.W., Yang, S.G. and Harn, C.H. (2002) Anew selection system for pepper regeneration by mannose.Journal of Plant Biotechnology 4, 129–134.

Kim, S., Kim, S.R., An, C.S., Hong, Y.N. and Lee, K.W. (2001)Constitutive expression of rice MADS box gene using seedexplants in hot pepper (Capsicum annuum L.). Moleculesand Cells 12, 221–226.

Kim, S.J., Lee, S.J., Kim, B.D. and Paek, K.H. (1997) Satellite-RNA-mediated resistance to cucumber mosaic virus in

JWBK232-Kole k0603 July 21, 2008 20:8

PEPPERS 95

transgenic plants of hot pepper (Capsicum annuum cv.Golden Tower). Plant Cell Reports 16, 825–830.

Kumar, S., Rai, S. K., Banerjee, M.K. and Kalloo, G. (2001)Cytological mechanisms of male sterility in a nuclear-cytoplasmic line of chili pepper (Capsicum annuum L).Capsicum and Eggplant Newsletter 20, 64–67.

Lee, D.H. (2001) Studies on unstable fertility of CGMS(cytoplasmic-genic male sterility) in Capsicum annuum L.Ph.D. Thesis, Seoul National University, Republic of Korea.

Lee, J.M., Nahm, S.H., Kim, Y.M. and Kim, B.D. (2004a)Characterization and molecular genetic mapping ofmicrosatellite loci in pepper. Theoretical and AppliedGenetics 108, 619–627.

Lee, S.J., Kim, B.D. and Paek, K.H. (1993) In vitro plantregeneration and Agrobacterium-mediated transformationfrom cotyledon explants of hot pepper (Capsicum annuumcv. Golden Tower). Korean Journal of Plant Tissue Culture20, 289–294.

Lee, Y.H., Kim, H.S., Kim, J.Y., Jung, M., Park, Y.S., Lee,J.S., Choi, S.H., Her, N.H., Lee, J.H., Hyung, N.I., Lee,C.H., Yang, S.G. and Harn, C.H. (2004b) A new selectionmethod for pepper transformation: callus-mediated shootformation. Plant Cell Reports 23, 50–58.

Lefebvre, V., Daubeze, A.M., Rouppe Van DerVoort, J.,Peleman, J., Bardin, M. and Palloix, A. (1995) Constructionof an intra-specific integrated linkage map of pepper usingmolecular markers and doubled-haploid progenies. Genome38, 112–121.

Lefebvre, V. Daubeze, A.-M., Rouppe Van DerVoort, J.,Peleman, J., Bardin, M. and Palloix, A. (2003) QTLs forresistance to powdery mildew in pepper under natural andartificial infections. Theoretical and Applied Genetics 107,661–666.

Li, D., Zhao, K., Xie, B., Zhang, B. and Luo, K. (2003)Establishment of a highly efficient transformation systemfor pepper (Capsicum annuum L.). Plant Cell Reports 21,785–788.

Lippert, L.F., Smith, P.G. and Bergh, B.O. (1966) Cytogeneticsof vegetable crops. Garden pepper, Capsicum spp. BotanicalReview 32, 24–55.

Liu, L., Parrott, W.A., Hildebrand, D.F., Collins, G.B.and Williams, E.G. (1990) Agrobacterium induced gallformation in bell pepper (Capsicum annuum L.) andformation of shoot-like structures expressing introducedgenes. Plant Cell Reports 9, 360–364.

Livingstone, K.D., Lackney, V.K., Blauth, J.R., Van Wijk,R. and Jahn, M.K. (1999) Genome mapping in capsicumand the evolution of genome structure in the Solanaceae.Genetics 152, 1183–1202.

Manoharan, M., Sree Vidya, C.S. and Lakshmi Sita, G.(1998) Agrobacterium-mediated genetic transformation inhot chilli (Capsicum annuum L. var. Pusa jwala). PlantScience 131, 77–83.

Martins, T.R. and Barkman, T.J. (2005) Reconstruction ofSolanaceae phylogeny using the nuclear gene SAMT.Systemic Botany 30, 435–447.

Mihalka, V., Balazs, E. and Nagy, I. (2003) Binarytransformation systems based on “shooter” mutantsof Agrobacterium tumefaciens: a simple, efficient anduniversal gene transfer technology that permits marker geneelimination. Plant Cell Reports 21, 778–784.

Mihalka, V., Fari, M., Szasz, A., Balazs, E. and Nagy, I. (2000)Optimised protocols for efficient plant regeneration andgene transfer in pepper (Capsicum annuum L.). Journal ofPlant Biotechnology 2, 143–149.

Mityko, J., Andrasfalvy, A., Csillery, G. and Fari, M. (1995)Anther-culture response in different genotypes and F1hybrids of pepper (Capsicum annuum L.). Plant Breeding114, 78–80.

Mityko, J. and Fari, M. (1994) Problems and results of doubledhaploid plant production in pepper (Capsicum annuumL.) via anther- and microspore culture. Third InternationalSymposium on In Vitro Culture and Horticultural Breeding.Acta Horticulturae 281–288.

Mityko, J. and Juhasz, A.G. (2006) Improvement in thehaploid technique routinely used for breeding sweet andspice peppers in Hungary. Acta Agronomica Hungarica 54,203–219.

Moury, B., Pflieger, S., Blattes, A., Lefebvre, V. and Palloix,A. (2000) A CAPS marker to assist selection of tomatospotted wilt virus (TSWV) resistance in pepper. Genome 43,137–142.

Murashige, T. and Skoog, F. (1962) A revised medium forrapid growth and bioassays with tobacco tissue cultures.Physiological Plant 15 (3), 473–497.

Nahm, S.H. (2001) QTL mapping of Phytophthora root rotresistance in chili pepper (Capsicum annuum L.). Ph.D.Dissertation, Seoul National University, Republic of Korea.

Ogundiwin, E.A., Berke, T.F., Massoudi, M., Black, L.L.,Huestis, G., Choi, D., Lee, S. and Prince, J.P. (2005) Con-struction of 2 intraspecific linkage maps and identificationof resistance QTLs for Phytophthora capsici root-rot andfoliar-blight diseases of pepper (Capsicum annuum L.).Genome 48, 698–711.

Olmstead, R.G., Sweere, J.A., Spangler, R.E., Bohs, L. andPalmer, J.D. (1999) Phylogeny and provisional classificationof the Solanaceae based on chloroplast DNA. In: Nee, M.,Symon, D.E., Jessup, J.P. and Hawkes, J.G. (eds.) SolanaceaeIV: Advances in Biology and Utilization. Royal BotanicGardens, Kew, pp. 111–137.

Onus, A.N. and Pickersgill, B. (2004) Unilateral incompatibilityin Capsicum (Solanaceae): occurrence and taxonomicdistribution. Annals of Botany 94, 289–295.

Palevitch, D. and Craker, L.E. (1995) Nutritional and medicalimportance of red pepper (Capsicum spp.). Journal of Herbs,Spices and Medicinal Plants 3, 55–83.

Paran, I., Rouppe Van DerVoort, J., Lefebvre, V., Jahn,M.M., Landry, L., Van Schriek, M., Tanyolac, B.,Caranta, C., Ben Chaim, A., Livingstone, K., Palloix,A. and Peleman, J. (2004) An integrated genetic linkagemap of pepper (Capsicum spp.). Molecular Breeding 13,251–261.

Perera, K.D.A., Hartman, G.C. and Poulos, J.M. (1992)Development of screening technique for evaluation ofresistance to Pseudomonas solanacearum. Capsicum andEggplant Newsletter 10, 31–32.

Pernezny, K., Roberts, P.D., Murphy, J. and Goldberg, N.P.(2003) Compendium of Pepper Disease. APS Press.

Pickersgill, B. (1997) Genetic resources and breeding ofCapsicum spp. Euphytica 96, 129–133.

Pierre, M., Noel, L., Lahaye, T., Ballvora, A., Veuskens, J.,Ganal, M. and Bonas, U. (2000) High-resolution genetic

JWBK232-Kole k0603 July 21, 2008 20:8

96 TRANSGENIC VEGETABLE CROPS

mapping of the pepper resistance locus Bs3 governingrecognition of the Xanthomonas campestris pv vesicatoraAvrBs3 protein. Theoretical and Applied Genetics 101,255–263.

Pochard, E. (1977) Localization of genes in Capsicum annuumL. by trisomic analysis. Annales De Amelioration Des Plantes27, 255–266.

Pochard, E. (1982) A major gene with quantitative effect on twodifferent viruses: CMV and TMV. Capsicum and EggplantNewsletter 1, 54–56.

Pochard, E. and Daubeze, A.M. (1982) Comparison of threedifferent sources of resistance to Phytophthora capsici inCapsicum annuum. Capsicum and Eggplant Newsletter 1,59–61.

Pochard, E., Dumas De Valuix, R. and Florent, A.(1983a) Linkage between partial resistance to CMV andsusceptibility to TMV in the line “Perennial”: analysis onandrogenetic homozygous lines. Capsicum and EggplantNewsletter 2, 34–35.

Pochard, E., Gebre Selassie, K. and Marehoux, G. (1983b)Oligogenic resistance to potato virus Y pathotype 1-2 inthe line “perennial”. Capsicum and Eggplant Newsletter 2,137–138.

Poulos, J.M. (1994) Pepper breeding (Capsicum spp.):achievements, challenges and possibilities. Plant BreedingAbstracts 64, 143–155.

Pozueta-Romero, J., Houlne, G., Canas, L., Schantz, R. andChamarro, J. (2001) Enhanced regeneration of tomatoand pepper seedlings explants for Agrobacterium-mediatedtransformation. Plant Cell, Tissue and Organ Culture 67,173–180.

Pruthi, J.S. (2003) Chemistry and quality control of Capsicumand Capsicum products. In: De, A.K. (ed.) Capsicum: TheGenus Capsicum. Taylor & Francis, New York, pp. 25–70.

Qin, X. and Rotino, G.L. (1993) Anther culture of severalsweet and hot pepper genotypes. Capsicum and EggplantNewsletter 12, 59–62.

Rosello, S., Diez, M.J., Jorda, C. and Nuez, F. (1996) Screeningof Capsicum chacoense accessions for tomato spotted wiltvirus resistance by mechanical inoculation. Capsicum andEggplant Newsletter 16, 58–60.

Ruffel, S., Dussault, M.H., Palloix, A., Moury, B., Bendah-mane, A., Robaglia, C. and Caranta, C. (2002) A naturalrecessive resistance gene against Potato virus Y in peppercorresponds to the eukaryotic initiation factor 4E (eIF4E).Plant Journal 32, 1067–1075.

Shifriss, C. (1997) Male sterility in pepper (Capsicum annuumL.). Euphytica 93, 83–88.

Shifriss, C. and Cohen, S. (1990) Test for resistance to cucumbermosaic virus (CMV) in Capsicum annuum L. germplasm.Capsicum and Eggplant Newsletter 8–9, 52–53.

Shifriss, C. and Guri, A. (1979) Variation in stability ofcytoplasmic-genic male sterility in Capsicum annuum L.Journal of the American Society for Horticultural Science104, 94–96.

Shin, R., Han, J.H., Lee, G.J. and Peak, K.H. (2002a) Thepotential use of a viral coat protein gene as a transgenescreening marker and multiple virus resistance of pepperplants coexpressing coat proteins of cucumber mosaicvirus and tomato mosaic virus. Transgenic Research 11,215–219.

Shin, R., Park, J.M., An, J.M., and Paek, K.H. (2002b) Ectopicexpression of Tsi1 in transgenic hot pepper plants enhanceshost resistance to viral, bacterial, and oomycete pathogens.Molecular Plant–Microbe Interactions 15, 983–989.

Sopory, S.K. and Munshi, M. (1996) Anther culture. In: Jain,M., Sopory, S.K. and Veilleus, R.E. (eds.) In Vitro HaploidProduction in Higher Plants. Volume 1: Fundamental Aspectsand Methods. Kluwer, Dordrecht, pp. 145–176.

Stewart, C., Jr., Kang, B.C., Liu, K., Mazourek, M., Moore,S.L., Yoo, E.Y., Kim, B.D., Paran, I. and Jahn, M.M. (2005)The Pun1 gene for pungency in pepper encodes a putativeacyltransferase. Plant Journal 42, 675–688.

Stoner, A.K. (2004) Preservation and utilization of Capsicumgermplasm. [Book of Abstracts.] The 17th InternationalPepper Conference. Florida, USA. p. 29.

Supena, E.D.J., Suharsono, S., Jacobsen, E. and Custers, J.B.M.(2006) Successful development of a shed-microspore cultureprotocol for doubled haploid production in Indonesianhot pepper (Capsicum annuum L.). Plant Cell Reports25, 1–10.

Tai, T., Dahlbeck, D., Stall, R.E., Peleman, J. and Staskawicz,B.J. (1999) High-resolution genetic physical mapping ofthe region containing the Bs2 resistance gene of pepper.Theoretical and Applied Genetics 99, 1201–1206.

Tai, T.H. and Staskawicz, B.J. (2000) Construction of a yeastartificial chromosome library of pepper (Capsicum annuumL.) and identification of clones from the Bs2 resistance locus.Theoretical and Applied Genetics 100, 112–117.

Tanksley, S.D. (1984) Linkage relationships and chromosomallocations of enzyme-coding genes in pepper (Capsicumannuum L.). Chromosoma 89, 352–360.

Tewksbury, J.J., Manchego, C., Haak, D.C. and Levey, D.J.(2006) Where did the chili get its spice? Biogeographyof capsaicinoid production in ancestral wild chili species.Journal of Chemical Ecology 32, 547–564.

Tewksbury, J.J. and Nabhan, G.P. (2001) Seed dispersal.Directed deterrence by capsaicin in chilies. Nature 412,403–404.

Thabuis, A., Lefebvre, V., Bernard, G., Daubeze, A.M., Phaly,T., Pochard, E. and Palloix, A. (2004a) Phenotypic andmolecular evaluation of a recurrent selection program fora polygenic resistance to Phytophthora capsici in pepper.Theoretical and Applied Genetics 109, 342–351.

Thabuis, A., Palloix, A., Pflieger, S., Daubeze, A.-M., Caranta,C. and Lefebvre, V. (2003) Comparative mapping ofPhytophthora resistance loci in pepper germplasm: evidencefor conserved resistance loci across Solanaceae and for alarge genetic diversity. Theoretical and Applied Genetics 106,1473–1485.

Thabuis, A., Palloix, A., Pflieger, S., Daubeze, A.M., Caranta,C. and Lefebvre, V. (2004b) Marker-assisted introgressionof 4 Phytophthora capsici resistance QTL alleles into a bellpepper lines: validation of additive and epistatic effects.Molecular Breeding 14, 9–20.

Thompson, J.T. (1995) The Great Hot Sauce Book. Ten SpeedPress, Berkeley.

Thorup, T.A., Tanyolac, B., Livingstone, K.D., Popovsky, S.,Paran, I. and Jahn, M. (2000) Candidate gene analysis oforgan pigmentation loci in the Solanaceae. Proceedings ofthe National Academy of the Science in the USA 97, 11192–11197.

JWBK232-Kole k0603 July 21, 2008 20:8

PEPPERS 97

Tong N. and Bosland, P.W. (1999) Capsicum tovarii, a newmember of the Capsicum baccatum complex. Euphytica 109,71–77.

Valera-Montero, L.L. and Ochoa-Alejo, N. (1992) Novelapproach for chili pepper (Capsicum annuum L.) plantregeneration: shoot induction in rooted hypocotyls. PlantScience 84, 215–219.

Varshney, R.K., Graner, A. and Sorrells, M.E. (2005)Genomics-assisted breeding for crop improvement. Trendsin Plant Science 10, 621–630.

Voinnet, O. (2001) RNA silencing as a plant immune systemagainst viruses. Trends in Genetics 17, 449–459.

Voorrips, R.E., Finkers, R., Sanjaya, L. and Groenwold, R.(2004) QTL mapping of anthracnose (Colletotrichum spp.)resistance in a cross between Capsicum annuum and C.chinense. Theoretical and Applied Genetics 109, 1275–1282.

Walsh, B.M. and Hoot, S.B. (2001) Phylogenetic relationshipsof Capsicum (Solanaceae) using DNA sequences from twononcoding regions: the chloroplast atpB-rbcL spacer region

and nuclear waxy introns. International Journal of PlantSciences 162, 1409–1418.

Yano, M. and Sasaki, T. (1997) Genetic and moleculardissection of quantitative traits in rice. Plant MolecularBiology 35, 145–153.

Yazawa, S., Yoneda, H. and Hosokawa, M. (2002) A new stableand available cytoplasmic male sterile line of Capsicum.Capsicum and Eggplant Newsletter 21, 52–55.

Yeam, I., Kang, B.C., Lindeman, W., Frantz, J.D., Faber,N. and Jahn, M.M. (2005) Allele-specific CAPS markersbased on point mutations in resistance alleles at the pvr1locus encoding eIF4E in Capsicum. Theoretical and AppliedGenetics 112, 178–186.

Zhang, B.X., Huang, S.W., Yang, G.M. and Guo, J.Z. (2000)Two RAPD markers linked to major fertility restorer genein pepper. Euphytica 113, 155–161.

Zhu, Y., Ou-Yang, W., Zhang, Y. and Chen, Z. (1996)Transgenic sweet pepper plants form Agrobacteriummediated transformation. Plant Cell Reports 16, 71–75.