Agric.Re~,22(3/4): 163- 182, 2001

ROLE OF BIOTECHNOLOGY IN HYBRID SEED PRODUCTIONOF VEGETABLE CROPS - A REVIEW

Rajinder Kumar Dhall and D.S.Cheema

Department of Vegetable Crops,Punjab Agricultural University, Ludhiana - 141 004, India

ABSTRACTThe review emphasises the role of biotechnological tools like micropropagation, molecular

markers, anther culture, cybridization, induced male sterility and transgenics in the production ofspecific parental lines or hybrids in vegetables. Micropropagation can be used for maintenance ofmale sterile lines either controlled by recessive genes (tomato, muskmelon, chilli) or dominantgenes (cabbage); maintenance of self-incompatible lines in cole crops and maintenance of hybridsas such through tissue culture. Molecular markers can be used for assessment of genetic diversity,construction of linkage maps, varietal identiHcaton and marker assisted selection for traits ofinterest. Anther culture techniques can be utilized for development of self-incompatible lines incole vegetables and also to develop inbred lines in cross-pollinated vegetables. Cybridization isused for single step transfer of cytoplasmic male sterility from potato to tomato by protoplastfusion and generation of noval cybrids in tomato. Induction of male sterility by the use of 'Barnase­Barstar' systerm of hybrids seed production, is universally applicable for economic hybrid seedproduction especially in those vegetable crops where male sterility is not available (e.g. okw).Genetic transformation techniques can be used for trait specific transgenic parental lines forhybrids.

The development of hybrid varietiesis most important achievement of applicationof genetics in crop improvement. In the com­ing years biotechnological tools have to playvery crucial role in various ways including de­velopment of specific parental lines or hybridsin vegetables. Some of the potential thrust ar­eas where. biotechnology can have additionalaid to the conventional hybrid breeding are asfollowing.

MicropropagationMicropropagation can be used for

msms x(malester~

Msms x(male fer7'

50 lJi) Msms :(male fertile)

we have to remove 50% male fertile plants inthe filed. So it is very labour intensive and 50%of plants get wasted.

maintenance of male sterile lines either con­trolled by recessive genes (tomato. muskmelon.chilli) or dominant genes (cabbage): mainte­nancz of self-incompatible lines in cole crops:maintenance of hybrids as such through tissueculture (e.g. tomato: Negi and Joshi, 1996):clonal hybrid seed production and all male Flhybrid seed production in Asparagus.

a) Maintenance of male sterility dueto recessive genes (e.g. tomato, chilli, musk­melon):

MsMs(male fertile)

msms(male sterile)

50lYt) msms(male sterile)

b) Maintenance of male sterility dueto dominant genes (e.g. cabbage): Advance­ment of biotechnology has made it possible to

164 AGRICULTURAL REVIEWS

utilize dominant male sterility for F1 seed pro­duction through micropropagation, in thosevegetable crops where vegetative part is con­sumed.

Male sterile lines with homozygous

dominant genes can be multiplied by clonesand then crossed as female parent with malefertile sister line to obtain large amount of seedsof male sterile line.

MSMS (male sterile)

I Clonal propagation.. (for multiplication)

MSMS x msms(male steril~ (male fertile)

Msms (male sterile)(To be used in hybrid Seed production)

c) Maintenance of self-incompatiblelines in cole crops: To maintain inbred linesin cole crops, tissue culture is of great impor­tance, as maintenance of self-incompatible linesby bud pollination is expensive because eachflower bud has to be opened and pollinated byhand.

Anderson and Carstens (1977) usedtissue culture for maintenance of self-incom­patible lines in Broccoli. These self-incompat­ible lines produced through tissue culture areused for development of hybrids (single cross,sister line single cross and modified single cross)shown in Fig. 1.

Single Cross

Inbred A Blines si si

Crosspollination bybees in thegreen house

®

Abbreviations:si=self-incompatabilitysc=self compatability

Sister Line Single Cross Modified Single Cross

Isogenic Al A2 Bl B2 Isogenic Al A2 Binbred si si si si inbred si si silines lines

Cross Crosspollination by pullination bybees in the bees in thegreen house green house

AIA2 BIB2 AIA2si si

lCross

lJCross

pollination pollinationin the field in the field

®®

Fig. 1. Breeding schemes of Fl hybrids in Brassica oleracea L. (Reinert & Bajaj. 1977)

d) Maintenance of hybrid as such can be possible in any vegetable crop in whichthrough micropropagation: This technique hybrid is already developed. We can maintain

Vol. 22, No. 3/4, 2001 165

the hybrid as such through tissue culture but of tissue culture. Cytological studies revealedfirst we have to see that which explant is more normal chromosomal number and behaviourresponsive to hybrid production. which confirms the chromosomal homogene-

This technique can be commercially ity of hybrid plants raised in tissue culture. Wefeasible in those crops where wider spacing is ~ay also .conclude th~t the n.umber of seed-

. d' .all . f rb·t B hngs obtamed from thiS techmque are depen-require I.e. especi y m case 0 cucu IS. e- d t th b f'l1 'dsides it is a rapid method eliminating the need ben huponbt .e dnumt er

h°t aXI F~rYllor 'tShl e

f I I · II" . ranc eso ame a eac sage. ma y, I aso manua emascu atlon, po mation, mamte- b· b d th t t f It· I' t' .

f teen 0 serve a ra e 0 mu IP Ica IOn m

nance 0 res orer. h b 'd t t b thO t h' f dy noma 0 y IS ec mque was oun ap-Negi and Joshi (1996) selected a hy- proximately three fold within 27 days.

brid tomato after observations on its perfor- Micropropagation of triploid watermelonmance in hilly climatic conditions and it was through induction of multiple shoots from seed­then multiplied successfully through the tech- ling tips has been achieved. In-vitro propaga­nique of tissue culture (Fig. 2). It was also ob- tion of tetraploid and diploid parental lines ofserved that the desired characters of this het- watermelon to facilitate the large scale produc­erozygous tomato were retained intact in the tion of 3n watermelon seeds is being carriednewly formed seedling through the technique out.

Axillary or sidebranches

~Repeat sub culture

Shoot tips(Explant)

---I~. M.S. medium (1 ppm ----t~. Axillary buds orkinetin+0.5 ppm I.AA) side branches

~Parent explant•Sub culture to root Sub culture to M.S.+medium (M.S.+ 1 ppm 1 ppm kinetin+0.5 ppm

1AT5 ppmW",U~~p.l1

Seedling ready fortransplanting

Fig. 2. Growing hybrid tomato through tissue cultl\re (Negi & Joshi, 1996)

Molecular MarkerOver the past decades, a linkage map

has been constructed containing loci for mor­phological markers, resistance genes and mu­tants affecting physiological functions. Thoughthis map has served well in genetic studies, its

application in plant breeding programs is ratherlimited. Therefore more recently, particular at­tention is paid to the molecular markers.Among the molecular markers, isozyme mark­ers have proven the usefulness already in ge­netic studies and breeding programs of vari-

166 AGRICULTURAL REVIEWS

ous crops. In isozyme analysis, crude plant terization, varietal identification, sex determi­extracts are subjected to gel electrophoresis on nation, assessment of genetic diversity and dis­starch on polycrylamide gels. In spite of the tinguishing zygotic from nucellar seedlings.advances made in DNA-marker technology in Some examples of applications of isozymerecent years, isozyme markers have been de- markers in vegetable crops are given in Tableveloped in several vegetable crops for charac- 1.

Table 1. Isozyme markers used in vegetable crops

Crop

AmaranthAsparagusChinese Cabbage

CucumberRadish

Tomato

Objective

ClassificationSex determinationCultivars identification andSeed purity testingInheritance and linkageCultivars identification andSeed purity testingNematode resistance (Mi)

Reference

Okeno and Ayieicho, 1996Maestri et al. 1991Taiyoung et al. 1995

Meglic and Staub, 1996Taiyoung et al. 1995

Rick and Fobes, 1974

Nevertheless, isozyme analysis has itsinhereni disadvantages like limited number ofenzyme loci and developmental and seasonaldependent enzyme expression. With the ad­vent of molecular biology techniques, DNAbased I narkers replaced the enzyme markersin Slermplasrn identification and characteriza­hem as well as in gene tagging. All the modernmOlecular techniques belong to the basic ap­proaches: Southern hybridization or poly­merase chain reaction (PCR). The southern hy­bridization based approach involves restrictiondigestion of genomic DNA: size fractionationof di~Jested DNA through gel electrophoresis;transfer of fractionated DNA fragments onnylon membranes; and finally hybridization withlabeled probe sequence to visualize DNA poly­morphism. RFLP (Restriction Fragment LengthPolymorphism) is the most widely used tech­nique under this approach. PCR based ap­proach involves amplification of specif:e/ran­dorn re~Jions of genomic DNA using primersof different specificity's through polymerasechain reaction followed by visualization of am­plified products on gel after staining withethidium bromide or fluorescence. RAPD.AFLP and STRs are the widely used techniquesuncleI' this approach. These DNA markers(HFLP. R/\PD) have been used for (a) assess-

ment of genetic diversity, (b) construction oflinkage maps, (c) varietal identification and (d)marker assisted selection for traits of interest.

Assessment of Genetic Diversity:An important component for efficient and ef­fective management of plant genetic resourcesas well as their utilization is characterization ofthe germplasm. Such a characterization is es­sential not only for the identification of variousspecies but also to determine genetic related­ness among t!lcm. The information generatcdcould be used successfully in breeding programswherever possible. Vegetable crops in whichDNA markers have been developed for the as­sessment of genetic diversity and constructionof Iinkuge maps are Allium species (Berget al., 1996), Amaranlhus spp (Lanoue et al..1996), common beans (Noclari et al.. 1993),crucifers (James et al., 1993; Santos ct al.,1994; Camargo and Osborn, 1996: Chcunget aI., 1997: Phippen et al., 1997), eggplant(Karihaloo et al., 1995), melons (Baudraccoand Pitrat, 1996), onion (James and Michael.1995: Pich et al., 1996), pea (Timmermanet al., 1993: Hoey et al., 1996), peppcr (Princeet al., 1993), sugarbeet (Lorenz et aI., 1996:Morchen et al.. 1996), sweet potato (Paulet al.. 1997). tomato (Smulders (:t al.. 1997)and watermelon (Lee et aI., 1996).

Vol. 22, No. 3/4, 2001 167

Varietal Identification: Molecular and diseases during selection is fairly high. Inmarkers are useful in identification of genic male perennial crops the breeder has to wait a longsterility at seedling stage; identified male sterile period for evaluation of a phenotypic trait.plants can serve the purpose of ovule parent in Therefore availability of molecular markersFl seed production field. For this purpose, poly- (isozymes and DNA markers) which can be usedmerase chain reaction (PeR) based markers seem for indirect screening of progeny at seedlingfeasible particularly in vegetables having less plant stage or even at seed stage can increase effi­population per hectare especially in cucurbits but ciency of selection process. The MAS is beingbefore utilization cost effectiveness of such tech- increasingly adopted in breeding programsniques needs to be worked out. James and since this technique permits the breeder toMichael (1995) used DNA markers for varietal make earlier decisions about his selections whileidentification in onion. Molecular markers like examining fewer plants. An added advantageisozymes cou~d be used to purify the hybrid seeds in breeding for disease resistance is that this(assessment of the proportion of sibs in F1 hy- could be done in the absence of pathogen oncebrids of Brussels sprout by seed isozyme analy- marker information is available. This opens newsis; Wills et aI, 1979). possibilities to breeders since quarantine rcgu-

Marker Assisted Selection (MAS): lations re.stricting. pathogen existing is a majorThe success of modern plant breeding program obstacle m breedmg for dIsease resIstance.depends largely on the experience and the ef- Initially markers were being developedficiency of the breeder to select the ideal par- only for monogenic traits. Several disease re­ents and their offsprings during a prolonged sistance genes have been identified in vegetableperiod of painstaking evaluation. For e.g., over crops (Table 2). Martin et aI, (1991) were theone million seedlings are usually screened for first to demonstrate the feasibility of usingdeveloping a new commercially successful po- HAPD markers to tag the pta gene, which can­tato cultivar (Plaisted et aI., 1994). This tech- fers resistance to Pseudomonas syri17gae pl.!.nique of offspring selection ;s a highly subjec- tomato in tomato, Michelmore et aJ, (1991)live process, which largely depends on the ex- identifier! the first HAPD markers linked toperience of the breeder. The process becomes downy mildew resistance gene (DrnS/S) in let­more difficult when the interaction of two liv- tuce using a technique called bulked segreganting organisms is involved, Le. in case of selec- analysis, However, recently markers have beention for resistance to pests and diseases, In such developed for several quantitative traits that arecases possibilities of escape of plants from pests governed by multigenes.

Table 2. Identification of markers linked to disease resistance in Vegetable Crops (Maior ct a/.. 19')X)~~=~~~~~~_______ Pathogen Gene-Type l;f;~~I\-;;;------

TOlllilto MeloJdogyne lncognitaMi -RAPl5-------------Cladosporium fulvum Cf9 AFLPLevell/ula taurlca Lv RFLP.RAPDPhytophthora lnfestans Ph2 RFLPVertidllium dahliae Ve HAPDYellow leaf curl virus Tyl RELP

Potato Globodera rostochiensis HI RFLPPhytophthora iniestans R1 & R3 RFLP

Pea Pea seed borne mosaic virus Sbm1 RFLP,RAPDPea comlTlon mosaic virus Mo RFLPErvsiphe polygone Er RAPDfusarium oxysf-Jorum Fw USAT

Bean Uromyces appendiculatLls Up2 RAPDCommon bean mosaic virus 1 . RAPD

168 AGRICULTURAL REVIEWS

Most of the important agronomic char- ferent vegetable crops. Illustrative examples are·acters controlled by several genes are yield and given in Table 3. While the DNA markers haveyield components, plant height and days to unquestioned benefits for basic research, theirflowering. The first report on the development utility for plant breeding remains to be estab­of the markers for QTL (Quantitative Trait Loci) lished and verified in several aspects. The ma­in vegetable crops has been on development jor limitation in popularizing DNA markers inof RFLP markers for soluble solid content in this selection process, relate to DNA technol­tomato (Osborn etai. (1987). Molecular marker ogy (cost and capacity), plant biology (GxE,studies using bulked segregant analysis of near genes and traits) and relative research experi­isogenic lines (Martin et al., 1991) or recombi- ence (lack of empirical evidence for MAS andnant inbred lines (Miklas et al, 1993) have efficiency of current breeding methods).accelerated the mapping of many genes in dif-

Table 3. Illustrative examples of association of molecular markers with traits of interestin different vegetable crops

Crop Trait of interest Reference

CapsicumCrucifersLettuceMelonPea

Potato

Sugar beet

Tomato

Stunted growthFlowering time in Brassica oleraceaDowny mildew resistanceDisease resistanceGreen seed colourPea mosaic virus resistanceFusarium wilt resistancePowdery mildew resistancePowdery mildew resistancePotato virus Y resistanceResistance to Phytophthora infestansCyst nematode resistance HI geneMale sterilityNematode resistancePest resistance in L.pinnelliResistance against F. oxysporumLsp. LycopersiciSpotted wilt virus resistanceTm-llocusTm-2locusPseudomonas resistanceInsect resistanceNematode resistance Mi geneCladosporium fulvum resistanceResistance to powdery mildewJoint lessSoluble solid content

Inai etal. 1933Camargo and Osborn, 1996Paran and Michelmore, 1993Baudracco-arnas and Pitrat, 1996McCallus et al. 1997Timmerman et al. 1993Dirlewanger et al. 1994Dirlewanger et al. 1994Timmerman et al. 1994Brigneti et al. 1997Meksem et aI. 1995Pineda et al. 1993Lorenz et al. 1996Heller et al. 1996Mutschler et al. 1996Sarfatti et al. 1991

Chaque et al. 1996Ohmori et al. 1996Young and Tanksley, 1989Martin et al. 1991Nienhuis et al. 1987Kiein-Iankhorst et al. 1991Dickinson et al. 1993Chungwongse et al. 1994Wing et al. 1994Osborn et aI. 1987

Anther CultureAnther culture technique can be uti'­

lized for development of self-incompatible linesin cole vegetables and also to develop inbredlines in cross-pollinated vegetables. Anotherculture is also used for production of Haploidsand used for hybrid sorting.

a) Development of self-incompat­ible lines: The development of self-incompat­ible lines is the most crucial step in hybrid seedproduction of cole crops. The development ofself-incompatible breeding lines by conventionalmethod is costly and time consuming becauseinitial step of stabilizing inbred lines for seed

Vol. 22, No. 3/4, 2001 169

ture method' followed by chromosome dou­bling. This technique has been applied to colecrops (Fig. 3) vegetable crops amenable to hy­brid sorting are cole crops, potato and pepperbut relative success is more in potato than colecrops and pepper.

parents require many years of selfing.

Development of Self incompatiblelines through Biotechnology and HybridSorting Techniques: True breeding homozy­gous lines with all the attributes of inbreds canbe produced in several species by 'Anther Cul-

I Plant A I x.. IPlant B I

These true breeding homozygouslines are used for hybrid production

F1 hybrid

~.Microspores into culture

IHaploid plants I

lChrom=, doob'09

True breeding recombinant lines

+sexual crosses

IEvaluate for hybrid seed production I

IR lines with best of plants A & B I"'self-fertilize and field test

Cross anyVerify stability ... ..-, Determine genetic basis I

.. Segregators

INew parent line II

.. Self fertilize

IEvaluate performance of new OP line IFig. 3. Breeding strategy for the use of gametocional variation, using a hybrid sorting technique,

for the development of new plant varieties (Evans et aI., 1984)

b) Production of Haploids: There are .duced via pollen 'calli were haploids, diploids,two methods by which haploids are produced triploids, tetraploids and aneuploids. Other veg­from anthers Le. by direct method and by cal- etables in which anther culture is successfullyIus formation method. In potato, anther cul- used for production of haploids are Brusselsture is successful method for the production of sprout, chillies, tomato, brinjal, summer squashhaploids. Direct plantlets produced from pol- arld Chinese cabbage.len were almost haploids, whereas plantlets pro-

170 AGRICULTURAL REVIEWS

CybridizationCybrids or 'cytoplasmic hybrids' are

cells (;ontaining nucleus of one specie but cy~

topt3sm from both the parental species andthe process of development of hybrids is calledcybridization.

Use of cybridizationa) Generation of novel nuclear-cytoplasmiccombinations in several crops (Zelcer et al..] 9(0)b) Transfer of cytoplasmic traits (e.g. C.M.S..Melchers et al. 1(92).

a) Cybridization in tomato (Zelceret al, 19(0). The transfer of cytoplasmic ge­nornes from Lycopersicon and Solaunum spe­cies to L.esculentum was attempted by means

of the 'donar-recipient' method. A cytoplas­mic-albino large red cherry mutant (ALRC) wasutilized as the recipient genotype. Protoplastsfrom cytoplasm donar genotype were irradi­ated with X-rays, in order to induce chromo­some elimination and transfer of cytoplasmicgenophores upon fusion.

Putative cybrid colonies were isolatedand subsequently regenerated. Characterizationof A:""'RC x L.peruvianum cybrids revealedisozyme patterns identical to L.esculentum.Cybrid plants exhibit a recombinant mt-DNApattern, containing parental as well as novelfragments. The experimental rationale is de­picted in (fig. 4).

Cybridization in tomato:experimental design

IMock Fusion II _~-t_~/~~~~:.•.

Albino recipient X-ray .. •

parent(ALRq ~Irradiation '"'::~-~I~,'1 ,;--~

/ \ / \~/" . \.

\ . 0 ~ '-..-t:::sD 0 0 Q Doner parent'_ 0 0 (tl 0 (Lycopersicon or

~ (~~g / SOlanutspp., \ ---~--

Fig. 4. Experimental rationale for the use of cytoplasmic albino as recipient genotype in tomato cybridization.(Zelcer et al., 1990)

Vol. 22, No. 3/4, 2001 171

b) Transfer of Cytoplasmic Male male sterility using 'arg E' gene under the con­Sterility (CMS): Single step trafJsfer of CMS trol of tapetum specific promoter. The 'agr E'from potato to tomato by protoplast fusion gene product (N-acetyl-L-ornithine deacetylase)(Melchers et al., 1992). In this method, meso- of Escherichia coli which convert the non toxicphyll protoplast of lycopersicon esculentum product (N-ac-pt) to a cytotoxic herbicide,were treated with iodoacetamide to inactive phosphinotricine (Kriete et al., 1996).mitochondria; and protoplasts of Solanum a) Barnase Barstar System: This sys­tuberosum and Solanum acaule were irradiated tern was developed by Mariani et a1. in 1990with or X-rays to inactivate nuclei. Mixtures of and is considered as one of the finest examplesprotoplasts thus modified were trea~ed with of the application of genetic engineering andCa2+ and polyethylene glycol to obtam heter- recombinant DNA technology in applied plantologous fusion products. Among the fusion biology. In this system male sterility is createdproducts were some tomat? plants ~hat w~re by conferring the expression of cytotoxin geneindistinguishable from the ongmal cultlvars WIth to cells in developing anthers (Fig. 5). Chimericrespect to morphology, phySiolog~ ~nd chr~- genes used in the original study consisted ofmosome number (2n=24) but exhibited van- the following components:ous degrees of male sterility (MS); complete • A trapetal _specific promoter, TA 29, oflack or malformation of anthers, shrunkern tobaccopollen and normal-looking stainable pollen than • Either of the two different ribonucleasec,auld n~t germi~ate. The MS thus ind~ced in genes:fIVe cultlvars of different growth types, mclud- (i) RNase T1 from Aspergillus oryzaeing one of subspecies L. esculentum (ij) Barnase gene from Bacilluscerasi!orme,. was inh~rited maternally over se~- amyloliquefacienseral generatIOns and IS, therefore, c~oplas~ll- • A selectable marker gene.cally determined MS (CMS). AnalYSIS of mlto- .chondrial DNA revealed that the mitochondrial Both chimeric TA29-RNase along Withgenome of the CMS hybrids does not contain ~el~c~able mark~r (ba~) genes were int~od~ced

all elements of the mitochondrial DNA of ei- mdlVldually by TI-medIated transformation mtother parent but includes sequences of a recom- tobacco and oilseed rape plants.binational nature not present in either parent. TA29-RNase T1 and TA29-barnaseThe CMS cybrids, therefore, posSess a true transformant plants were identical to each otherhybrid mitochondrial genome, the advantage and to untransformed control plants, with re­of this method over others for generating MS sped to growth rate, height, morphology, veg­are as follow; (i) only one step is required; (ii) etation and floral organ systems, time of flow­the nuclear genotype of the cultivar is unaf- ering and floral coloration pattern. But in con­fected; (iii) the prospect that cytoplasmic de- trast to mature untransformed plants, anthertermination allows generation of 100% CMS of transformed plants failed to shed pollen.

progenies. Further study of transf~rmed plantsInduction of Male Sterility showed that the sterile anthers lacked a de-"Barnase-Barstar" system of hybrid tectable tapetum and had a collapsed pollen

seed production developed by Mariani et al. sac without visible microspores or pollen grains.(1990), is universally applicable for economic These were mainly due to expression of thehybrid seed production especially in those veg- chimeric RNase genes during anther develop­etable crops where male sterility is not avail- ment (Fig. 5). (Mariani etal., 1990).able (e.g. okra). In another study, induction of

172 AGRICULTURAL REVIEWS

Transgenic plant carrying barnase-bar genes..

1 MALE FERTILE: 1 MALE STERILEIIHerbicideI ~ ..

IElimination of male fertile plantsIIMale sterile plant~ V-- x ITransgenic plant carrying barstar genel

+

IExpression of barnase and bar genes in plants I...

Destruction of tapetumMALE STERILITY

x

+IUntransformed counter partI

y

ICo-expression of barnase/barstar genes I...

IBarnase/barstar protein complex I

Fig. 5 .. Hybrid seed production ushlg barnase/barstar system

The barnase gene of Bacillusamyloiiquefaciens contains the coding sequencefor the extracellular RNase . This gene has cor­responding inhibitor protein called barstar.Barstar is produced interacellulary and protectsthe bacteria from the lethal effects of barnaseby forming a stable complex with barnase inthe cytoplasm (Mariani et ai., 1992).

The barstar gene has been used toprevent the activity of barnase gene leading tomale fertility of plant tarrying both of the genesLe. the barstar gene can be used as a domi­nant gene for fertility restoration.

Mariani et ai. (1992) crossed betweenmale fertile plants containing a TA29-barstargene and male sterile plants carrying TA29­barnase gene. The progeny plants containedboth the genes and were male fertile. The fer­tility of F1 plants was due to co-expression ifTA29-barstar and TA29-barnase genes in an-

ther (Fig. 5).

It was indicated that TA29-barstargene is a dominant supressor of cytotoxicTA29-barnase gene activity and fertility resto­ration was due to the formation of tapetal-spe­cific barnase/barstar protein complex (Marianiet ai., 1992).

To maintain the male sterile line,transgenic plant carrying MS gene and select­able marker genes (barnase-bar) in hemizygouscondition is crossed with untransformed coun­terpart plant. This results in 1:1 segregationof male fertile but herbicide sensitive (as it doesnot carry transgenes) and male sterile (carryingboth the genes for male sterility and herbicideresistance). Male fertile plants can be eliminatedby spraying herbicide. To compensate elimi­nation of the susceptible plants, female parentis planted at twice the normal density (Fig. 5).

Because the TA29 gene promoter is

•

IMale Sterilityl ...4111----------

Vol. 22, No. 3/4, 2001 173

regulated correctly in a wide range of dicot and acetyl-L-phosphinotricine (N-ac-Pt, and aminomonocot crop plants (Mariani et aJ., 1992), acid analogue and herbicide derivative that car­the barnase/barstar system has the potential ries an acetyl group) into a herbicidefor universally applicable for hybrid seed pro- phosphinotricine by deacetylation mechanism.duction in a wide variety of crop plants. Tissue specific expression of the gene leads to

b) argEGene Product: Another strat- selective destruction of the tissue after exter­egywas developed by Kriete etal. (1996). They nal application of N-ac-L-Pt (Fig. 6). Theydeveloped an inducible system for destruction showed that pollen formation in transgenicof specific plant tissue (Anther/tapetum). This tobacco plants expressing the argEgene in thesystem is based on the argE gene product (N- tapetum is blocked by application of N-ac-L­acetyl-L-ornithine decetylase) of Esherichia coli, Pt.which converts the nontoxic compound N-

ITransgenic plant carrying argE g~ne I

./ ~No application of N-ac Pt Application of N-ac-Pt

MUltiPIiCatiotof parent~l female line Activatio!f argE gene atappropriate time in anthe~

(tapetum layer) ....

~N-acetyle-L-ornithine deacetylase•N-ac-Phosphinotricine ----------.~ Phosphinotricine

(Non-toxic) (Cytotoxic)

tDestruction of tapetal cells

Fig. 6. Induction of male sterility using argE gene under the control of a tapetum specific promoter.The argE gene product converts a non-toxic compound (N-ac-Pt) to a herbicide, phosphinotricine.

A chimeric gene consisting of tape­tum-specific TA29 promoter and the argE cod­ing region was construced. Nicotiana tabacumplants was transformed with a fusion of aboveconstruct showed no differences tountransformed control plants in growth, devel­opments, and time of flowering. However,transformed plants treated with N-ac-Pt, didnot contain any pollen, while untreated flowerof the same plant produced normal pollen. The

male sterile flowers such obtained, did not showany differences in their appearance comparedto untreated control.

The compound N-ac-Pt applied to theleaves, is distributed throughout the wholeplant, but mainly accumulated in the upper partof the plant. N-ac-Pt is non-phytotoxic up to adosage which is 100 times higher than thatrequires for induction of male sterility, more­over it remains unchanged for more than 14

174 AGRICULTURAL REVIEWS

weeks in unmodified plants (Droge et al.,1992). The compound N-ac-Pt may thereforeapplied in higher concentration in the earlygrowing season of the crop and should stH! bepresent in sufficient amount at the time tape­tum cells start to develop and express thedeacetylase.

Additionally, since the deacetylase isonly present in the tapetum cells of transgenicplants changes in the protein content of eat­able parts should not be expected. Moreoverthe substrate N-ac-Pt is only used by breeder,therefore the amount of N-ac-Pt which has tobe applied is relatively low and no undersirableresidue have to be expected in the crop.

TrangenicsGenetic transformation techniques can

be used for trait specific transgenic parentallines for hybrids. However, it is now relativelysimple to transform potato, tomato (Fig. 7) andmany other commercially important plants.Methods are rapidly being developed also for

genetic transformation of monocotyledonousspecies and we can expect many developmentin this area in the near further. The simplesttype of genetic engineering involves the pro­duction of a transgenic plant that expresses aforeign gene in many different organs and tis­sues throughout the life cycle. This is so calledconstitutive expression, is suitable for geneticengineering of single gene resistances to, forexample, insects, viruses or herbicides. Tran­scription promoters such as the CaMV 355promoter (from cauliflower, mosaic virus), arefrequently used to give this type of expressionpattern. However, it may sometimes be neces­sary to arrange for cell-specific or developmen­tal regulation of added genes to obtain expres­sion in, for example, the epidermis or in fruits,seeds, tubers or flowers. In addition to addingnew genes, it is also possible to reduce or evenabolish the effect of undersirable existing geneusing antisense RNA technology. Fisk andDandekar, (1993) reported some spps whichtransformed into complete transgenic.

Detailed sllldy ofphysiologlc,,1 andbiochemicalpropelt:e!. ofthe fruit

TRANSGENIC TOMATO

Study expression vf gene inripening fruit of genetic,Jllyengineered plant~

Evaluation of commercialadvantZages of geneticallyengineered plants

Investigate stability andinheritance of gene r 1,F2 generations

Gene is transferred fromAgrobacterium plasmid tothe plant cells which areallowed to grow

tMicropropagation ofgenetically engineeredplants t

l *' ~ J

l 1'" -J_1Infect plant cells

in culture withAgrobucterium

~

)S23\\\,-j/

THE PROCESS OF TRANSFERRING A GENE INTO A TOMATO PLANT

Mature plant ingreenhouse

~

Transfer plasmid toAgrobcldcriUTll

l'.~~~ :~~s~:l~thC /. engineered"\J gene

/,/ '-----~~.-.--

Bdctenal LchromosonlC

Take gcnctlCi:Jlly engineered!lCne and illSCli it into dbaclcriul plasmid

IS/,,/VI

Fig. 7. Transferring genes to tomato plants using Agrobacterium Ti plasmids. DNA sequences locatedwithin the T-DNA of the plasmid are transferred to the nucleus of the plants cells and integrated

in one or more of the chromosomes. Generally, a selectable marker gene for antibioticresistance is added with the DNA to be transferred. (Don Grierson, 1991)

Vol. 22, No. 3/4, 2001 175

a) Engineering for Herbicide Resis- step, or 2) adding a gene that directs the syn­tance: The criteria for an ideal herbicide are thesis of an enzyme that detoxifices the herbi­that it should have no toxicity towards humans, cide thus preventing it from inhibiting its tar­animals or soil organisms and should kill selec- get (Fig. 8). The former strategy has been usedtively weeds and not crop species. Unfortu- to produce plants tolerant to glyphosate,nately, many herbicides effectively kill crop suifonylureas, and imidazolinones by identify­plants and weeds. Genetic engineering has in~ gene~ for the appropriate en~yme in bac­enabled a new approach to this area by mak- t~r:a or hlgh~r J;>lants that are resIstant to her­ing it possible to confer on crop plants resis- blcldes, by taJ1on~g, ~here necessary, the ge~es

t t 'f' h b"d th t th b to be expressed m hIgher plants, and by usmgance a specl IC er ICI es a may en e" J.'

d t k'll t' d TI plasmlds to transfer the resIstant genesuse a I compe mg wee s. (Stalker, 1990), Examples of the second ap-The generation of transgenic plants proach include the transfer to higher plants of

resistant to these herbicides can be achieved the 'bar' gene from sterptomyceseither by 1) transferring a gene enabling plants hygroscopicus, conferring on transgenic plantsto synthesize an enzyme not inhibited by the the ability to acetylate ilnd thus detoxifyherbicide to catalyze the essential metabolic phosphinothricin (De Block et ai" 1987),

Transgenic plants resistant to herbicide

A

8

Herbicide target is an essential plant enzymeX+VHerbicide

~Add gene encoding an enzyme that detoxifies the herbICide

C

X---I"~ V Target reaction no longer inhibited

Add gene encoding an enzyme resistantto the herbicide that catalyses samereaction

Herbicide inhibits plant enzyme

Fig. 8. Strategies for generating herbicide-resistant transgenic plants by adding a gene

encoding a detOXifying enzyme (8) or a resistant enzyme (C). (Don Grierson, 1991)

b) Engineering for Virus Resistance:Three approaches have been used in attemptsto engineer virus resistance. The first methodaims at introducing genes encoding viral coatprotein to produce transgenic plants resistantto plant virus infection (Fig. 9). The viral coatprotein produced by the transgenic plants ap­pears to block a receptor in the plant cell thatis required for uncoating the viral nucleic acid,

or to interfere with viral replication or expres­sion of viral genes, Resistance is manifested b~1

delay in symptom development or escape forinfection altogether,

A second approach to engineering vi­rus resistance in plants has centered on satel­lite RNA's. It has been observed that expres­sion of satellite RNA's in transgenic plants canreduce infection with cucumber mosaic virus(Harrison et aJ., 1987),

176 AGRICULTURAL REVIEWS

Transgenic plant resistant to virusesPlant Cell

Nucleus

0 0

.0000 7 000 0 0

o

A

Virus

~QQQQOQC"o8~00<:00(0000

~

[~~ \) ~ )Replication

;;t Gene---+ expression

BVirus

:")OQQQOQC"o~~O°COOC'OOgO

Nucleus

~) [~~~iJ-00 0 0 0 0 0O"'Q;l0 ,0 0

0 0 000 Virus geneo0 ~ expressionCO)~ blocked

Plant Cell Expression VirusCoat Protein Gene

Fig. 9. Diagrammatic representation of the role of virus coat protein expressed in transgenic plantsin reducing symptoms of infection by virus particles. (Don Grierson, 1991)

and molecular mechanisms that confer transgenemediated resistance may lead to a second gen­eration viral genes that improve resistance be­yond current levels and extend its applicationsto still more viruses and crop plants. The ex­amples of successful transgenic vegetables againstviruses ani presented in Table 4.

A better understanding of the cellular

Table 4. Examples of successful transgenic vegetables against viruses (Major et aJ., 1998).

The third approach aims at usingantisense virus RNA to govern transgenic resis­tance. Following the successful field testing ofcoat protein mediated resistance (CPMR) againstTMV in tomato plants (Nelson et aJ., 1988) ad­ditional testing of a number of plant lines hasoccurred.

Purpose of genetic manipulation against Transgene product Origin of transgene Transformed plantToMV TomatoCMV CucumberPVX PotatoPVY PotatoToMV TomatoCMV TomatoTSWV TomatoTYLCV Tomato

Tomato mosaic virus (ToMV)Cucumber mosaic virusPotato virus X (PVX)Potato virus Y (PVY)Tomato mosaic virusCucumber mosaic virus (CMV)Tomato spotted wilt virus (TSWV)Tom<lto yellow leaf curl virus(TYLCV)Tomato yellow leaf curl virus(TYLCV)

Viral coat proteinViral coat proteinViral coat proteinViral coat proteinAntisense RNASatellite RNAN-geneCoat protein

Turncated version ofCI gene of TYLCV

TYLCV Tomato

Vol. 22, No. 3/4, 2001 177

C) Engineering for Bacterial Resis- (orinthine carbamoyl transferase) gene was iso­tance: Most of the crop plants are susceptible lated from Pseudomonas syringae pv.to bacterial diseases, which have been difficult physeolicola and introduced to bean to observeto control through plant breeding. Bactericides total resistance against the pathogen (Herrera­are not a complete solution, because bacteria Estrella and Simpson, 1995). This simple andquickly evolve resistance to them. Using mo- useful strategy for molecular breeding of dis­lecular biological tool, a novel approach for en- ease resistant plants by detoxification of patho­gineering resistance against bacteria was un- genic toxins has a great potential to be exploiteddertaken to isolate the tabtoxin resistant genes against bacterial and fungal diseases in veg­(ttr) from bacteria Pseudomonas syringae pv. etable crops. The examples of successfultabaci and subsequently to introduce into sus- transgenic vegetable crops against bacteria areceptible plants, for tnngenic resistance. In a presented in Table 5.similar way Phaseolotoxin- insensitive OCTase

Table 5. Example of successful transgenic vegetables against bacteria (Major et al., 1998)

Resistant against

Erwinia carotovaraE. carotovoraPseudomonas syringae pv.

. phaseolicolaWinia carotovora

E. carotovara

Transgene product

LysozymeTachyplesinPhaseolotoxininsensitive OCTasePectate lyaseGlucose oxidase

Origin of transgene

T4 bacteriophageHorseshoe crabPsyringae pv.phaseolicolaE.carotovora

Aspergillus niger

Transformed plant

PotatoPotatoBean

Potato

Potato

d) Engineering for Fungal Resis- e) Engineering for Insect Pest Re-tance: One of the major approaches for engi- sistance: Today insect resistant transgenes,neering plants resistant to fungal includes use whether of plant bacterial or other origin, canof chitinase gene of plant and/or bacterial ori- be introduced into plant to increase the levelgin. Chitinase enzyme degrades chitin-rich cell of insect resistance, a technology that has dra­walls of fungi and delays symptom develop- matically extended the scope of resistancement. An effective antifungal protein from genes available to plant breeders. The first re­other antifungal protein genes includes port of transgenic insect resistant plants wereglucanase, thionin, peroxidase etc. Recently, published only in 1987 but technological ad­from barely seeds called ribosome inactivating vancement has been swifted since then. Thisprotein (RIP) has been isolated. RIP acts by new technology is seen as an additional toolinhibiting protein synthesis via N-glycosidase for the control of crop pests and could offermodification of fungal 28 S rDNA. Transgenic certain advantages over conventional insecti­crop plants expressing RIP proteins are now cides, such as more effective targeting of in­being tested against many fungi. sects protected within plants, greater resilience

Scientists have found that deliberately to weather conditions, fast biodegradability,disarmed fungal pathogen can trigger the nec- reduced operator exposure to toxins and finan­essary response in susceptible (i.e. slow re- cial savings.sponding) plants before the virulent pathogen The insect resistance genes transferredarrives. This approach, the use of pathogen into plant to date mainly target the insect di­derived 'bio-control' agents to induce resistance·. gestive system. Most have been derived from ain susceptible plants has opened a new avenue" single species of bacteriym, or a range of h'ojl1erin disease control. plants, in addition some insect resistance genes

178 AGRICULTURAL REVIEWS

resistant transgenic vegetable crops are scantyas compared to other crops (Table 6).

from animals and other microorganisms havealso recently been introduced into crop plants.The number of genetically engineered insect

Table 6. Transgenic plants expressing insect resistance gene (Major et al., 1998).

Noval genes Origin of transgene Target insects Transformedplants

Genes from Microorganisms~MB &~~~~~~~

~MB &w~~~~km~

cry IN &cillus thuringiensiscry 3A Bacillus thuringiensiscry 9C Bacillus thuringiensisGenes from higher plants proteinase inhibitorsCpT/(cowpea trypsin inhibitor) Cowpea

Tomato proteinase inhibitor-I

Tomato proteinase inhibitor-IIAmylase inhibitorAI-Pv(Amylase inhibitor of the common bean)LectinsSnowdrop lectins (GNA)Wheat germ aggultinin (WGA)

Jacalin

Rice lectin

Tomato

Tomato

Common bean

LepidopteraLepidopteraLepidopteraColeopteraLepidoptera

ColeopteraLepidopteraLepidoptera

Lepidoptera

Coleoptera

LepidopteraColeopteraLepidopteraColeopteraLepidopteraColeopteraLepidoptera

TomatoTomatoCornEggplantCorn

Tomato

Tomato

Tomato

TomatoCorn

Corn

Corn

Lepidoptera

LepidopteraOthersTobacco anionic peroxidase

Genes of animal originBovine pancreatic trypsinInhibitor (BPT1)

Bt corn has been cleared for commercial release in the USA, Canada. Argentina, Japan and Europe.

Tomato

Lettuce

f) Engineering Parthenocarpic tophan to indolacetamide, which slowly con­Plants: In plants able to develop fruits in the verted to IAA) from Pseudomonas syringae pv.absence of fertilization (parthenocarpic fruits), savastanoi, under the control of the ovule-spe­seeds are absent, a feature that can increase cHic 'DefH9; gene from Antirrhinum majorfruit acceptance by consumers. Parthenocarpy showed parthenocarpic fruit development. Thehas been achieved by genetic engineering in achievement of parthenocarpic developmenteggplant. Transgenic eggplants expressing the in eggplant bearing fruit indicate the potential­coding region of the 'iaah' gene (codes for a ity of this tool to engineer parthenocarpic linestryptophan mono-oxygenase that converts tryp- in other commercial plants species (Table 7).

Table 7. Examples of sllccessful transgenic------_._---_.. R~sistance against Tran>gene

Tomato ral B geneBrinjal iaah gene

vegetables for parthenocarpic fruits (Major et aI., 1998)

Origin of transgene Transformed plant

A-rhizogens Tomatops.syringae Brinjalpv.savastanoi

Vol. 22, No. 3/4, 2001 179

g) Engineering for Post Harvest range of enzymes thought to be required toTraits: One of the major objectives over the bring about changes leading to ripe fruit. To­past few years has been to identify and clone mato being a climacteric fruit, shows a dra­plant genes and then modify the expression in matic increase in respiration at the onset oftransgenic plants. Polygalacturonase (PG) is an ripening, accompanied by increased synthesisenda-acting polygalacturonic acid hydrolase of phytoharmone ethylene, production ofthat is synthesized specifically during ripening which appears to be involved in the initiation,of some fruit (for example, tomato) and it de- modulation and co-ordination of expression ofgrades the pectin fraction of fruit cell walls, many of the ?enes req~ired for ripening pro­thus plays a role in softening. It has been shown cess. The ant1~~ense RN.A approach has beenthat expression of tomato 'pg' gene during rip- successfully ut.lh~ed to .brmg about r~duced et~­ening could be inhibited by antisense RNA ylene synthesIs 10 frUit by expressIOn of antl­gene (Sheehy et ai., 1988; Smith et aI., 1988). sense RNA for ACC oxidase (Hamilton et a/. ,

O e of th f· t t . d tid 1990) or ACC synthase RNA and to disruptn e Irs ransgenIc pro uc s re ease f" . b . hf 'a! uit' t' . USA' 'R S ' rUit pigmentation y down-regulation of t eor commerci c Iva Ion In· IS avr- avr .t t ·th· d ' h If I'f' Eth I phytoene synthase gene (Bird et aI., 1988).oma 0 WI Increase s e - 1 e . y ene Th Iff I .

d f . h'b't d b 93()1' .. e examp es 0 success u transgenIc veg­pro uc IOn was In I ley 0 10 npenIng etables for post harvest traits are presented intransgenic tomatoes expressing anti-sense Table 8. Wijbrandi and De both (1993) reportedgene. some vegetable crops for which genetic trans-

The 'ripening related' genes encode a formation has been accompalished.

Table 8. Examples of successful transgenic vegetable for post harvest traits (Major et aI., 1998).

Purpose of genetic Transgene product Origin of Transformed Ref.manipulation transgene plant

Hamilton et al. 1990Bird et al. 1991

Sheehy et al. 1988

TomatoTomato

TomatoTomato

Antisense ACC oxidasePhytoene synthase gene

Improved storage/ Antisense polygalacturonase Tomato .Tomatoshelf-lifeRipeningFruit pigmentation

Commercial releases of transgenicvegetable crops

Eleven transgenic in tomato mainlywith delayed fruit ripening & virus resistancetraits have been so for released for commer­cial cultivation in the world. These include 6 inUSA, 2 in China and one each in EuropeanUnion, Australia and Mexico, China was the

first country in the world to conduct field traitsof transgenic tobacco and tomato as early as1989. Of the tomato transgenic approved forcommercialization all have delayed fruit ripen­ing trait except one each developed in china(Cheng, 1998) and Australia (Millis, 1998),which have virus resistance (Table 9),

Table 9 . Transgenic vegetable crops released for commercialization

Country Crop Trait Company/Variety

1 234USA Tomato Delayed fruit soft8ning Calgene (1994)/

(PG antisense) 'Flavr Savr'Tomato Delayed fruit ripening DNAP (1995)/

(ACC synthase antisense) 'Endless Summer'Tomato Delayed fruit ripening Monsanto (1995)

(Contd.

180

1

China

MexicoAustraliaEuropean Union

AGRICULTURAL REVIEWS

2 3 4

(ACC synthase antisense)Tomato Altered ripening Agritope (1996)Tomato Delayed ripening Zeneca (1995)

(PGL antisense)Tomato Virus resistance (Bt) Monsanto (1997)

(Lepidoptera)Squash Virus resistance Asgrow (1995)

(WMV2/ZYMV) 'Freedom II'Tomato Virus resistance Public Sector (1994)Tomato Delayed ripening Huazhong Agric. Univ. (1997)Tomato Delayed ripening Calgene (1995)/'Flavr Savr'Tomato Insect resistance Public Sector (1997)Tomato Delayed ripening tomato Zeneca (1995)

Source: Medley (1998), Millis (1998) and Cheng (1998).

Somatic HybridizationThe results of the fusion of two so­

matic cells of different species, genera or fam­ily are considered in somatic hybridization. Thisis also known as protoplast fusion or parasexualhybridization. Somatic hybridization has a greatadvantage over sexual method for inter spe­cific and intergeneric hybridization. A numberof species and genera are incompatible to eachother due to barriers in sexual hybridization invegetable crops. The common presyngamicand post-syngamic sexual hybridization barri­ers are overcome by this technique.

The protoplasts from leaves, roots andcell cultures are used to produce somatic hy­brids. Several workers attempted the protoplastfusion technique to produce somatic hybrids.A classical example of protoplast fusion or so­matic hybridization is solanaceae, which hasamply demonstrated by Melchers (1982) andthe phenomenon of somatic hybrid production

is mainly limited to this family, as a large num­ber of somatic hybrids have been reported inthis family. The genera so/anum and/ycopersicon are not sexually crossable, butthere is the fusion of protoplasts of these gen­era, which is of great promise for breeding. Inthis case, Melchers (1982) experimentally fusedthe mesophyll protoplasts of /ycopersicon witha potato "chlorophyll deficient" mutant. Theplants were recovered and grew outside whengrafted onto tomato and produced partheno­carpic fruits. The recovered plants were hybridand exhibited variation in the chromosomenumbers. Morphologically, they were mal­formed, grew slowly, produced double flow­ers, had thickened roots, no stolon and weresterile. No fertile hybrid was reported among alarge number of plants produced by Melchers(1982). Somatic hybridization in vegetablecrops involving distant species and genera ispresented in Table 10.

Table 10. Experiment on Protoplast Fusion Involving Intergeneric and Interspecific Somatic Hybridization

Sr.No. Crop Reference

(Contd.G.max + Zea Mays5.

1 2 3

1. Solanum tuberosum + Lycopersicon esculentum Melchers (1982)2. Daucus carota + Aegopodium pedagararia Dudits et al.• (1979)3. Glycine max + Pisumsativum Kao eta!., (1974)4. Gmax+ Viciahajastana Kaoetal.,(1974);

Constabel etal., (1976)Kao eta!., (1974)

1

6.7.8.9.10.

2

50nigrum + S. tuberosum5. tuberosum + S. chacoense5otuberosum + S.brividensB.oleracea + B. compestrisL.esculentum + L.pennellii

Vol. 22, No 3/4, 2001

3

Binding et ai, (1982)Butenko ard Kuchko (1980)Barsbay ('tal., (1984)Schenck and Robbelen (1982)O'Cannell and Hamon 'J 985)

181

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