‘ _I —u I. 1;. ~ " . ‘1' ' ‘2‘: with“ . .tzis.‘ '. f. '. ' I In; It; If. ‘ "'>.T.I‘VII:','II,II.‘I.' .’; ‘: ‘L EI‘ " :n‘f. .‘é..l':‘§$1tyf I ' ”w -I' W‘ - I 'w-wmI {i ’ “KP-3: '- It; HII-I'h I I | “I I o’_‘\"’I. 'I) LI .I .4... ‘ y'1 I ”It? 1:11? ; ', '1' 1. I54 a - .- "lintifI'lL;‘I'l'I.‘ .‘TL Li‘IIiIs'; 5:12.: syn} . .j,[' I AIv'I; '7’“ I. 'I'IW i If “I‘m .. “I; 2._ ;, ‘I‘I I! v‘ .n . I I ‘l o.“ v - ’ n '7‘- n.— ”d . or I 'm'nj III-‘4, ~ H II 6'5 ‘I 3151‘.» r ~I~-,I. I .1. I. ' :5I ‘7”!qu ‘ZI‘I; m'I‘“ ‘, II 9 “ MIII'Z” ‘ ->.;';‘~M:,.' ) :I III} ”I”: ‘ _ .‘53‘- 5351.3 ‘ 4“ nle‘ '1’ . 2 ‘I‘l'l ”‘3“ “a 1.3..“ 3mm } .I iz"“'I-'I '. If?! II‘gNI: " I" . ‘2 II “III 41.1. .|IIUI1 \ ,1 I.” I] <5‘- . 44,." ‘ Isiah: I“ ‘1'" hi“ I my; “a.“ I5 I; 3321‘. 41' wH‘H {3'3}: (“Iii :lfii‘ " 5‘, II‘o‘v 3”,: It “:6 ..‘ In: If. ‘I‘ :1 (1‘1de . ~5. . I ”I. IzI‘ilfig y: .2 I1 I" ; “at?“ n I'- c .I N - l‘l‘I‘Irtfil. I I: jun. I'é' ‘I . . 'IEIBI- I . "I: n 'I; ‘I‘ M f 3131. . I' ’v. I A '. . . I '. ' L" 1‘ ;'.: ; : ."l‘ N‘:? ,5 H5 ‘. 21': ’v- !j-:“" [1523:5- ‘I'! I - I.“ 7%" "1'. a~ma»I- 3~ 133‘}; . ‘33,; I :5'5' In)”: . div"; 2.].1 Q' ~' .Inl ., -_ . .. -;_-a .t'q ' M >2 L:':;\ (-3", . '1!— 34:33; (1.. \ 3:. 4.» 0 Jun ._,.,.... ._. -. .—- III “XXIII" :55?- 53,4“ ' Psi; "Ifluazm “:8 31' Ir.’ . . Wt; I E .‘ . EII , '1 I-IW‘I‘IHQIT I.3 ‘tflIb‘ . I :j'I'I 1' i "" 41'“ . W} - ’E‘"‘E;t;:.u I"? I ‘II ”'3 ’- “I‘III'II "I““IIIttuIi'iI‘I'II‘”"’I“‘I " "I” If] I‘I; I'MI ,I I I .. I I; n I .' 3;. .. J”.- II i-I'.;I.It:“IIi;i- "“45: " fig II III I III“? i $125311.IL'iii-‘IIIZEIIII’IIie-h ”II 'i‘ I . * 3' . t III-3* - ' ...,!.t=.::s"-‘=I'«I~i 25,, “*INIIII'I W . II I". . Ian-{w 3*! 3* .. ’ Ie15:**I.'I‘:'I‘.i2IIIII'IIIII‘III'I'IIIIII‘III II 1‘ .‘ '°* I II IIIZWIIWW 233' III, - X '1‘...)in .l '1 . “I; ' "" ' "' , 1' "II ,1 I H ' - 4‘; -= IIIIi'Ii‘II 'IIIII‘I’I III II I; I ‘: 'IIII --" ‘5' 325;, III ‘ ,. I, ',I"II,II ' {In ‘3“ .IIII I It .,I I“ v IIIII Irui'III’III ‘ ‘ :;~;IJ;}1‘4R.AII, a.” ., h I; I III.“ [IIIWIIII 'IIIIII'IIII as III II II;- . III I M“ "I. “3' I 5' 'IIIIIIIILI {III *‘IIIIHI‘IIII'HI I I m: V" ' ,‘IE'II‘II ’III‘AI'I" III I L—«——— __...A Iflisfl ‘ THESSS )‘1/2 ’/ dlllilllll‘l’il lllllllll'llillillllllllflllillll ' 3 1293 01691 4297 This is to certify that the thesis entitled STUDY OF THE INHERITANCE OF THE CRY I A(c), PVY COAT PROTEIN AND NPT II TRANSGENES IN POTATO presented by Rafael Oduardo Mendez Ospino has been accepted towards fulfillment of the requirements for M. S. degree in Plant Breeding & Genetics/Crop & Soil Sciences 321%? Major professor Date Dr. A .1 HQ? 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Mlchlgan State Unlverslty PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. DATE DUE MTE DUE DATE DUE use comm“ STUDY OF THE INHERITANCE OF THE CRY I A(c), PVY COAT PROTEIN AND NPT II TRANSGENES IN POTATO By Rafael Oduardo Mendez Ospino A THESIS Sumitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER IN SCIENCES Department of Crop and Soil Sciences 1 997 ABSTRACT STUDY OF THE INHERITANCE OF THE Bt-CRY I A(c) AND PVY COAT PROTEIN TRANSGENES IN POTATO By Rafael Oduardo Mendez Ospino The production of transgenic plants has recently become a routine practice for many crop species. In potato breeding, genetic transformation plays an important role since traditional breeding is difficult due to the tetraploid nature of its genome. In this study, the inheritance patterns for Bt-Cry [A(c), PVY coat protein (PVY cp) and neomycin phosphotransferase II (NPT II) transgenes in crosses between FL1607-A30 (Bt clone) and ATL-PVYS (clone with the coat protein gene) and non-transgenic plants (cv. Atlantic and line MS702-80) were determined through PCR , Southern and kanamycin analyses. Southern analysis and the segregation pattern of the Bt gene suggested a single gene locus insert in clone FL1607-A30. In the case of the PVY cp gene and NPT 11, Southern analyses indicated the presence of 2 inserted copies of the gene, but the segregation rates suggested tight linkage between the inserted copies. The kanamycin assay corroborated the segregation pattern of the NPT II gene found in the PCR analysis. It indicated a stable integration and expression of the introduced NPT II gene, that behaved as a single dominant allele. F orty-seven individuals that have both the Bt-Cry [A(c) and PVY cp genes were yielded. However, further research is needed to determine if the resistance observed in the transgenic parents is expressed in this progeny. To my wife, Rosario E. Mendez Whose patience, support, understanding and love make me a rich man. To my son, Rafael Jesus, and my future son, Ricardo Jose (-1 moth old) I hope this achievement will give you enough inspiration to reach for high goals in your life iii ACKNOWLEGMENTS , I would like to express my deep gratitude to all those who helped me to complete my Master program: Dr. David Douches, my academic advisor, for his patience and understanding while I walked thought the long learning process called "The Graduate School" Dr. Kenneth Sink and Dr. Ray Hammerschmidt, for serving in my guidance committee and giving their support and encouragement to face all problems (technical, financial, visa status and so on) during my research. The potato group at MSU, Kelly Zarka, Peter Hudy, Kaz Jastrzebski, Kim Walters, Wenbi Lee, Chris Long Joseph Coombs and Dilson Bisognin, for their assistance and their friendship. Meylin Zimmerman, visitor student, for her help in the isolation of my samples. The wheat breeding program and the soy bean breeding program at MSU for allowing me to use their own equipment. Dr James Kelly, Joan Whalon, Richard Ward, who originally gave me the opportunity to study in Michigan State University. My mother, Gisela Ospino, and my mother-in-law, Rafaela de Valera, who encouragement to take this huge challenge. My family: my wife, Rosario, for her tireless support, understanding and sacrifice through the period of my study and my son, Rafael Jesus, for encouraging me to work hard ("go to study, Daddy"). iv Latin American Scholarship Program of American University (LASPAU), Venezuelan Agriculture department ("Misnisterio de Agricultura y Cria") Fundacion Gran Mariscal de Ayacucho (Fundayacucho), the office of International Scholars and Students, the dean office of the College of Agriculture and Natural Resources, the dean office of the Graduate School, for the financial support needed in this expensive project. The Venezuelan group in MSU: family Rodriguez-Lozada, Uzcategui, Pineda- Bennudez, Mindiola, Rodriguez-Mindiola, Sorzano and Juan Hernandez for their friendship. Last but not least, thank you Lord, who gave me enough strength in the most difficult time. TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... vii LIST OF FIGURE ....................................................................................................... viii LIST OF ABBREVIATIONS ....................................................................................... ix INTRODUCTION ....................................................................................................... 1 Potato tuber moth... .......................................................................................... 3 Bacillus thuringiensis ........................................................................................ 4 Bacillus thuringiensis (Cry) genes .................................................................. . 7 Cry I gene ........................................................................................................ . 8 Bacillus thuringiensis (Cry) toxin in plants ...................................................... 9 Resistance problems ......................................................................................... l4 Poty virus Y (PVY) .......................................................................................... 16 Coat protein mediated resistance ..................................................................... . l8 Mechanism of protection in coat protein transgenic plants .............................. 20 Constrains in the use of CP-mediated protection .............................................. 25 OBJECTIVES ............................................................................................................ . 27 MATERIALS AND METHODS ............................................................................... . 28 Plant materials ................................................................................................. . 28 Southern blot analysis .................................................................. 32 Isolation of DNA ............................................................................................. . 32 Quantification of DNA ..................................................................................... 34 Polymerase chain reaction (PCR) ...................................................................... 34 Kanamycin assay ................................................................................................ 37 Statistical analysis ............................................................................................ . 39 RESULTS ...................................................................................................................... 39 DISCUSSION ............................................................................................................... 53 BIBLIOGRAPHY .......................................................................................................... 58 vi LIST OF TABLES Table 1- Crosses used in the PCR analysis ............................................. 31 Table 2- Primers used in the PCR analysis ............................................. 35 Table 3- Test of Goodness of fit for binomials in the segregating population .................................................... 49 Table 4- Chi-Square analysis to test individual probabilities of Bt-Cry L4(c) and PVY cp genes .............................. 52 Table 5- Kanamycin assay results ........................................................ 55 vii LIST OF FIGURE Figure 1- Diagram of the plasmid pWBl39 Bt/Kan ............................................. 29 Figure 2- Diagram of the plasmid PVY cp gene/ PBI 121 ..................................... 30 Figure 3- PCR amplification products ........................................................... 38 Figure 4- Southem blot for Bt-Cry IA(c) digested with HindIII ..................................................... 40 Figure 5- Southern blot for ‘ Bt-Cry IA(c) digested with BamHI ...................................................... 41 Figure 6- Southem blot for PVY cp gene digested with BamHI and EcoRI ........................................ 42 Figure 7- Southern blot for PVY cp gene digested with BamHI ...................................................... 43 Figure 8- PCR amplification of NPT II gene ................................................... 45 Figure 9- PCR amplification of Bt-Cry IA(c) gene ............................................ 46 Figure 10- PCR amplification of PVY cp gene ................................................. 47 viii LIST OF ABBREVIATIONS Bt ............................................. Bacillus thuringiensis PVY .......................................... Potato virus Y PTM .......................................... Potato tuber moth PVY cp gene ............................... Potato virus Y coat protein gene NPT II ....................................... Neomycin phosphotransferase CaMV 35$ promoter ....................... Cauliflower mosaic virus 358 promoter PCR .......................................... Polymerase chain reaction RFLP ......................................... Restriction fragment length polymorphism DNA .......................................... Deoxyribonucleic acid RNA .......................................... Ribonucleic acid A, T, C, G .................................... Adenine, Thymine, Cytosine, Guanine FM ............................................. Fully modified PM ............................................. Partially modified WT ............................................. Wild Type TMV ........................................... Tomato mosaic virus AIMV .......................................... Alfalfa mosaic virus ArMV ........................................... Arabis mosaic virus CMV ............................................ Cucumber mosaic virus GCMV .......................................... Grapevine chrome mosaic virus PLRV ........................................... Potato leafroll virus PVS ............................................ Potato virus S ix PVX ............................................. Potato virus X PVYc ........................................... Potato virus Y, stipple streak strain PVYn ........................................... Potato virus Y, tobacco venial necrosis strain PVY0 ............................................. Potato virus Y, common strain RSV .............................................. Rice stripe virus SMV ............................................. Soybean mosaic virus TEV .............................................. Tobacco etch virus TSV ............................................. Tobacco streak virus TSWV ........................................... Tomato spotted wilt virus TYLCV .......................................... Tomato yellow leaf curl virus INTRODUCTION Disease resistant cr0p plants have long been produced by identifying resistant genotypes and then crossing such individuals exhibiting resistance with cultivars that possess the requisite array of agriculturally valuable traits (Grumet et al. 1993). However, in potato because of the tetraploid nature of the genome, classical approaches to breeding and selection for improved properties are especially difficult and laborious when compared with diploid crop species (MacKenzei et al. 1991). In addition, the desired resistance is not always available: it may exist in species that are not fertile with the crop, the gene for resistance may be tightly linked to undesirable traits, or the resistance may be polygenic and thus difficult to transfer (Grumet et al. 1993). Genetic engineering techniques may be used to overcome some of the factors limiting traditional plant breeding such as fertility barriers among species. This means that we do not have only genes transferred between different plant families, but among individuals of different kingdoms (Grumet et al. 1993). The application of biotechnology may serve as a key element in the concept of integrated pest management, enhancing the trend toward environmental-compatible pest and disease control strategies (Williams et al. 1992). The high incidence of diseases and pests on crop plants as compared to wild plant species is most likely due to uninterrupted monoculture production in combination with a 2 selection for characteristics such as yield rather than for disease and pest resistance in the breeding process. To control these diseases and pests, the industry has developed many chemical products (van den Elzen et al. 1989). However, there is pressure by consumers to reduce pesticide use in all crop production and to minimize the amount of pesticide control. This force drives the development of alternative technologies, including genetic engineering (Martin et al. 1994). The production of transgenic plants has recently become a routine practice for many. species, and an array of genes for resistance is now available. Since crops are often damaged by more than a single pest or disease, lines expressing multiple resistance genes are needed for improved crop protection (Liang et al. 1994). Some major constraints to commercial potato production are insect pests and viruses. Among the insect pests, Phthorimaea opercullela (Zeller) or potato tuber moth (PTM) is an important pest both in the field and in storage in tropical and sub-tropical regions (Trivedi et al. 1992). In addition, virus problems in potato are well recognized, and virus control by chemical products has been ineffective. Viruses make the seed certification scheme expensive in potato as in vegetatively propagated crops (van den Elzen et al. 1989). Fortunately, some genes are available to control these problems such as the Cry IA(c) gene that codes for a lepidoteran specific Bt protein toxin, and PVY coat protein gene that confers resistance to homologous and heterologous viruses (van den Elzen et al. 1989, Barton and Miller 1993, Liang et al. 1994). The practical application of plant transformation technology to potato improvement can have two directions. First, transgenes can be introduced directly into 3 suitable breeding lines and these lines can be used to generate new varieties by standard crossing and selection procedures. Second, transgenes can be introduced directly into existing cultivars to confer specific genetic changes. The second approach has the advantage of producing transgenic lines within established backgrounds (Belknap et al. 1994). POTATO TUBER MOTH, Phthorimaea opercullela (Zeller) The potato tuber moth (PTM), Phthorimaea opercullela (Zeller) (Lepidoptera:Gelechiidae) is an oligophagous insect widely distributed in almost all tropical and subtropical regions. The species is a serious pest of potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.), and sporadically strikes tomato (Lycopersicum esculentum Mill) (Das et al. 1994). Potato tuber moth is a serious pest of potato under both field and storage conditions. The infestations start in the field on the leaves and tubers, and infested tubers are carried to storage and act as the initial source of infestation (Das et al. 1994). Feeding damage caused by this pest drastically reduces the quality and market value of tubers and creates sites for infection by pathogens that cause complete destruction of the tubers (Das et a1. 1994). Losses up to 50% and more have been recorded in commercial crops in Peru, storage losses may be up to 100% in India and Philippines, 90% in Kenya, 86 % in Tunisia, Algeria and Turkey (Das et al. 1995). The larvae of PTM mine in the mesophyll layer of the leaves and shoots, and additionally potato tubers. Tunneling damage in tubers caused by PTM is a severe problem, especially in traditional, non-refrigerated storage systems (Jansens et al. 1995). 4 Under field conditions, the pest deposits 3 high proportion of eggs in the soil adjacent to host plants rather than directly on the plants themselves and some eggs are laid under the skin of partially exposed tubers. Post-harvest infestation also occurs if the tubers are left unprotected in the field. The larvae hatch under the tuber skin and feed inside until the 5‘“ instar when they exit through holes to pupate in the soil. The bored tubers are then contaminated with fungi and bacteria, leading to rotting and destruction (Siddig 1988, Fenemore 1988). Until recently, the use of insecticides was a widely accepted practice to control PTM; however, the use of insecticides (especially for stored table potatoes) is a potential health risk, resistance to commonly used insecticides is likely to develop, and chemical control in storage has been inefficient (Siddig 1988, Das et al. 1995 and Kroschel et al. 1996). Bacillus tlruringiensis Commercial formulations of Bacillus thuringiensis (Bt) have demonstrated insecticidal effect against PTM in potato storage (Kroschel et al. 1996). However, Bt sprays have limited field efficacy, since they may be washed off the leaves by rain and are most effective on young larvae. Therefore, the topically applied Bt protein must be synchronized with good weather conditions and susceptible larval stages. These drawbacks of Bt sprays make the introduction of Bt genes into plants a better use of this biological pest control (Stewart et al. 1996, Cheng et al. 1992). Bt is a gram-positive, free-living soil bacterium that accumulates high levels of insecticidal proteins during sporulation. The bacteria have been utilized for several 5 decades in insecticidal formulations, which have shown high specificity toward certain insect pests. Such high specificity of Bt for target pests presents a potential ecological advantage over many chemical insecticides: Bt toxins are not toxic to beneficial insects, plants and vertebrates, and chemical insecticide may be persistent well afier application (Barton and Miller 1993, Salamitou et a1. 1996, Stewart et al. 1996, Adang et al. 1993). Reports on bacteria as causative agents of insect disease date back to the 19008. Their use as control agent began in the 1930S. For example, Bacillus popilliae has been used to control Japanese beetle grubs. Bt was originally isolated from infected silkworm colonies in 1901 in Japan and was initially named B. sotto (B. thuringiensis subsp. sorta, in the current nomenclature). The identification of intracellular proteinaceous inclusions as a major source of the insecticidal agents produced by these bacteria dates back to the 1940's (Aronson 1994, Whitely and Schnepf 1986). The introduction of Bt as a biological pesticide started in the early 1960's (Baum et al. 1994). The Bt group produces protoxins designated delta (5) endotoxins and in some cases secrete a or B exotoxins (Aronson 1994). These designations refer to the type of toxic activity involved. While a or B exotoxins are toxic either to a variety of insect orders or to many cell types, the crystal protein, or (8) endotoxins, have a more limited host range (Whitely and Schenepf 1986). The crystalline proteins are accumulated in the cytoplasm of the cells during the stationary phase or sporulation to form crystal inclusions which can account for 25% of the dry weight of the cell, and B. thuringiensis can synthesize one or more crystal inclusions (Salamitou et al. 1996, Bora et al. 1994). At the end of the sponrlation process, crystals and spores are liberated by lysis of the cell (Salamitou et al. 1996). These crystals are composed of proteins called 8-endotoxins or 6 Cry proteins. A single crystal may contain one million protein subunits held together by interchain disulfide bonds, and thus the cleavage of those bonds is a critical step in crystal solubilization. Many Bacillus thuringiensis strains have multiple Cry genes; however, there are some exceptions such as B. thuringiensis HD-73, which produces crystals from only a single CryIA (c) gene (Du et al. 1996, and Herrera et al. 1994). In addition, some strains show non-toxic effects toward known susceptible insects, such as the isolated LBIT-113 which has a flat, square parasporal crystal composed of two proteins with sizes of 88 and 54 kDa (Lopez-Meza et a1. 1996). Cry proteins are synthesized as protoxins, which must be solubilized and activated through proteolytical cleavage by trypsin-like proteases in the alkaline environment of the insect gut. The activated toxin binds to receptors, which are located in the apical microvilli of susceptible larval midgut epithelial cells. After binding, the toxin inserts itself into plasma membrane and forms a pore or lesion by a process that is not yet fully understood. Pore formation allows net uptake or leakage of ions and water leading to osmotic lysis of the epithelial target cells, cessation of feeding and finally insect death (Ceron et al. 1995, Adang et al. 1993, Orduz et al. 1994, Meza et al. 1996, de Maagd et al. 1996, Dean et al. 1996). The specificity of a particular toxin depends on the efficiency of each of the steps mentioned above. Hence, the ability to solubilize and activate protoxins has been shown to influence toxicity. The activity of the protease-activated toxin is dependent on the type of gut protease involved and the present of a specific receptor in the brush border membrane (de Maagd et al. 1996). Bacillus thuringiensis (CR)? genes . Many thousands of Bacillus thuringiensis strains have been isolated, and they exhibit a great diversity in the spectrum of their toxicities including toxic activity against lepidopteran, dipteran, coleopteran insects and against nematode and aphids (Lopez- Meza et al. 1996). To date, nearly 100 distinct crystal protein gene sequences have been published either in the general scientific literature or in patent applications (Du et al. 1996, Lambert et al. 1996, Baum et al. 1996). To search for and to characterize novel Cry genes is a worldwide project (Ceron et al. 1995). These Cry genes have been classified into six different groups based on their amino acid sequence similarities and ranges of specificities (Ceron et al. 1995, Barton and Miller 1993, Du et al. 1996). They are: Cry 1, lepidopteran-specific; Cry H, lepidopteran— and dipteran-specific; Cry III, coleopteran-specific; Cry IV, dipteran- specific; Cry V, coleopteran and lepidopteran-specific; and Cry VI, specific against nematodes, but the origin of its protein is different from those of the other Cry proteins (Ceron et al. 1995, Kenneth et al. 1993). Each class of Cry genes is grouped into subclasses (A, B, C...; and a, b, c...) according to amino acid sequence (Agaisse and Lereclus 1995, Kuo et al. 1996). The Cry genes code for proteins with molecular masses from approximately 50 to , 140 kDa, which release insecticidal crystal proteins (delta-endotoxins) of 27 to 140 kDa in the larval midgut (Grochulski et al. 1995, Blizzard et al. 1991). Cry I, Cry III, Cry IV proteins are closely related and their activated toxins share five segments of significant homology, and the Cry 11 and Cry V proteins are more distantly related to the other Cry proteins and homology in these five regions is weaker, while Cry VI proteins display no 8 detectable homology sequences at all. In spite of the differences in insecticidal spectra and divergence in amino acid sequences among the Cry proteins, their mode of action appears to be very similar (Grochulski et al. 1995). To identify the different Cry gene classes, several methods have been employed. One of them is the multiplex PCR Cry gene typing method (Ceron et al. 1995). However, this method is not sharpen to identify novel Cry type genes. So far, a new method termed the PCR-RFLP typing system has demonstrated to be efficient in detecting both known and novel Cry genes. In this method, two pairs of universal oligonucleotide primers, which have the most highly conserved nucleotide sequences, based upon published gene sequences, were designed for PCR amplification. Following PCR amplification, restriction fragment length polymorphism (RF LP) analysis was employed to identify the origin of the Cry-type genes (Lopez-Meza et a1 1996). CRY I GENE Cry I genes have lepidopteran insecticidal activity and are the most thoroughly studied of all 6-endotoxin genes. The Cry I genes encode proteinaceous protoxins of 130- 140 kDa, which are assembled as bipyrarnidal crystal inclusions within the bacterial spore. The proteins of these crystals would disperse when exposed to alkaline medium (pH 9-12), as found in the gut of lepidopteran insects. Proteolytic activity from various sources, including both bacterial residue and insecticidal gut juices, is capable of degrading the high molecular weight protoxins by undergoing specific trypsin cleavage to smaller, insecticidally active peptides or the N-terminal toxin of 65-70 kDa (Barton and Miller 1993, Regev et al. 1996). 9 The resulting toxin comprises three structural domains. The N-terminal domain disrupts the midgut epithelial cell membrane by forming ion pores. The immediate terminus is contained within amino acids 1-29, and the carboxil terminus is contained approximately within amino acids 603-1179 on Cry I gene products. The two latter domains are involved in specific folding and interaction with membrane receptors, which is a prerequisite for pore formation and dictates toxin specificity (Barton and Miller 1993, Regev et al 1996). ‘ The individual Cry I genes generally display broad toxicity toward a number of different lepidopteran species. In spite of the substantial relatedness of the Cry I genes, variations in the Cry I protein sequences contribute to differing levels of toxic activity against susceptible lepidopteran insects (Kenneth and Miller 1993, Von Tersch et al. 1991). Six different genes coding for lepidopteran-specific protein have been identified on the basis of DNA sequence. They are Cry IA(a), Cry IA(b), Cry IA(c), Cry [8, Cry IC and Cry ID. The first three genes are closely related, the remaining three genes are not as widely distributed (Blizzard et al. 1991 ). Bacillus tlruringiensis (Cry) Toxin in Plants Formulations of B. thuringiensis crystals and spores have been used as biopesticides for over 20 years, but their instability in the environment and relatively high cost incurred by multiple applications have limited grower acceptance (Barton and Miller 1993, Herrera et al. 1994, Li et al.1995). On the other hand, major insect pests are developing resistance to most classes of chemical insecticides and these insecticidal chemicals cause serious environment problems (Li et al. 1995). These facts make the 10 production of protein with insecticidal activity by the plant itself an attractive alternative for protection (Barton and Miller 1993, Herrera et al. 1994, Li et al. 1995, Wunn et al. 1996). Recently, some researchers have introduced the crystal protein genes (Cry genes) into different plants such as tobacco (Nicotiana tabacum L.), tomato (Lycopersicum esculentum Mill.), cotton (Gossypium hirsutum L.), potato (Solanum tuberosum L.), rice (Oryza sativa L.), maize (Zea mays L.), American cranberry (Vacciniurn macrocarpon Ait), plants of rutabaga (Brassica napobrassica L.), apple ( Malus pumila cv. Greensleeves), strawberry (F ragaria spp. ), canola (Brassica napus L.), amelanchier (Arnelanchier laevis), populus (Populus spp.), walnut (Juglans regia L.), cabbage (Brassica oleracea var. capitata), white clover (Trifolium spp), common bean (Phaseolus vulgaris L.), Chrysanthemum (Chrysanthemum morifoliurn), soybean (Glycine max [L.]Merr.), alfalfa (Medicago sativa L.) eggplant (Solanurn melongena L.), rapeseed in order to produce genetically engineered insect resistance plants (Li et al. 1995, Serres et al. 1993, James et al. 1993, Sims et al. 1996, Iannocone et al. 1995, Dolgov et al. 1995, Kleiner et al. 1995, Strizhov et al. 1996, Parrot et al. 1994, Barton and Miller 1993, Herrera et al. 1994, Stewart et al. 1995, Von Tersch et al.1991, Van Aarssen et al. 1995). A common problem encountered by most research groups has been the difficulty in achieving a high level of expression of these prokaryotic Cry genes in higher plants (Barton and Miller 1993). Without exception, typically, chimeric Cry plant genes have about 1000-fold lower levels of protein than chimeric bialophos resistance (BAR), neomycin phosphotransferase II (N PT 11), chloramphenicol acetyltransferase (CAT), or glucoronidase (GUS) genes even being flanked by the same plant regulatory expression ll signal (Van Aarssen et al. 1995). Although, the underlying mechanism restricting Cry expression is still not understood, it is believed that plants do not have many of the tRN As required by bacterial gene codons, and also the low level of Cry gene expression in plants is due to a higher A+T (adenine-thymine) content in native Cry genes than in plants. These multiple A+T rich motifs resembling plant introns and ATTTA sequence (polyadenylation signal sequence) have been shown to destabilize mRNA in other systems. In addition, the presence of transcription predetermination signals in the B. thuringiensis system and the codon usage of native Cry-coding sequence, that is very unlike the preferred plant codon usage or rarely used in plants, are among the factors that restrict Cry gene expression (Steward et al. 1996, Van Aarssen et al. 1995, Regev et al. 1996, Perlak et a1. 1991). In addition, the problem appeared to be a result of either inefficiency in the synthesis of gene products, or rapid degradation of either the B. rhuringiensis mRNA or protein following synthesis (Barton and Miller 1993). However, Aarssen et al. (1995) provided some evidences that the level of cytoplasmic import rather than mRNA instability is responsible for the inefficient cytoplasmic accumulation of Bt gene mRNA. He suggested that the increase in mRN A level in transgenic tobacco plants expressing Cry IA(b) with a replacement of aberrant native codons by preferred codons and increase in the G-C content, is not necessarily linked to an improved efficient and/or mRN A stability, but could be due to improved nuclear processing and/or transport. The low expression of wild Cry gene in transgenic plants could be sufficient for an insecticidal effect to susceptible insects, but for adequate control of less susceptible insects in the field several approaches have been demonstrated to increase the expression 12 of these prokaryotic genes (Wunn et al. 1996, Blizzard et al 1991). Those strategies include the truncated form of the gene, modification of the coding sequence (codon- optirnized synthetic Bt genes), and the use of strong promoters (Barton and Miller 1993, Steward et al. 1996, Wunn et al. 1991, Blizzard et al. 1991, Wong et al 1992, Carozzi et al. 1992). Schnepf and Whiteley (1986) reported that a truncated form of the Cry gene, encoding only the amino-terminal portion of the protoxin, showed insecticidal activity. Transgenic tobacco and tomato plants carrying a truncated (1.8 kb) Cry I gene, expressing the 60 kDa toxin, produced slightly higher levels of Bt protein than transgenic tobacco carrying the full length (3.5kb) Cry I gene (Wong et al. 1992, Fishhoff et al. 1987). Transgenic tobacco carrying a truncated form of the Cry gene coding 645 amino acids showed levels of toxins generally at 0.001 % of the total cell protein or lower. The range. of expression of these genes was typical of chimeric gene expression, with variation in levels of expression due to insertion into differing chromosomal locations (Barton and Miller 1993). The truncated version of Bt genes has a significant toxicity to insect; however, the levels of expression of these genes were too low to provide practical levels of resistance toward less sensitive insect pests such as Spodoptera exigua or Heliothis virescens (Barton and Miller 1993, Corozzi et al 1992). Recognizing the high content of A+T in native Bt-Cry gene compared with typical plant genes, and the presence of ATI‘TA as a destabilizing element of mRNA, Perlak et al. (1991) proposed two approaches to enhance the expression of Bt genes (Cry IA(b) and Cry IA(c)) in transgenic plants of tobacco and tomato through modification of the coding sequence. These two approaches selectively removed DNA sequences from 13 the truncated wild Cry genes by site-directed mutagenesis. In the first, called partially modified synthetic gene (PM), it was done without changing the amino acid sequence. The other strategy, called fully modified synthetic gene (FM), was a more rigorous application of the principle used, taking codon usage in plants, potential secondary ' structure of mRNAs, and potential regulatory sequences under consideration. Both FM genes and PM genes were synthetic versions of the Bt-Cry I genes. The PM gene differs from a truncated wild type (WT) gene by 3% of the nucleotide, but this change was enough to increase more than 10-fold in the insecticidal protein levels in tobacco compared with the truncated WT gene. The FM genes encode proteins nearly identical in amino acid sequence to that of WT gene, eliminating AT'ITA sequences, and almost all potential polyadenylylation sequences. In the transgenic plant, the FM genes appeared to be expressed 5- to 10 fold higher than the PM genes and up to 100-fold higher than the WT gene. The higher level of insect control protein in these transgenic plants was directly correlated to increased insecticidal activity (Perlak et al. 1991). Similar results were obtained using modified Cry III A (Perlak et al. 1993). The other strategy to increase the expression of Bt genes is the use of the most efficient promoters. The cauliflower mosaic virus 358 promoter (CaMV 35S) has been a strong promoter in transgenic plants, shown to be active in most plant organs and is considered to be expressed throughout development (Carozzi et al. 1992, Fishhoff et al. 1987). The CaMV 35$ promoter has been used in almost all examples of Bt transgenic plants. Carozzi et al. (1992) showed the levels of protein coded by a truncated native Cry IA(b) were higher in older than younger tissues, and speculated that flowering may induce higher levels of expression from the CaMV 358 promoter. The expression of this l4 truncated gene was unaffected by the different environmental conditions found between the field and greenhouse. Wong et al. (1992) reported that in transgenic tobacco that the ats l A promoter with its transit peptide sequence firsed to the truncated fully modified Cry IA(c) protein provided a 10— to 20-fold increase in Cry IA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus promoter with a duplication of the enhancer region (CaMV-EnBSS) was used to express the same Bt gene. The majority of the transgenic plants analyzed with the ats 1 A promoter and a modified transit peptide expressed the Cry IA(c) as at least 0.4 % of the total leaf protein (Wong et al. 1992). So far, the truncated PM and FM genes have profound levels of insect control in transgenic plants (Perlak et al. 1991). However, it is difficult to directly compare the expression levels of these Bt genes in the mentioned reports without knowledge about when. the samples were assayed because levels of Cry protein are related to the developmental phase of the plant and the age of the leaf tissue (Carozii et al. 1992). RESISTANCE PROBLEMS The use or high application of Bacillus thuringiensis in the laboratory or in the field has led to isolation of resistance colonies of various insect, such as Plodia interpunctella (Hubner), Cadra cautella, Plutella xylostella (L.), Spodoptera exigua (Hubner) and Heliothis virescens (F.). The number of alternative ways to achieve resistance to Bt is as high as the given steps between inclusion indigestion and toxin insertion to the membrane, such as proteolysis of protoxins, activated toxin, binding to the plasma membrane and pore 15 formation (Perlak et al 1991, Williams et al. 1992, Moar et al. 1995). Changes in solubility and activation properties of crystal protein; as well as decreased affinity of receptor binding account for the development of resistance to Bt (de Maagd et al. 1996). It has been clearly demonstrated that insects can develop resistance to a crystal protein by changing the binding characteristics of the midgut receptor. Therefore, the resistance to one crystal protein does not necessarily cause resistance to other crystal proteins (Peferoen 1992). The use of transgenic plants that express Bt genes can increase the selection of Bt- resistant insects. However, some strategies such as combining Bt genes with other host resistance factors, pyramiding Bt with proteinase inhibitor transgenes, use of insect- friendly refuge in space and time may delay the time for insects to acquire resistance to Bt (Williams et al. 1992). Regained sensitivity of a resistant lepidopteran larval populations (resistance attributed to a loss of specific midgut receptors), lead to the strategy of maintaining a certain proportion of wild-type refuge plants or rotation with crops that do not express Cry proteins in field of transgenic Bt plants. In addition, co- applications of Bt and chitinase have significantly increased the insecticidal effect of Bt. Therefore, pyrarniding Bt gene with some chitinase genes seems a good strategy to avoid resistance in transgenic Bt plants (Regev et al. 1996) Another strategy to minimize the potential problems of resistance to Bt Cry protein is to identify novel Cry genes, with the aim of obtaining new proteins that bind alternative membrane receptors on the insect midgut cells. Increasing the diversity of genes available for transgenic plants should increase the efficiency of pest control and delay the emergence of resistance (Gleave et al. 1992). POTY VIRUS Y (PVY) Potato virus Y (PVY) is a type member of the family Potyviridae, which constitutes the largest known, economically. most important and the most widely distributed group of plant viruses (Thole et al. 1993, Dolja et al. 1994, Andy et al.1993, Dar et al. 1994, Van den Elzen et al.1989, Dinant et al. 1993, Singh et al. 1994, Singh et al. 1995). The Potyvirus group has three distinct genera (Potyvirus, Bymovirus, Rymovirus) and one possible genus (Ipomovirus). Potato virus Y (PVY), which is a type species of the genus Potyvirus, has three distinct strains based on host range and symptomology. These strains are PVY° (common strain), PVYn (tobacco venial necrosis strain) and PVYc (stipple streak strain) ( Singh et al. 1995 and Dhar et al. 1994). PVY can cause serious damage in several plant species belonging to the Solanaceae group, it can cause yield losses of up to 70% or 80% in sensitive potato cultivars (Vallejo et al. 1994, Pehu et al. 1995, Dinant et al. 1993). PVY° causes various degrees of mosaic symptoms. However, among the three main groups of PVY, the tobacco venial necrosis strain of PVY (PVY“) can cause the most devastating losses and because of these potential losses, PVYn have been considered a quaranteen species in North America. However, in recent years, outbreaks of PVY" in tobacco and in potato have been reported from North America (Van de Elzen et al. 1989). PVY" first appeared in European seed potato growing areas in 19508 causing epidemics, but it probably originated in South America from where it was introduced to Europe, with wild potato species or native cultivars imported for breeding purposes (Van den Elzen et al. 1994). The PVY genome is a single-stranded messenger-sense RNA molecule of approximately 10 kb with a 5’-terminal genome-linked protein (V pg) and a poly(A) tract at the 3’ end encapsidated into flexuous rods 680 to 900 nm in length and 12 to 15 nm in diameter. The viral RNA of potyviruses is translated into a large polyprotein precursor l6 17 (340-360 kDa) which is co- and post-translationally processed by at least three viral proteases (Thole et al. 1993, Dolja et al. 1994, Ohschima et al. 1993, Dougherty and Canington 1988, Dinant et al. 1993). The PVY non-segmented positive-sense RNA strand is 9.7 kb in length and encodes a large protein, co- or post-translationally processed by viral-encoded proteases into at least eight mature functional proteins. The polyprotein contains the necessary protease activity to cleave itself into eight mature proteins (Van den Elzen et al 1989, Andy et al. 1993, Pehu et al. 1995). PVY is transmitted by aphids in a non-persistent manner fiom an infected plant to a healthy plant giving a primary infected plant. At the end of the season the virus migrates down to the tubers. In the next season, seed tubers from the primary infected plant will be the source for secondary infections (Malnoe et al. 1994). The severity of potyvirus epidemic is determined by the activity of the aphid population early in the season, the presence of primary infected plants serving as viral source and the presence of volunteer potato plants, other solanaceous crop and weeds (V alkonen et al. 1994, Malnoe et al. 1994). The use of insecticides to eliminate the aphids has been inefficient to control PVY virus because the virus is transmitted in a non-persistent manner. In addition, classical breeding for virus resistant varieties is possible, but difficult because the cultivated potato varieties are tetraploid and highly heterozygous. Therefore, the introduction of coat protein gene in potato plant has improved the traditional methods for obtaining resistant plants against viral diseases (Malnoe et al. 1994). COAT PROTEIN-MEDIATED RESISTANCE . Sanford and Johnston proposed the concept of pathogen-derived resistance (Audy et al. 1993) defined as the capacity of a host expressing particular parasite sequences to overcome a disease. This concept was applied in plant virology by Powell and collegues who reported that transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein showed resistance to TMV infection. The use of pathogen genes is based on the principle that in any given set of host-pathogen interactions, if one of any pathogen- specific functions is disrupted by expressing a pathogen gene at the wrong time, in the wrong amount or in a counterfunctional form, the pathogen process should be stopped (Grumet 1990). However, the underlying mechanism(s) of virus resistance in transgenic plants expressing viral genes is not yet completely understood, and it is likely that there are different mechanisms of resistance in different host/virus combinations and that there may be more than one mechanism in a given system (Reimann-Phlipp and Beachy 1993). Genetically engineering virus resistance has two potential advantages over the use of host-derived resistance genes; these are: 1. the source of resistance genes would not be limited, as the genome of each pathogen would provide resistance genes, and 2. the smaller genomes and shorter life cycles of a pathogen than those of hosts make the isolation of such genes much simpler (Grumet 1990). Genetically engineering virus resistance or pathogen-derived resistance has been reported in several plant systems (Grumet 1990). This resistance approach is constituted by several strategies that use viral coat protein gene, movement protein gene, satellite sequence of viruses, viral antisense RNAs, replicase sequence and the complete viral 18 l9 genome of a mild strain (Grumet 1990, Audy et a1 1993, Reimann-Philipp and Beachy 1993). So far, among these distinct strategies the coat-protein-mediated resistance has been generally applicable and is a successful strategy (Grumet 1990). Several groups have demonstrated that expression of viral capsid genes by the host results in either prevented, delayed, or reduced symptoms and systemic spread of infection (Grumet 1990, Grumet and Fang 1993). Coat protein-mediated resistance has been termed as genetically engineered cross protection, in reference to the hypothesis that the protection conferred by a mild virus strain against subsequent infection by a more severe strain is due to the presence of coat protein form from the mild strain. This role of coat protein in cross protection is supported by the fact that classical cross protection can be overcome by inoculating with viral RNA, and the protection of coat protein transgenic plants is also overcome by inoculating with viral RNA. However, the mechanism of cross protection is not well understood, and some evidence using coat protein mutants, transencapsidated viruses and viroids (which do not have a coat protein) have shown that coat protein is not necessary to confer cross protection. In addition, the classical cross protection as well as the resistances of transgenic plants expressing coat protein of TMV, AIMV and PVX, but not CMV were overcome by increasing the amount of inoculum (Grumet 1990). MECHANISM OF PROTECTION IN COAT PROTEIN TRANSGENIC PLANTS Since the initial demonstration that the expression of the coat protein (cp) gene of tobacco mosaic virus (TMV) in transgenic plants could provide effective protection in transgenic plants by Powell et al. (1986), the use of viral coat protein (cp) genes has been 20 the most widely used strategy for genetically engineered virus resistance to date (Farinelli and Malnoe 1993, Grumet 1994). This strategy for engineering virus resistance has been reported in at least 20 different RNA viruses which vary in particle morphology, genome organization, and mode of transmission (Kavanagh and Spilane 1995). The mechanism(s) of virus resistance in transgenic plants is not yet completely understood; however, several mechanisms have been proposed, including prevention of uncoating of the incoming virus, interference with viral translation and/or replication, and interference with cell-to-cell and/or long distance movement (Grumet 1994, Reimann- Philipp and Beachy 1993). So far, it is clear that there are different mechanisms of resistance in different host/cp/virus combinations and that there is more than one mechanism in a given system (Reimann-Philipp and Beachy 1993). These mechanisms could operate in the initial interaction between cp, plant and the challenge virus, an early event in infection prior to replication of the incoming virus (“inhibited-uncoating” hypothesis), or a latter stage during viral movement which can delay disease development and systemic spread (Hackland et al. 1994). Viral infection starts upon the introduction of plant viruses into its host with release of the nucleic acid for translation by host ribosome. In the first hypothesis, it is believed that coat protein gene expression interferes with the disassembly of the virus (Hackland et al. 1994). Register et al. (1989) postulated two models whereby cp could inhibit uncoating of virions, in one he specified that a site for virus uncoating within the cell would be blocked by endogenous cp. The second model states that virus disassembly is inhibited by shifting the disassembly-assembly reaction in favor of assembly, thereby impeding virus infection. If this were limited to the exchange of cp subunit of the virion 21 with the endogenous cp in the transgenic cell, then the endogenous cp would be required to mediate this resistance (Clark et al. 1995). These models are valid in the case of tomato mosaic virus (TMV), rice stripe virus (RSV), alfalfa mosaic virus (AIMV), potato virus X (PVX) and tomato yellow leaf curl virus (TYLCV). The strength of resistance correlated positively with the levels of coat protein in transgenic plants, and plants that accumulated only coat protein transcripts and non-detectable coat proteins were not resistant (Grumet 1995, Grumet 1994, Kavanagh and Spillane 1995). In addition, some studies with TMV, AIMV and rsv showed that resistance was overcome by inoculation with viral RNA. This proposes that cp interferes with the initial uncoating of the virus (Grumet1994, Hackland et al. 1994). However, for other studies with viruses such as PVX (potato virus X), PVS (carlavirus), and ArMV and GCMV (nepoviruses), the cp-mediated protection is effective against viral RNA as well as whole virions. This suggests that cp affects some other step in addition to uncoating (Grumet 1994). In the case of potato virus Y (PVY) and potato leafroll (PLRV), resistance correlated with the levels of coat protein transcripts and not with levels of coat protein which was undetectable for some virus resistant transgenic lines. But the mechanism of resistance is more complex in the case of TEV and TSWV, virus resistant plants were obtained when an intact nucleocapsid protein transgene was rendered untranslatable through removal of the initiating ATG codon (Kavanagh and Spillane 1995). Nejidat and Beachy (1989) examined the levels of TMV cp in transgenic tobacco plants under different temperature conditions and concluded that increase in continuous temperature resulted in a decline in cp accumulation, but not in the accumulation of cp 22 mRNA, and reduction in the protection against TMV. This involves the cp and not the mRNA in this protection mechanism. However, in transgenic tomato under the same condition, protection did not decrease. This indicates that the mechanisms of protection can differ with the different plant host (Nejidat and Beachy 1989, Hackland et al. 1994). To test the inhibiting uncoating hypothesis, Osbum et al. (1989) encapsidated the chimeric reporter mRNA encoding B-glucuronidase (GUS) in TMV cp to form “pseudovirus” particles. When the TMV-like particles were introduced into protoplasts they uncoated and expressed their non-replicating “seudo-genomes” transiently. In this way, the level of expression was a direct measure of the extent of pseudovirus disassembly. The fact that GUS particles were expressed 100-fold less efficiently in cp(+) transgenic protoplasts and unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently showed that the endogenous cp must interfere with the inhibition of the (GUS) nucleocapside disassembly and a later stage of infection involving the viral RNA (Hackland et al. 1994). Another manifestation of cp-mediated protection is the delay of the spread of virus (known as interference of later events of infection) from inoculated leaves to upper leaves. In a systemic viral infection in plants, viruses replicate and move short distances from cell to cell through the plasmodesmata requiring viral encoded movement proteins (MP). For long distance movement, viruses move through the vascular system, and viral genes and their products seem to be required for it. Therefore, several groups reported a delay in the development of systemic disease symptoms afier inoculation of cp(+) transgenic plants with the respective viruses. This is evidence that the cp interferes with: 1) the spread of viruses from cell to cell in the inoculated tissue; 2) the movement of 23 viruses from inoculated leaf into the vascular tissue; 3) the movement through the vascular tissue, and 4) the movement into upper or lower non-inoculated leaves. To determine whether long distance movement was reduced in cp(+) tissue, Wisniewski et al. (1990) initiated several grafting experiments. Using non-transgenic tissue as rootstock, and apical section from cp(+) plants with and without leaves, and cp(- ) plants, he reported that the long distance movement of TMV was reduced and the symptoms were fewer in grafted plants that contained a cp(+) stem section with leaves compared with those with the cp(-) stem section. However, grafied plants with cp(+) apical section without leaves showed no effect on TMV spread. It was suggested that the leaf in cp(+) tissue acted as a virus sink. After entering these leaves, these viruses may not be able to establish an infection, reducing the virus titer and disabling further spread. Wisniewski et al. (1990) also reported that the spread of TMV to closely adjacent tissues (1-3 mm) was similar in cp(+) and cp(-) sections, but spread to more distant tissues (5-10 mm) was significally reduced. Since the cp-mediated resistance can be overcome with RNA in the case of TMV, the adjacent cells should become infected. This evidence suggests that the mechanisms of preventing initial infection in cp-mediated resistance differs from those of preventing long distance movement of infection within the plant. Clark et al. (1990) compared plant lines expressing similar levels of TMV cp from rch and CaMV 358 promoters and found that those whole plants with the CaMV 35S:cp constructs exhibited higher resistance than those with the rch:cp constructs. This result suggests that whatever the transport form, it is likely that the presence of cp in the phloem and associated cells interferes with viral long-distance spread, since the CaMV 24 35S promoter is highly active in phloem-associated cells. Therefore, there is more than one mechanism in cp(+) plants, and these proposed mechanisms active during early events in virus infection could provide further protection against virus multiplication; therefore, no rule can be made for the spectrum of resistance delivered by cp genes (Hackland et al. 1994). On the other hand, protection in transgenic plants given by viral coat protein can extend to related viruses. It has been reported that trangenic plants expressing the coat protein (cp) gene of a given virus were protected against infection by that virus (homologous virus) and this protection was extended to related strain or viruses (heterologous virus) (Grumet and Fang 1993). For example, transgenic tobacco expressing the coat protein gene of soybean mosaic virus (SMV) to which tobacco is a non-host species showed protection to PVY and tobacco etch virus (TEV). Similar results have been reported with viruses which share approximately 60 % or greater anrino acid sequence homology with the TMV coat protein (Kavanagh and Spillane 1995). Dinant et al. (1993) reported that transgenic tobacco plants, Nicotiana tabacum cv. Xanthi, containing a modified coat protein gene of LMV showed heterologous resistance to five different strains of potato virus Y. This group observed different phenotypes, including lines with complete resistance, delay and attenuation of symptoms in some lines, and delay in symptoms with no modification of symptoms in one line. In the two latter cases, the accumulation of PVY and symptoms were reduced. The PVY and LMV cp's are clearly distinct, with a 66% amino acid sequence homology; however such high resistance was achieved (Dinant et al. 1993). CONSTRAINTS IN THE USE OF CP-MEDIATED PROTECTION , The scientific community and the public in general have expressed a number of concerns about the release of genetically engineering organisms for crop production. Field testing has followed strict USDA-APHIS guidelines, and these field trials try to assess the possible impact of these engineered plants on agricultural ecosystems (Grumet 1994). These experiments have focused on the spread of transgenes into related species (mainly by pollen), and on the transfer of the transgenes to microorganisms. However, the possible appearance of new viral strains and diseases by heterologous encapsidation or by template switching (recombination) are other concerns in the case of cp-mediated protection also need to be taken into consideration (Grumet 1994, F arinelli et al. 1992). ' Transencapsidation or heterologous encapsidation refers to the potential for altered vector-specificity due to the encapsidation of an incoming virus in the coat protein being produced by the transgenic plant (Grumet 1994). Heterologous encapsidation is a natural process that takes place when two virus strains infect the same plant and the genomic RNA of one strain becomes totally (transencapsidation) or partially encapsidated (mixed encapsidation or phenotypic mixing) by cp's of the other strain. The transcapsidated particle can have altered ways of transmission (Farinelli et al. 1992). There are several examples of transencapsidation. The RNA from cp minus mutant TMV was inoculated onto transgenic TMV-cp expressing plants, the progeny viruses were capable of spreading further within the transgenic plants than within inoculated control plants. From the transgenic plants, but not the control, encapsidated viral particles were isolated and visible by electron microscope (Grumet 1994). 25 26 In another study, Farinelli et al. (1992) infected PVYn-cp transgenic tobacco plants that were completely or partially resistant to PVYn with PVY°. Electron microscopy of virus particles isolated from transgenic plants showed a mixture of viral particles with the two cp types. The potential risk associated with this phenomenon must be evaluated carefully. However, the transencapsidation is a natural process that seems far more likely occur in non-transgenic plants infected with several viruses than in transgenic plants. In fact, infected non-transgenic plants have higher levels of cp than cp-transgenic plants (Grumet 1994). To circumvent transencapsidation, cp-mediated resistance could use mutant cp genes from non-transmissible virus strains or use truncated cp genes lacking transmission-important domains (Grumet 1994). The other potential risk in the cp—mediated resistance is termed template switching, copy choice or recombination. By this mechanism, the transgenic RNA could become incorporated into viral genome of the incoming virus. The incorporation of cp sequences into the genome of RNA virus could in theory lead to appearance of new viral strains with altered virulence, host range or transmission (Grumet 1994, Farinelli et al. 1992). It is believed that polymerase begins on one strand of RNA and then moves to another. Several groups have isolated recombinant viruses from transgenic plants inoculated with a virus mixture under strong selection (Grumet 1994). However, to test for recombination in non-selective conditions could reflect the real situation of transgenic plants. Under this consideration, Angenent et al. (1989) failed 27 to detect recombinants using tobaviruses. This suggests that the strong selection pressure favors. recombination. Recognizing that recombination is the incorporation of transgenic RNA into viral RNA in transgenic plants, the potential risk could be even greater in viral mixed-infected non-transgenic plants, because they contain higher viral RNA levels than those observed in transgenic plants (Grumet 1994). OBJECTIVES The main goal of this research was to study the inheritance of the PVY0 coat protein and the truncated wild type Bt-Cry IA(c) transgenes, in tetrasomic potato lines. The specific objectives of this approach were: 0 Confirm the incorporation of these foreign genes, II’VYO coat protein gene and the truncated Cry IA(c), in potato clones (cv. Atlantic and FL-1607) through PCR and Southern blot analysis. 0 Determine if the segregation of Bacillus thuringiensis_(Cry IA(c)) gene carried by FL1607 potato lines, and PVY coat protein genes carried by Atlantic, follow tetrasomic ratios. 0 Determine if crosses between transgenic plants carrying Bt genes and plants carrying the PVY coat protein gene and cultivated varieties yield progeny carrying these genes. MATERIALS AND METHODS Plant materials All potato clones were obtained from the MSU Potato Breeding and Genetics Program. The transgenic plants contain either a truncated wild type version of Bt-Cry IA(c) gene (FL1607-A30) or one or more copies of PVYO coat protein genes (ATL- PVY5). Figures 1 and 2 show the two constructs carried by the Bt-transgenic plants and the PVY cp transgenic plants, respectively. The plants were grown in vitro in GA-7 Boxes (Magenta Corporation) before transplanting, and six plants of each transgenic clone were transplanted to planting medium in the greenhouse. After one month, 4 plants per clone were selected and each of these plants was transplanted to a 4 liter plastic pot. Controlled crosses and self-pollinations were made between Bt-transgenic plants (FL1607-A30), PVY CP transgenic plants (ATL-PVYS), and the non-transgenic plants (Lemhi Russet and MS702-80). The fruits were harvested, seeds were collected, cleaned and planted in the greenhouse. The progeny plants were used for segregation analysis of the transgenes (Table 1). Leaf tissue from the transgenic plants was also used for DNA isolation. 28 29 .28 .m 2852.: 03889 e388. ”.m E Eek «5. 82m = Engagefimesq £93802 5 .52 .ExS ém we we 05 move“. €23 28m 05 we comma? 3325; ”$3 rowan .83an 25> 38o:— ..o>5§3a0 ”mmm >330 sewage. 0355?. 25302 28 m0 Z 38:85 035:? 25.80 Z 65 mOZ .mmga 05 no woman mm 2833 £5. .02 £64 am $33363 seem—HEB? .«o 3.55 .2 can? an 80¢ vofiafio mm? .2538 2:. .aaoa 3 cos 53:5 35% ease on. Co Samoa ._ can; m m M w m 0 d 0 a m a W a. ma ace 82 .m a sat = 22 ES been an >25 1 ea 82 mm Uh>m we 28w £38m 88 32332 “meow m0 o>>m gages—ea "25> 3808 335230 ”mmm >330 .meh Sea a gougenqgna £93502 5 .52 eczema—c8 89:8? 2:3an £8 mOZ .58889 0358? 8:302 ”ek— wOZ AQ in 05 5 25m mDO 05 mo com—a E wotomfi we? 8% mo >>m 2E. damage—2 8 5383? we $803.5 .EBefiam 23E we “cognac .5830 H .5 88m woe—mane we? 883:8 2E. .aaoa o. 855 5.5% 2883 as .a cause .N 2:5 Pvu II Sac I Xba I Sma I BamH I Puv 11 K——' PstI a as .6 sea as >26 seaeeI e .52 IE aele.V mm..— mm Uh! 1 Hooter I 18d 1 qu 111 pmn 31 ' Table 1. Crosses used in PCR analysis Crosses Number of (female x male) Segregating transgenes progeny FL1607-A30 x ATL-PVYS Rt 67 PVY CP 80 NPT H 66 ATL-PVYS x FL1607-A30 Br 74 ' PVY CP 69 NPT II 0 ATL-PVYS x MS702-8O PVY CP 66 NPT II 66 ATL-PVYS x Lemhi Russet PVY CP 31 NPT H 31 Lemhi Russet x ATL-PVYS PVY CP 35 ‘ NPT II 35 ATL-PVYS (x) PVY CP 35 NPT II 31 Southern blot analysis: Genomic DNA was extracted by the CTAB method (Saghai-Maroof et al. 1984) modified by adding 2% beta-mercaptoethanol to the extraction buffer. DNA (20 pg) from ATL-PVYS clone was digested with BamHI and EcoRI to cut the PVY gene and copy number was analyzed by digesting with just BamH] . The fragments were separated by electrophoresis through a 1.0% agarose gel, eluted onto a nylon membrane (Hybond N, Amersham, England) using a capillary transfer procedure (Sambrook et. al.1989). Nucleic acids were fixed to the membrane by auto-cross- linking. Prehybridization was conducted for 2 h at 42°C in the solution containing 5X SSC (0.75 M of NaCl, 0.075 M of NaCitrate, at pH 7), 1% skim milk, 0.1% N-lauroylsarcosine, 0.02% SDS, 50% forrnamide and 125 ug/ml sheared salmon sperm DNA. Hybridization was performed at 42°C overnight in fresh solution with a non-radioactive random primed DIG- labeled probe (1.2kb SmaI\SacI fragment of the PVY coding region was inserted into a Bluescript SK+ vector [Stratagene Inc. La Jolla CA] amplified by PCR using the T7\T3 primers) according to manufacture's instructions (BMB, Indianapolis, IN). Following hybridization, the membrane was washed twice in 2X SSC, 0.1% SDS for 15 min at room temperature, twice in 0.5 X SSC, 0.1% SDS for 20 min at 65°C. CSPD (BMB, Indianapolis, IN) was used for chemiluminescence detection by following the manufacturer’s instructions. The membrane was then exposed to X-ray film (Kodak X-OMAT) for 15-30 min. Isolation of DNA for PCR analysis DNA was extracted from fresh potato leaves following the quickprep isolation procedure, a modified version of Edwards et al. (1991). Two or three disks of leaf tissue 32 33 were punched out with the lid of a sterile 1.5 ml Eppendorf tube. CTAB (400 pl) extraction buffer (0.1M Tris HCl pH 8.0, 1.4 M NaCl, 0.02 M EDTA and 2% cetyltrimethylarnmonium bromide) and 1% of beta-mercaptoethanol were added. The tissue was macerated with a sterilized pestle, and then incubated at 65 °C for 20-30 minutes. Sample DNAs were extracted adding 400 pl of chloroformzisoamyl alcohol (24:1) to each tube and mixing the two layers for 1 min. Then, the tubes were centrifuged in a microfuge (14,000 rpm) for 5 minutes. The top (aqueous) layer was transferred to a new sterile Eppendorf tube, and the nucleic acid was precipitated by mixing the aqueous phase with 400 pl of cold isopropanol. The nucleic acids were recovered by centrifugation in a microfuge (14,000 rpm) for 5 minutes, the supernatant was discarded and the small white pellet at the bottom of the tube was dried in a Speed-vac for 10 minutes and resuspended in 100 pl of TtoEo,1(10 mM tris- acetate and 0.1 mM EDTA) . Single-strand RNA was digested with 4 pl of Rnase A from bovine pancreas (10 mg/ml stock solution) for 15 minutes at room temperature. The DNA is then precipitated with 300 pl of cold 95% ethanol, mixing gently the tubes by inversion, and the tubes were centrifuged in a microfuge (14,000 rpm) for 10 minutes. The supernatant was discarded and the pellet was dried in the Speed-vac for 10 minutes and resuspended in 50 D] of TIOEOJ. The samples were maintained in a freezer at -20 °C . Quantification of DNA The amount of DNA in each sample was measured by the flouromeuic method, using- a Hoefer Scientific Miniflourometer Model TKOIOO (Hoefer Scientific, San Francisco, California). 34 The 10X TNE Buffer (100 mM of Tris, 10 Mm of EDTA-N32, 1.0 M of NaCl and pH to 7.4 with cone. HCl) was diluted to 1 X and 10 pl of fluorescence dye, Hoeschst 33258 (0.1 pg/ml stock solution) was added to form the dye solution. The sample readings were taken adding 2 pl of sample DNA to the 2 m1 of dye solution. The cuvette was rinsed with filtered (0.22 pm) water between each reading. The extracted DNA samples were standardized to a uniform concentration of 100 pg/pl or to 50 pg/pl otherwise. Polymerase Chain Reaction (PCR) The presence of the transgenes was verified in the parental material through PCR and Southern analyses. In addition, potato plants within each segregating population were chosen at random for PCR-based analysis of segregation of the three transgenes (NPT II, Bt-Cry IA(c) and PVY coat protein genes). PCR reaction was performed in a volume of 25 pl, amplifying each transgene separately, following the recommendation for amplification with Taq DNA polymerase (Gibco BRL, Life Technologies Inc, MD, USA). For Bt-Cry IA(c) gene, the 25p] of PCR reaction contained 1X PCR buffer (20 mM of Tris-HCI pH 8.0 and 50 mM of KCl), 0.2 mM of dNTP mix (0.2 mM of each dNTP), 1.5 mM of MgClz, 0.625 unit of Taq DNA polymerase, 0.6729 pM of Btk-802 primer, 0.6688 pM of Btk-l634 primer, 150 ng of sample DNA template. The primer sequences (5’-3’) are shown in Table 2. For the PVY coat protein gene, the PCR reaction had 1X buffer buffer (20 mM of 35 mm O>m mm >m-mma Essa 2: use as» = as a. 5.8535. -s Ewen beam 253 x 8582: 05 5 .3223 a use 2....» ES beam ea =2§Eae< -e Oceania see 8% be S beam ea EEOEEE... am $.39. 265a 2: ace 8% me Ea eo =2§Eae< av .ae<->>._-mma 2883 2: 82.. 8% me >3 .6 838535. ..m 32.4% 83 25m .6 >3 eo SEES... ..N E E: as, 383 <23 -2 ”355 $2605 :oneongfim «Um .m 0.59m 39 number of germinated and non-germinated seeds, dead plants, and resistance plants after 6 weeks. Statistical Analysis Chi-square and goodness-of-fit for binomial analysis were used to test the segregating generation to tetrasomic ratios. The non-significant null hypotheses were not rejected when the probability was equal to or greater than 0.05. RESULTS The presence of Bt-Cry IA(c) in the clone FL1607-A30 was confirmed by Southern blot analysis. The labeled Cry IA (c) probe hybridized one fragment of genomic plant DNA digested with Hind III or with BamHI. This indicated the presence of one copy of the Bt-Cry IA(c) gene in FL1607-A30 (Figure 4 and 5). The Southern blot analysis of ATL-PVYS showed 2 copies of the PVY coat protein gene. When the DNA fiom ATL-PVYS was digested with BamHI and EcoRI, the labeled probe hybridized only one restriction fiagment. Therefore, the presence of one band on the revealed X-ray film indicated that these two restriction sites of the enzymes flank the PVY cp gene. However, the presence of two revealed bands, when only BamH] was used, suggested the insertion of two copies in the ATL-PVYS clone (Figures 6 and 7). i The potato clone ATL-PVYS was positive for the NPT II gene and the PVY cp 40 5148 4973 4268 3530 2600 2027 Figure 4. Southern blot for Bt-Cry IA (c) using HindIII . The labeled probe, a 256 bp fragment from the Cry [A (0) gene hybridized a fragment of 2.6 kb in the transformed FL1607-A30 Lane 1: FL1607-A30. Lane 2 to 5: Untransformed FL1607. Lane 6: Lambda DNA digested with HindIII and EcoRI to yield a molecular weight marker. All numbers to the right are base pairs. 41 .- C 2027 - I904 I584 3 I330 ‘ 983 Figure 5. Southern blot for Bt-Cry IA(c) using BamH]. The labeled 256 bp fragment from CryIA (c ) gene (the probe) hybridized a fragment of 18000 bp in the FL1607-A30 line and Two fragments in the line F L1607-A1 1. Lane 1: FL1605-A11 Lane 2: FL1607—A30 Lane 3: Un-transformed FL1607 Lane 4: Blank. Lane 5: Molecular weight marker: Lambda DNA digested with BamHI 42 Hybridized probes Figure 6. Southern blot for PVY cp gene using BamHI and EcoRI. The labeled probe hybridized a fragment of 1584 bp in the ATL-PVYS and Desiree- PVY5 Lane 1: Molecular weight marker: Lambda DNA digested with ....... Lane 2: Non-transgenic potato cv.“Atlantic” Lane 3: ATL-PVYS Lane 4: Desiree-PVY5 43 Two hybridized fragments in ATL-PVYS Figure 7. Southern blot revealed film for PVY cp gene using BamH]. Lane 1: Molecular weight marker: Lambda DNA digested with ...... Lane 2: Non-transgenic potato cv.“Atlantic” Lane3: ATL-PVYS Lane 4: Desiree-PVY5 44 gene, while the clone FL1607-A30 was positive for the NPT II gene and the Bt-Cry IA(c) gene based upon separate PCR amplification reactions. The non-transgenic potato cv. Lemhi Russet was negative for both genes (Figure 8, 9 and 10). i The cultivated potato is considered to be an autotetraploid with a genome of 2n=4x=48. Traits inherited in a tetrasomic manner from autotetraploids are much more complex than traits inherited in a disomic manner from diploids (Poehlman and Sleper 1995). In a two allele model, there are five possible genotypes at a locus in an autotetraploid. These can be defined on the basis of the number of dominant or recessive alleles present as nulliplex (no dominant alleles: aaaa), simplex (one dominant allele: Aaaa), duplex (two dominant allele: Aaaa), triplex (three dominant alleles: AAAa) and quadriplex (only dominant alleles: AAAA). If there are four alleles at a locus, the different combinations are termed nulliplex (aaaa), simplex (aaab), duplex (aabb), trigenic (aabc) and tetragenic (abcd). The gametes obtained from a tetraploid are diploid (2x), and the different gametes depend on the parental genotype. For example: only a gametes are expected from nulliplex; a and Aa gametes in a ratio of 1:1 are expected from a simplex; aa, Aa and AA gametes in a ratio of 1:4:1 are expected from a duplex; and so on. The segregation pattern is even more complex in transgenic potato plants that have multiple transgenes that segregate independently. One copy of a transgene should behave as a single allele in a specific locus (simplex), and two copies of a transgene should behave as two alleles at two different loci (several simplex loci), if there is no linkage between them. In this way, for one copy of any transgene, the expected segregation ratio would be 1:1 (non-transgenicztransgenic) in a progeny from the cross 45 5 6 7 8 91011121314151617181920 +++++++ +-— -+ Figure 8. PCR amplification for NPT II gene. Lanes: l.- ADNA digested by Hind III 2.- reaction without DNA template 3.- Transgenic ATL-PVYS 4.- Non-transgenic potato cv “Atlantic” 5.- Transgenic FL1607-A30 6 to 20.- Progeny from FL1607-A30xATL-PVY5 46 123 456 78 910111213141516171819202122 - -+-++-++++++++++++-+ Figure 9. PCR amplification for Bt-Cry IA (c) gene. Lanes: 1.- lDNA digested by Hind III 2.- Transgenic ATL-PVYS 3.- Non-transgenic potato cv “Atlantic” 4.- Transgenic FL1607-A30 5.- Reaction without DNA template 6 to 22.- Progeny from FL1607-A30xATL-PVY5 47 123 45 6 7 891011121314151617 --+-+++-+--+++-- 565 bp Figure 10. PCR amplification for PVY Coat protein gene. Lanes: l.- XDNA digested by Hind III 2.- Non-transgenic potato cv “Atlantic” 3.- Transgenic FL1607-A30 4.- Transgenic ATL-PVYS 5.- Reaction without DNA template 6 to l7.- Progeny from ATL-PVYS x Lemhi Russet 48 between transgenic and non-transgenic tetraploid potato plants, 1:3 (non- transgenicztransgenic) in a progeny from a self-pollinated transgenic tetraploid potato plants. To determine the expected ratio of any segregating population for any copy number, we need to consider each copy as an independent locus and the probability of a genotype in the offspring will be the product of the individual probabilities of each copy. To verify the presence of one copy of the Bt gene in FL1607-A30 clones, the progenies of ATL-PVYS x F L1607-A30 and of the reciprocal cross FL1607-A30 x ATL- PVYS were scored for the amplification of the Bt gene by PCR. The progeny of ATL- PVY5 x FL1607-A30 segregated in a ratio of 1:1 for presencezabsence of the amplified band of the Bt gene. However, progeny of the reciprocal cross had a ratio that deviated fi'om the expected 1:1 ratio for one copy in the donor parent but not from the 3:1 ratio (Table 3). These two reciprocal cross families were scored also for the presence of the PCR amplification band for the PVY cp gene. The ratio for presencezabsence of the PCR amplification band of the PVY cp gene in the ATL-PVYS x FL1607-A30 progeny, and the reciprocal cross progeny (FL1607-A30 x ATL-PVYS) fit the 1:1 ratio. However, both segregating populations had ratios that deviated significantly from the Mendelian ratio of 3 :1 expected for two independent copies of the PVY cp gene in the donor parent (Table 3). The segregation of NPT II gene should be 3:1 (presencezabsence) for progenies in which both parents have one copy of the gene or 7:1 for progenies in which one parent has one copy and the other has two copies. The FL1607-A30 x ATL-PVYS deviated significantly from the expected ratio (7:1) but fit the ratio 3:1 (Table 3). This indicated h“; 49 3.. 33$ 3.. N .mZ cod 2 _ 29 : HA2 3.. 3.5.9 7m N .mZ cod 2 _ 2 ”2 mo gm 83.3 383 x m>>mHF< 3.. meme: fin N .mZ vNEd E _ oNNm = .52 a... w~w— .m— Tm N .mZ $8.0 Z _ Nmém mo >>m 8-852 x m>>méh< i. mmmof in N .wZ 33.0 E _ 3.”? mo >>m I 3K2: fim N .mZ ES; 2 _ Rune am Swallow—Am x m>>AJH< .I VNmmE fin 850 2: Eoc N use E28 25 Eat 2 .mZ N350 7m E83 some ONnov = #2 Eon.“ _ 3.. NE: in N .m.Z 32.0 E _ mvsm m0 >>m t. 30.8 fin m .m2 9 _ ._ rm N 23: 1. 3m.» 3 _ New am mm>>QJH< x om<-\.oe:m Eamodnx 60an 63% no.3 con—=32 .300 32.8% 25$ch 39: x 295.: mEEoEm 5m owed 380qu 62.823 nose—30m ECO mmoceooo 23% 82030 wcuewonwom 9.23.: 0 can e em 2: E nip—~25 no.“ E mo $2680 mo «mob .m 032. 50 555% Asyx 0.2;» 58.28%; :2h 2: ace sesame 58%»; :3: H.2. Hzmodax Aooax 205$ .m_mo£o%; :3: of Eat 8:2er ufiomimmm H_._ Emodnx Voonx 82$» nae—#893 =5: 9: Eat oococobme anEcwmm 0 Z ”m. Z .qxoknfidédxo-.mnocvwnoowx mama—05m new 9.8:..— .«o mmocuoow mgwa .I. Seems Tm ~ .w.Z cod _H_ <\Z DUE = HA2 ..:.. hNNv Tm fl .mZ no: ._ H; <\Z .2 Hmm m0 >>m m>>mih< x m>>m-..:.< .1. 300.: in N .mZ 2.36 fig ~ ON“: = HAZ .mZ owmv.m Tm N .mZ mwNn: fifi _ E u _N no >>m m>>mHEr< x 833% E804 meocsaom mcuawocwom 2: 5 $2825 .8 E .«o mmocuoow mo 50,—. €253 m 033. 51 the presence of one copy of the gene or a possible linkage between copies. The proportion of individuals that had both PVY cp and Bt transgenes in ATL- PVYS x FL1607-A30 progeny fit in the 25% of the total segregating population expected for one copy of PVY coat protein gene or a linkage between the copies of the cp gene and one cOpy of the Bt-Cry IA(c) gene, and it also fit in the 37.5% expected fi'om one copy of one gene and two copies of the other. However, the proportion (26 out of 67) of the reciprocal progeny with the two genes differs from the 25% proportion expected if there are one copy of Bt-Cry I A(c) gene and one copy of PVY cp gene, but not from the 37.5% (Table 4). Progeny from crosses between ATL-PVYS and non-transgenic parents (clone MS702-80 and cv. Lemhi Russet) were used to verify if the Mendelian 1:1 ratios (presencezabsence) expected for one copy or the linkage between copies of the PVY cp gene in the donor parent appears in the segregation of the PVY cp and NPT II genes. The observed ratios (presencezabsence) in the segregation of PVY cp gene were 15:16 for ATL-PVYS x Lemhi Russet progeny, 21 :14 for Lemhi Russet x ATL-PVYS progeny and 34:32 for ATL-PVYS x MS702-80 progeny. All these segregations fit a 1:1 ratio expected for one copy. Similar results were observed in the segregation of NPT II. The segregations of this gene were 16:15 for ATL-PVYS x Lemhi Russet progeny, 15:20 for Lemhi Russet x ATL-PVYS progeny and 37:39 for ATL-PVYS x MS702-80 progeny. In addition, the segregation of the PVY cp gene in the self-pollinated progeny supports the above results. The observed ratio of the PVY cp was 23:12 and fits in the ratio 3:] expected for the segregation of one copy of PVY cp gene or co-segregation of these copies or linkage between copies in the selfed progeny of ATL-PVYS (Table 3). 52 .86.... .8... 2.; .88. a... .5 a... 3.....8 .82.... 8... Enqoux Acoux 82:5 28500.3 :0: 2: 82.. 858% .583qu H... 33.8% vooNVn 08:3 285093 :0: 05 80b 85.8% “gouge. 02 ”m2 982%3.22.8238. 2 ”8:82: 8: 22:: 8 .8880 .. 3.2.: 28.2 $2.8 2:“: 28m 88 2:12 .8 82:8 N 2.0": 8:8 2: 233.2 0 8300 N .32 :82 .o 222 $8.2 28 x Doug m0 .800 _ .. £82. 2.2 $82 2 no": 28: 88 8 09383.. no": .8 .28 _ .. 28.2 22.2 $2.8 2:": 28m 88 2:12 :o 8&8 2 2:": 2:0 2: 3283.2 no 22000 N .mz 822 2:2 $8.2 :o .38 _ x .mz 223 2.: $8.2 2 no": 28: 88 2 ages? Wong m0 .300 2 8830 :8 883m :8 88m 8282 8822: 2.5.8: 8:858: meta :8 :8 8:88.: meta :00 :2, 82:8 .809 :o 8838: .852 28:53 8.892 8882”... 882:5 8:28:22: 28.:o&m 828: :23 2.88.8: .8 88808 . , 838:0 8:285 :22 , 8%. £205 “:00 gm 0:: 33 20.5 0 835305 36365 05 «no: 8 2:38: 2:55-20 .v 038. 53 Some progenies showed resistance to the antibiotic kanamycin. Therefore, the kanamycin assay indicated stable integration and expression of the introduced NPT II gene in the segregating progeny of these transgenic clones (F L1607-A30 and ATL- PVYS). The kanamycin assay corroborated the segregating pattern of NPT 11 observed by PCR analyses. The ratios for presencezabsence of resistance plants fit the ratios of 3:1 in the ATL-PVYS x FL1607-A30 progeny, of 1:1 in the ATL-PVYS x MS702-80 and ATL- PVYS x Lemhi Russet progenies and of 1:1 in the selfed progeny of ATL-PVYS. Except for the selfed progeny, these ratios suggested the presence of one copy of the NPT II gene or a tight linkage between the two inserted copies (Table 5). Discussion The production of transgenic plants has increased the diversity of germplasm sources that can be used by the breeder. However, such transgenic plants only will be of value if their phenotype is faithfully transmitted in a predictable manner through subsequent generations (Finnegan and MacElroy 1994). The segregating ratios for Bt, PVY cp and NPT 11 genes were assessed in several offspring from crosses using transgenic lines (ATL-PVYS and FL1607-A30) and non- transgenic lines (MS702-80 and cv. Lemhi Russet). These ratios can be used to explain how the transgenic loci act. In the case of the Bt gene in F L1607-A30, the observed PCR segregation pattern of the ATL-PVYS x FL1607-A30 progeny confirmed the data from the Southern blot analysis. Therefore, the gathered data support the contention that there is one copy of the 54 88398885 ".39 .85 Srodnx Aooux 0855 88500.3 :0: 2: 80¢ oococobau «80%—ma $35 ”1. Emodux 82.x 8239 88500.3 :0: 05 80¢ 888b€ Edemamm H .. Hamodnx 82.x 08:3 88500.»: :0: 05 80: 888mm“. “80%—ma 02 ”m2 .qxoknfi.0-_.nxo-.30§”wu8vx 2282.5 com 205E .«0 8050006 3.38.. >88 50.2883 .m 033. 55 mm mm 2 2 vm vm £05000 ofiowmcmbéocv $2 $2 5 mm <2 <2 5 mm 5220 .>0 .1. mummem fim Q Q vm .mZ 803.0 2 G 0 nm 2 M3 vm m>>méh>mih< 3 2 vn 2 2 mm .3. $3.0m fin m. 2 vm .m.Z 290.0 E 3 0 3 M: 3 VN 830M EEoAxm>>th< 2 9 on S m _ mm I 20.3 7m 3 2 vm .m2 082 5.0 2 vv 2 mm 2 mm 3 owéonmEmeEJH/x 0 2 VN o M: vm 3.. v3.3.2 3. m 2 mm .mZ mmmohmod in rm 5 3 m _ mm mm om<$oosmxm>>mfih< Aoovxv A3? .803 mug—W080 820 8 38.0 820 80 £08m 80E Any—«E8380: 238005 00% 0:8 .«0 80:50 38.3 080 38mg“: 383 0800:0000 8 80m cog—anon 8055 8on 30H m0 80:52 00 8080: 00:098.: 00 0200800 wcgmoumom MO mmOGUOOO Each. “0 HODEDZ hobs—3: Each. MO hon—832 $38. >88 EGEEEM .m 033. 56 Bt gene in the clone FL1607-A30. However, the FL1607-A30 x ATL-PVYS progeny showed segregation distortions from the expected Mendelian segregation ratio. This distortion could be due to chance, linkage between the gene of interest and other genes that affect gametic or zygotic selection, or linkage to lethal alleles (Aron et al. 1997). The observed inheritance patterns for the PVY cp gene of the progenies in which ATL-PVYS clone was involved, are incongruent with the Southern blot analysis. One way to explain the observed pattems'is that the two copies are linked in coupling. This conclusion is also verified by the segregation pattern for the NPT II gene in the same progenies. The selectable marker gene used, neomycin phosphotransferase II (N PT 11) detoxifies neomycin, kanamycin and G418 by phosphorylation. Our selection medium was enriched with the antibiotic kanamycin, which is the most widely used selection agents in dicot transformation systems (Webb and Morris 1992). The kanamycin assay indicated stable integration and expression of NPT II gene, and provided evidence that the inserted NPT II gene is inheritance as a single dominant alleles. It also corroborates the segregation pattern found in the ATL-PVYS x FL1607-A30, ATL-PVYS x MS702- 80, ATL-PVYS x Lemhi Russet progenies using PCR. However, the selfed progeny showed a ratio of 1:1 both in the kanamycin assay and the PCR analysis that deviated from the Mendelian ratios expected either for one or two inserted copies of the gene. This distortion from the Mendelian ratios may be explained for bias due to the low number of samples used, or a linkage of these copies to factor involved in self-incompatibility. Since the same ratio appears in the PCR analysis and in the Kanamycin assay and this distortion from the Mendelian ratio appears only in the selfed progeny of ATL-PVYS and not in 57 other progenies with ATL-PVYS as parent, the second hypothesis is more likely. Currently, the selectable marker is used to distinguish the transgenic genotypes after the transformation event. Since the selectable marker (NPT II) and PVY cp gene from ATL-PVYS seem not to segregate independently, a strategy to produce plants with multiple resistance using this clone could be selection in kanamycin medium of germinating seed. In this way, we assure to discard those plants that do not have the gene(s) of interest, shortening the selection process. In this study, we also produced plants that carry the combined genes for resistance against potato tuber moth and PVY virus, the proportion of these plants can be determined by the combination of the individual probabilities of these genes, when no distortion was found. A total of 47 individuals with both resistant genes were obtained. Further research is needed to determine the value of this multiple resistance, since the loss of transgene expression does not always correlate with the loss of the transgene, but rather with its inactivation (Finnegan and McElroy 1994). Therefore, greenhouse and field studies are needed to evaluate the level of resistance to potato tuber moth and virus. REFEREN Adang M. J., Brody M. S., Cardineau G., Eagan N., Roush R. T., Shewmaker C. K. Jones A., Oakes J. V. and McBride K. E. 1993. The reconstruction and Expression of a Bacillus thuringiensis Cry IIIA gene in protoplasts and potato plant. Plant molecular biology 21:1131-1 145. Agaisse H., and Lereclus D. 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? . Journal of Bacteriology 177(21):6027-6032. Aron Y., Czosnek H. and Gazit S. 1997. Segregation Distortion and Linkage of Mango Isozyrne Loci. HortScience 32(5):918-920. Aronson A. 1994. Bacillus thuringiensis and its use as a biological pesticide. In: Plant Breeding Reviews pp. 19-45. Vol. 12. New York: John Wiley & Sons, Inc. Audy P., Palukaitis P., Slack SA. and Zaitlin M. 1993. Replicase-mediated resistance to potato virus Y in transgenic tobacco plants. Molecular Plant-microbe Interactions 7(1): 15-22. Barton K. A. and Miller M. J. 1993. Production of Bacillus thuringiensis Insecticidal Proteins in Plants. In: Transgenic plants, vol 1, Engineering and utilization pp.297-315. Academic press Inc. San Diego, California Baum J .A., Coley D.M., Gilbert M.P., Jany CS. and Gawron-Burke C. 1994. Novel cloning vector for Bacillus thuringiensis, American Society for Microbiology 56(1 1):3420—3428. Baum J ., Kakefuda M. and Gawbron-Burke C. 1996. Engineering Bacillus thuringiensis biopesticide with and Indigenous site-specific recombination system. American society for Microbiology 62(12):4367-4373 58 59 Belknap W., Corsini D., Pavek J ., Snyder G., Rockhold D., and Vayda M. 1994. Field performance of transgenic Russet Burbank and Lemhi Russet potatoes. American Potato Journal 71: 285-295. Blizzard B.L., Schnepf HE. and Kronstad J .W. 1991. Expression of Cry IB crystal protein gene of Bacillus thuringiensis. Mol. Gen. Genet. 231:59-64. Bora R.S., Murty M.G., Shenbagarathai R. and Vaithilingam S. 1994. Introduction of a lepidopteran-specific insecticidal crystal protein gene of Bacillus thuringiensis subsp. kur'staki by conjugal transfer into a Bacillus megaterium strain that persists in the cotton Phyllosphere. Applied and Environment Microbiology 60(1):214- 222. Carozzi N.B., Warren G.W., Desai N., Jayne S.M., Lotstein R., Rice D.A., Evola S. and Koziel M.G. 1992. Expression of a chimeric Camv 3SS Bacillus thuringiensis insecticidal protein gene in transgenic tobacco. Plan Molecular Biology 20:539- 548. Ceron J ., Ortiz A., Quintero R., Guereca L. and Bravo A. 1995. Specific PCR primer directed to identify Cry I and Cry 111 genes within a Bacillus thuringiensis Strain collection. America society for microbiology 61(1 1):3826-3831. Cheng, J ., Bolyard,M., Saxena, R., and Sticklen, M. 1992. Production of insect resistant potato by genetic transformation with a a-endotoxin gene from Bacillus I thuringiensis var Kurstaki. Plant Science 81: 83-91. Clark W.G., F itchen J .H. and Beachy RN. 1994. Studies of coat protein-mediated resistance to TMV: I. the PM2 assembly defective mutant confers resistance to TMV. Virology 208: 485-491. 60 Das G.P. 1995. Plants used in controlling the tuber moth, Phthorimaea opercullela (Zeller). Crop Protection l4(8):63 1-636. Das G.P. and Raman K.V. 1994. Alternate host of the potato tuber moth, Phthorirnaea operculella (Zeller), review. Crop Protection 13(2):83-86. Dean D.H., Rajamohan F., Lee M.K., Wu S.-J., Chen X.J., Alcantara E. and Hussain SR. 1996. Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis- a minireview. Gene 179:111-117. de Maagd R.A., Kwa M.s., van der Klei H.,Yamamoto T., Schipper B., Vlak J. M., Stekema W. J. and Bosch D. 1996 Domain III substitution in Bacillus thuringiensis Delta-Endotoxin Cry IA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. American society . for microbiology 62(5): 1 537—1 543. Dhar A.K., Singh RF. and Boucher A. 1994. Molecular cloning and sequencing of the capsid and the nuclear inclusion protein genes of a North American PVY“ isolate. Can. J. Microbiol. 40:798-804. Dinant S., Blaise F., Kusiak C., Astier-Manifacier S. and Albouy J. 1993. Heterologous resistance to potato virus Y in transgenic tobacco plants expressing the coat protein gene of lettuce mosaic virus. Phytopathology 83(8):818-824. Dolgov S.V., Mityshkina T.U., Rukavtsova EB. and Buryanov Y.I. 1995. Production of transgenic plants of Chrysanthemun morifolium ramat with the gene of B. thuringiensis 6-endotoxin. Acta Horticultirae 420:46—47 Dolja V.V., Haldeman, Robertson N.,Dougherty W. and Carrington J.C. 1994. Distinct functions of capsid protein in assembly and movement of tobacco eth potyvirus in a .;. plants. The EMBO journal 13(6):]482-1491. Dougherty W.G. and Canington J .C. 1988. Expression and function of potyvirus gene products. Ann. Rev. Phytopathol. 26:123—143 Du C. and Nickerson K. W. 1996. Bacillus thuringiensis HD-73 Spore have surface- localized Cry Ac Toxins: Physiological and Pathogenic Consequences. American society for Microbiology 62(10):3722-3726. Edwards KC. and Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nuc. Acids Res. l9(6):1349. Farinelli L. and Malnoe P. 1993. Coat protein gene-mediated resistance to potato virus Y in tobacco: Examination of resistance mechanisms- is the transgenic coat protein required for protection?. Molecular Plant Microbe Interactions 6(3):284-292 F arinelli L. and Malnoe P. and Collet GR 1992. Heterologus encapsidation of potato virus Y strain 0 (PVY°) with the transgenic coat protein of PVY strain N (PVYn) I in Solanum tuberosum cv. Bintje. Bio/technology 10: 1020-1025. F erh W.R. 1993. Polyploidy. In: Principles of cultivar development pp 59-65. Macmillan Publishing Company, Iowa Fenemore P.G. 1988. Host-plant location and selection by adult potato moth, Phthorimaea opercullela (Lepidoptera: Gelechiidae): A review. Journal of Insect Phisiology 34(3):175-177 Finnegan J. and McElroy D. 1994. Transgene inactivation: Plants fight back!. Bio/Technology 12:883-888. F ischhofl' D.A., Bowdish K.S., Perlak F.J., Marrone P.G., McCorrnik S.M., Niedermeyer , J .G., Dean D.A., Kusano-Kretzmer K.,Mayer E.J., Rochester D.E., Roger S.G. 62 and Frayley RT. 1987. Insect tolerant transgenic tomato plants. Bio/technology 5:807-813. Gleave A.P., Hedges R.J. and Broadwell AH. 1992. Identification of an insecticidal crystal protein form Bacillus thuringiensis from previously described toxins. Journal of General Microbiology 138:55-62 Grochulski P., Masson L., Borisova S., Pusztai-Carey M., Schwartz J .-L., Brousseau R. and Cygler M. 1995. Bacillus thuringiensis_ Cry Ia(a) insecticidal toxin: Crystal structure and channel formation. J mol. Biol. 254:447-464 Grumet R. 1990. Genetically Engineered Plant Virus Resistance. Hort Science 25: 509- 5 l3. Grumet R. 1994. Development of virus resistant plants via genetic engineering. In: Plant Breeding Reviews pp. 47-79. Vol. 12. New York: John Wiley & Sons, Inc. Grumet R. 1995. Genetic engineering for crop virus resistance. Hort Science 30(3): 449- 456. Grumet R.and Fang G. 1993. Genetic engineering of potivirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gen. Molecular Plant-Microbe Interaction 6: 358-367. Hackland A.F., Rybicki RP. and Thomson J .A. 1994. Coat protein-mediated resistance in transgenic plants: Brief review. Archives of virology 139: 1-22 Hartl D.L. 1994. Principles of heredity and variation. In: Genetics pp 1-27. Jones and Bartlett publisher, Boston .3 J1 Herrera G., Snyman S. J. and Thomson J. 1994. Construction of a bioinsecticidal Strain of Pseudomonas flourescens active against the sugarcane borer, Eldana saccharina. American society for Microbiology 60(2):682-690. Hoekema A. and Comelissen B.J.C. 1989 Engineering virus resistance in agricultural crops. Plant Molecular Biology 13:337-346. Iannacone R., Fiore M.C., Macchi A., Grieco P.D., Arpaia S., Perrone D., Mennella G., Sunseri F., Cellini F. and Rotino G.L. 1995. Genetic engineering of eggplant . (Solanurn melongena L.). Acta Horticuturae 392:227-233. James D.J., Passey A.J., Dandekar A.M. and Uratsu S.L. 1993. Transgenic apples and strawberries: Advance in transformation, introduction of genes for insect resistance and field tissue cultured plants. Acta Horticulturae 336:179-184. Jansens S., Comelissen M., De Clercq R., Reynaerts A. and Peferoen M. 1995. Phthorimaea opercullela (Lepidoptera: Gelechiidae) resistance in potato by expression of the Bacillus thuringiensis Cry IA(b) Insecticidal crystal protein. Journal of Economic Entomology 88(5):]469-1476. Kalman S., Kiehne K. L., Libs J. L. and Yamamoto T. 1993. Cloning of a novel cry IC- type gene from a strain of Bacillus thuringiensis subsp. galleriae. Applied and Environmental Microbiology 59(4): 1 13 1 -1 137. Kavanagh T.A. and Spillane C. 1995. Strategies to engineering virus resistance in transgenic plants. Euphytica 85: 149-158. Kleiner K.W., Ellis D.D.,McCown B.H. and Kenneth FR. 1995. Field evaluation of transgenic Popplar expressing a Bacillus thuringiensis cry IA(a) 8-endotoxins gene against forest tent Carterpillar (Lepidoptera:Lasiocarnpidae) and Gypsy 64 moth (lepidoptera: Lymantriidae) following winter dormancy. Entomological Society of America 24(5):]358-1364. Kroschel J. and Koch W. 1996. Studies on the use of chemicals, botanicals and Bacillus thuringiensis in the management of the potato tuber moth in potato stores. Crop Protection 15(2): 197-203. Kuo W.-S. and Chak K.-F. 1996. Identification of novel cry type genes from Bacillus thuringiensis strain on the bases of the restriction fragment length polymorphism of the PCR-Amplified DNA. American society for Microbiology 62(4):1369- 1377. Lambert B., Buysse L., Decocko C.,Jansens J ., Piens C., Saey B., Seurinck J .S., van Audenhove K., van Rie J ., van Vliet A. and Peferoen M. 1996. A Bacillus thuringiensis Insecticidal crystal protein with a high activity against members of the family Noctuidae. American society for Microbiology 62(1):80-86. Li X.-b., Mao H.-Z. and Bai Y.-Y. 1995. Transgenic plants of rutabaga (Brassica napobrasssica) tolerant to pest insects. Plant Cell Reports 15:97-101. Liang X.-Y., Zhu Y.-X., Mi J.-J. and Chen Z.-L. 1994. production of virus resistance and insect tolerant transgenic tobacco plants. Plant Cell Reports 14: 141-144. Lopez-Meza J .E. and Ibarra J.E. 1996. Characterization of a Novel Strain of Bacillus thuringiensis. American society for Microbiology 62(4):1306-1310. Mackenzei, D., Tremaine, J. and McPherson, J. 1991 . Genetically engineered resistance to potato virus S in potato cultivar Russet Burbank. Molecular Plant-Microbe Interaction 4: 95-101. 65 Malnoe P., Farinelli L., Collet G.P. and Reust W. 1994. Small-scale field test with transgenic potato, cv. Bintje, to test resistance to primay and secondary infections with potato virus y. Plant Molecular Biology 25:963-975 Martin RR. 1994. Genetic engineering of potatoes. America potato journal 71:347-358. Meza R., Nunez-Valdez M.-E., Sanchez J. and Bravo A. 1996. Isolation of Cry 1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system. FEMS 145:333-339. Moar W.J., Pusztai-Carey M., van Faassen H., Bosch D., Frutos R., Rang C., Luo K. and ‘ Adang MJ. 1995. Development of bacillus thuringiensis CryIC resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae). Applied and Environmental Microbiology 61(6): 2086-2092. Nejidat A. and Beachy RN. 1989. Decreased levels of TMV coat protein in transgenic tobacco plants at elevated temperatures reduce resistance to TMV infection. Virology 173:531-538. Ohshima K., Inoue AK. and Shikata E. 1993. Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene. Archives of Virology 128:15-28. Orduz S., Diaz T., Thiery 1., Charles J .-F . and Rojas W. 1994. Crystal proteins from Bacillus thuringiensis serovar. medellin. Applied Microbiology and Biotechnology 40:794-799. Parrot W.A., All J .N., Adang M.J., Bayley MA. and Steward C.N. 1994. Recovery and evaluation of soybean plant transgenic for Bacillus thuringiensis var. Kurstaki insecticidal gene. In vitro Cell. Dev. Biol. 30:114-149. 66 Peferoen Marnix. 1992. Engineering of insect-resistant plants with Bacillus thuringiensis Cristal protein genes. In: Plant genetic manipulation for crop protection. Edited by A.M.R. Gatehouse, V.A. Hilder, D.Boulter. UK CAB International pp135-153 Pehu T.M., Maki-Valkama T.K., Valkonen J .P.T., Koivu K.T., Lehto K.M. and Pehu ER 1995. Potato plants transformed with a potato virus Y P1 gene sequence are resistant to PVY°. American Potato journal 72:523-533 Perlak F.J., Fuchs R.L., Dean D.A., McPherson S.L. and Fischhofi‘ DA. 1991. ‘ Modification of the coding sequence enhances plant expression of insect control protein genes. American society for Microbiology 88:3324-3328. Perlak F.J., Stone T.B., Muskopf Y.M., Petersen L.J., Parker G.B., McPherson S.A., Wyman J ., Love 8., Reed 0., Biever D. and Fischhoff D. A. 1993. Genetically improved potatoes: protection from damage by colorado potato beetles. Plant Molecular Biology 22:3 13-321. Perlak F., Stone T., Muskopf Y., Petersen L., Parker 6., McPherson S., Wyman J ., Love 8., Reed G., Biever D. and F ischhoff D. 1993. Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Molecular Biology 22: 313-321. Poehlman J .M. and Sleper DA. 1995. Breeding Potato. In: Breeding field crops pp 419- 433. Iowa State University Press, Iowa. Rarnan R Booth R and Palacios M. 1987. Control of the potato tuber moth, Phthorimaea opecullela (Zeller), in rural potato store. In "Integrated Pest Management for Tropical Root and Tuber Crops".pp 95-107. American printing group Co. Ltd., Bangkok, Thailand. 67 Jl I Rarnan K. V. and Altman D. W. 1994. Biotechnology initiative to achieve plant pest and diseases resistance. Crop protection 13(8): 591-596 Regev A., Keller M., Strizhov N., Sneh B., Prudovsky E., Chet I., Ginzberg I., Koncz- Kalman Z., Koncz C., Schell J. and Zilberstein A. 1996. Synergistic activity of a Bacillus thuringiensis 6-Endotoxin and 3 Bacterial Endochitinase against Spodoptera Iittorallis larvae. American society for Microbiology 62(10):3581- 3586. Register J .C. and Beachy RN. 1988. Resistance to TMV in transgenic plants results from interference with an early event in infection. Virology 166: 524-532. Reimann-Philipp U. and Beachy RN. 1993. Coat protein-mediated resistance in transgenic tobacco expressing the tobacco mosaic virus coat protein from tissue- specific promoters. Molecular Plant-Microbe Interactions 6(3):323-329. Salamitou S., Agaisse H., Bravo A. and Lereclus D. 1996. Genetic analysis of Cry IlIa gene expression in Bacillus thuringiensis. Microbiology, 142:2049-2055. Sambrook J ., Fritsch ER and Maniatis T. 1989. Molecular Cloning: a laboratory manual. 2'“1 ed. Cold Spring Harbor, NY. Sandford J .C. and Johnston SA. 1985. The concept of pathogene derived resistance: 1 Deriving resistance genes from the parasite own genome. J. theor. Biol. 113:395- 405. Serres R.A., McCown B.H. and McCabe DE. 1993. Application of biotechnology to cranberry: a model for fruit crop improvement. Acta horticulturae 345:??? 68 Siddig SA. 1988. Cultural means of controlling potato tuber moth (Phthorimaea opercullela Zell.) and improvement of potato yield and quality in the sudan. Acta Horticulturae 218:281-287 Sims S.R., Berberich S.A., Nida D.L., Segalini L.L., Leach J.N., Ebert CC. and Fuschs R.L. 1996. Analysis of expressed proteins in fiber fractions from insect-protected and Glyphosate—tolerant cotton varieties. Crop Science 36: 1212-1216. Singh M. and Singh RP. 1995. Host dependent cross protection between PVYn, PVYo, and PVA in potato cultivars and Solanum brachycarpum. Canadian Journal of plant pathology 17:82-86. Singh M., Singh RP. and Somerville TH. 1994. Evaluation of tuber-bearing Solanum species for symptomology, as diagnostic host, and sources of immunity to potato virus Y necrotic strain (PVY"). American Potato Journal 71 :567—579. Sokal RR and Rohlf F.J. 1995. Amalysis of frequencies. In: Biometry pp 685-793. W.H. Freeman and Company, New York. Stewart C. N., Adang M.J., All J .N., Boerma H.R., Cardineau G., Tucker D. and Parrott W. A. 1996 Genetic transformation, recovery, and characterization of fertile V soybean transgenic for a synthetic Bacillus thuringiensis. Plant Physiol. 112:121- 129. Sticklen, M., Ebora, R., Cheng, J ., Ebora, M., Bolyard, M., Saxena, R. and Miller D. 1993. Genetic transformation of potato with Bacillus thuringiensis H0 73 Cry I A(C) gene and development of insect resistant plant. Biotechnology in Agriculture. Page 233-236. Kluwer Academic Publishers. 69 Strizhov N., Keller M., Mathur J .,Koncz-Kalman Z., Bosch D., Prudovsky E., Schell J ., Sneh B., Koncz C. and Zilberstein A. 1996. A synthetic Cry IC gene, encoding a Bacillus thuringiensis d-endotoxin, confer Spodoptera resistance in alfalfa and tobacco. American society for Microbiology 93:15012-15017. Thole V., Dalmay T., Burgyan J. and Balazs E. 1993. Cloning and sequencing of potato virus Y (Hungarian isolate) genomic RNA. Gene 123:149-156. Trivedi T.P. and Rajagopal D. 1992. Distribution, biology, ecology and management of potato tuber moth, Phtorimaea opercullela (Zeller) (Lepidoptera:Gelechiidae): a review. Tropical Pest Management 38(3):279-285. Valkonen J .P.T., Slack SA. and Plaisted RL. 1994. Use of the virus strain group concept to characterize the resistance to PVX and PVY0 in the potato cv. Allegancy. American Potato Journal 71 :507-516. Vallejo R. L., Collins W.W., Schiavone R.D., Lommel SA. and Young J.B.1994. Extreme resistance to infection by potato virus Y and potato virus X in an advanced hybrid Solanum phureja-S. stenotonum diploid potato population. American Potato journal 71:617-628. Van Aarssen R., Soetaert P., Starn M., Dockx J .,Gossele V., Seurinck J., Reynaerts A. and Comelissen M. 1995. Cry IA(b) transcript formation in tobacco is inefficient. Plant Molecular Biology 28:513-524. Van den Elzen P.J.M., Huisman M.J., Posthumus-Lutke D., Jongedijk E., Hoekema A. and Comelissen B.J.C. 1989. Engineering virus resistance in agricultural crops. Plant Molecular Biology 13:337-346. 70 Von Tersch M.A., Robbins H.L., Jany CS. and Johnson T.B. 1991. Insecticidal toxins from Baccillus thuringiensis subsp. kenyae: Gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki Cry IA(c) toxins. American society for Microbiology 57(2):349-358. Webb K.J. and Morris Phillip 1992. Methodologies of plant transformation. In: Plant Genetic Manipulation for crop protection. Edited by A.M.R. Gatehouse, V.A. Hilder, D.Boulter. UK CAB International pp 7-44. Whiteley HR. and Schnepf HE. 1986. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annual reviews of microbiology 40:549-576 Williams S., Friedrich L., Dincher S., Carozzi N., Kessmann H., Ward E. and Ryals J. 1992. Chemical regulation of Bacillus thuringiensis 8—endotoxins expression in transgenic plants. bio/technology 10:540-543. Wisniewski L., Powel P.A., Nelson RS. and Beachy RN. 1990. Local and systemic movement of tobacco mosaic virus (TMV) in tobacco plants that express the TMV coat protein gene. Plant Cell 2:559-567. Wong E., Hironaka GM. and F ischhoff DA. 1992. Arabidopsis thaliana small] subunit . leader and transit peptide enhance the expression of Bacillus thuringiensis protein in transgenic plants. Plan Molecular Biology 20:81-93. Wunn, J ., Kloti, A., Burkhardt, P.K., Godhosh, G., Launis, K. and Iglesias V. 1996. Transgenic Indica Rice breeding line IR58 expressing a synthetic Cry 1 A(b) gene from Bacillus thuringiensis provides effective insect pest control. Bio/Technology 14: 171-176. MICHIGAN sran UNIV. LIBRARIES llHllllllHUIIWIHMIIllHlllllllllllllWWI“HI 31293016914297