Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

No description available.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Delaware, 2005. / Principal faculty advisors: John C. Bernard and John D. Pesek, Dept. of Food and Resource Economics. Includes bibliographical references.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular characterisation of four sugarcane lines, regenerated after transformation, confirmed that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST present in line 2153, and a minimum of five copies (or portions thereof) present in line 2121. The novel gene was successfully transcribed and translated in both lines, as confirmed by cDNA gel blot hybridisation and HPLC analysis respectively. Kestose production was stable under field resembling conditions and levels of this trisaccharide progressively increased with increasing internodal maturity from 7.94 ± 2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode 16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the oldest sampled internode hence the lines were referred to as high- and low-1-kestose producers. The production of 1-kestose did not reduce sucrose levels in the transgenics, instead they contained significantly higher levels of sucrose than the control line NCo310 (p<0.01, N=72). The production of this alternative sugar in addition to elevated sucrose levels significantly increased the total sugar content in the transgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer had statistically more total sugar than the low-1-kestose producer (p<0.01, N=72). Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC 3.2.1.26) from non-transgenic sugarcane internodal tissues were separated and partially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAI eluting at approximately 100 mM KCl in a linear gradient while NI eluted at approximately 500 mM KCl. The final specific activities of SAI and NI were 88.57 pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- fold purification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 and that for soluble acid invertase less than 5.0. Due to the broad pH activities of the invertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity of these invertases for 1-kestose hydrolysis was tested. The invertases displayed hyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Km values ranging from 50 - 247 mM. Furthermore, the presence of 200 mM 1-kestose had an inhibitory effect on SAI-mediated sucrose hydrolysis reducing activity to 51 % and 54 % for isoform 1 and 2 respectively. To determine whether carbon allocation had been altered by the expression and activity of 1-SST, 14C whole-plant radiolabelling experiments were conducted. Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation of carbon to different parts of the culm was assessed. There was no significant difference in the distribution of total radiolabel down the culm of the three sugarcane lines (p>0.05, N=72). However, the percentage of total radiolabel in the water-soluble fraction per internode in the high-1-kestose producer was significantly higher than the other two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsoluble fraction in this transgenic was concomitantly lower than in the other lines. Carbon was therefore redirected from the water-insoluble fraction to the water-soluble fraction to account for the additive production of 1-kestose. The expression of 1-SST in sugarcane therefore established an additional carbohydrate sink by the flow of carbon from the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrose pool, but rather stimulated carbon channelling into this pathway, thereby increasing the non-structural carbohydrate content of the plant in one of the transgenics. The work described in this study is the first to report on carbon partitioning in 1- kestose-producing sugarcane grown under field resembling conditions. It contributes significantly to an improved understanding of carbon partitioning in the culm, and demonstrates that an alternative sugar can be produced in sugarcane under field resembling conditions.

Spodoptera frugiperda (J. E. Smith) e Pseudoplusia includens (Walker) são importantes insetos-praga no algodoeiro (Gossypium hirsutum L.) devido às injúrias de desfolha e destruição de estruturas reprodutivas no caso de S. frugiperda. Os eventos de algodão Bt que expressam as proteínas Cry1Ac/Cry2Ab2 (Bollgard® II) e Cry1Ac/Cry1F (WideStrike(TM)) de Bacillus thuringiensis Berliner são ferramentas disponíveis para o controle dessas espécies-praga. A fim de subsidiar o Manejo da Resistência de Insetos (MRI) foram conduzidos estudos em laboratório para avaliar a sobrevivência e desenvolvimento de S. frugiperda e P. includens nos eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F. Em bioensaios com discos de folhas, a eficácia de controle de neonatas nos dois eventos de algodão Bt foi superior a 80% para S. frugiperda e de 100% para P. includens. Em bioensaios com brácteas com neonatas de S. frugiperda, a eficácia de controle de ambos os eventos de algodão Bt também foi superior a 80%. As lagartas de S. frugiperda sobreviventes em algodão Bt apresentaram severa inibição de desenvolvimento larval em folhas (> 75%) e brácteas (> 44%). Em bioensaios com simulações de alimentação larval, as quais consistiam em grupos de lagartas alimentadas com o algodão Bt aos 0, 3, 6, 9, 12, 15 e 18 dias após a inoculação (DAI), S. frugiperda e P. includens demonstraram que a suscetibilidade dessas espécies diminuiu com o avançar do desenvolvimento larval. Para S. frugiperda, em todas as simulações de alimentação com o algodão Cry1Ac/Cry2Ab2 houve lagartas que atingiram as fases de pupa e adulto. Por outro lado, quando expostas ao algodão Cry1Ac/Cry1F, somente algumas das lagartas de 5º e 6º ínstares atingiram as fases de pupa e adulto. Para P. includens, somente lagartas no 6º ínstar atingiram as fases de pupa e adulto quando alimentadas com os dois eventos de algodão Bt. Os parâmetros biológicos de S. frugiperda sobreviventes em algodão Cry1Ac/Cry2Ab2 foram afetados negativamente com aumento da duração da fase larval (? 9 dias), baixa viabilidade larval (1,4%) e de insetos que completaram o ciclo biológico (0,9%), aumento no intervalo entre gerações (? 9 dias) e redução da taxa intrínseca de crescimento populacional (? 83%). Os eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F são promissores no controle de S. frugiperda e P. includens. No entanto, a atividade inseticida dos dois eventos de algodão Bt em lagartas de S. frugiperda e P. includens diminui com o desenvolvimento larval e essa constatação deve ser considerada em programas de MRI, especialmente na disposição espacial do refúgio. / Spodoptera frugiperda (J. E. Smith) and Pseudoplusia includens (Walker) are important insect pests in cotton (Gossypium hirsutum L.) due to damage on leaves and reproductive structures in the case of S. frugiperda. The events of Bt cotton expressing proteins Cry1Ac/Cry2Ab2 (Bollgard® II) and Cry1Ac/Cry1F (WideStrike(TM)) from Bacillus thuringiensis Berliner are tools available to control these pest species. To develop an Insect Resistance Management (IRM), we performed laboratory studies to evaluate the survival and development of S. frugiperda and P. includens. In fresh leaf discs bioassays, the control efficacy of neonates in both Bt cotton events was greater than 80% mortality for S. frugiperda and 100% for P. includens. In fresh bracts bioassays to neonates of S. frugiperda, the control efficacy of both Bt cotton events was over 80%. The surviving larvae of S. frugiperda in Bt cotton showed severe growth inhibition (weight and instar) in leaves (> 75%) and bracts (> 44%). In simulations feed bioassays with larvae, which consisted of groups of larvae fed on Bt cotton at 0, 3, 6, 9, 12, 15 and 18 days after inoculation (DAI), S. frugiperda and P. includens showed that the susceptibility of species decreases with advancing larval development. For S. frugiperda, in all feed simulations with cotton Cry1Ac/Cry2Ab2 had caterpillars that reached pupae and adult stages. Moreover, when exposed to cotton Cry1Ac/Cry1F, only some caterpillars of 5th and 6th instars reached pupae and adult stages. For P. includens, only some caterpillars of 6th instar reached pupae and adult stages when fed with two events of Bt cotton. The biological parameters of S. frugiperda fed on cotton Cry1Ac/Cry2Ab2 were negatively affected with increasing duration of the larval stage (? 9 days), reduced larval viability (1,4%) and insects that completed the life cycle (0,9%), increased the generation time (? 9 days) and decreased the intrinsic rate of increase (? 83%). The events of cotton Cry1Ac/Cry2Ab2 and Cry1Ac/Cry1F are promising for the control of S. frugiperda and P. includens. However, the insecticidal activity of both events of Bt cotton in larvae of S. frugiperda and P. includens decreases through larval development and this finding should be considered in programs of MRI, especially in the spatial arrangement of the refuge.

A soja geneticamente modificada MON 87701 × MON 89788, Glycine max (L.) Merrill, que expressa genes que codificam a proteína Cry1Ac de Bacillus thuringiensis var. kurstaki Berliner e a proteína 5-enolpiruvilchiquimato-3-fosfato sintase de Agrobacterium sp., foi liberada para uso comercial no Brasil em 2010. Para subsidiar um programa de Manejo da Resistência de Insetos (MRI) foi realizada a avaliação de risco de evolução de resistência a Cry1Ac para as pragas-alvo primárias, Anticarsia gemmatalis Hübner e Pseudoplusia includens (Walker), a praga-alvo secundária Heliothis virescens (Fabricius) e as pragas nãoalvo Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) e Spodoptera frugiperda (J. E. Smith). Em bioensaios com a proteína Cry1Ac purificada incorporada em dieta artificial, verificou-se que Cry1Ac foi extremamente ativa para A. gemmatalis [CL50 (IC 95%) = 0,23 (0,15 - 0,34) g Cry1Ac/mL de dieta], P. includens [CL50 (IC 95%) = 3,72 (2,65 - 4,86) g Cry1Ac/mL de dieta] e H. virescens [CL50 (IC 95%) = 0,026 (0,021 - 0,033) g de Cry1Ac/mL de dieta]. Em contraste, na máxima concentração testada de 100 g Cry1Ac/mL de dieta, S. cosmioides e S. eridania apresentaram mortalidade < 13%, e S. frugiperda de » 50%. Em bioensaios com tecido liofilizado de soja MON 87701 × MON 89788, diluído 25 vezes em dieta artificial, houve 100% de mortalidade para A. gemmatalis e H. virescens e até 96% para P. includens. Entretanto, as lagartas sobreviventes de P. includens apresentaram enfezamento larval > 95%. Em bioensaios com discos de folha em laboratório (para A. gemmatalis, P. includens e H. virescens), vagens (para H. virescens) e elevada infestação em condições de casa de vegetação e de infestação natural em campo (para A. gemmatalis e P. includens) foram obtidas alta eficácia da soja MON 87701 × MON 89788 no controle das pragas-alvo primárias e secundária. Por outro lado, a soja MON 87701 × MON 89788 apresentou baixa eficácia para S. cosmioides e S. eridania (mortalidade < 7%) e atividade intermediária para S. frugiperda (32 a 47% mortalidade). A soja MON 87701 × MON 89788 não afetou os parâmetros biológicos de S. cosmoides e S. eridania. Para S. frugiperda houve prolongamento da fase larval (» 5 dias), menor viabilidade larval e total, aumento no intervalo entre gerações (» 8 dias) e redução na taxa intrínseca de crescimento (» 41%). No contexto do MRI, a soja MON 87701 × MON 89788 expressa a proteína Cry1Ac em níveis que constituem alta dose para A. gemmatalis e H. virescens, e muito próximo a alta dose para P. includens. Por outro lado, para as espécies de Spodoptera, a soja MON 87701 × MON 89788 é um evento de baixa dose, pois permite que indivíduos suscetíveis completem o ciclo biológico. Em termos de MIP, a soja MON 87701 × MON 89788 apresenta um elevado nível de controle de A. gemmatalis, P. includens e H. virescens. No entanto, a soja MON 87701 × MON 89788 não ocasiona controle efetivo de espécies de Spodoptera. As informações obtidas no presente trabalho contribuirão para subsidiar programas de MRI e preservar a durabilidade dessa tecnologia para o MIP-Soja no Brasil. / Genetically modified MON 87701 × MON 89788 soybean, Glycine max (L.) Merrill, expressing genes that code for the Cry1Ac protein of Bacillus thuringiensis var. kurstaki Berliner and the 5-enolpyruvylshikimate-3-phosphate synthase protein of Agrobacterium sp., has been registered for commercial use in Brazil in 2010. To develop a program of Insect Resistance Management (IRM) for this event, we conducted resistance risk assessment to the primary target pests, Anticarsia gemmatalis Hübner and Pseudoplusia includens (Walker), the secondary target pest Heliothis virescens (Fabricius) and the non-target pests Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) and Spodoptera frugiperda (J. E. Smith) to the Cry1Ac protein. In bioassays with purified Cry1Ac protein incorporated into artificial diet, Cry1Ac was highly active for A. gemmatalis [LC50 (95% FL) = 0.23 (0.15 - 0.34) g Cry1Ac/mL of diet], P. includens [LC50 (95% FL) = 3.72 (2.65 - 4.86) g Cry1Ac/mL of diet] and H. virescens [LC50 (95% FL) = 0.026 (0.021 - 0.033) g Cry1Ac/mL of diet]. In contrast, even at the highest concentration tested of 100 g Cry1Ac/mL of diet, the mortality observed to S. cosmioides and S. eridania was < 13% and to S. frugiperda was » 50%. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue, diluted 25 times in artificial diet, there was 100% mortality to A. gemmatalis and H.virescens and up to 96% for P. includens. However, the surviving P. includens larvae showed > 95% larval stunting. In bioassays with leaf discs in the laboratory to (A. gemmatalis, P. includens and H. virescens), pods (H. virescens), high pest infestation studies under greenhouse conditions and in natural infestation in the field (to A. gemmatalis and to P. includens) showed a very high level of efficacy against these primary and secondary target pests. On the other hand, soybean MON 87701 × MON 89788 showed low efficacy to S. cosmioides and S.eridania (mortality < 7%), and intermediate activity to S. frugiperda (32 to 47% of mortality). MON 87701 × MON 89788 soybean did not affect the biological parameters of S. cosmoides and S. eridania. For S. frugiperda, the larval stage was prolonged (» 5 days), with reduction on larval and total viability, increase in generation time (» 8 days) and reduction in the intrinsic rate of increase (» 41%). In the context of IRM, MON 87701 × MON 89788 soybean expresses the Cry1Ac protein at levels that are high-dose for A. gemmatalis and H. virescens, and near to the highdose for P. includens. On the other hand, for the Spodoptera species, MON 87701 × MON 89788 soybean is a low-dose event, because it allows susceptible individuals to complete larval developement. In terms of IPM, MON 87701 × MON 89788 soybean provided a high level of control of A. gemmatalis, P. includens and H. virescens. However, MON 87701 × MON 89788 soybean was not effective to control Spodoptera species. The information obtained in this research will contribute to develop IRM programs and to preserve the durability of this technology for soybean IPM in Brazil.

O presente estudo objetivou identificar as interações do Algodão Bt que expressa Cry1Ac (Bollgard®), com o predador Podisus nigrispinus Dallas (Hemiptera: Pentatomidae), no manejo da resistência de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) a lambda-cyhalothrin em duas condições: ausência e presença do inseticida imidacloprid. Foram utilizadas lagartas de S. frugiperda provenientes das seguintes condições: linhagens resistentes (1) e suscetíveis (2) a lambda-cyhalothrin alimentadas de folhas de algodoeiro Bollgard® (DP 404 BG); e linhagens resistentes (3) e suscetíveis (4) a lambda-cyhalothrin alimentadas de folhas de algodoeiro não transgênico (cultivar DP4049). Os resultados dessa pesquisa revelaram que na ausência de imidacloprid, independente do tratamento, o comportamento de predação foi melhor representado pelo tipo III de resposta funcional, pois a taxa de ataque aumentou linearmente em todas as condições estudadas (a= bN). Houve diferenças entre o tempo de manipulação (Th) de fêmeas do predador que receberam lagartas suscetíveis a lambda-cyhalothrin, previamente alimentadas de algodão não transgênico, em relação aos demais tratamentos. Na densidade de 16 lagartas/predador, o número de lagartas predadas por fêmeas de P. nigrispinus foi significativamente superior em lagartas resistentes a lambda-cyhalothrin, alimentadas de algodão Bt ou não Bt, em relação às lagartas suscetíveis alimentadas de algodão não Bt. Além do mais, se constatou que quando foram ofertadas 16 lagartas de S. frugiperda ao predador, o número de indivíduos predados foi significativamente inferior em lagartas suscetíveis a lambda-cyhalothrin que foram alimentadas de algodão não Bt, em relação as que receberam lagartas previamente alimentadas de algodão Bt. Na presença de imidacloprid, constatou-se que o comportamento de predação de P. nigrispinus foi afetado pelo neonicotinóide imidacloprid, sendo a curva assintótica do tipo II, a que melhor descreveu os dados da sua resposta funcional. Na presença de imadacloprid, o tempo de manipulação (Th) de fêmeas do predador não diferiu entre os tratamentos estudados. Todavia, a taxa de ataque foi representada por um decréscimo em função do aumento da densidade de lagartas ofertadas. Independente do tratamento (linhagem de S. frugiperda ou cultivar de algodão), o número de lagartas de S. frugiperda predadas por fêmeas de P. nigrispinus quando na exposição ao imidacloprid, foi significativamente inferior, especialmente na densidade de 16 lagartas/predador. Diante destes resultados, a pesquisa reforça que o custo adaptativo associado à resistência a lambda-cyhalothrin, assim como a cultivar de algodão Bt afetaram a taxa de predação de lagartas de S. frugiperda por fêmeas de P. nigrispinus, somente na maior densidade testada (16 lagartas/predador). O comportamento de predação de lagartas de S. frugiperda por fêmeas de P. nigrispinus foi negativamente afetado pelo inseticida imidacloprid. / This study aimed to identify the interactions of Bt cotton expressing Cry1Ac (Bollgard®), with the predator Podisus nigrispinus Dallas (Hemiptera: Pentatomidae), in resistance management of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) to lambda-cyhalothrin in two conditions: absence and presence of the insecticide imidacloprid. Larvae of S. frugiperda were used from the following conditions: resistant (1) and susceptible (2) strains to lambda-cyhalothrin fed Bollgard® cotton leaves (DP 404 BG); and resistant (3) and susceptible (4) strains to lambda-cyhalothrin fed non-transgenic cotton leaves (cultivar DP4049). The results of this study revealed that in the absence of imidacloprid, independent of treatment, the behavior of predation was best represented by the type III of functional response, because the attack rate increased linearly in all conditions studied (a = bN). There were differences between the handling time (Th) of females of the predator who received larvae of S. frugiperda susceptible to insecticides previously fed non-transgenic cotton in relation to other treatments. The density of 16 larvae/predator, the number of larvae preyed by female of P. nigrispinus was significantly higher in larvae of S. frugiperda resistant to lambda-cyhalothrin, fed on Bt cotton or non-Bt compared to susceptible larvae fed non Bt cotton. Moreover, when we offered 16 larvae of S. frugiperda to the predator, the number of larvae predate were significantly lower in larvae susceptible to lambda-cyhalothrin that were fed non-Bt cotton, compared to larvae that were previously fed on transgenic cotton. In the presence of imidacloprid, the predatory behavior of P. nigrispinus was affected by the neonicotinoid imidacloprid, and the asymptotic curve type II was the one that best described the data of the functional response. In the presence of imadacloprid, handling time (Th) of females of the predator did not differ among treatments. However, the attack rate was represented by a decrease due to the increase of the density of larvae offered. Regardless of the treatment (strain of S. frugiperda or cultivar of cotton), the predation larvae of S. frugiperda by females of P. nigrispinus when exposed to imidacloprid was significantly lower, especially at density of 16 larvae/predator. Given these results, the research reinforces the fitness cost associated to lambda-cyhalothrin resistance as well as Bt cotton affected the rate of predation on larvae of S. frugiperda by females of P. nigrispinus, only at the highest density tested (16 larvae/predator). The behavior of predation on larvae of S. frugiperda by females of P. nigrispinus was affected by insecticide imidacloprid.

No Brasil, doenças causadas pelo Papaya ringspot virus - type Watermelon (PRSV-W) e Zucchini yellow mosaic virus (ZYMV) reduzem a produção e a qualidade dos frutos de abobrinha-de-moita (Cucurbita pepo) e melancia (Citrullus lanatus), assim como em outras cucurbitáceas. O objetivo deste trabalho foi a obtenção de plantas transgênicas de abobrinha-de-moita e melancia resistentes ao PRSV-W e ao ZYMV. Um sistema eficiente de regeneração in vitro é necessário para a obtenção de plantas transgênicas. O sistema de organogênese in vitro de abobrinha-de-moita foi desenvolvido utilizando como explantes a região basal do cotilédone e um segmento do hipocótilo obtidos a partir de sementes germinadas in vitro. Os explantes foram cultivados em meio de cultura MS (MURASHIGE; SKOOG, 1962), suplementado com diferentes concentrações de BAP (benzilaminopurina). A indução de gemas adventícias foi mais eficiente nas concentrações de 1,0 e 1,25 mg/L de BAP. Este protocolo foi usado para regenerar plantas em experimentos de transformação genética de abobrinha-de-moita cv. Caserta e melancia cv. Crimson Sweet, via Agrobacterium tumefaciens. O vetor binário pCAMBIA2201, contendo fragmentos dos genes da proteína capsidial do ZYMV e do PRSV-W, numa construção gênica do tipo hairpin e o gene de seleção nptII, sob controle do promotor 35S, foi usado nos experimentos de transformação genética. Após 2 dias de co-cultivo, em meio de cultura MS, suplementado com BAP (1 mg/L), os explantes foram transferidos para meio de cultura de seleção, suplementado com BAP (1 mg/L), timentin (400 mg/L) e canamicina (100 mg/L) e cultivados por 3 a 4 semanas, sob fotoperíodo de 16 horas de luz. Plantas regeneradas foram analisadas por PCR, usando primers específicos para detecção dos fragmentos dos genes da proteína capsidial do PRSV-W e ZYMV. Foram utilizados 1050 explantes de abobrinha-de-moita e de 973 explantes de melancia, resultando em 36 e 59 plantas PCR positivas, respectivamente. A eficiência de transformação foi de 3,4% para abobrinha-de-moita e 6,1% para melancia. Plantas PCR positivas foram aclimatizadas, gradualmente, em sala de luz e transferidas para casa-de-vegetação. Pela análise de Southern blot foi confirmada a integração dos fragmentos dos genes da proteína capsidial do ZYMV e PRSV-W em 3 plantas de abobrinha-de-moita. Depois de desenvolvidas, flores femininas foram polinizadas manualmente e sementes foram coletadas de frutos maduros. Plantas R1 de abobrinha-de-moita e melancia foram inoculadas com o PRSV-W e o ZYMV por meio de Myzus nicotianae virulíferos. Não foram identificadas, até o momento, plantas resistentes aos patógenos em estudo / Diseases caused by the potyviruses Papaya ringspot virus - type Watermelon (PRSV-W) and Zucchini yellow mosaic virus (ZYMV) significantly reduce the yield and fruit quality of zucchini squash (Cucurbita pepo), watermelon (Citrullus lanatus), as well as other cucurbit crops in Brazil. The purpose of this work was to obtain zucchini squash and watermelon transgenic plants resistant to PRSV-W and ZYMV. An efficient in vitro regeneration system which can be associated with the protocol is necessary to obtain transgenic plants. In vitro organogenesis system was successfully developed using comprised of distal region of hypocotyl and the base of cotyledon of a germinated seed. The explants were cultured in MS medium (MURASHIGE; SKOOG, 1962), supplemented with different concentraction of BAP (benzylaminopurine). The induction of adventitious buds was more efficient at concentrations of 1.0 and 1.25 mg.L-1 BAP. This protocol was used to regenerate plants from genetic transformation experiments with zucchini squash cv. Caserta and watermelon cv. Crimson Sweet via Agrobacterium tumefaciens. For transformation, the binary vector pCAMBIA 2201, containing sequences of the coat protein coding regions of ZYMV and PRSV-W in a hairpin construct and the nptII gene, driven by 35S promoter was used. After 2 days of co-culture in MS medium supplemented with BAP (1.0 mg.L-1), explants were transferred to the MS selection culture medium, supplemented with BAP (1.0 mg.L-1), timentin (400 mg.L-1) and kanamycin (100 mg.L-1), and incubated for 3 to 4 weeks at 27 oC, under 16 h photoperiod. Regenerated plants were analyzed by PCR, using specific pairs of primers for the detection of the coat protein gene segments of PRSV-W and ZYMV. A total of 1,050 zucchini squash and 973 watermelon explants were used in the transformation experiments, resulting in 36 and 59 PCR positive plants, respectively. The genetic transformation efficiency was 3.4% for zucchini squash and 6.1% for watermelon. The PCR positive plants were slowly acclimatized in the culture room and transferred to the greenhouse for further growth. Southern blot analysis confirmed the genome integration of the the ZYMV and PRSV-W coat protein gene fragments in three zucchini squash plants which survived the acclimatization step. Later in development, female flowers were were manually pollinated and seeds were collected from mature fruits. R1 transgenic zucchini squash and watermelon plants were inoculated with PRSV-W and ZYMV by means of viruliferous Myzus nicotianae. Resistant plants were not yet observed among the R1 plants available