Transgenic Papaya Plants Research Articles

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Optimization process for de-novo organogenesis and regeneration of transgenic papaya plants using leaf tissue as explants

Resistance of transgenic papaya trees to papaya ringspot in Brazil

Abstract: The objective of this work was to evaluate the resistance of transgenic papaya populations (PTPs) to Papaya ringspot virus-P (PRSV-P). 'Sunrise Solo' transgenic papaya plants were produced with the gene of the PRSV-P protein coat, and PRSV was mechanically inoculated in plants in greenhouse conditions. The presence of the CP/PRSV gene and homozygosis were evaluated by polymerase chain reaction (PCR). Selected plants and the 'Sunrise Solo' control were transplanted to the field for agronomic evaluations. The plants evaluated in greenhouse conditions showed resistance between 96.3 and 5.8%, without variation of symptoms. The PTPs 1/6, 1/7, 1/9, 1/10, 1/15, 2/38, 2/41, 2/56, 2/65, 3/27, 3/46, 3/48, 4/9, 4/27, 8/4, 8/23, 8/33, 18/3, 18/4, 18/8, 18/22, 18/27, 28/97, 28/104, and 28/110 showed no symptoms, they were ELISA negative, and most of them contained the CP and NPT II genes. PTPs 1/6 and 3/46 had the CP gene in homozygosis and in double insertion. PTPs 1/6/20, 1/6/59, 1/6/64, 1/6/90, 3/46/44, 3/46/52, and 18/27/97 had a well-formed fruit cluster, piriform fruit weighing approximately 500 grams, orange pulp, and less than 10% carpelloidy. PTPs 1/6/59 and 3/46/52 show resistance to PRSV, good agronomic characteristics, and the CP gene in homozygosis.

Characterization of Papaya ringspot virus isolates infecting transgenic papaya \u2018Huanong No.1\u2019 in South China

In 2006, the release and cultivation of the genetically modified papaya cultivar ‘Huanong No.1’ successfully controlled the destructive papaya ringspot disease caused by Papaya ringspot virus (PRSV) in South China. However, some transgenic papaya plants from Guangdong and Hainan are found infected by PRSV. In this study, Field investigation was carried out and susceptible transgenic papaya samples were collected during 2012–2016. Twenty representative isolates were artificially inoculated into Cucurbita pepo and commercialised ‘Huanong No.1’ papaya, and results indicated that the plants showed obvious disease symptoms. Phylogenetic analysis of CP genes of 120 PRSV-infected isolates showed that PRSV can be divided into three groups. Isolates from Guangdong and Hainan belong to Group III, which is further divided into two subgroups. The isolates collected in this study have greatly diverged from the previously reported dominant strains Ys, Vb and Sm in South China, indicating that they belong to a new lineage. Further analysis showed a highly genetic differentiation between isolates, and 27.1% of the isolates were identified as recombinants on the basis of CP nucleotide sequences. These results indicate that the genetic variation of PRSV and the formation of the new virus lineage may explain the loss of transgenic papaya resistance in South China.

Transformasi Gen Antisens ACC Oksidase pada Pepaya dengan Teknik Penembakan Partikel

Papaya (Carica papaya L.) is a climacteric fruit that exhibit a<br />very fast ripening rate. Ethylene controls the ripening event<br />in the papaya fruit. 1-aminocyclopropane-1-carbocxylic acid<br />(ACC) oxidase gene encodes a specific enzyme for ethylene<br />biosynthesis. The gene had become a target for manipulation<br />to make a gene construct of an antisense ACC oxidase<br />to regenerate transgenic papaya that has a characteristic of<br />delayed ripening. The objective of the experiment is to engineer transgenic papaya that has a delayed ripening characteristic by transforming papaya with the antisense ACC oxidase gene through particle bombardment technique. The immature embryos of papaya variety Burung<br />were used for the explants. Antisense ACC oxidase and reporter (gus) genes were co-transformed to papaya calli. Four hundreds eighteen calli were bombarded by the antisense ACC oxidase gene. The transformation experiment resulted 25 putatives transgenic plants out of fifty plants<br />acclimatized in a greenhouse. Gus gene expression assay observed at 9 days after bombardment showed that the papaya explants bombarded twice at 9 cm shoot distance had 53.3% transformation rate of gus positive and 5.25 blue spots number in average. The results of PCR analysis showed that four out of 25 transgenic putative papaya plants (TR6, TR9, TR20, dan TR24), indicated a positive PCR of the antisense ACC oxidase gene with the amplified fragment DNA size of 800 base pair.

USING MANNOSE AS A POSITIVE SELECTION OF TRANSFORMED CARICA PAPAYA L. VAR ‘EKSOTIKA’

The main objective of this study is to develop marker-free transgenic papaya plants via positive selection using phosphomannose isomerase (pmi) gene. Phosphomannose isomerase (PMI) is an enzyme that converts mannose-6-phosphate to fructose-6-phosphate, a glycolysis intermediate that supports the growth of plant cells. To establish a marker-free positive selection system using this PMI, the effect of mannose on the growth and development of embryogenic ‘Eksotika’ papaya callus was evaluated. One-month old embryogenic calli were cultured on Murashige and Skoog (MS) medium in which 60 g/L sucrose in the original recipe was replaced with different concentrations of mannose and sucrose. Mannose was supplied as the sole carbon source or in combination with sucrose at 0, 5, 10, 15, 20, 25 or 30 g/L. Embryogenic calli cultured on medium supplemented with a ratio of 0:60 g/L mannose: sucrose was used as a control. The results after six sub-cultures showed that most of the embryogenic calli transferred on media containing only mannose turned brown. Higher concentrations of mannose resulted in higher percentage of brown calli (dead). Mannose at 30 g/L mannose was found to be effective for screening transformed embryogenic calli. Evaluation of papaya transformation efficiency using this positive selection system was pursued using 650 one-month-old embryogenic calli Agrobacterium-transformed with pNOV2819 harboring the pmi gene. Only transformed cells are capable of utilizing mannose as a carbon source to grow. After five months on mannose selection, all 67 putative transformants obtained were PCR-positives for the pmi gene.

Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

Variation of Neomycin Phosphotransferase II Marker Gene in Transgenic Papaya Plants Under the Field Cultivation

The transgenic “Huanong No. 1” papaya, which harbors the replicase gene (rp) of Papaya ringspot virus (PRSV) and the neomycin phosphotransferase II (npt II) gene, is widely commercially cultivated in China. In this study, the expression and mutation of the marker gene npt II in the transgenic papaya were investigated. By analysis via DAS-ELISA, PCR, RT-PCR, and Southern blot, five transgenic papaya plants expressing rp normally and npt II abnormally were obtained from 305 transgenic papaya plants that were cultivated in fields for 5 years. Sequence analysis of the npt II fragments amplified by PCR or RT-PCR from the 5 variant plants indicated point mutations in the sequence of the npt II region from 3 plants, leading to the formation of the termination codon to stop protein translation earlier. The mRNA of the npt II could not be transcribed in one plant because of the destroyed CAAT box in the NOS promoter region of the npt II while another plant showed the absence of the npt II expression cassette. These results indicate that selective marker genes in transgenic plants can be naturally deleted by mutation and that marker-free transgenic plants can be obtained for basal plant materials to develop desirable transgenic crops for commercialization.

DEVELOPING TRANSGENIC PAPAYA WITH IMPROVED FUNGAL DISEASE RESISTANCE

Papaya (Carica papaya L.), one of the more important fruit crops of the tropics, has limited resistance to a range of fungal pathogens that may kill the plant or, at a minimum, reduce productivity and fruit quality. Phytoalexins have been shown to be important natural components in the defense of plants against pathogen infection. Several fruit crops, including grapes and peanuts, synthesize the stilbene-type phytoalexin, resveratrol, (trans-3,4', 5-trihydroxy-stilbene), when attacked by fungal pathogens such as, Botrytis circerea or Plasmopara viticola. The level of resistance to P. viticola was positively correlated with the capacity of Vitis spp. to synthesize stilbene. We conducted in-vitro pathogen inhibition assays to show that resveratrol also inhibited fungal pathogens of a tropical plant, papaya. Resveratrol at 1.0 mM in V8 agar culture medium inhibited mycelial growth of Phytophthora palmivora by up to 50%, as compared to the control. The compound was active against P. palmivora at concentrations as low as 100 μM. Resveratrol was not as effective against the anthracnose pathogen, Colletotrichum gloeosporiodes. Further, we transformed papaya embryogenic cultures with the stilbene synthase gene cloned from grapevine and driven by its own inducible promoter along with the hygromycin resistance or kanamycin resistance gene under the control of a CaMV35S promoter. The presence of the transgenes was confirmed by PCR and Southern blot analysis. Transgenic papaya plants were challenged with P. palmivora under greenhouse conditions. Data from greenhouse studies showed that the disease levels in transgenics plant were reduced to 35% of the disease levels in non-transformed control plants. Our findings indicated that metabolic engineering Carica papaya with heterologous phytoalexins, either under its own inducible promoter or a constitutive promoter, can improve plant defenses against fungal pathogens.

Papaya transformed with the Galanthus nivalis GNA gene produces a biologically active lectin with spider mite control activity

Snowdrop ( Galanthus nivalis) lectin has previously been shown to have anti-feedant and insecticidal activity towards sap-sucking insects. However, its effectiveness against plant-parasitic mites has not been demonstrated. In this study, the commercial papaya ( Carica papaya L.) cultivar Kapoho, which is highly susceptible to mites, was transformed with the snowdrop lectin ( G. nivalis agglutin [ GNA]) gene. Polymerase chain reaction confirmed the presence of the transgene and six independent transformed lines were selected for expression analysis. Western blot analysis showed that the lines expressed a recombinant protein with a molecular weight similar to that of the native snowdrop lectin. Leaf extracts containing the recombinant GNA protein agglutinated trypsinized rabbit erythrocytes thus, showing the GNA protein to be biologically active. ELISA and indirect measurement from the agglutination assay showed there to be variation in GNA expression among the lines produced. A laboratory bioassay using carmine spider mites ( Tetranychus cinnabarinus) suggested improved pest resistance in the transgenic papaya plants. This is the first report that a transgenic plant expressing the GNA gene possesses enhanced resistance to a plant-parasitic mite.

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization, fruit characters, and nutrients were analyzed for T4 generation of transgenic papaya. All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field. The RP transgene can be steadily inherited to, and expressed at RNA level, the progenies. The growth characteristics of transgenic papaya were much better than non-transgenic papaya in the field. The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV. They died quickly and never grew to produce fruit. The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time, while most of transgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV, and produced more, bigger, and high quality fruits. Compared with non-transgenic plants, the fresh fruit length of T4 generation of transgenic papaya increased 2.6%–5%, and the diameter decreased 0.6%–1.5%. The flesh thickness of fresh fruit increased 12%–15%, which made it fitter for eating. Although the fresh fruit quality changed, there was no significant difference between transgenic and non-transgenic papaya. The quality characteristics of dry fruit including the contents of water, lipid, N, protein, reduced sugar, vitamin A, vitamin C, and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents. This means that transgenic plants and non-transgenic plants are substantially equivalent, and the transgene has no effect on dry fruit quality. In this study, we found that vitamin A and vitamin C in red-fleshed papaya were 1.4–1.8 and 1.78–2.07 times more than the yellow-fleshed ones, respectively, while N and protein were only 84.2%–92.1% and 82.1%–98.9% of the yellow-fleshed ones.

Resistance of transgenic papaya plants to Papaya ringspot virus

The coat protein gene isolated from Papaya ringspot virus, Thai isolate, was used to generate transgenic papayas. A binary vector containing the coat protein gene under the control of a 35S promoter, was constructed and transformed into somatic embryos of papaya cultivar Khak Dum by microprojectile bombardment. Eight transgenic lines were identified from 1980 bombarded calli of papaya somatic embryos under kanamycin selection. Integration of the transferred genes into kanamycin resistant papaya calli was verified by PCR amplification of the coat protein gene, GUS assays and Southern blot hybridization. Although the coat protein gene was detected in all transgenic lines, only line G2 was found to be highly resistant to virus. This resistant line showed high degree of rearrangement of the inserted coat protein expression cassette while the coat protein gene itself had a deletion of 166 bp on the 3' end of its sequence. Although the transcription of the coat protein gene was detected in all transgenic lines by RT-PCR, only two transgenic papayas expressed the intact coat protein. Moreover, in the resistant line G2 the amount of the truncated coat protein mRNA was significantly decreased. These results point to an RNA mediated mechanism of coat protein mediated resistance in papaya, probably based on post-transcriptional gene silencing.

INCREASED DISEASE RESISTANCE IN PAPAYA BY TRANSFORMING A PATHOGEN-INDUCIBLE STILBENE SYNTHASE GENE

Phytoalexins have been shown to be important natural components in the defense of plants against fungal infection. Resveratrol is a major phytoalexin in grapevine. We conducted in vitro pathogen inhibition assays to show that Resveratrol inhibited fungal pathogens of papaya. Resveratrol at 1.0 mM in V8 agar culture medium inhibited mycelia growth of P. palmivora up to 50% of control. The compound was active against P. palmivora as low as 100 μM. Resveratrol was not as effective against the anthracnose pathogen, Colletotrichum gloeosporiodes. We transformed papaya embryogenic cultures with the stilbene synthase gene cloned from grapevine and driven by its own inducible promoter along with the hygromycin resistance or kanamycin resistance gene under the control of a CaMV35S promoter. The presence of transgenes was confirmed by PCR and Southern blot. Twenty lines of transgenic plants were propagated from tissue cultures for greenhouse assays. Transgenic papaya plants were challenged with oomycete pathogen in greenhouse conditions. Data from greenhouse studies showed that the disease level in transgenic plants was reduced to 35% of the disease level in non-transformed control plants. Papaya lines produced with increased resistance to diseases became available for crossing with SunUp to produce hybrids that have resistance to both virus and fungal diseases.

Effective selection of transgenic papaya plants with the PMI/Man selection system

The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations.

Etiologia e estratégias de controle de viroses do mamoeiro no brasil

As viroses constituem o principal grupo de doenças do mamoeiro (Carica papaya), ocasionando grandes perdas na produção, podendo chegar à destruição total das plantações afetadas. Embora mais de dez vírus tenham sido constatados infetando naturalmente o mamoeiro, em todo o mundo, no Brasil, até o presente, foram assinaladas apenas as ocorrências do vírus da mancha anelar do mamoeiro (Papaya ringspot virus, PRSV), do vírus do amarelo letal do mamoeiro (Papaya lethal yellowing virus, PLYV) e do vírus da meleira que se encontra em fase de caracterização. A mancha anelar causada pelo PRSV é, inquestionavelmente, o mais importante problema sanitário do mamoeiro. O controle do PRSV mostra-se imprescindível, apesar de bastante difícil, em razão da sua forma de disseminação rápida e eficiente por diversas espécies de afídeos e ausência de resistência genética em C. papaya. Na tentativa de controlar o PRSV, várias medidas já foram testadas, não existindo, até o momento, nenhuma estratégia eficiente e duradoura para seu controle no Brasil. O desenvolvimento de plantas transgênicas de mamoeiro expressando o gene da capa protéica (cp) do PRSV, imunes ao mesmo, abriu nova possibilidade para solução do problema.

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems.

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.

Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus.

Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.

Regeneration of transgenic papaya plants via somatic embryogenesis induced byAgrobacterium rhizogenes

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, β-glucuronidase activity (GUS), and Southern hybridization analysis. Over one-third of the infected leaf explants produced transformed roots with GUS activity, from which 10% spontaneously produced somatic embryos. Histological analysis ofA. rhizogenes-transformed embryos showed that they have an altered symmetry between the shoot apex and the root meristem when compared to somatic embryos induced with hormone treatment from control explants. Transgenic papaya plants containingA. rhizogenes rol genes were more sensitive to auxins, developed wrinkled leaves, and grew slower than nontransformed plants.

Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots

In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and β-glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.

Transgenic papaya plants from Agrobacterium -mediated transformation of petioles of in vitro propagated multishoots

Transgenic papaya plants from Agrobacterium -mediated transformation of petioles of in vitro propagated multishoots

Somatic Embryogenesis using Immature Zygotic Embryos from Developing Fruits of Papaya (Carica papaya)

An efficient protocol has been developed for the in vitro multiplication of papaya (Carica papaya L.) through somatic embryogenesis utilizing immature zgotic embryos. Somatic embryos were initiated on MS basel media supplemented with 5 mg·liter–1 2,4-D, 400 mg·liter–1 glutamine, and 6% sucrose. After culturing for 2 months, 65% of the explants became highly embryogenic. Each explant produced 50 to 80 embryos in 4 months on culture induction medium. Frequency of embryogenesis was increased (75 to 150 somatic embryos on 80% explants) upon supplementing medium with 4% maltose as a carbon source and 100 mg·liter–1 L-asparagine. The embryogenic callus appeared yellow and embryos at different stages of development were well-organized. On regular subculturing, these cultures continued to produce secondary embryos. Following their transfer to the hormone-free medium supplemented with 4% maltose, these embryos germinated. The somatic embryogenesis system is rapid, repetitive, and highly proliferative. Thus, this system may have a potential use in the development of synthetic seed and transgenic papaya plants. Details of important factors affecting somatic embryogenesis will be discussed.