Previous PageTable Of ContentsNext Page

Towards Development of PRSV Resistant Papaya by Genetic Engineering

Pablito M. Magdalita, Lolita D. Valencia, Anna T.I.D. Ocampo, Reynaldo T. Tabay and Violeta N. Villegas

Institute of Plant Breeding, College of Agriculture, University of the Philippines at Los Baos, College, Laguna 4031,
Philippines. Email pmmagdalita@ipb-uplb.org.ph, pablitomagdalita@yahoo.com

Abstract

The development of genetically engineered crops is one of the options in controlling papaya ringspot virus (PRSV), a major disease of papaya worldwide. This research made use of the coat protein (cp) gene technology and microprojectile bombardment to develop transgenic papaya that are resistant to PRSV infection. A 0.9kb fragment of the cp gene was cloned from the Philippine PRSV, sequenced, characterized and inserted in a vector construct suitable for transformation into papaya. Blast search analysis revealed that the cp gene is 92%, 89%, 88% and 86% homologous to the Thai, Vietnamese, Australian and Hawaiian, and Puerto Rican and Sri Lankan PRSV cp genes respectively. The cp gene was inserted into the p2K7 cloning vector and plasmid DNA of pCP-LBP construct was used for microprojectile bombardment of papaya somatic embryos. Using transient expression, a pressure of 1200 kPa and a distance of 12.5 cm were established as best conditions for bombardment. Eighteen thousand two hundred thirteen (18,213) zygotic embryos were isolated, of which, 7,845 (43%) produced somatic embryo clusters that were used for transformation. Two hundred thirty two (232) individual targets were bombarded and 68 individual transformation events survived kanamycin selection and eventually regenerated into putative transgenic plantlets. Three hundred fifty nine R0 transgenic plants were produced and some were planted in a BL2 glasshouse. The putative R0 transgenic plants are morphologically similar to the non-transgenic control. They are moderately susceptible to PRSV but resistant R1 lines (15) were derived from the R0 transgenic plants.

Media summary

Genetically engineered PRSV resistant papaya lines were developed using coat protein gene technology and microprojectile bombardment.

Key Words

particle inflow gun, primers, embryogenesis, serology, pollen fertility

Introduction

Papaya production in Southern Luzon, Philippines used to be a lucrative business until the outbreak of the papaya ringspot virus (PRSV). It is a major disease of papaya worldwide (Yeh and Gonsalves, 1994). In the Philippines, PRSV decreased papaya production by as much as 80% in 1995 in Region IV alone. It is now widespread all over Luzon and the Visayas and a few in Mindanao (Herradura et al. 2001). There are no resistant Carica papaya varieties, but there is resistance among the wild Carica species (Magdalita et al.1988). There are a few PRSV tolerant varieties. PRSV resistant interspecific hybrids were produced but varieties could not be developed due to strong incompatibility barriers (Magdalita et al. 1996). The use of genetic engineering is the most promising approach to develop PRSV resistant papaya. The commercialization of the genetically modified (GM) Rainbow papaya of Hawaii containing the coat protein (cp) gene of PRSV (Lius et al. 1997) was a major breakthrough in managing this disease. However, this GM papaya is resistant only to the Hawaiian PRSV because of the highly specific nature of resistance. Therefore, it is necessary for the Philippines to develop its own GM papaya using the local cp gene hence, this study.

Methods

Cloning of PRSV cp gene, vector construction and characterization

PRSV infected leaves were collected and the RNA extracted following the phenol-glycine method. The cloning of PRSV cp gene and the construction of the plasmid vector pCP-LBP containing the Philippine PRSV isolate was done at the Kasetsart University in Thailand. The DNA was analyzed by PCR using primers MB 11 and MB 12 specific for PRSV-P cp gene. The DNA was also digested with Bam H1 and Xba 1 to detect the presence of the insert. In addition, the PRSV cp gene was purified and sequenced. The sequence was compared with the other known PRSV cp sequences of other countries through blast search analysis.

Optimization of bombardment conditions by transient expression

The protocol developed by Magdalita (2002) in Australia was adopted in this work. Microprojectiles were prepared by mixing 50 l of 100 g/l tungsten with 20 l (1 g/l) plasmid DNA of pBI 121, 50 l CaCl2 (2.5 mM), 20 l spermidine (100 mM). Four microliters of the suspension were used for each bombardment. A protective baffle nylon mesh (Franks and Birch, 1991) was placed over the tissue during bombardment. Various pressures of 800, 1000, 1200, 1400, and 1600 kPa and distance ranging from 12.5 to 17.5 cm with a pulse duration of 50 m/sec were used. The bombarded embryos were incubated for 48 hours on half strength MS medium and Gus activity was assayed histochemically by incubating the embryos in X-gluc solution overnight at 37oC (Jefferson, 1987). Transient expression was assayed 12 hours after incubation and measured as total blue foci count per shot area.

Stable Transformation and Regeneration of Transgenic Plants

Zygotic embryos from 90-120 day old ‘Davao Solo’ papaya fruits were inoculated onto the Somatic Embryo Induction medium containing half strength MS and vitamins, 100 mg/L L-glutamine, 50 mg/L myo-inositol, 100 mg/L thiamine-HCl, 10 mg/L 2,4-D (Fitch et al. 1990). Eight weeks after induction, the somatic embryo clusters were squashed on top of a filter paper overlaid onto the SEIM and then transferred onto the osmoticum medium (3-4 hours) prior to bombardment (Gonsalves et al. 1998). Following the protocol of Magdalita (2002), the tissues were bombarded with the plasmid DNA of pCP-LBP using the particle inflow gun from Australia. Tissue were placed on selection media containing 150 mg/L kanamycin for one month and then with 300 mg/L kanamycin for another month. They were then transferred to a regeneration medium with 75 mg kanamycin until plantlets developed.

Morphological characterization, pollen fertility test, fruit quality evaluation and PRSV reaction of R0 transgenic plants

The plant height, petiole length, leaf length, number of leaves, number of nodes, number of days to fruit set, sex type, petiole color, total number of flowers, and number of flowers per node of the putative transgenic plants were recorded. Pollen fertility was determined using I2Kl staining and the percent pollen fertility was computed. Fruits of R0 transgenic plants were evaluated for fruit weight and shape, flesh color, texture, flavor, total soluble solids, percent edible portion, seed number and PRSV infection. PRSV infection on the fruit's surface and on the vegetative parts was rated using a defined rating scale.

Generation of advance lines and PRSV reaction of R1 lines

Hermaphrodite flowers of T0 putative transgenic lines were self-pollinated. The pollinated fruits were grown to maturity and seeds were extracted to constitute the T1 generation. Screening of the T1 and controls was conducted in the BL2 containment glasshouse. They were mechanically inoculated with sap of the PRSV isolate and infection was assessed after 2 weeks. Re-inoculation and ELISA assay were also done.

Results

Cloning of PRSV cp gene, vector construction and characterization

A 900 bp fragments representing the PRSV-P coat protein gene was inserted into p2K7 to generate pCP-LBP (Philippine PRSV). Digestion analysis showed two bands: a 5.8 kb fragment representing the plasmid vector and a 0.9 kb fragment representing the cp gene of the PRSV-P. Sequence analysis of the Philippine cp showed 92% and 89% homologous to Thai and Vietnamese PRSV isolates, respectively implying that the Philippine PRSV cp gene is largely similar to the Thai and Vietnamese strains. In addition, it was 88% homologous to Australian and Hawaiian isolates and 86% homologous to Puerto Rican and Sri Lankan isolates implying some degree of similarity of these isolates to the Philippine cp isolate.

Transient expression and stable transformation

The dispersion of the microprojectiles on the target filter varies with each bombardment and with each of the pressure level used. An even distribution of the microprojectiles on the surface of the target was observed at a pressure of 1200 to 1400 kPa. Transient expression was variable with each bombardment using the four pressures at a distance of 12.5 cm from the target somatic embryos. Expression of gus on the bombarded somatic embryos was very strong using a pressure of 1200 kPa. This result indicates that this pressure level is optimal for delivering the microprojectiles into the target tissues. In addition, the results suggest that the number of individual transformation events could be highest at this pressure level.

A total of 18,213 `’Davao Solo’ immature zygotic embryos were isolated of which only 7,845 (43%) produced somatic embryos and were consequently used for transient and stable transformation. A total of 232 individual targets were bombarded with the plasmid DNA of pCP-LBP construct. After 4-5 months incubation in selection and regeneration media, green somatic embryos emerged into plantlets with some root initials and dark green leaves. Three hundred fifty-nine (359) putative R0 transgenic plantlets were produced.

Morphological characterization, pollen fertility test, fruit quality evaluation and PRSV reaction of R0 transgenic plants

The putative transgenic plants are similar to each other and to the non-transgenic control suggesting that no somaclonal variation occurred during the transformation process. Plant height ranges from 101 - 128 cm, while stem diameter ranges from 2.88-4.36 cm. They all have the green to brownish petiole and greenish stem. Generally, the reaction of PRSV infection ranges from moderate to the highly susceptible. Pollen fertility ranges from 47.3 - 77.0%. The fruit qualities (27 fruits) of the putative transgenic R0 plants are typical of ‘Davao Solo’ like the non-transgenic control, again indicating that somaclonal variation has not occurred during the transformation process. They are small-fruited (46.8-288.0 grams), sweet (9.0-16.6oBrix) and have high edible portions (48.04-84.41%). The color of the flesh is yellow to yellow orange. The various putative R0 transgenic lines have varied reaction to PRSV ranging from high to moderate susceptibility. However, a few lines (Pb 12, Pb 13 and Pb 23) show symptoms of mild PRSV infection. The result suggests that these putative transgenic R0 plants are hemizygous for the introduced PRSV cp gene, thus selfing is necessary to get a full complement of the gene.

Generation of advance lines and PRSV reaction of R1 lines

A total of 141 hermaphrodite flowers were self-pollinated while 85 were sib-pollinated. Out of these, 65 set fruits (28.76%) but only 27 fruits were harvested. Twenty-seven R1 lines were derived from the R0 transgenic plants. Sixty-one progenies coming from 15 R1 lines are PRSV resistant based on symptomatology and serology by ELISA.

Conclusion

The coat protein (cp) gene of the Philippine PRSV was cloned, sequenced, characterized and placed in p2K7 transformation vector. The cp sequence is 92% homologous to the Thai cp, 89% to the Vietnamese, 88% to the Australian and Hawaiian and 86% to the Puerto Rican and Sri Lankan cp. The transformation parameters for microprojectile bombardment were established at 1200 kPa at a distance of 12.5 cm, by transient gus gene expression. For stable transformation, 18,213 immature zygotic embryos were isolated, of which 7,845 (43%) produced somatic embryos. Two hundred thirty-two individual targets were bombarded with the cp gene. Out of this, 359 putative transgenics plantlets were produced. The putative transgenic plants are morphologically similar to each other and to the non-transgenic control. All the putative R0 transgenic lines have high to moderate susceptibility to PRSV. However, PRSV resistant R1 lines were derived from infected R0 lines.

Acknowledgement

The PCARRD-DOST and ISAAA provided funds for this project.

References

Fitch MMM Manshardt RM Gonsalves D Slightom JL and Sanford JC (1990). Stable transformation of papaya via microprojectile bombardment. Plant Cell Reports 9, 189-194.

Franks T and Birch RD (1991). Gene transfer into intact sugarcane cells using microprojectile bombardment. Australian Journal of Plant Physiology 18, 471-480.

Gonsalves C Wenqi C Tennant P and Gonsalves D (1998). Effective development of papaya ringspot virus resistant papaya with untranslatable coat protein gene using a modified microprojectile transformation method. Acta Horticulturae 461, 311-314.

Herradura LE Magnaye LV and Bajet NB (2001). Occurrence of papaya ringspot in Mindanao. Philippine Phytopathology 33, 52-58.

Jefferson RA (1987). Assaying chimeric genes in plants: the gus gene fusion system. Plant Molecular Biology Reporter 5, 387-405.

Lius S Manshardt RM Fitch MMM Slightom JL Sanford JC Gonsalves D (1997). Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus. Molecular Breeding 3, 161-168.

Magdalita PM Adkins SW Godwin ID and Drew RA (1996). An improved embryo-rescue protocol for a Carica interspecific hybrid. Australian Journal of Botany 44, 343-353.

Magdalita PM Laurena AC Yabut-Perez BM Mendoza EMT and Villegas VN (2002). Progress in the development of transgenic papaya: Transformation of Solo papaya using ACC synthase antisense construct. Acta Horticulturae 575, 171-176.

Magdalita PM Villegas VN Pimentel RB and Bayot RG (1988). Reaction of papaya (Carica papaya L.) and related Carica species to ringspot virus. Philippine Journal of Crop Science 13, 129-132.

Yeh SD and Gonsalves D (1994). Practices and perspective of control of papaya ringspot virus by cross protection. In: Harris KF (ed) Advances in disease vector research (pp 237-257). New York: Springer-Verlag.

Previous PageTop Of PageNext Page