Development of PRSV-P resistant papaya genotypes by introgression of genes from wild Carica species

A fertile PRSV-P resistant plant was produced in a previous project when a C. papaya x C. quercifolia hybrid was backcrossed to C. papaya clone 2.001. This plant is numbered clone 54 for ease of identification. In the previous 12 months clone 54 has been used to backcross to C. papaya clone 2.001 (2nd backcross) and to C. papaya lines 16 (a SE Qld variety), Solo (developed in Hawaii and used extensively in the tropics) and GD 319-2 (the parent of a hybrid seed line from North Queensland). Clone 54 and seed of these crosses has been sent to the Philippines. Problems have been experienced with seed germination of these crosses in both countries (particularly the 2nd backcross to 2.001), however seedlings populations of each cross have been established in a glasshouse at Griffith University.

Sub-Project 2. Selection of backcross generations for PRSV-P resistance and good agronomic traits.
Two hundred plants of a backcross generation between clone 54 and papaya line 16 have been grown in a glasshouse at Griffith University. These plants have been manually inoculated 5 times with PRSV-P over the 12-month period. Twenty-six plants have remained free of symptoms after 12 months, and no virus has been detected in them by ELISA. Virus infected all control plants after 2 inoculations. They will be planted in the field in 2003 where they will be subjected to aphid transmission of the virus. Other populations representing backcrosses between 54 and other C. papaya lines will be inoculated with PRSV-P in 2003.

Sub-Project 3. Backcross fertile resistant C. papaya x C. quercifolia F1 hybrids to C. papaya.

Australia
Of the large number of F1 hybrids between C. papaya x C. quercifolia, generated in a previous ACIAR project (CS1/1991/007), 4 have been identified as being PRSV-P resistant and having some pollen fertility. These have been given the clone numbers 410, 468 [parent of 54], 469 and 507. Pollen from these hybrids has been used to pollinate female flowers of C. papaya clone 2.001 and other C. papaya varieties in SE Qld. Embryo rescue will be attempted on seeds from resultants fruit between Jan and April in 2003.

Philippines
Seven inbred lines developed by the Institute of Plant Breeding and 5 F1 interspecific hybrids (401, 410, 468, 469 and 507) from Australia were planted in the field last Oct. 18, 2002. Table 1 shows the fruit characteristics and reaction to PRSV of the different inbred lines. Flowering has commenced and backcrossing activities have been started. Tissue culture activities to mass propagate the F1 interspecific hybrids through nodal cuttings is also in progress. These materials will be used to set-up duplicate hybridisation blocks in the islands of Davao in Mindanao, and Guimaras Island in the Visayas.

Table 1. Reaction to papaya ringspot virus (PRSV) and fruit characteristics of selected inbred lines developed at the Institute of Plant Breeding used in the experiment.

Inbred Line Reaction to PRSV Fruit flesh color Height to flower (cm) TSS (B)
Py 2 (4108) moderately tolerant red orange 67.30 10.8
Py 3 (4172) moderately tolerant red orange 121. 35 11.6
Py 4 (4308) severe red orange 74.54 11.1
Py 5 (5648) moderately tolerant yellow orange 71.46 11.8
Py 6 (5893) moderately tolerant red orange 73.70 11.0
Py 7 (3878) severe yellow orange 67.54 11.4
Py 10 (F2- derived) moderately tolerant red orange 105.80 11.7

Sub-Project 4. Development of molecular markers for PRSV-P resistance.
We decided to work initially on the C. pubescens x C. parviflora population as we have produced a large F2 population (300 plants in the glasshouse) that is segregating for resistance to PRSV-P. DNA has been extracted from F1 and F2 plants, and from plants of C. pubescens and C. parviflora, and is being compared using RAF markers. RAF is a new technique that produces more polymorphisms than AFLPs. A genetic map of the two species is being developed. Multiple primers will be screened across two F2 populations (one resistant and one susceptible to PRSV-P) using the RAF marker technique during the first 6 months of 2003. We hope to develop a marker for PRSV-P resistance using this approach. If a marker linked to PRSV-P resistance is developed, it will be used to screen the C. papaya x C. quercifolia and backcross populations.

Sub-Project 5. Micropropagation of elite papaya genotypes.
Clones of all C. papaya genotypes, fertile resistant C. papaya x C. quercifolia hybrids, and some backcross plants (including 54) are being maintained in vitro. Clones have been transferred to the Philippines and are being multiplied in the tissue culture laboratory at IPB.

Sub-Project 1. Incorporation of PRSV-P resistance genes into elite papaya genotypes, using fertile resistant backcross plants.

Year 2 (01/01/2003-31/12/2003)
Subproject 1: Incorporation of PRSV-P resistance genes into elite papaya genotypes, using fertile resistant backcross plants
Australia: A fertile PRSV-P resistant plant was produced in a previous project when a C. papaya x C. quercifolia hybrid was backcrossed to C. papaya clone 2.001. This plant is numbered clone 54 for ease of identification. Second generation backcross plants (BC2) from V. quercifolia have been produced when 54 was crossed with 4 papaya lines. These second-generation backcross plants are being screened for virus resistance both in Australia and the Philippines
Philippines: Clone 54 and seed of these crosses were sent to the Philippines. Both 54 and all the backcross plants were susceptible to PRSV-P in Los Banos however there was variation in symptom expression. The fertile PRSV-P resistant plant clone 54 succumbed to PRSV-P two months after transplanting in the field, with symptoms ranging from chlorotic leaves to oily streaks on the trunk.
Subproject 2: Selection of backcross generations for PRSV-P resistance and good agronomic trait.
Australia: Of the first 200 backcross (BC2) plants screened in Queensland (a backcross generation between clone 54 and papaya line 16), 26 remained symptomless after 5 manual inoculations of virus in a glasshouse. All control plants produced PRSV-P symptoms after 2 inoculations. These 26 plants were planted in the field in the presence of heavy PRSV-P infection. After 8 months, one male and one female plant remain symptomless. The other 26 vary in symptom expression from severe to mild symptoms of PRSV-P. More recently, more second-generation backcross plants have been planted in the glasshouse at GU and inoculated with PRSV-P. They represent 120 plants of 54 backcrossed to Solo, 60 of 54 backcrossed to GD319-2 (from North Queensland) and 70 plants of 54 backcrossed to papaya clone 2.001. It is too early for symptom development at this stage. To determine agronomic traits of backcross plants, 1 row (34 plants) of 54 crossed to 2.001 and 1 row of 54 crossed to Solo have been planted in the field in Southeast Queensland.
Philippines: Three BC2 populations (2.001 x 54, Sunrise Solo x 54, and GD 319-2 x 54) were planted in Mainit experimental station to evaluate the reaction of the segregating populations against PRSV-P. Symptom expression was observed two weeks after artificial inoculation. Symptom expression of ringspot disease varies in terms of onset of disease and severity of infection in each plant. Another batch of the same BC2 generations were evaluated in the glasshouse. One hundred ninety five BC2 generation between clone 54 and papaya line 2.001, 36 BC2 generation between line 54 and line GD 319-2 and 26 BC2 generation between line 54 and Sunrise Solo papaya were grown in the glasshouse at IPB. The plants were manually inoculated with PRSV-P. From the first inoculation, 74 of BC2 2.001 x 54 plants, 10 of BC2 GD 319-2 x 54 plants and two of Sunrise Solo x 54 plants remained free of symptoms and no virus has been detected by ELISA . Virus infected all control and susceptible plants two weeks after inoculation.
Subproject 3: Backcross fertile resistant C. papaya x C. quercifolia F1 hybrids to C. papaya
Australia: Of the large number of F1 hybrids between C. papaya x C. quercifolia, generated in a previous ACIAR project (CS1/1991/007), 5 were identified as being PRSV-P resistant and having some pollen fertility. These have been given the clone numbers 401,410, 468 [parent of 54], 469 and 507. Pollen from these hybrids has been used to pollinate female flowers of C. papaya clone 2.001 and other C. papaya varieties in Southeast Queensland. Seeds from 1077 fruit (from 1302 crosses) of C. papaya clone 2.001 have been dissected. From these 1077 fruit, 35,626 seed have been dissected and 185 embryos cultured in vitro. Of these, 131 have produced plants and they will be tested for resistance to PRSV-P next year.
Philippines: Four susceptible IPB inbred lines (4108, 4172, 5648 and 5893) as females and resistant F1 interspecific hybrid lines 410 and 401 as males were used in the backcrossing program to transfer PRSV resistance from Carica quercifolia to elite Philippines papaya genotypes. A total of 280 fruits were harvested from crosses generated using these inbred and F1 hybrid lines. Majority of the fruits came from 5648 (154) and 5893 (76) while the least number came from 4108 (30) and 4172 (20). Ninety to 120 day -old fruits were harvested for embryo rescue and culture. A total of 41, 553 seeds were dissected from the different crosses. Inbred 5648 produced the highest seed number of 20,395 but despite these number of seeds only 99 embryos were rescued. Ten well developed embryos were recovered from 10,877 seeds involving inbred line 5893. Twenty six embryos were rescued from 160 seeds involving inbred line 4108. On the other hand, inbred line 4172 produced only 15 seeds, however despite this number eight fully developed embryos were recovered. From the 143 embryos that were rescued from the different crosses, 90 embryos developed into plantlets and are now being maintained in the laboratory.
Subproject 4: Development of molecular markers for PRSV-P resistance
We decided to work initially on the C. pubescens x C. parviflora population as we have produced a large F2 population (300 plants in the glasshouse) that is segregating for resistance to PRSV-P. Good progress has been made on mapping of V. pubescens and V. parviflora using RAF markers. A genetic map of the two species has been developed. In recent research on a F2 population that is segregating for virus resistance, 5 molecular markers have been linked to PRSV-P resistance. Two of these, one each side of the gene are being sequenced.
Subproject 5: Micropropagation of elite papaya genotypes
Clones of all C. papaya genotypes, fertile resistant C. papaya x C. quercifolia hybrids, and some backcross plants (including 54) are being maintained in vitro. Plants obtained from the 131 backcross embryos (reported under objective 3) are being multiplied in Australia. In the Philippines, clones of fertile resistant C. papaya x C. quercifolia hybrids and backross plants which were embryo rescued are being maintained in vitro.

Sub-Project 1. Incorporation of PRSV-P resistance genes into elite papaya genotypes, using fertile resistant backcross plants.
Australia
All second backcross (BC2) plants produced from line 54 eventually succumbed to PRSV-P after 9 months in the field. However, there was large variation between plants in terms of degrees of severity to the virus symptoms and time to develop symptoms (2 to 9 months). Thus line 54 had a higher but variable level of tolerance to PRSV-P .

Philippines
Line 54 and BC2 plants produced from it have been susceptible to PRSV-P in the field. As in Australia, variation in rate and severity of symptom expression was observed.

Sub-Project 2. Selection of backcross generations for PRSV-P resistance and good agronomic traits.
A large number of BC2 plants between 54 and other papaya genotypes were tested for PRSV-P resistance. In Australia, 200 plants of 54 backcrossed to line 16, 120 plants of 54 backcrossed to Solo, 60 of 54 backcrossed to GD319-2 (from North Queensland) and 70 plants of 54 backcrossed to papaya clone 2.001. Similarly, a large number of progeny were tested in the Philippines. All were susceptible to PRSV-P.

To determine agronomic traits of backcross plants, 1 row (34 plants) of 54 crossed to 2.001 and 1 row of 54 crossed to Solo were planted in the field in SE Qld. Plants of 54 x Solo and 2.001 x 54 showed good agronomic traits. Plants tended to be more like C. papaya. High yields, good fruit shape and size and high brix levels were recorded ~14 (can be linked back to Vasconcella quercifolia, which has fruit with high sugar levels). Preliminary molecular marker analyses showed these plants had approx. 2% quercifolia DNA. This suggests that only a one or two more backcrosses would be needed to produce a commercial papaya.

Sub-Project 3. Backcross fertile resistant C. papaya x V. quercifolia F1 hybrids to C. papaya.

Australia
We have continued to backcross from 5 F1 hybrids that are PRSV-P resistant and having some pollen fertility, (401, 410, 468 [parent of 54], 469 and 507). 131 BC1 plants are currently being screened for PRSV-P in the field. The first 50 plants have developed viral symptoms, again with differing degrees of severity. The other 81 clones are being monitored.

Philippines
Six F1 intergeneric hybrid lines (401, 404, 410, 468, 469 and 507) of C. papaya x V. quercifolia from Australia were used as paternal parents to incorporate PRSV-P resistant gene/genes to five elite papaya inbred lines (4108, 4172, 5648, 5893 and 3878) of IPB. Intergeneric hybrid lines 410, 401, 404 and 468 had been extensively used in the backcrossing work. Pollen viability ranged from 1.2 to 2.2% under Philippine conditions. The ratio of male flower in cross-pollinating female flower was increased from 10:1 to 20:1 to compensate the low pollen viability of the hybrids. To date, fifteen backcrosses were completed with varying success in terms of viable embryos produced. Nine hundred forty (940) fruits were harvested in which 114,839 seeds were dissected under laboratory conditions. Ten backcrosses (4172 x 410, 4108 x 410, 5648 x 410, 5893 x 410, 5893 x 404 , 5648 x 404, 5648 x 468, 4108 x 468, 4172 x 404 and 3878 x 410) produced a total of 1011 embryos. Seven hundred thirty three (733) embryos had germinated into normal plantlets, each one was considered as a unique genotype. BC1 5648 x 410 comprised 46.66% of the genotypes with 342 embryos germinated, the other BC1 with embryos germinated were 4108 x 410 (132), 5893 x 410 (60), 4172 x 410 (60), 5648 x 404 (76), 3878 x 410 (44), 5893 x 404 (5), 4108 x 468 (6) and 4172 x 404 (8). Most of the genotypes were subjected to clonal micropropagation. As of now 427 genotypes had been potted out and were evaluated for PRSV-P resistance in the screenhouse. One genotype, BC1 5648 x 410 showed putative resistance to PRSV after three mechanical inoculations. This was planted in the field on Oct 6, 2004. This plant's reaction to natural infection of PRSV is currently being monitored in the field. It is still symptom and virus free as of now (5 months in the field). ELISA confirmed the resistance of this promising genotype to the presence of PRSV. This putative resistant plant is a male tree and has 65% pollen fertility. Backcrossing activities using pollen from this plant is in progress. In addition, 52 different interspecific hybrid plants were established in the field this year.

Sub-Project 4. Development of molecular markers for PRSV-P resistance.
Good progress has been made on mapping of V. pubescens and V. parviflora using RAF markers. A genetic map of the two species has been developed. Five molecular markers have been linked to PRSV-P resistance. Two of these have been sequenced, and a co-dominant SCAR marker has been developed from one. The other segregates 100% with PRSV-P resistance, and will be used in an attempt to isolate the gene for resistance in 2005.

Sub-Project 5. Micropropagation of elite papaya genotypes.
Clones of all C. papaya genotypes, fertile resistant C. papaya x V. quercifolia hybrids, and some backcross plants (including 54) are being maintained in vitro both in Australia and the Philippines. Considerable effort has been given to maintaining the 131 backcross plants (reported under objective 3) in Australia until they can be evaluated for virus resistance.

Philippines

During the three years of implementation of the project, we have reported one genotype, BC1 5648 x 410 that showed putative resistance to PRSV-P after three manual inoculations and under field condition for less than two years before it succumbed to bacterial crown rot. Backcrossing activities using the pollen from this plant were intensified during the last phase of the project.

One hundred and thirty seven (137) BC2 03R lines were generated and inoculated for screening against PRSV-P. After three (3) manual inoculations in a screenhouse, 32 plants (21 female and 11 male) were symptom-free and were transplanted to the field where susceptible papaya varieties are currently being grown. Mild PRSV symptoms were observed a month later. These observed mild symptoms were confined to the older leaves and did not progress to the younger leaves. Symptoms were localized. ELISA test was conducted and negative ELISA test results showed in 21 plants despite the mild and confined symptoms in the older leaves.

Sib-pollination of selected (male and female) BC2 03R line plants are currently in progress towards the advancement of a backcross generation. Likewise, efforts to incorporate and cross the resistant BC2 03R lines to Davao Solo is also in progress. The BC2 03R lines are also being crossed to inbred line 4172 (the male parent of hybrid 'Sinta') to reconstitute 'Sinta' and test the resulting hybrid with 'Sinta' in terms of PRSV resistance. 'Sinta' is tolerant to PRSV-P and is a cross between inbred line 5648 x 4172.

This putative resistant PRSV-P BC plant is the first conventionally bred resistant line in the Philippines.

Australia

A fertile PRSV-P resistant plant (54) was produced in a previous project when a C. papaya x C. quercifolia hybrid was backcrossed to C. papaya clone 2.001. All second backcross (BC2) plants produced from line 54 eventually succumbed to PRSV-P after 9 months in the field. However, there was large variation between plants in terms of degrees of severity to the virus symptoms and time to develop symptoms (2 to 9 months). Thus line 54 had a high level of tolerance to PRSV-P but was not resistant. BC2 plants showed good agronomic traits: high yields, good fruit shape, flavour and high brix levels. These results show that commercially acceptable fruit could be produced after 2 or 4 backcrosses.

Five F1 plants (C. papaya x V. quercifolia) that were PRSV resistant and had low pollen fertility were used to produce 131 BC1 plants. All were cloned and multiples of each clone were field tested in the presence of PRSV-P. All developed viral symptoms however symptom severity and time to infection varied between clones.

A BC2 population developed from the resistant BC1 plant produced in Los Banos have been field tested against an Australian strain of PRSV-P in SE Queensland. After 6 months in the field, 100 plants are segregating for PRSV-P resistance.

V. pubescens and V. parviflora and an F1 and F2 population derived from them were used as a mapping population. The F2 segregated in a 3:1::resistant: susceptible ratio indicating a single dominant gene controlling PRSV-P resistance in Vasconcellea. A genetic map of the two species has been developed using RAF markers. Twelve molecular markers have been linked to PRSV-P resistance. Two of these have been sequenced, and a co-dominant SCAR marker has been developed from one. The other segregates 100% with PRSV-P resistance, and is being used to identify the PRSV-P resistance gene.

Clones of all C. papaya genotypes, Vasconcellea species, fertile resistant C. papaya x V. quercifolia hybrids, and some backcross plants (including 54) are being maintained in vitro. This collection is essential for future research.

Sub-Project 1. Incorporation of PRSV-P resistance genes into elite papaya genotypes, using fertile resistant backcross plants.

Extensive backcrossing of the F1 intergeneric hybrids and inbred lines has resulted in one PRSV-P resistant backcross plant (BC1). This plant is a crossed between inbred line 5648 and intergeneric hybrid 410. This male fertile BC1 was used for further backcrossing to generate BC2 and BC3 plants.

Sub-Project 2. Selection of backcross generations for PRSV-P resistance and good agronomic traits.

A total of 137 BC2 plants were manually inoculated three times at two-week intervals in the screenhouse. Fifty plants or 38% showed typical symptoms (ranging from chlorosis to shoe-stringed leaves ) of papaya ringspot virus after inoculations. Surviving healthy plants (30 plants) were transplanted in the field and were exposed to aphid vectors from susceptible papaya (inbred 4172 and Davao Solo) plants that had a high inoculum level of PRSV-P. ELISA test results revealed that only ten out of the thirty plants tested were positive to the virus. It was also observed that four plants which showed mild infection of the virus based on visual inspection were negative to the virus. The mild infection during the first few months after field transplanting did not progress in these four plants, hence the negative ELISA result. In Southeast Queensland, approx. 25% of BC2 plants are still virus free in a field planting after >12 months in the field in the presence of PRSV-P. All controls were infected after 2 months in the field.

A total of 354 BC2 sib crosses and 124 BC3 plants which survived three manual inoculations in the screenhouse were transplanted in the field. Evaluation against natural infection to PRSV-P is in progress.

Sub-Project 3. Backcross fertile resistant C. papaya x V. quercifolia F1 hybrids to C. papaya.

IPB inbred lines (4108, 4172, 5648 and 5893), resistant BC1 and intergeneric hybrid 410 were planted in the field last Nov. 2006 for further backcrossing program.

Sub-Project 4. Development of molecular markers for PRSV-P resistance.

An attempt to develop a molecular marker is currently in progress. To confirm introgression, genetic fingerprinting of several genotypes of C. papaya and some of its wild relatives was done using simple sequence repeats (SSRs).

Preliminary result indicated introgression of a putative nucleotide fragment (150bp) from V. quercifolia, hypothesized to mark for PRSV resistant attribute of both V. quercifolia and F1 intergeneric hybrids (410, 468 and 469). In the resistant backcross progenies, a putative nucleotide fragment (100 bp) was observed to be consistent. This 100 bp could be a fragment of that putative nucleotide fragment from V. quercifolia which could
explain the resistance of the backcross plants. Sequencing of said fragments is in progress to further validate the marker.

Sub-Project 5. Micropropagation of elite papaya g
Micropropagation of some BC3 (5648 x BC2 03R-73) plantlets and clonal propagation of the remaining F1 intergeneric hybrid 410 is in progress.

Sub-project 6. Evaluation on Grower's Properties

Reconstituted 'Sinta' (BC2 03 R lines crossed with 4172) is now being tested along with the original 'Sinta' in the farmer's field in Batangas.

Sub-Project 1. Selection of backcross generations for PRSV-P resistance and good agronomic traits.
Field performance of BC3 and BC2 sib-cross plants against papaya ringspot virus (PRSV-P) was evaluated. BC3 plants are the product of introgressing the PRSV-P trait from the papaya wild type Vasconcellea quercifolia to Carica papaya. BC2 sib-crosses were developed by sib-crossing selected female and male BC2 plants. Selection was based on ELISA test result and symptom development in the field.
A total of 634 plants (88 BC3, and 546 BC2 sib-crosses) were inoculated three times at two weeks interval in the screenhouse. Three hundred twenty-five (46 BC3 and 279 BC2 sib-cross) or 51.3% showed typical symptoms, which ranged from distortion of young leaves, mosaic, chlorosis to shoe-stringed on older leaves. Plants that remained healthy and symptom free together with the susceptible check, Davao Solo, were then transplanted in the field and were assessed for resistance/susceptibility to the Philippine strain of PRSV-P. A total of 81 BC2 sib-crosses and 34 BC3 plants were planted in Mainit, Bay, Laguna. Initial results showed that, there was variation in the rate of symptom development in BC3 and BC2 sib- crosses from the control. Davao Solo produced severe symptoms after 1-2 months of transplanting in the field while there were BC3 and BC2 sib-cross plants that remained symptom free for about 7-8 months. The difference between backcross papaya and Davao solo was also evident in the ability of the trees to bear good quality fruit. Davao Solo produced a few small and unmarketable fruits. Backcross plants in contrast to the Davao Solo had the ability to recover from early infection based on visual inspection and serological test (ELISA).
Fruit qualities of some backcross plants and DS were evaluated. Fruit weight of backcross plants ranged from 834.53-754.92 grams in contrast with DS's 202.67 grams. Fruits have firm yellow orange flesh, with mild papaya aroma. TSS (0B) values of BC3, BC2 sib-cross lines and DS were 10.0, 12.2, and 9.3 respectively which corresponds to sweet taste for backcross lines and not so sweet for DS.
A total of 401 BC3 sib crosses and 355 BC4 plants are now being grown and will be inoculated manually three times in the screenhouse. The surviving plants will be transplanted in the field where its resistance/susceptibility against Philippine strain of PRSV-P will be tested.
Sub-Project 2. Backcross fertile resistant C. papaya x V. quercifolia F1 hybrids to C. papaya.
IPB inbred lines (4172, 5648 and 5893) were planted in the field last April 15, 2007, to be used in the continuing effort of introducing PRSV-P resistance gene(s) in other elite papaya lines.
Sub-Project 3. Development of molecular markers for PRSV-P resistance.
An attempt to develop a molecular marker was successfully completed. To confirm introgression, genetic fingerprinting of several genotypes of C. papaya and some of its wild relatives was carried out using simple sequence repeats (SSRs).
Preliminary result indicated introgression of a putative nucleotide fragment (150bp) from V. quercifolia, hypothesized to mark for PRSV resistant attribute of both V. quercifolia and F1 intergeneric hybrids (410, 468 and 469). In the resistant backcross progenies, a putative nucleotide fragment (100 bp) was observed to be consistent. This 100 bp could be a fragment of that putative nucleotide fragment from V. quercifolia which could explain the resistance of the backcross plants.
Sub-Project 4. Micropropagation of elite papaya genotypes.
Micropropagation of some BC3 (5648 x BC2 03R-73), BC3 sib-cross plantlets, F1 intergeneric hybrids (404, 410, 468, 469, and 507) and RR papaya cultures (cross between two Vasconcellea species) from Australia is continuing. Clonal micropropagation of BC1 is also in progress.
Sub-project 5. Evaluation on Grower's Properties
Re-constituted "Sinta" (BC2 03R lines crossed with 4172) is now being tested along with the original "Sinta" in the farmer's field in Batangas. Evaluation of resistance of these Re-constituted "Sinta" is still on-going. Fruit evaluation of this line was also Completed.