Screening of Carica Papaya x Vasconcellea Cauliflora Hybrids for Resistance to Papaya Ring Spot Virus (PRSV)

Carica papaya x vasconcelleacauliflora and intergeneric F₁ hybrids of these species were screened for resistance to severely infected papaya ringspot virus isolates of papaya ringspot virus. Artificial screening for papaya ringspot virus was carried out 27 days after sap inoculation. Out of twenty-nine F₁ hybrid plants of CO 7 x Vasconcelleacauliflora, only six plants were found free from PRSV symptoms. Similarly, out of fifty-five F₁ hybrid plants of PusaNanha x Vasconcelleacaulifloraonly twenty-three were found free from the symptoms and seventy plants out of 335 plants of CP50 x Vasconcelleacauliflora were found free from PRSV symptoms. The resistance of the hybrids and parents and their hybrids viz, CO 7 x Vasconcelleacauliflora, PusaNanha x Vasconcelleacauliflora and CP50 x Vasconcelleacauliflora were subjected to DAS ELISA test. Molecular marker viz, ISSR markers were used to check and verify the hybridity. ISSR markers showed confirmity on three hybrid progenies viz, CO7V3, CO7V5 and CO7V6 from CO 7 xVasconcelleacauliflora.

Keywords: Carica Papaya, V.C. (Vasconcelleacauliflora), CO7V3 (CO7 x V.c), PNV1(Pusa Nanha x V.c) CPV1(CP 50 x V.c) Intergeneric Hybrids, Papaya Ringspot Virus

Papaya (Carica papaya L.), a delicious fruit tree, is affected by number of diseases caused by various pathogens and viruses. At present, it is cultivated throughout the world. Besides Central America, papaya is important as a commercial plant in Hawaii, South Africa, Australia, India, Ceylon, the Philippines and South-East Asia. The names papaw, pawpaw, paw-paw, melon pawpaw, papaya and papita are applied to Carica papaya L, the most commonly used being papaya and papaw. The papaya plant has short life, hence the area under cultivation varies greatly in different years. In India it is cultivated over an area of 97.7 thousand hectares with annual production of 3628.9 thousand MT. (NHB, 2020). In India, it is commercially cultivated in Andhra Pradesh, Gujarat, Maharashtra, Karnataka, West Bengal, Assam, Orissa, Madhya Pradesh, Manipur, Tamil Nadu and Bihar and certain extent in Kerala.

The papaya is popular as a backyard tree in many developing countries but increasingly becoming more important in commercial plantings for domestic markets and for export in countries like Mexico and Malaysia. The advantage in papaya cultivation is the rapid return of investment due to its early maturation, intensive cultivation and high yield. Most papayas in the tropics can be harvested 8 or 9 months after sowing and yields can range from 60 to 100 t/ha/year for improved varieties. The ripe fruit has a delicate aroma and sweetness and has high contents of vitamins A and C. One medium-sized papaya exceeds the Dietary Reference Intakes (DRI) of 3000 IU for vitamin A and 90 mg for vitamin C, established by the U.S. Food and Nutrition Board (OECD 2004). There is great diversity in the size, shape and quality of the fruit. In unselected germplasm or backyard trees, fruits are usually very large and not very palatable, but varieties such as ‘Solo’ and ‘Eksotika’, specifically selected for export or up-markets, are usually small for convenience in packaging and have much better taste and storage attributes. Papaya is usually eaten fully ripe when the flesh is soft and succulent. However, it can also be eaten raw, sliced into thin strips and eaten as vegetable or processed into various products such as candy, pickle or puree. The ‘Eksotika’ papayas imported by China are served as a delicacy in high-end restaurants: the half-cut fruit with seed scooped out is filled with ‘sharks-fin’ or ‘birds-nest’ and steamed before serving. The latex from unripe fruit and leaves contains a proteolytic enzyme papain, which can be used for tenderizing meat, chill-proofing beer, tanning leather and for making chewing gum. In pharmaceutics, papain is used for suppression of inflammation, treatment of gangrenous wounds and for various digestive ailments. As a proteolytic enzyme, it has exfoliating property that removes the dead surface cells of the skin, giving it a rejuvenated feeling. It is therefore popularly used in soaps, creams, shampoos and lotions in the cosmetic industry.

Papaya is affected by number of diseases caused by various pathogens and viruses. Nowdays the most destructive disease of C. papaya worldwide is papaya ring spot caused by papaya ring spot virus-type P Litz, (1984), Manshardt, (1992), a definitive potyvirus species in the Potyviridae (Shukla et al, 1994). PRSV is grouped into two types, Type P (PRSV - P) infects cucurbits and papaya and type W (PRSV-W) infects cucurbits but not papaya (Gonsalves, 1998). Almost all cultivated varieties are highly susceptible. Carica cauliflora J, a wild species having non-edible fruits is known to be resistant for this viral disease (Jimenez and Horovitz, 1957). Now the species cauliflora has been grouped under the genera Vasconcellea (Vegas et al, 2003).

Control measures to check the viral incidence against PRSV-P include cultural practices, cross-protection and planting of tolerant cultivars (Gonsalves, 1994). None of these has been very successful and the development of virus resistant cultivars through conventional breeding is the only reliable tool for long term control. None of the Carica papaya cultivars has natural-resistance to PRSV-P. Even though interspecific hybridization of Carica papaya with other species attempted, a very little work has been done using Vasconcellea cauliflora which has the desirable gene for PRSV resistance (Jayavalli et al, 2015). Selection and sibmating of intergeneric progenies of papaya and evaluation of intergeneric progenies (F6) for fruit characteristics and PRSV tolerance (Vasugi,2022), Disease resistances that have been identified in Carica species for PRSV-P resistance are C. cauliflora, C. pubescens, C. quercifolia and C. stipulate (Conovar, 1964, Horovitz and Jimenez, 1967). Papaya breeding in India can be broadly classified into three phases. Work carried out at Tamil Nadu Agricultural University, Pusa, Pantnagar, Pune and at Bangalore has resulted in the development of new varieties suitable for papain extraction and for table purpose. Information on the inheritance pattern has helped in identifying the parents as gene donors for several characters. In recent times the breeding is being carried out with the objective of developing lines resistant to PRSV (Dinesh, 2 010). Papaya ringspot virus type P(PRSV-P) is a major threat to the papaya industry worldwide. F1 hybrids have been produced when Carica papaya L. female flowers have been pollinated with pollen of the PRSV-P resistant species Vasconcellea quercifolia. A single dominant gene for PRSV-P resistance in V. pubescens has been mapped by use of dominant, polymorphic randomly amplified DNA fingerprint (RAF) markers in F2 interspecific population of V. parviflora (PRSV-P susceptible) and V. pubescens (PRSV-P resistant) Drew et al, 2007. Hydrogel capsules are a potential candidate for drug delivery and an interesting alternative to polyelectrolyte multilayer capsules which are under investigation in the last 20 years. Recently introduced polyelectrolyte complex capsules produced by spraying are non-biodegradable and not biocompatible, which limits their practical application, while biodegradable alginate capsules require complex coaxial electrospray ionization jetting. biodegradable alginate capsules cross-linked by calcium are successfully produced by hydrodynamic electrospray ionization jetting with the assistance of low frequency ultrasound. The size and shape of most capsules show significant differences with respect to different spraying distance, spraying mode, electrode shape and spraying concentration. Capsules in the shape of vase, mushrooms and spheres were successfully produced. Average capsule size can be adjusted from 10 μm to 2 mm. These capsules are used to encapsulate a model drug. Encapsulated paramagnetic particles enable defined directional motion under the propulsion of a rotating magnetic field, while model drugs can be released by ultrasound (Rutkowski et al, 2019

Disease resistance, increased yields and improved quality and storage traits are important objectives for breeding programmes of any crop. While significant improvements have been made with conventional hybridization techniques, programmes incorporating methods of genetic engineering offer opportunities for the transfer of genetic variability from other gene pools. Much of the review addresses transgenic virus resistance, which is the major application. Approaches related to improved quality traits and pharmaceutical productions are also examined (Melaine Randle, Paula Tennant, 2020 Hence, the development of virus resistant cultivars through conventional breeding is the only reliable tool for long term control and cost of production is very low compare to production of transgenic papaya. Under these circumstances, screening of Carica papaya x Vasconcellea cauliflora hybrids for resistance to papaya ringspot virus (PRSV) is attempted.

Present investigations on the breeding for papaya ring spot virus (PRSV) resistance in papaya (Carica papaya L.) were carried out in the College Orchard, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore.

One gram of infected leaves was ground in a pre-chilled mortar and pestle using 1 ml of 0.1M chilled sodium phosphate buffer (pH 7.2) containing β-mercaptoethanol and 0.01 M EDTA. The sap was rub inoculated using the pestle or glass rod on the young leaves of seedlings at 3 leaves stage previously dusted with carborundum powder 600 meshes. After 5 minutes, the excess sap was washed off by distilled water. The disease incidence and intensity score were given using the scale developed by Dhanam (2006 Details of the disease incidence and intensity score scale is presented in Table 1.

Antibody for PRSV and their positive samples were provided from DSMZ, Braunschweing, Germany.

DAS-ELISA was performed for the detection of PRSV by following the manufacturer’s instructions (DSMZ Gmbh, Braunschweig, Germany). Purified IgG was diluted in coating buffer (1:1000) and 200 μl was added to each well of a micro titer plate (Grainer). The plates were then incubated at 37oC for 2 to 4 hours and thereafter plates were washed with PBS-T using wash bottle, soaked for a few minutes and repeat washing for twice. Plates were blotted by tapping upside down on tissue paper. 200 μl aliquots of the test sample (extracted in sample extraction buffer) were added to duplicate wells. The plates were incubated overnight at 4oC. The plates were washed as in earlier and added with 200 μl of the anti-virus conjugate (1:500) to each well and incubated at 37oC for 2 hours. Then the plates were washed three times as done earlier. Finally, 200 μl of freshly prepared substrate (10 mg ρ-nitro phenyl phosphate (Sigma 104-105) dissolved in 10 ml of freshly prepared substrate buffer) was added to each well and incubated in dark at room temperature for 20 to 45 minutes or as long as necessary to obtain clear reactions. Spectrometric measurement of absorbance was then read at 405 nm (EL 800, BIO-TEK Instrument Inc, and USA The reaction was stopped by adding 50 μl of 3 M NaOH. Buffer served as negative control.

DNA extraction from leaves of parents and F1’s was carried out following CTAB method (Doyle and Doyle, 1987).

PCR reaction was performed using 10 (SSR) and 6 (ISSR) primers. The reagents that required for performing PCR reaction are as follows. The details of the primers are presented in APPENDIX I.

PCR reaction was carried out in total volume of 10 μl in 96 tubes PCR plates. Following was the master mix of solution for one reaction.

Touch down protocol was followed for all the primers.

Electrophoresis was performed in 1.5 per cent agarose with 120V for 2 hours. PAGE electrophoresis was carried out for SSR’s silver staining protocol as performed following Benbouza et al. (2006).

Carica papaya and Vasconcellea cauliflora were produced via intergeneric hybridization. Intergeneric hybrid seedlings along with parents were raised and artificially inoculated with PRSV under glass house conditions for screening. Observation for PRSV was done 27 days after inoculation. Out of 29 intergeneric hybrid seedlings involving CO 7 x V.c six were found to be apparently free from the disease. Similarly in the cross-combination Pusa Nanha x V.c out of 55 seedlings, 23 seedlings were found to be apparently free from PRSV. In the cross-combination CP 50 x V.c out of 335 seedlings, 70 seedlings were apparently free from PRSV disease. However, all the parents except Vasconcellea cauliflora showed typical PRSV symptoms after artificial inoculation (Table 2). In a perennial crop like papaya, field screening for diseases is very difficult since, it requires a larger area for planting. Hence, screening in glass houses in the nursery stage proved quick and rapid method.

Touch down protocol was followed for all the primers.

Typical PRSV symptom of mottling of leaves and water-soaked lesions on stems were observed in the susceptible parents and the hybrids. However, six out of 29 seedlings in CO 7 x V.c, 23 out of 55 in Pusa Nanha V.c and 70 out of 335 in CP 50 V.c were found to be completely free from PRSV symptoms (Plate 1). Regarding the female parents, all were found to exhibit the virus symptoms uniformly after sap inoculation. Symptom free F1 hybrids were transplanted in the main field for further evaluation. The failures of PRSV symptoms to develop on the manually inoculated hybrid plants indicate the incorporation of genes resistant to PRSV. Further, the wild genus V. cauliflora was found to be completely resistant to the strain PRSV prevalent in Coimbatore area of Tamil Nadu, India (Manoranjithamet al, 2008).

The Enzyme Linked Immunosorbent Assay (ELISA), a powerful immunological test (Clark and Adams, 1977), is extensively used for detecting, identifying and quantifying viruses in many plant species (Clark, 1994). Parents and their hybrids viz, CO 7 x V.c, Pusa Nanha x V.c and CP50 x V.c were subjected to DAS- ELISA test.

Parents and F1 progenies involving CO 7 and Vasconcellea cauliflora were subjected to DAS- ELISA test ELISA titre value varied from 0.216 to 0.972. Among the parents, the resistant male parent Vasconcellea cauliflora had recorded the lowest titre value of 0.216. However, the susceptible female parent CO 7 recorded the highest titre value of 0.972, followed by PusaNanha (0.952) and CP 50 (0.942) (Plate 2).

Among the hybrids involving CO7 and V.c, ELISA titre value varied from 0.243 to 0.266 (Table 3). Among the hybrids involving Pusa Nanha x V.c, ELISA titre value varied from 0.218 to 0.286 (Table 4). Among the hybrids involving CP50 x V.c, ELISA titre value varied from 0.218 to 0.299 (Table 5).

The cross combinations namely CO7V3, CO7V5 and CO7V6 were found to record lower titre values proving their tolerance to PRSV. Similarly, cross combination involving crosses viz, PNV1, PNV3, PNV9, PNV6, PNV8, PNV13 and PNV21 were found to record lower titre values proving their tolerance to PRSV. F1 progenies namely CPV1, CPV12, CPV23, CPV31, CPV39, CPV26 and CPV56 were found to record lower titre values proving their tolerance to this virus (Plate 3, 4). This observation confirms the earlier report of Manshardt (1992) who studied the intergeneric hybrids involving C. cauliflora x C.papaya hybrids. Similar studies using ELISA test had been conducted previously to identify PRSV-P infected C. papaya (Gonsalves and Ishii, 1980, Thomas and Dodman, 1993).

The main objective of resistance breeding is the introgression of one or more resistant genes from the donor parent into the elite variety. Precise identification of plants using morphological markers to distinguish the true hybrid and out cross seeds is difficult as the phenotypic marker to differentiate male and female plants should be available (Zamir and Tadmore, 1986). In the absence of that proceeding for further generations to tag the useful genes conferring resistance, molecular marker will be a reliable tool to discriminate the hybrids and the parental lines. In the present investigation, to verify the hybridity and the level of resistance derived from V. cauliflora, a study was carried out using Inter- simple sequence repeats (ISSR). To detect hybridity, there must be polymorphism between the parents. Polymorphic bands which are present in male parent should be present in all the hybrids and should not be present in female parent (Magdalita et al, 1998).

The primer UBC - 856 produced unique banding patterns in Vasconcellea cauliflora (male parent) in which five bands were prominent, out of which third and fifth were absent in female parent (Figure.1) but present in CO 7 x Vasconcellea cauliflora (CO7V3). The same primer produced distingusible band between PusaNanha xVasconcellea cauliflora (PNV9) which was used for the identification of true hybrid (Figure.2).

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Ruas et al. (2003) used Inter-simple sequence repeat (ISSR) markers and successfully evaluated the genetic divergence among the eight Coffea species. To confirm the hybridity of intergeneric hybrids involving Carica papaya x V. cauliflora, Praveen (2005) also used ISSR markers and confirmed successfully.

In case of UBC- 807 primer, one prominent band was observed in male parent which was absent in female parent but present in CP 50 xVasconcellea cauliflora (CPV23) hybrid (Figure.3). These primers were helpful to identify F1’s in cross (CO7V3, CO7V5 and CO7V6), (PNV1, PNV3, PNV6, PNV8, PNV9, PNV11, PNV13 and PNV21) and (CPV1, CPV23, CPV12, CPV26, CPV31, CPV39 and CPV56). The hybridity confirmed F1 plants were forwarded to F2.