Analysis of Papaya Intergeneric Hybrids for Morphological Traits

Successful crosses have been achieved in intergeneric hybridization between Carica papaya and wild Vasconcellea cauliflora , to break post zygotic barriers by using various nutrients. Among the nutrients used, sucrose 5 per cent, sucrose 5 per cent + boron 0.5 per cent and sucrose 5 per cent + CaCl 2 0.5 per cent improved the fruit set and seed set percentage. A total of 1197 flowers were pollinated resulted 308 fruits. On extraction, 721 seeds were obtained from CO 7, Pusa Nanha and CP 50. Out of twenty nine F 1 hybrid plants of CO 7 x Vasconcellea cauliflora, only six plants namely CO7V1 to CO7V6 were found free from PRSV symptoms. Similarly, out of fifty five F 1 hybrid plants of Pusa Nanha x Vasconcellea cauliflora only twenty three plants namely PNV1 to PNV23 were found free from the symptoms and seventy plants namely CPV1 to CPV70 out of 335 plants of CP50 x Vasconcellea cauliflora were found free from PRSV symptoms. F 1 hybrids exhibited very clear morphological marker in the form of pink vein colour. The number of days for first flowering, first flowering height (cm), plant height (cm), stem girth (cm), number of leaves, first fruiting height (cm), fruit set (%), days to first harvest, fruit yield per tree (kg), were intermediate between the two parents.

Papaya, also called papaw or pawpaw, is an edible fruit of tropical softwood tree (Carica papaya) of the family Caricaceae. It is native to Central America and has spread to many other tropical and sub-tropical lands including India, the Philippines, and parts of Africa by way of early trade routes. Papaya is considered one of the most economically important and nutritious fruits for rich source of antioxidants, carotenes, vitamin C and flavonoids; the B vitamins folate and pantothenic acid; the minerals potassium and magnesium; and fiber. In addition, papaya is the source of the digestive enzyme papain, which is an industrial ingredient used in brewing, meat tenderizing, pharmaceuticals, beauty products and cosmetics. India and Indonesia have consistently been the top producers, contributing more than 71% of the total world production (FAOSTAT, 2004). At present, it is cultivated throughout the world. India is one of the largest producer of papaya in the world has an area of about 98, 000 ha with an annual production of about 3629 million tonnes (NHB, 2009). 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.
Papaya crops suffer from several diseases and pests, the most ubiquitous and widespread of which is papaya ringspot virus (PRSV). Almost all cultivated varieties are found to be highly susceptible. This virus affects production and productivity in every region of the world by decreasing photosynthetic capacity of the plants, which subsequently display stunted growth, deformed and made it fruit inedible and eventually, plant mortality.
Incidence of PRSV has been reported to be more than 90 per cent in India (Varma, 1996) and rendering papaya orchards economically unviable (Hema and Theertha Prasad, 2003). In Tamil Nadu, the disease was first noticed in Coimbatore during 2003(Jyoti Sharma et al., 2004. Existing management technologies are no way successful to check the disease. The conventional breeding is the only reliable tool for long term control of the disease. None of the Carica papaya cultivars has natural-resistance to PRSV-P. Several related wild species of Carica have been reported as resistant to PRSV-P. However, several wild Carica species such as C. cauliflora, C. pubescens and C. quercifolia are resistant to PRSV. Conventional interspecific hybridization of Carica papaya with other species has been difficult because of interspecific reproductive barriers (Manshardt and Wenslaff, 1989). In Australia, hybrids between C.papaya and Vasconcellea cauliflora were produced via embryo rescue technique. Dinesh et al. (2007) produced intergeneric hybrids in Carica papaya and Vasconcellea cauliflora using sucrose 5 % as an agent to break reproductive barrier. Keeping the above points in consideration the present study was contemplated to produce the F1 hybrids between different varieties of C. papaya and Vasconcellea cauliflora for the resistance against PRSV.

Source of parental material and Production of F1 hybrids
Different varieties of C. papaya and a PRSV resistant male parent, Vasconcellea cauliflora were obtained from the Department of Pomology, Horticultural College, Coimbatore. F1 hybrids were developed through intergeneric hybridization by adopting the procedure given by Dinesh et al. (2007).

Glass house screening of F1 hybrids and Parents
A total of three hybrids, one male parent and three female parents were screened for PRSV resistance under glass house conditions by sap inoculation method. 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 â-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 was given using the scale developed by Dhanam (2006).

Field evaluation
The cross combinations and parents were subjected to field evaluation for resistance against PRSV. Six F1 hybrid seedlings of CO 7 x Vasconcellea cauliflora, 23 F1 hybrid seedling of Pusa Nanha x Vasconcellea cauliflora and 70 F1 hybrid seedlings of CP50 x Vasconcellea cauliflora along with parents (6 seedlings each) were transplanted in the main field for evaluation. Seedlings were planted in main field in Randomized Block Design and standard practices were adopted. The disease intensity in each combination was scored according to the scale developed by Dhanam (2006).

DAS ELISA of hybrids and parents
Apparently virus free 99 plants from different hybrid combinations and parents were subjected for ELISA confirmation using PRSV specific antibody. Antibody for PRSV and their positive samples were provided from DSMZ, Braunschweing, Germany. AS-ELISA was performed for the detection of PRSV by following the manufacturer's instructions (DSMZ Gmbh, Braunschweig, Germany).

Morphological analysis
Intergeneric F1 hybrids exhibited very clear morphological marker in the form of vein colour and colour of petiole was observed by visual appearance. Morphological characters (Days to first flowering, 167 First flowering height (cm), Plant height (cm), Stem girth (cm), Number of leaves , First fruiting height (cm), Fruit set (%),Days to first harvest, Fruit yield per tree (kg), were intermediate between the two parents) were examined. Days taken for the first flower to appear from the date of planting was counted and expressed in numbers. Height of the plant was measured from ground level to the node of first flower and expressed in centimeters. Stem girth was measured 15 cm above the ground level and expressed in centimeters. Fully developed, opened leaves were counted from the tip to the base of the tree and expressed in numbers. Fruit set per cent was calculated by the formula.

No. of fruits harvested
Fruit set per cent = ------× 100 No. of fruit set The days taken from the date of planting to the date on which the fruit was first harvested from the tree at colour break stage was counted and expressed in number. The fruit yield per tree was arrived at by multiplying number of fruits harvested with mean fruit weight and expressed as kilograms.

Results and Discussion
Inter-generic hybridization of Carica papaya with other genus Vasconcellea cauliflora was started with the aim to transfer the desirable genes for PRSV resistance into the cultivars of papaya. In the present investigation, a large scale crossing programme was initiated using nine papaya varieties viz., CO 1, CO 2, CO 4, CO 5, CO 6, Pusa Dwarf, Pusa Nanha, CP 50 all dioecious and CO 7 a gynodioecious type, as females with Vasconcellea cauliflora as male. Among the nine varieties used for crossing, only three cross combinations viz., CO 7 x Vasconcellea cauliflora, Pusa Nanha x Vasconcellea cauliflora and CP 50 x Vasconcellea cauliflora had produced viable seeds and the rest produced only parthenocarpic fruits. Though the pollination was enhanced by using different nutrient solutions, the success of hybridization varied considerably with the varieties/ genotypes used. Litz and Conover (1983) also reported that the success in intergeneric hybridization between C.papaya and V.cauliflora varied depending on the genotype used for crossing. Manshardt and Wenslaff (1989), Sawant (1958), and Zamir and Tadmore (1986) also confirmed the same.
In the present study, out of 721 F0 seeds sown in the nursery, 419 seed germinated with a percentage of 57 due to intergeneric crosses. Intergeneric cross involving CO 7 x Vasconcellea cauliflora had 29 hybrid seedlings out of 45 seeds, Pusa Nanha x Vasconcellea cauliflora had 55 hybrid seedlings out of 67 seeds and CP50 x Vasconcellea cauliflora had 335 hybrid seedlings out of 609 seeds. Thirugnanavel (2010) also obtained six seedlings Data not statistically analyzed, Disease scoring was 0 to 5 (0 = no disease symptoms; 1 = slight mosaic on leaves; 2 = mosaic patches and/or necrotic spots on leaves; 3 = leaves near apical meristem deformed slightly, yellow, and reduced in size; 4 = apical meristem with mosaic and deformation; 5 = extensive mosaic and serious deformation of leaves, or plant dead).
from 222 seeds (two from CO2 and four from CP 50).

Screening of F1 progenies through artificial inoculation against PRSV under glass house conditions
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 Vasconcellea cauliflora, six (CO 7V1 to CO 7V6) were found to be apparently free from the disease. Similarly in the cross combination Pusa Nanha x Vasconcellea cauliflora, 23 out of 55 seedlings (PNV1 to PNV23) were found to be apparently free from PRSV. In the cross combination CP 50 x Vasconcellea cauliflora, 70 out of 335 seedlings (CPV1 to CPV70) were apparently free from PRSV disease. However, the female parent showed typical PRSV symptoms and the male parent V. cauliflora was free from PRSV after artificial inoculation (Table 1). 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. 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  Manoranjitham et al., 2008).

Hybrid confirmation by morphological markers
The morphological markers identified by the breeders to confirm the hybridity in the male parent Vasconcellea cauliflora has very distinctive character of pubescent petiole with light pink colour and five pink colour veins found in Vasconcellea cauliflora against seven to eight veins with light yellow to green found in C. papaya (Table 2). 169 Among the hybrid combinations, the crosses involving Pusa Nanha x Vasconcellea cauliflora exhibited very clear morphological markers in the form of vein colour. The crosses had light pink veins against light yellow to green found in female parent. Similarly, the colour of the petiole and texture of the petiole were intermediatery in nature in the hybrids (Fig.1). Morphological characters could be used as a reliable visual marker for preliminary identification of the hybrid prior to genetic analysis. Intermediate morphological characters have been used previously for the identification of C. papaya x C. cauliflora interspecific hybrids by Chen et al. (1991). In the present study, among the cross a -Female; b -Hybrid; c -male

Fig. 1. Morphological marker in the F1 intergeneric population
combinations, Pusa Nanha x Vasconcellea cauliflora crosses were found to be early in flower and fruit production, flowers at lowest height with maximum leaves at first flowering and at harvest. Early vigour is an important morphological character which is considered to be a necessary trait for disease resistance, because, before the plant is fully infested, it can able to give a reasonable yield (Thirugnanavel 2010).Among the cross combinations, Pusa Nanha x Vasconcellea cauliflora had recorded the maximum fruit set percentage, however for days to harvest, not much difference was observed among the crosses. (Table 3, 4 and 5) Manshardt et al. (1995) also evaluated several F1 interspecific hybrids and found better growth and vigoures among them. Hybridization of V. cundinamarcensis and V. parviflora produced vigorous F1 plants (Drew et al., 1998). These

Conclusion
Intergeneric hybrids in Carica papaya and Vasconcellea cauliflora using sucrose 5 per cent, sucrose 5 per cent +boron 0.5 per cent and sucrose 5 per cent + CaCl2 0.5 per cent improved the fruit set and seed set percentage. Morphological characters could be used as a reliable visual marker for preliminary identification of the hybrid prior to genetic analysis. Pusa Nanha x Vasconcellea cauliflora exhibited very clear morphological markers. Based on the disease intensity score, reaction to the PRSV and performance, the cross combinations viz., CO 7 x Vasconcellea cauliflora (CO7V3), Pusa Nanha x Vasconcellea cauliflora (PNV9) and CP 50 x Vasconcellea cauliflora (CPV23) were advanced to F2 generations.