Slide1: Speaker : Nishit V. Soni Genetic improvement in papaya ( Carica papaya L.) 1
OUTLINE: OUTLINE INTRODUCTION ORIGIN AND DISTRIBUTION IMPORTANCE OF PAPAYA TAXONOMICAL STATUS FLORAL BIOLOGY GENETICS OF SEX INHERITANCE BREEDING OBJECTIVES AND METHODS CASE STUDIES VARIABILITY & HERITABILITY STUDIES CONVENTIONAL METHODS INTRODUCTION MASS SELECTION HYBRIDIZATION BACKCROSSING MUTATION STUDY NON-CONVENTIONAL METHODS TISSUE CULTURE TRANSGENICS ACHIEVEMENT CONCLUSION FUTURE PROSPECTS 2
ORIGIN AND DOMESTICATION: Cultivated papaya Wild species 1525 to Costa Rica 1550 to Philippines during Spanish exploration 1611 to India , Africa and Nepal (1650 ) Villegas, 1997 ORIGIN AND DOMESTICATION 3
Slide4: Why papaya is Important ? Rich source of vitamins, minerals, fiber and abundant source of carotene in the daily diet All the parts of papaya can be used for medicinal purpose Ripe fruits as ingredient in jam, jellies , candy and as vegetable Source of the enzyme papain , which is used in making beer, tenderizing meat, and in producing certain pharmaceutical and cosmetic products First plantation fruit tree genome sequenced: 372Mbp 4
Table 1: Nutritional value: Table 1: Nutritional value India Constituents Green papaya (100 gram) Ripe papaya (100 gram) Protein 0.7 g 0.6 g Fat 0.2 g 0.1 g Minerals 0.5 g 0.5 g Fibre 0.9 g 0.8 g Carbohydrates 5.7 g 7.2 g Energy 27 kcal 32 kcal Total carotene 0 2740 µM Beta carotene 0 888 µM 5 Krishna et al ., 2008
Slide6: Area (Lakh ha) Production (Lakh tonnes ) Productivity ( tonnes /ha) World 4.06 115.81 28.51 India 1.04 41.80 40.07 Gujarat 0.185 ha 10.60 57.35 6 FAO STAT, 2013 & NHB 2012 India having highest share in the world production of 38.61%
Terminologies: Terminologies 7 Term Meaning Staminate flower : Male flower Pistillate flower : Female flower Hermaphrodite : Male and female in one flower. Also called bisexual Monoecious : Male and female flowers on one plant at different position Dioecious : Male and female flowers on separate plants Andromonoecious : A species having male and hermaphroditic flowers on the same individual Gynodioecious : Species or p opulation having female and hermaphrodite flowers on different individuals Trioecious : Species or population having male, female, and hermaphroditic flowers on different individuals
TAXONOMICAL STATUS: TAXONOMICAL STATUS Taxonomy : Six genus in these family (Badillo, 2002) 35 species, and 32 of them are dioecious (Manshardt and Wenslaff, 1989 ). 2n = 2X =18 (Bennett & Leitch, 2005) Kingdom : Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Rosids Order: Brassicales Family: Caricaceae Genus: Carica Species: C. Papaya L. 1. Carica 4. Jarilla 2. Cyclimorpha 5. Vasconcellea 3. Jacaratia 6. Horovitzia 8
Table 3 : Importance of different species: Species Characteristics 1 Carica papaya ( Linn.) Trioecious , Cultivated papaya 2 Vasconcellea cundinamarcensis ( Hook) Trioecious , tolerant to frost 3 Vasconcellea monoica (Desf) Monoecious , susceptible to virus 4 Vasconcellea quercifolia (bant and Hook) Dioecious, Oak Leaved Papaya , papain production with high proteolytic activity, resistant to distortion ringspot 5 Vasconcellea cauliflora (Jacq) Dioecious, resistant to PRSV virus, Mountaineous papaya, species is grown in higher altitude 6 Vasconcellea pubescens (Lenne et Koch) Dioecious, resistant to distortion ringspot 7 Vasconcellea stipulata (Badillo) Dioecious, resistant to distortion ringspot 8 Vasconcellea candicans (Gray) Dioecious, resistant to distortion ringspot 9 Vasconcellea pennata (Heilborn, Siensk) Dioecious, resistant to frost 10 Vasconcellea pentagona (Heilorn) Dioecious, resistant to frost 11 Vasconcellea microcarpa (Jacq) Dioecious, papain production 12 Vasconcellea goudotiana (Solms-Lauback) Dioecious, papain production 13 Vasconcellea parviflora (Solms) Dioecious, papain production 14 Vasconcellea horovitziana (Badillo) Dioecious, papain production Table 3 : Importance of different species 9
Sex forms in trioecious papaya Carica papaya (Linn.) Vasconcellea cundinamarcensis (Hook): Sex forms in trioecious papaya Carica papaya (Linn.) Vasconcellea cundinamarcensis (Hook) Female flower Hermaphrodite flower Male flower Female fruit Hermaphrodite fruit Male tree 10
Slide11: Female flower Male flower Mature fruit 11 Sex forms in monoecious papaya Vasconcellea monoica (Desf.)
Examples of distinct leaf morphology: Vasconcellea quercifolia (bant and Hook) Oak Leaved Papaya Vasconcellea cauliflora (Jacq) Castor type leaves 12 Examples of distinct leaf morphology
Floral biology: Floral biology Storey (1941) classified papaya flowers into five basic types based on Flower types and sexes Type I Pistillate or female flower devoid of stamens, with a distinct ovoid ovary terminating in a five lobed stigma TypeII Hermaphrodite (Pentandria ) flower having five functional stamens and a globose five furrowed ovary TypeIII Hermaphrodite ( Carpelloid ) flower having six to nine functional stamens and an irregular ridge ovary 13
Slide14: TypeIV Hermaphrodite (Elongata) flower having ten functional stamens ( 5 long + 5 short . most commercially important type ) and elongated, smooth ovary TypeIV+ Hermaphrodite (Barren) flower having ten functional stamens but the pistil aborts , becomes vestigial and lacks a stigma TypeV Staminate flower having ten functional stamens only. The ovary is completely absent and flower are bunched in an inflorescence Storey (1941) classified papaya flowers into five basic types 14
Genetics of sex inheritance: Genetics of sex inheritance Hofmeyr, 1938 explained genetics of sex in papaya, determined by monogenic inheritance involving three alleles ‘M 1 ’ male, ‘M 2 ’ bisexual & ‘m’ female M 1 M 1, M 1 M 2 and M 2 M 2 homozygous dominant are letha l M 1 m = male M 2 m = bisexual mm = female Pollination combination Segregation ratio Female (mm) Bisexual (M 2 m) Male (M 1 m) Non-viable Lethal (M 1 M 1, M 1 M 2 and M 2 M 2 ) mm X M 1 m 1 - 1 - mm X M 2 m 1 1 - - M 1 m (Self) 1 - 2 1 M 1 m X M 1 m 1 - 2 1 M 2 m (Self) 1 2 - 1 M 2 m X M 2 m 1 2 - 1 M 2 m X M 1 m 1 1 1 1 M 1 m X M 2 m 1 1 1 1 15
Slide16: Major objectives: Higher yield with better quality fruits Development of gynodioecious varieties with incorporation of resistance to Papaya ringspot virus ( PRSV ) Early and low bearing height with short Internodal length Minor objectives: Uniform fruit shape, texture and flavour for domestic and export trade Increasing latex yield for papain production. Resistance to biotic stress such as virus, fungi and bacteria Tolerant to abiotic stress like frost, excessive soil moisture and salinity 16 BREEDING OBJECTIVES
Slide17: Conventional method Introduction Mass selection Hybridization Intervarietal hybridization Interspecific hybridization Backcross Breeding Mutation Breeding Non-conventional method Tissue culture Somatic hybridization Transgenic technology 17 BREEDING METHODS
Variability: Variability 18 CASE STUDIES
Slide19: Trait Range GCV(%) PCV(%) h 2 bs (%) GA over mean (%) Plant height (cm) 139.47-237.77 13.58 14.54 87.29 26.14 Stem diameter (cm) 29.23-37.77 5.93 9.26 41.09 7.85 Fruit weight (g) 486.67-1380.33 21.60 27.85 60.12 34.49 Fruit length (cm) 12.33-23.80 20.01 20.88 91.86 39.53 Fruit breadth (cm) 7.97-15.23 16.25 17.93 82.13 30.27 Pulp thickness (cm) 2.10-3.27 15.04 16.05 87.79 28.96 TSS (˚Brix) 9.53-13.83 11.43 12.93 78.06 20.82 Total caretenoids (mg/100g) 1.53-3.27 20.74 22.26 86.83 39.68 Titratable acid (%) 0.13-0.33 20.42 25.69 63.21 33.33 Ascorbic acid (mg/100g) 36.27-69.00 16.15 17.55 84.66 30.60 Das and Dinesh, 2004 Bangalore, India Table 4: Genetic Variability, heritability and genetic advance in 12 papaya varieties 19
Slide20: Characters Range PCV (%) GCV (%) H 2 bs (%) GA (% of mean) Plant height (cm) 138.4-240.6 19.82 18.24 84.84 34.32 Plant girth (cm) 28.2-36.8 9.28 5.95 41.10 7.87 Days to first flowering 122-188 14.94 14.32 92.60 28.24 Node at first fruiting (no.) 28.6-84.8 23.80 18.67 61.52 30.12 Leaf length(cm) 32.0-54.2 20.88 10.58 25.62 24.26 Fruiting height (cm) 46.4-142.4 22.22 21.46 92.62 42.60 Fruit length (cm) 44.4-104.2 26.84 25.24 92.68 50.42 Fruit weight (kg) 0.48-2.20 40.26 39.00 96.00 78.24 No. of fruits / plant 32.20-62.40 29.24 20.72 50.44 30.84 Fruit yield/ plant (kg) 20.8-38.40 44.00 42.26 92.80 81.22 20 Singh and Kumar, 2010 Bihar, India Table 5 : The range, coefficient of variation, heritability and genetic advance for 10 characters in 18 papaya genotypes
Slide21: 1 2 3 4 5 6 7 8 9 10 1 No. of fruits/ plant G 1.000 0.412* -0.402* -0.194 -0.214 0.421* 0.712 0.452 0.316 -0.418 P 1.000 0.336* -0.317* -0.121 -0.162 0.310* 0.646 0.321 0.276 -0.388 2 Fruit yield / plant G 1.000 0.724 -0.498* -0.434* 0.386 0.320 0.516 -0.314* -0.534* P 1.000 0.686 -0.446 -0.358* 0.242 0.240 0.441 -0.232 -0.462 3 Fruit weight G 1.000 -0.396* -0.312 0.214 -0.232 0.242 -0.454 -0.274 P 1.000 -0.362* -0.272 0.100 -0.151 0.111 -0.392 -0.182 4 Fruiting height G 1.000 0.814 0.426* 0.124 -0.116 0.712 0.452* P 1.000 0.786 0.314* 0.072 -0.072 0.602 0.362* 5 No. of n odes at first fruiting G 1.000 0.342 -0.054 -0.024 0.628 0.462 P 1.000 0.221 -0.032 -0.011 0.542 0.371 6 Plant girth G 1.000 0.442 0.236 0.312 0.124 P 1.000 0.386 0.168 0.226 0.059 7 Fruiting length G 1.000 0.421* 0.616 -0.242 P 1.000 0.338* 0.566 -0.157 8 Leaf length G 1.000 0.422* -0.416 P 1.000 0.326* -0.388 9 Plant height G 1.000 0.032 P 1.000 0.018 10 Days to flowering G 1.000 P 1.000 *,** Significant at 5% and 1% levels, respectively P = Phenotypic correlation coefficient G = Genotypic correlation coefficient 21 Singh and Kumar, 2010 Bihar, India Table 6: Correlation coefficient at the phenotypic (P) and genotypic (G) levels with respect to growth, yield and yield attributes of papaya
INTRODUCTION: INTRODUCTION Sr no. Exotic cultivars Country 1 Red lady USA 2 Taiwan-785 Taiwan 3 Taiwan-786 Taiwan 4 Washington USA 5 Solo Barbados 6 Sunrise Solo USA 7 Sunset USA 8 Waimanalo USA 9 Higgins USA 10 Wilder USA 11 Thiland Thiland 12 Eksotika Malasiya 13 Kapoho Solo USA 14 Kamiya USA 22
Mass selection: Mass selection Cultivar name Selection from Specific trait Pusa Dwarf Pusa Giant Low fruiting height (40cm) CO 5 Washington Papain production (1500-1600 kg/ha of dried) CO 6 Pusa Majesty Dwarf stature, large fruit, both papain and table purpose Coorg Honey Dew Honey dew Gynodioecious type Pink Flesh Sweet Local material medium size fruit with pink flesh, TSS 12-14˚ Brix 23
Slide24: 24 Heterosis and combining ability HYBRIDIZATION
Method of pollination in papaya: Method of pollination in papaya 25
Slide26: *,** Significant at 5% and 1% levels, respectively 26 Kamalkumar et al ., 2010 Tamilnadu, India Table 7 : Relative heterosis (di) and heterobeltiosis (dii) for plant and fruit attributes of dioecious crosses Crosses Days to flowering No. of fruits at first harvest Mean fruit weight (kg) Fruit length (cm) Dioecious di dii di dii di dii di dii CO 2 X Pusa Giant -4.32** -0.64 32.26** 7.89** -0.15 -5.51 -6.56** -11.85** CO 2 X CO 5 11.95** 14.1 -4.18** -8.74** 41.02** 37.65** 11.03** 8.13** 9-1 (D) X CO 5 -0.63 2.09** 11.64** 10.31** 2.5 0.24 11.88** 10.61** CO 5 X 9-1(D) -2.30** 0.37 -0.75 -1.93* -4.6 -6.71 3.69** 2.52 Pusa Dwarf X 9-1 (D) 1.68** 2.48** 11.58** -2.02* 2.89 -14.25** -14.39** -15.44 Fruit circumference (cm) Cavity index (%) Flesh thickness (cm) Papain recovery (g/fruit) Dioecious di dii di dii di dii di dii CO 2 X Pusa Giant -11.04** -19.20** 21.84* 21.08 -0.97 -1.92 -53.69** -54.87 CO 2 X CO 5 16.09** 12.63** 7.58 8.78 5.00** 2.61 -46.18** -49.08 9-1 (D) X CO 5 4.40** 1.48 4.85 5.19 0.17 -2.05 1.46 -6.36 CO 5 X 9-1(D) 0.94 -1.88 2.67 3.01 8.9 6.48** -18.70** -24.97** Pusa Dwarf X 9-1 (D) -9.37 -18.06** 7.94 8.28 9.30** 2.86 41.23** 24.17**
Slide27: *,** Significant at 5% and 1% levels, respectively 27 Kamalkumar et al ., 2010 Tamilnadu, India Table 8 : Relative heterosis (di) and heterobeltiosis ( dii) for plant and fruit attributes of gynodioecious crosses Crosses Days to flowering No. of fruits at first harvest Mean fruit weight (kg) Fruit length (cm) Gynodioecious di dii di dii di dii di dii CO 3 X CO 7 8.50** 12.50** -2.02* -4.36** 57.64** 33.99** 28.43** 7.45** CO 3 X IIHR 37 -2.54 3.85 6.85** 4.31** 105.75** 97.93** 12.16** -6.16** CO 7 X IIHR 37 -6.10** -3.59* 3.05** 3.05** 59.89** 40.51** 2.94** 2.94** CO 7 X Exotica -4.10** -1.94 50.00** 50.00** 89.09** 58.06** 25.38** 16.35** IIHR 37 X Coorg Honey Dew -5.66** -3.52 38.82** 26.68** 38.75** 24.22** -10.25** -18.79** Fruit circumference (cm) Cavity index (%) Flesh thickness (cm) Percentage of carpelloidy fruits Gynodioecious di dii di dii di dii di dii CO 3 X CO 7 25.00* 11.81** 42.63** 62.60** 12.28** 10.34** 183.83** 221.45** CO 3 X IIHR 37 -5.75 -13.56** -7.68** 11.47** -0.19 -6.07 -22.80** -14.52** CO 7 X IIHR 37 -1.4 -4.09 -3.27 1.82 -2.42 -9.66** -40.38** -24.40** CO 7 X Exotica 22.68** 8.18** -13.67** -11.09** -11.38** -11.38** 125.57** 175.84** IIHR 37 X Coorg Honey Dew -9.59** -13.41** -22.64** -5.94 -17.14** -25.88** 2.81 34.37**
Slide28: Crosses Acidity (%) Ascorbic acid (mg / 100g ) Carotene (mg / 100g) Sugar acid ratio Dioecious di dii di dii di dii di dii CO 2 X Pusa Giant -10.34** 0.00 -28.48** -28.93** 36.36** 25.00** -15.38** -19.96** CO 2 X CO 5 -10.34** 0.00 -43.31** -43.31** -22.58** -42.86** 20.70** -2.78** 9-1 (D) X CO 5 -53.85** -40.00** -10.82** -25.11** 15.79** 4.76* 96.66** 43.58** CO 5 X 9-1(D) -53.85** -40.00** -35.57** -45.89** -36.84** -42.86** 127.87** 66.36** Pusa Dwarf X 9-1 (D) -23.08** 0.00 55.73** 26.41** -43.18** -45.05** 22.49** -7.38** Gynodioecious di dii di dii di dii di dii CO 3 X CO 7 -37.50** -16.67** 6.82** -5.53* 16.13** -10.00** 35.79** 11.06** CO 3 X IIHR 37 0.00 0.00 -54.07** -58.64** -44.44** -50.00** 6.47** 4.69** CO 7 X IIHR 37 -25.00** 0.00 -50.77** -51.76** -18.52** -31.25** 34.18** 8.33** CO 7 X Exotica 25.00** 0.00 -10.95** -13.68** 14.29** -5.88** -37.77** -49.11** IIHR 37 X Coorg Honey Dew -25.00** 0.00 33.96** 11.52** -3.03** -5.88** 1.17 -26.18** 28 Kamalkumar et al ., 2010 Tamilnadu, India Table 9 : Relative heterosis (di) and heterobeltiosis (dii) for fruit quality attributes in dioecious and gynodioecious crosses *,** Significant at 5% and 1% levels, respectively
Table 10 : General combining ability, specific combining ability and reciprocal effect of 4 parents: Table 10 : General combining ability, specific combining ability and reciprocal effect of 4 parents Parents/ crosses Plant height (cm) plant girth (cm) Height to first flower (cm) Days to firs flower Days to harvest No. of fruits / plant Fruit weight (g) Fruit yield/ plant (kg) Acidity (%) Total sugars (%) Ascorbic acid (mg / 100 g) Total carotenoids Pusa Nanha (PN) 4.91* 0.66* -2.37 -5.50* -7.96* -8.41* 379.38* 4.55* 0.01 -1.75* 2.48* 0.13* Pusa Dwarf (PD) -22.82* -0.83* -18.49* -6.16* 6.28* -6.50* 34.06 -2.95* 0.02 -1.52* 3.49* -0.14* Coorg Honey Dew (CHD) 14.60* 0.85* 13.45* 4.11* -3.44* 11.53* -413.75* -5.01* -0.01 2.15* -1.28 0.06* Sunrise Solo (SS) 3.30 -0.68* 7.41* 7.55* 5.13* 3.38* 0.31 3.41* -0.02 1.12* -4.69* -0.04* PN x PD 14.48* -0.07 0.66 -1.16 -3.15* 6.62* -29.38 4.23* 0.01 0.37* -1.17 0.22* PN x CHD -16.88* -2.38* -8.52* -4.30 4.13* -9.66* 24.69 -0.81 -0.01 0.08 -3.41* -0.08 PN x SS -1.15 -1.37* 10.51* 1.95 -9.32* 4.50* 479.38* 2.82 -0.02* -0.49* 2.44* 0.10* PD x CHD -3.02 -1.67* 2.54 -4.95* -8.47* -3.59* 420.00 7.31* -0.02* -0.62* 1.66* 0.09 * PD x SS -17.10* -1.19* -13.05* -4.08* -5.62* 0.22 43.44 4.58* -0.02* 0.20 -1.13 -0.19* CHD x SS 1.10 0.25 -10.62* 5.59* 7.54* 12.81* -131.25* 6.69* -0.01 0.02 1.46 -0.01 PD x PN 0.81 -1.50* -9.31* 3.69* -13.88* -1.50 100.00 -0.16 0.02* -0.34 0.81 -0.04 CHD x PN -16.75* -1.31* -10.56* -1.56 -8.69* 0.75 431.25* 12.99* 0.01 -1.10* 1.82 0.02 CHD x PD -7.25 -1.03 -15.00* 0.62 0.95 -11.00* 1131.25* 23.72* -0.02* -1.78* 2.88* -0.29* SS x PN -3.69 1.16 -2.31 -6.75 7.44* -6.50* 650.00* 0.72 -0.01 -2.33* 7.50* -0.12* SS x PD -3.00 0.53 -19.00 9.56* 3.62* -9.12* 193.75* -8.24* -0.02* -1.84* -8.56* 0.06 SS x CHD -22.12* -2.91* -7.38 -8.62* -3.06* -16.75 603.75* 21.86* 0.01 0.50* -5.38* -0.01 29 Nair et al ., 2010 Kerala, India *,** Significant at 5% and 1% levels, respectively
Slide30: б 2 GCA б 2 SCA б 2 a/ б 2 d Plant height 178.35 237.65 1.50 Plant girth -1.39 7.47 0.37 Height to first flower 144.10 159.84 1.80 Days to first flower 36.49 37.46 1.90 Days to harvest 15.98 115.17 0.27 No. of fruits/plant 60.09 94.82 1.27 Fruit weight 57195.02 177858.14 0.60 Fruit yield / plant -9.60 106.63 0.18 Acidity (%) 0.72 0.29 4.96 Total sugars 3.66 0.15 48.80 Ascorbic acid 11.80 6.28 3.75 Total carotenoids 0.004 0.03 0.26 30 Nair et al ., 2010 Kerala, India Table 11 : Components of gca and sca variance in papaya
Slide31: 31 Interspecific hybridization
Table 12 : % Fruit set of intergeneric hybrids with different combination of nutrient solutions: Table 12 : % Fruit set of intergeneric hybrids with different combination of nutrient solutions Nutrient solutions Crosses CO 1 x VC CO 2 x VC CO 4 x VC CO 5 x VC CO 6 x VC CO 7 x VC Pusa Dwarf x VC Pusa Nanha x VC CP 50 x VC Sucrose 5% 30.00 20.00 20.66 41.17 24.00 12.72 12.50 22.22 20.00 Sucrose 5% + Boron 0.5% 25.00 27.27 20.83 26.66 31.03 16.00 27.27 13.33 26.66 Sucrose 5% + Cacl 2 0.5% 26.67 30.76 33.33 33.33 19.23 21.42 40.00 22.72 28.57 Sucrose 5% + Boron 0.5% + Cacl 2 0.5% 40.00 25.71 37.50 36.00 26.92 20.00 36.36 46.15 45.83 SE 3.02 1.74 3.54 2.37 2.73 1.66 5.58 5.47 4.59 SD 6.75 3.88 7.92 5.31 6.11 3.72 12.48 12.24 10.26 Confidence level (95%) 8.38 4.82 9.84 6.59 7.58 4.62 15.50 15.19 12.74 Conventional breeding strategies for PRSV resistance by intergeneric hybridization with Vasconcellea cauliflora 32 Jayavalli et al., 2011 Tamilnadu, India
Slide33: Nutient solutions CO 7 x VC Pusa Nanha x VC CP 50 x VC Number of seeds obtained Sucrose 5% 45 67 400 Sucrose 5% + Boron 0.5% - - 200 Sucrose 5% + Cacl 2 0.5% - - 9 Total number of F 0 seeds = 721 33 Jayavalli et al., 2011 Tamilnadu, India Table 13 : Number of seeds, germination percentage and number of seedlings obtained from intergeneric crosses Number of seedlings obtained Sucrose 5% 29 55 228 Sucrose 5% + Boron 0.5% - - 107 Total number of seedlings = 419 Seedling germination percentage Sucrose 5% 64.44 82.08 57.00 Sucrose 5% + Boron 0.5% - - 53.35
Slide34: Figure 1 : Seed set in intergeneric hybridization using sucrose 5% nutrient solution CO 7 x V. cauliflora Pusa Nanha x V. cauliflora CP 50 x V. cauliflora 34 Jayavalli et al., 2011 Tamilnadu, India
Slide35: Figure 2 : Confirmation of PRSV resistance in F 1 seedlings 27 days after inoculation Without symptoms With symptoms Table 14 : Screening of F 1 progenies through artificial inoculation against PRSV under glass house Parents / hybrids Total no. of plants inoculated Disease scoring (number of plants in each catogary) No. of plants without symptoms* % resistance plants 0 1 2 3 4 5 CO 7 5 0 0 0 0 0 5 0 - Pusa nanha 5 0 0 0 0 0 5 0 - CP 50 5 0 0 0 0 0 5 0 - VC 5 5 0 0 0 0 0 5 100 CO 7 x VC 29 6 0 0 0 10 13 6 20.69 Pusa Nanha x VC 55 23 0 0 0 15 17 23 41.82 CP 50 x VC 335 70 0 0 0 100 165 70 20.90 35 Jayavalli et al., 2011 Tamilnadu, India
Slide36: Parents /hybrids Number of fruits per plant Mean Fruit weight (kg) Fruit yield per tree (kg) Total Soluble solids Total sugars (%) Titrable Acidity (%) Ascorbic acid (mg/ 100g) Carotene (mg/ 100g) Sugar Acid ratio Parents CO 7 17.00 0.98 16.66 13.5 7.83 0.11 30.33 1.93 71.21 Pusa nanha 22.00 0.92 18.98 11 5.14 0.14 39 1.44 36.16 CP 50 21.00 1.82 38.27 9.73 4.35 0.21 40.66 1.43 20.71 VC 19.00 0.11 2.66 5.83 3.95 0.24 43.33 1.38 16.49 Hybrids CO 7 x VC 19.00 0.74 13.99 10.83 7.63 0.12 31.73 1.86 65.53 Pusa Nanha x VC 35.00 1.11 38.85 10.33 5.07 0.15 41 1.41 33.33 CP 50 x VC 26.00 1.21 31.38 9.47 4.32 0.22 41.67 1.39 19.66 General mean 22.71 0.98 22.97 10.1 5.47 0.17 38.23 1.55 37.58 S.Ed. 0.90 0.01 0.73 0.59 0.03 0.01 0.72 0.01 1.74 CD (p=0.05) 1.97 0.03 1.61 1.29 0.06 0.02 1.58 0.02 3.78 36 Jayavalli et al., 2011 Tamilnadu, India Table 15 : Mean performance of parents and F 1 hybrids for fruit yield and quality traits
Slide37: Figure 3 : Pusa Nanha x V . cauliflora 37
Slide38: Genetic parameters Cross / parents First flowering height (cm) First fruiting height (cm) Plant height at first harvest (cm) Stem girth at first harvest (cm) Number of fruits per tree Mean fruit weight (kg) Fruit yield per tree (kg) Mean Pusa Nanha (PN) 43.05 52.50 127.76 27.95 21.83 0.93 21.66 CP 50 106.40 121.59 232.29 27.50 21.33 1.82 28.82 CO 7 94.68 114.01 218.86 29.76 16.90 0.98 15.42 V. cauliflora (VC) 31.53 37.00 106.00 31.66 19.00 0.15 2.85 Mean PN x VC 38.49a 46.93a 117.17a 30.38a 30.26a 1.17b 35.61a CP 50 x VC 93.08c 100.19c 185.76c 30.02a 26.25b 1.27a 32.00b CO 7 x VC 73.19b 94.68b 169.03b 25.87a 16.69c 0.83c 13.31c PCV (%) PN x VC 27.18 30.4 25.60 48.38 84.13 21.02 71.81 CP 50 x VC 26.98 92.66 45.84 66.42 33.68 17.42 57.72 CO 7 x VC 17.02 47.37 83.12 24.41 35.19 14.60 49.54 GCV (%) PN x VC 24.93 29.57 23.22 48.03 83.97 20.97 71.66 CP 50 x VC 16.31 90.10 40.98 57.58 31.74 16.10 57.50 CO 7 x VC 13.28 41.36 79.81 16.67 30.15 13.81 42.34 Heritability (%) PN x VC 84.17 94.26 82.25 98.57 99.61 99.55 99.56 CP 50 x VC 36.53 94.55 79.95 75.16 88.81 85.43 99.26 CO 7 x VC 60.89 76.263 92.21 46.63 73.39 89.45 73.05 GAM (%) PN x VC 7.60 8.63 4.01 17.82 31.39 39.93 24.68 CP 50 x VC 2.10 18.03 5.54 18.77 12.03 27.15 20.27 CO 7 x VC 2.50 7.64 12.14 4.61 13.03 29.51 20.44 38 Sudha et al., 2013 Tamilnadu, India Table 16 : Genetic parameters for morphological and yield traits in F 2 population
Slide39: Genetic parameters Cross / parents TSS (˚ Brix) Total sugars (%) Carotene (mg/ 100g) Sugar acid ratio Papain recovery (g / fruit) Proteolytic activity (TU/mg) Mean Pusa Nanha (PN) 10.88 5.28 1.49 36.38 6.44 144.50 CP 50 9.94 4.75 1.41 22.39 7.80 190.96 CO 7 13.10 7.43 2.03 73.18 - - V. cauliflora (VC) 5.75 3.90 1.35 16.24 - - Mean PN x VC 9.55b 4.71b 1.42b 32.60b 5.80b 126.91b CP 50 x VC 9.03c 4.13c 1.36c 22.81c 7.10a 157.64a CO 7 x VC 9.93a 5.87a 1.82a 63.25a - - PCV (%) PN x VC 42.45 26.10 5.51 85.18 25.23 42.27 CP 50 x VC 24.13 11.49 5.07 73.91 26.17 37.32 CO 7 x VC 20.90 18.54 20.39 69.77 - - GCV (%) PN x VC 40.55 24.34 3.53 84.95 25.15 42.03 CP 50 x VC 23.27 11.15 2.52 60.89 26.44 36.71 CO 7 x VC 16.14 17. 09 20.09 68.34 - - Heritability (%) PN x VC 91.23 86.99 41.06 99.46 99.31 98.90 CP 50 x VC 93.03 94.07 24.61 67.89 97.96 96.80 CO 7 x VC 59.64 84.96 96.99 95.95 - - GAM (%) PN x VC 25.82 21.55 3.91 30.57 21.43 7.64 CP 50 x VC 15.39 10.96 2.21 21.64 20.15 5.93 CO 7 x VC 8.15 13.39 30.19 17.34 - - 39 Sudha et al., 2013 Tamilnadu, India Table 17 : Genetic parameters fruit quality traits and papain yield in F 2 population
Slide40: 40 Backcrossing
BREEDING STRATAGIES FOR RED PULPED FRUITS: BREEDING STRATAGIES FOR RED PULPED FRUITS CO 2 population segregated occasionally into red pulp fruits and light yellow to deep yellow pulped fruits. CO 2 (red pulp) CO 2 (deep yellow) M 1 R(D) F 1 plants M 1 R(D) BC 1 plants [9-1(D) plants] Repeated three times to obtain BC 3 F 1 plants Red pulped with better sized and quality fruits 426 BC 3 F 1 plants + 20 CO 2 plants for evaluation 426 plants 308 females 198 yellow pulped 110 red pulped 118 male 41 Cynthia et al ., 2003 Tamilnadu, India
Table 18 : Mean performance, range and coefficient of variation in yellow and red fruited population of 9-1(D): Table 18 : Mean performance, range and coefficient of variation in yellow and red fruited population of 9-1(D) Character Yellow Red CO-2 Mean Range CV (%) Mean Range CV (%) Mean Range CV (%) Fruit bearing height (cm) 100.85 62-138 15.10 97.70 70-150 13.79 105.47 73-138 18.11 Thickness of pulp (cm) 2.66 2-3.5 11.28 2.84 2-3.5 11.49 2.66 2-3.3 12.04 Number of Fruit/tree 55.98 41-86 11.57 62.91 40-71 9.17 55.60 47-68 9.63 Fruit weight (kg) 1.30 0.7-2.7 29.61 1.40 0.65-2.95 33.51 1.30 0.85-2.05 26.82 Yield per plant (kg) 72.89 32.9-159 32.49 88.33 30.55-191.75 35.91 72.60 46.55-139.40 31.79 42 Cynthia et al ., 2003 Tamilnadu, India
Slide43: Dioecious nature of papaya !!! Is it necessary to transplant 3 plants per hill ??? Rouging of male plants !!! 43
DNA MARKER ASSISTED SEX CONVERSION IN DIOECIOUS PAPAYA VARIETY: DNA MARKER ASSISTED SEX CONVERSION IN DIOECIOUS PAPAYA VARIETY Cariflora (Dioecious ) Recurrent parent Sunrise Solo 783 (Hermaphrodite) Donor parent of M 2 allele F 1 plants Cariflora Recurrent BC 1 plants 94 hermaphrodite plants selected based on morphology similar to recurrent line BC 2 plants 87 hermaphrodite plants selected Morphological Based on genetic similarity using RAPD Cariflora Recurrent Figure 5 : Sex conversion by backcross breeding 44 Silva et al ., 2007 Brazil
Figure 4 : Dendrogram of genetic dissimilarity of 87 BC2 tree and recurrent (1) and donor parents (2) based on GD by RAPD: 48.3% genotypes Cariflora Recurrent SS 783 Donor 51.7% genotypes Figure 4 : Dendrogram of genetic dissimilarity of 87 BC 2 tree and recurrent (1) and donor parents (2) based on GD by RAPD 74 69 84 76 83 55 25 50 56 88 79 8 66 72 67 70 14 7 11 37 13 62 63 28 21 45 32 16 41 19 49 89 39 60 44 52 75 30 6 64 58 81 20 17 1 15 18 33 40 82 85 24 5 38 42 48 12 34 35 54 57 43 31 46 77 51 2 3 80 36 5 61 9 73 26 68 65 4 23 87 27 29 10 53 78 22 47 86 71 84% 76% 81% Cariflora 45 Silva et al ., 2007 Brazil
SEX DETERMINATION AT SEEDLING STAGE THROUGH MORPHOLOGICAL AND RAPD MARKERS: SEX DETERMINATION AT SEEDLING STAGE THROUGH MORPHOLOGICAL AND RAPD MARKERS Ratna, Washington , Honeydew, CO 6 , and CO 2 . Figure 9 : RAPD profiles with OPL-13 primer Lane 1: papaya three lobed leaf plant Lane 2: matured male plant Lane 3 : papaya five lobed leaf plant Lane 4: matured female plant Figure 5 : Three lobed papaya seedlings Figure 6 : Five lobed papaya seedlings Figure 7 : Mature male plant Figure 8 : Mature female plant 46 Reddy et al ., 2012 Hydrabad, India
Slide47: 47 Mutation breeding
INDUCED GAMMA RADIATION IN “EKSOTIKA” CULTIVAR: INDUCED GAMMA RADIATION IN “EKSOTIKA” CULTIVAR Figure 10 : Variation in height of M 2 seedlings Figure 11 : Variation in leaf length of M 2 seedlings Hermaphrodite Female Male Total Number % Number % Number % Wet seeds ( 42.5Gy) 257 64.1 143 35.7 1 0.2 401 Dry seeds (525Gy) 229 65.1 119 33.8 4 1.1 352 Control 35 70.0 15 30.0 0 0.0 50 Table 19 : Sex segregation in M 2 population 48 Chan et al., 2007 Malasiya
Slide49: Figure 12 : Distribution of M 2 population for tree height 49 Chan et al., 2007 Malasiya Figure 13 : Distribution of M 2 population for fruit weight Figure 14 : Distribution of M 2 population for yield Figure 15 : Distribution of M 2 population for total soluble solids
Slide50: 50 Tissue culture
SHORTENING THE BREEDING CYCLE BY CULTURING EMBRYOS TREATED WITH ETHREL: SHORTENING THE BREEDING CYCLE BY CULTURING EMBRYOS TREATED WITH ETHREL Female cultivar : Fruit tower Male cultivar: WI For ethrel treatment, 45, 60, and 75 - day-old fruits (DOF) after pollination were used. Distilled water (control) or a solution of 100µl l -1 ethrel for 10 or 20 days 45 DOF treated with water and ethrel for 10 & 20 days The rate of embryo germination from 105, 120, 140, 160 and 180 DOF in the control 51 Tamaki et al ., 2011 Japan ** significant difference at p<0.01 Figure 16 : Effect of ethrel treatments on in vitro embryo germination
Slide52: Table 20 : Effect of ethrel of immature fruits on the weight and growth of in vitro seedlings Treatment (DOF + DWT or DE T) Fresh weight (mg) Hypocotyl length (mm) Longest root length (mm) Control (45 + 10) 55 - - - Ethrel (45 + 10) 55 26± 6 1.5 ±0.4 6.2 ± 0.3 Control (45 + 20) 65 34 ± 4b 2.5 ± 0.2b 7.5 ± 0.5b Ethrel (45 + 20) 65 74 ± 5a 4.4 ± 0.4a 9.4 ± 0.7a Control (60 + 10) 70 61 ± 3b 7.3 ± 0.8b 11 .5 ±0.8a Ethrel (60 + 10) 70 82 ± 6a 10.1 ±0.8a 13.4 ± 1.0a Control (60 + 20) 80 56 ± 2b 10.3 ± 0.5b 9.4 ± 0.4b Ethrel (60 + 20) 80 85 ± 4a 13.1 ±0.7a 19.4 ± 1.3a Control (75 + 10) 85 80 ± 4b 12.6 ± 0.6b 12.4 ± 0.7b Ethrel (75 + 10) 85 91 ± 3a 15.4 ±0.4a 15.2 ± 0.5a Control (75 + 20) 95 78 ± 2b 15.6 ± 0.4a 13.5 ± 0.4b Ethrel (75 + 20) 95 86 ± 3a 15.9 ± 0.3a 16.2 ± 0.5a Values are means ± standard error. Values within the same duration of treatments with different letters are significantly different at p < 0.05 Seedlings derived from embryo of control 80 days (60 DOF plus 20 DET ) Seedlings derived from embryo treated with ethrel 80 days (60 DOF plus 20 DET ) Bar indicated 1cm 52 Tamaki et al ., 2011 Japan Figure 17 : In vitro seedling growth promoted by control and ethrel treatment of immature fruits.
Slide53: 53 Papaya leaf distortion mosaic virus ( PLDMV) Severe leaf narrowing, leaf distortion and apex stunting Shoe-stringing of leaves Papaya ringspot virus (PRSV) Yellowing and severe mosaic symptoms. Fruit will exhibit bumps and the classic “ringspot” Transgenic technology
FIRST TRANSGENIC PLANTATION TREE FOR PAPAYA RINGSPOT VIRUS (PRSV): FIRST TRANSGENIC PLANTATION TREE FOR PAPAYA RINGSPOT VIRUS (PRSV) Coat protein gene isolated from mild mutant strain PRV HA 5-1 , by Particle bombardment using immature zygotic embryo of cv. Sunset and Kapoho Transgenic papaya Southern analysis PCR Transcript verified by Gene expression analyzed by Northern analysis Vector pGA482GG Figure 19 : Total RNA from transgenic papaya using RNA probed with CP probe Lane 1 : untransformed control Lane 2 and 3 : Transgenic line S60-3 Lane 4 and 5 : Transgenic line S55-1 Transcript size: 1.35kb Figure 18 : PCR detection of PRV cp genes in transgenic papaya lines of GUS + & GUS - plants Lane 1 : untransformed control line Lane 2 to 9: transgenic papaya lines GUS 54 Fitch et al., 1992 Hawaii
Slide55: R 0 Line GUS PC19-1R cp Height when inoculated (cm) No. of infected/ no. of inoculated Percent infected Delay in symptoms* S55-1 + + 5-11 0/11 0 -- S55-1 + + 25-28 0/2 0 -- S60-3 + + 7-12 3/3 100 6-15 S60-3 + + 20-48 0/9 0 -- K19-1 + + 6-9 3/4 75 3-17 K19-1 + + 14-25 1/8 13 0 K39-1 + + 8-12 10/10 100 0-7 K39-1 + + 11-46 4/5 80 0-15 S33-2 - - 13-14 3/3 100 0 S54-1 - - 14-50 9/9 100 0 S62-1 - - 4-13 9/9 100 0 S62-5 - - 14-37 5/5 100 0 K44-1 + - 11-50 9/9 100 0 Control - - 8-28 35/35 100 0 Table 21 : Reaction of sub cloned transgenic R 0 papaya lines to inoculation with PRV-HA * The delay in symptoms is estimated by using the time of symptom expression in controls as “0” days. 55 Fitch et al., 1992 Hawaii
Slide56: Figure 22 : Untransformed line inoculated Figure 20 : Transgenic line S55-1 uninoculated Figure 21 : Transgenic line S55-1 inoculated Figure 23 : 6 months after artificial inoculation Left: Transgenic line S55-1 inoculated Right: Untransformed line inoculated 56 Fitch et al., 1992 Hawaii
GENERATION OF HERMAPHRODITE TRANSGENIC LINES WITH VIRUS RESISTANCE FOR TWO DISEASES: GENERATION OF HERMAPHRODITE TRANSGENIC LINES WITH VIRUS RESISTANCE FOR TWO DISEASES Transgenic papaya Southern analysis Transcript verified by Gene expression analyzed by Northern analysis Figure 25 : Total RNA from transgenic papaya using RNA probed with PLDMV- CP specific probe Figure 24 : Detection of transgne copy numbers of chimeric construct using PLDMV- CP specific probe. 57 Kung et al., 2010 Taiwan C himeric construct pYP08 containing truncated PRSV- cp and PLDMV- cp genes by Agrobacterium transformation using cv. ‘Tainung No. 2’, ‘Sunrise’, and ‘Thailand’
Table 22 : Evaluation of resistance of transgenic papaya lines by artificial inoculation with PRSV or PLDMV under greenhouse condition: Table 22 : Evaluation of resistance of transgenic papaya lines by artificial inoculation with PRSV or PLDMV under greenhouse condition Resistance level No. of plants showing symptoms after PRSV YK inoculation at day No. of plants showing symptoms after PLDMV inoculation at day Total number of transgenic lines 7 14 21 28 35 42 7 14 21 28 35 42 S (susceptible) 0 10 10 10 10 10 0 10 10 10 10 10 47 WR (weakly resistance) 0 4 5 7 9 10 0 0 4 6 8 9 15 MR (Moderately resistance) 0 0 0 1 2 2 0 0 0 1 2 2 18 HR (Highly resistance) 0 0 0 0 0 0 0 0 0 0 0 0 9 NT 0 10 10 10 10 10 0 10 10 10 10 10 - Figure 26 : Confirmation in green house A: Nontransgenic line inoculated for PRSV B: Nontransgenic line inoculated for PLDMV C: Transgenic line H2-1 inoculated for PRSV D: Transgenic line H2-1 inoculated for PLDMV 58 Kung et al., 2010 Taiwan
Slide59: Figure 27 : PCR analysis of hermaphrodite phenotypes of transgenic lines. PCR analysis with the hermaphrodite-linked primers, SDP-1 and SDP-2. Hermaphrodite phenotypes of transgenic lines. After planting in greenhouse for 1 year, plants of selected transgenic papaya lines SR 2-13-1, TN B1-4 and TL 3-5-1 showed hermaphrodite flowers ( upper panel, gray arrow = anther and white arrow = pistil ) and fruits ( lower panel ) similar to those of the original NT plants of TN, SR and TL lines 59 Kung et al., 2010 Taiwan
Slide60: Pharma papaya 60
ACHIEVEMENTS : ACHIEVEMENTS 61
Slide62: Sr no. Indigenous cultivars Country Sr no. Exotic cultivars Country 1 Pusa Dwarf India 19 Red lady USA 2 Pusa Delicious India 20 Taiwan-785 Taiwan 3 Pusa Giant India 21 Taiwan-786 Taiwan 4 Pusa Majesty India 22 Washington USA 5 Pusa Nanha India 23 Solo Barbados 6 Pant papaya-1 India 24 Sunrise Solo USA 7 Ranchi India 25 Sunset USA 8 Honey Dew India 26 Waimanalo USA 9 Coorg Honey Dew India 27 Higgins USA 10 CO 1 India 28 Wilder USA 11 CO 2 India 29 Thiland Thiland 12 CO 3 India 30 Eksotika Malasiya 13 CO 4 India 31 Kapoho Solo USA 14 CO 5 India 32 Kamiya USA 15 CO 6 India 16 CO 7 India 17 Surya India 18 Pink Flesh Sweet India 62
CONCLUSION: CONCLUSION Selection for dwarf plant progenies would give higher fruit yield at low bearing height. Vasconcellea genus can be utilized in plant breeding programme to develop lines for tolerance/resistance to biotic and abiotic stresses in C. papaya . Morphological markers at seedling level help to discriminate male and female plants and reduce laborious work of rouging in dioecious cultivars. Mutation and Intergeneric hybridization can be served as a valuable tool for creating variability in papaya improvement Breeding cycle of papaya could be shortened by in vitro embryo culture with good-quality embryos that have been excised from immature ethrel treated fruits. PTGS can be induced using coat protein ( cp ) gene of virus in construct to develop transgenic papaya lines resistant to viral diseases. 63
FUTURE PROSPECTS : FUTURE PROSPECTS Exploration, conservation and characterization of genetic variability. Development of stable and dwarf gynodioecious genotypes for papain production. Production of genetically pure seeds by controlled pollination. Breeding varieties resistant to biotic and abiotic stresses. Varieties resistant to biotic stresses like ‘PRSV’ and also can tolerate high temperature and frost. Gene introgression for resistance breeding using interspecific and intergeneric hybridization. Transgenic technology should be utilized for developing PRSV resistant lines in papaya. 64
MARADOL variety: MARADOL variety From cuba Red-orange skin, salmon red pulp and weighs 1.5-2.6kg. 95 % of total papaya production in USA being of the Maradol variety (FAOSTAT, 2012) . 65
Slide66: Country Varieties South Africa Hortus Gold, Honey Gold, Kaapmssider Sri Lanka Peradcniya Hawaii (USA) Solo, Solo-10, Higgins, Wilder, Waimanalo, Homestead. Cariflora Florida (USA) Betty, Washington, Honey Dew Trinidad Santa Cruz Giant (hermaphrodite), Cedros (dioecious), Singapore Mexico Verde, Gialla, Cera, Chincona Indonesia Semangka (Red Flesh Fruits); Thailand, Bangkok, Danpit Cuba Maradol Roja, Cubana, Paraguanera Australia Improved Peterson, Peterson, Guinea Gold, Airline 57 Surinam Santo 3, Santo 4, Santo 7 Improved papaya varieties of other countries… 66
Slide67: THANK YOU… 67