Breeding for seedlessness in fruit crops
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This document summarizes a seminar on breeding for seedlessness in fruit crops. The seminar covered various topics related to seedlessness including factors affecting it, parthenocarpy, breeding methods, case studies, and conclusions. Parthenocarpy, the development of seedless fruit without fertilization, can occur naturally or be induced. It is an important trait for many fruit crops as it allows for seedless fruits. Breeding approaches like hybridization and mutation as well as chemical treatments with plant growth regulators like auxins and gibberellins can induce parthenocarpy. Case studies on seedlessness in crops like citrus, grapes, and kiwifruit were also presented.
![• Triploid papaya plants were obtained by
immature endosperm culture
• Young fruits [10–13 days after pollination
(DAP)] of non transgenic papayas Carica
papaya L. ‘Sunrise’ were collected.
• The excised endosperm, with or without
embryo, was cultured in the callus
induction medium.
• Shoot buds were produced when the callus
was sub cultured (BA or TDZ + NAA).
• Over 75% of the endosperm-derived plants
were triploid with chromosome number 2n
= 3x = 27.
Comparison of the number of chloroplasts in
the guard cells and stoma density for diploid
and endosperm derived plant of Carica
papaya. a,c Epidermal peels from the diploid
parent plant. b,d Epidermal peels from the
endosperm-derived plant.
51
Sun et al., (2010)](https://image.slidesharecdn.com/breedingforseedlessnessinfruitcrops-220715163812-e6a38fca/85/Breeding-for-seedlessness-in-fruit-crops-54-320.jpg)
Breeding for seedlessness in fruit crops
- 2. Breeding for Seedlessness in Fruit Crops STUDENT S. Sharvesh Reg. No. – 207140008 M.Sc., Horticulture (Fruit Science) AnnamalaI University INTER MEMBER Dr. P. K. Karthikeyan, Assistant Professor Dept. Of Soil Science and Agricultural Chemistry Annamalai university INTRA MEMBER Dr. G. Samlind sujin Assistant Professor Dept. of Horticulture Annamalai university CHAIRPERSON Mr. S. Elakkuvan Assistant Professor Dept. Of Horticulture Annamalai University
- 3. • Introduction • Factors affecting seedlessness • Parthenocarpy • Breeding methods • Case studies • Future trust • Conclusion Content
- 4. Introduction ☻ A plant is considered to be seedless if it is able to produce fruit with no seed, traces of aborted seeds or a much reduced number of seeds. ☻ Seedlessness is a very desirable trait in edible fruit with hard seeds. ☻ Commercial cultivars of banana and pineapples are examples of seedless fruits. ☻ Up to 20% of the wild fruits is parthenocarpic. 1
- 5. ☻ Most commercially produced seedless fruits have been developed from plants whose fruits normally contain numerous relatively large hard seeds distributed throughout the flesh of the fruit. ☻ Evidence that seedless forms of grapes have been prized for many centuries as dried fruit is provided by Greek philosophers such as Hippocrate, Platon and in the writings of ancient Egypt of 3000 BC. ☻The oldest known cultivated plant is a parthenocarpic fig first grown atleast 11,200 years ago 2
- 6. Factors affecting of seedlessness Parthenocarpy Male or female sterility, self- incompatibility, defective ovules and embryo sac abortion Environmental factors Chemical treatments (PGR) Auxin, Gibberelins and Cytokinin Genetic factors Chromosomal aberrations 3
- 7. Parthenocarpy ☻ Term parthenocarpy coined by Null (1902) . ☻ Parthenos- VIRGIN, Carpy- FRUIT ☻ Parthenocarpy is the development of the ovary into a seedless fruit without the need of pollination and fertilization. ☻ The parthenocarpic fruit is therefore seedless. 4
- 8. ☻ Parthenocarpy literally means “virgin fruit” is the natural, artificially induced, or genetically modified production of fruit without fertilization. ☻ A plant is considered to be seedless if it is able to produce fruit with no seed, traces of aborted seeds or much reduced number of seeds. ☻ Seedlessness is a very desirable trait in edible fruit with hard seeds. 5
- 9. ☻ Its desirable in fruit crops that may be difficult to pollinize and fertilize ☻ Increased production under adverse environment ☻ Seedless fruits ☻ Improved quality ☻ Improve shelf life due to reduce ethylene ☻ Off season production ☻ Protected cultivation ☻ Reduced cost of cultivation ☻ Achieved easily 6 Importance of Parthenocarpy in fruit crops Varoquaux et al., (2002)
- 10. ☻ Seed and fruit development control by phytohormones. ☻ GA3, Auxin and Cytokinin involve signaling process after fertilization for seed and fruit development. ☻ Increase endogenous hormones during parthenocarpic fruit set not from source of seed. ☻ Trigger the expression of auxin biosynthetic gene . 7 Mechanism of parthenocarpy Carmi et al., (2003)
- 11. 11 Types of Parthenocarpy Natural or Genetic Obligatory Parthenocarpy e.g. Banana, Pineapple Facultative Parthenocarpy e.g. Citrus cvs. Grape Vegetative Parthenocarpy e.g. Apple, Banana, Pineapple Stimulative parthenocarpy e.g. Grape cvs. Black Corinth Stenospermocarpy e.g. Black Monukka & Sultania grapes, Mango cvs. sindhu Artificial or induced 8
- 12. ☻ Obligative : No external Influence Eg. Banana and pineapple ☻ Facultative : Adverse conditions for pollination and fertilization. Eg. Citrus cvs. and Grapes 9 Obligative and facultative parthenocarpy
- 13. ☻ If a fruit developed even without the stimulus of pollination and fertilization, then the phenomenon is referred to as vegetative parthenocarpy (automatic). Eg. Banana, Pinapple, Fig 10 Vegetative Parthenocarpy
- 14. ☻ Term coined by Stout, 1936 ☻ If a fruit develops from the stimulus of the pollination (But without fertilization), the phenomenon is known as stimulative parthenocarpy. Eg. Grapes var. Black corianth, Litchi, Breadfruit. 11 Stimulative Parthenocarpy
- 15. ☻ Normal pollination and fertlization occurs, but the embryos abort when they are young. ☻ Often, remnants of seeds can be seen in the fruits. Eg. Mango cvs. sindhu , Black Monukka, Black kishmish & Sultania grape, 12 Stenopermoscarpy
- 16. ☻Stenospermocarpy may also produce apparently seedless fruit, but the seeds are actually aborted while still small. ☻Parthenocarpy (stenospermocarpy) occasionally occurs as a mutation in nature, but if it affects every flower, then the plant can no longer sexually reproduce but might be able to propagate by vegetative means. 13
- 17. Induced parthenocarpy is when seedless fruits are produced from the ovary by giving with special treatment to the flower. Causes of induced parthenocarpy ☻ Irradiated pollen ☻ Natural or synthetic auxin ☻ Gibberelline Eg. Lemon, Apple, Orange 14 Artificial / Induced parthenocarpy Gustafson (1942)
- 18. Causes for Parthenocarpy ☻ Self incompatibility – e.g. Mandarine cv. Clementine ☻ Dioecious ☻ Absence of pollinator and pollinizer ☻ Adverse environmental condition ☻ Male sterility ☻ Pollen maturation and fertilization are affected by environmental factors such as light, temperature particularly nocturnal temperature (The low temperature slows down pollen tube growth and pollen tube fails to reach the ovary), relative humidity and fog. 15
- 19. ☻ In some cultivars, where the level of parthenocarpy is high, pollination is not required for fruit formation. e.g. Tahiti lime, Satsuma mandarin. ☻ In other cases, with a low level of parthenocarpy, pollination is required to set fruit. e.g. Star ruby - grapefruit - to obtain seedlessness, it is then necessary to pollinate with dead pollen or preferably, to combine genetic seedlessness with self- incompatibility. 16
- 20. Gametophytic Self-incompatibility Causes Seedlessness in ‘Kagzi Kalan’ Lemon (Citrus limon) 17 Kakade et al., (2017) IARI, New Delhi
- 21. Figure 1: Pollen viability of Kagzi Kalan lemon (C. limon) (a), Kagzi lime (C. aurantifolia) (b), and Marsh Seedless (C. paradisi) (c) assessed by acetocarmine test and FDA test V: viable pollen; NV: non-viable pollen. 18
- 22. Table 1: In vivo pollen germination and pollen tube growth under different pollination treatments in lemon cv. Kagzi Kalan. Values are means ± SE (n = 15). Different letters in the same column indicate significant differences at P ≤ 0.05. 19 Treatments Pollen germination on stigma (%) At mid style (%) At base style (%) Kagzi Kalan ( Hand pollination) 53.51 ± 1.76c 3.00 ± 0.58c 0.00 ± 0.00b Kagzi Kalan × Kagzi lime 72.32 ± 3.91a 51.33 ± 3.18a 15.33 ± 2.40a Kagzi Kalan × Marsh Seedless 65.36 ± 0.82ab 44.33 ± 3.48b 14.33 ± 2.33a Bagging without emasculation 70.37 ± 2.57a 03.67 ± 0.33c 0.00 ± 0.00b Open pollination 59.09 ± 0.81bc 2.67 ± 0.33c 0.00 ± 0.00b LSD (P ≤ 0.05) 8.07 6.48 4.36 IARI, New Delhi Kakade et al., (2017)
- 23. Table 2: Effect of different pollination treatments on number of normal and shrunken seeds in Kagzi Kalan fruits. Values are means ± SE (n = 15). Different letters in the same column indicate significant differences at P ≤ 0.05. 20 Treatment Number of normal seed(s)/fruit Number of shrunken seed(s)/fruit Kagzi Kalan ( Hand pollination) 0.00 ± 0.000b 0.00 ± 0.00b Kagzi Kalan × Kagzi lime 16.67 ± 3.84a 3.00 ± 1.16a Kagzi Kalan × Marsh seedless 15.00 ± 1.73a 3.00 ± 0.58a Bagging without emasculation 0.00 ± 0.00b 0.00 ± 0.00b Bagging with emasculation 0.00 ± 0.00b 0.00 ± 0.00b Open pollination (solid block) 0.00 ± 0.00b 0.00 ± 0.00b LSD (P ≤ 0.05) 5.64 1.62 IARI, New Delhi Kakade et al., (2017)
- 24. Causes for Parthenocarpy in Citrus ☻Male sterility – e.g. Citrus cultivars ☻Chromosomal aberrations – induce pollen sterility in Mukaku yuzu developed by mutation ☻Reciprocal translocation – e.g. Sweet Orange cv. Valencia ☻Spindle formation failure – e.g. Grapefruit cv. Marsh seedless ☻Anther abortion – Satsuma citrus hybrids ☻Pollen mother cell degeneration – e.g. Tahiti lime, Sweet Orange cv. Washington navel 21
- 25. Causes and Induction of Parthenocarpy in Fruit Crops Fruit crops Causes Guava e.g. Allahabad seedless Due to triploidy failure of fertilization of well fertilized ovules degeneration of fertilized ovules Grape Due to embryo abortion (Stenospermocarpy) Citrus Due to self-incompatibility (Mandrins) Cytoplasmic male sterility (Stasuma mandarin) Chromosomal irregularities (Tahiti lime, oroblanco, Melogold) Apple Lack of pollination Banana Due to triploidy 22
- 26. Methods to Induce Seedlessness ☻ External application of PGR ☻ Breeding approaches Hybridization Polyploidy Mutation Embryo rescue Endosperm culture ☻ Biotechnological approach 23
- 27. External Application of PGR’s ☻ Auxin, gibberellins and cytokinins or mixture of these harmones have been proven to be effective in inducing fruit development in the absence of fertilization in several crop species ☻ Auxin and gibberellin may act in parallel or in a sequential way on fruit set. Auxin ☻ Exogenous auxin application to flowers for induction of parthenocarpy was first reported by Gustafson (1936). ☻ Natural and artificial auxins supplied exogenously to unpollinated flowers induce fruit growth. ☻ It can replace the signals provided by pollination and fertilization. 24
- 28. Exogenous auxin application Triggers the auxin bio-synthetic genes in ovaries and ovules Development of parthenocarpic fruit Genes Treatment Underlying pathway Species Sources Auxin biosynthetic genes Exogenous application Auxin Strawberry (Fragaria ananassa and F. vesca) Kang et al., (2013) YUCCA Pre blossom application Auxin Eriobotrya japonica Mesejo et al., (2010) Table 3. Genes and phytohormones involved in parthenocarpic fruit development 25
- 29. Gibberellins • Active gibberellins (GA1 or GA3) are able to induce fruit set without pollination. • Auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. • GA3 along with auxin seems to be playing an important role in parthenocarpy. • The pollen produce GA3, while the exogenous application of GA3 – increase auxin level in the ovary of an unpollinated flowers. • which trigger fruit set in the absence of pollination and fertilization 26
- 30. Role of Plant Harmones in Inducing Parthenocarpy Plant Harmones Crop Reference GA3 – 1500ppm Custard apple cv. Gefner Rayane et al., (2016) GA3 - 250 and 300 ppm on emasculated flowers loquat Aslmoshtaghi and Shahsavar, (2013) GA4, GA7 500mg/l and CPPU 2, 4-D at 50 ppm at the time of anthesis Japanese pear cv. ‘Akibae’ and cv. ‘Iwate yamanashi’ Zhang (2008) GA3 applied at 0.15 mM concentration before and after anthesis Swenson red seeded grape Fellman et al., (1991) GA3 @ 600, 800 and 1000 ppm GA3 @ 400 ppm. Grape cv. Flordasun cv Sharbati Saini et al., (1981) GA3 @ 500 ppm cut surfaces of the emasculated flowers at their bloom stage Peach I. Kiyokawa and Nagakawa S. (1972) 27
- 31. Study of Parthenocarpy in Three Kiwifruit Commercial Cultivars Treatment details Murakami, (2020) SRIAF, Shizuoka, Japan 28
- 32. Table 4: Effect of CPPU induces parthenocarpy in kiwifruit cultivar 29 Fruit quality at harvesting time Variety Treatment Fruit weight (g) a Seed number per fruit a Number of Fruits sampled Soluble solid Content (Brix) Flesh firmness (N) Titratable acidity (%) ‘Rainbow Red’ Pistil removal + CPPU 29 ± 1 b 0 ± 0 c 9 14.4 ± 0.6a 20.5 ± 0.8b 1.71 ± 0.07b CPPU + Natural pollination 66 ± 3b 209 ± 58b 10 7.4 ± 0.6b 26.9 ± 0.7a 1.94 ± 0.06a Artificial pollination 79 ± 3a 680 ± 29a 10 6.2 ± 0.1b 28.7 ± 0.2a 1.86 ±0.03ab ‘Kaimitsu’ Pistil removal 26 ± 4 c 0 ± 0 c 5 8.7 ± 0.6bc 25.0 ± 0.5a 2.47 ± 0.03a Pistil removal + CPPU 41 ± 2b 0 ± 0 c 5 20.0 ± 1.9a 13.2 ± 2.6b 2.48 ± 0.16a CPPU + Natural pollination 74 ± 4b 46 ± 19b 10 11.1 ± 1.1b 21.6 ± 2.1a 2.32 ± 0.05a Artificial pollination 126 ± 1a 900 ± 29a 10 6.8 ± 0.1 c 26.8 ± 0.3a 2.32 ± 0.05a ‘Hayward’ Pistil removal + CPPU 80 ± 2a 0 ± 0 c 10 7.1 ± 0.1b 21.8 ± 0.4b 1.83 ± 0.02a CPPU + Natural pollination 67 ± 3b 31 ± 10b 10 7.3 ± 0.4b 22.5 ± 0.3b 1.61 ± 0.09b Artificial pollination 79 ± 3a 1114 ± 50a 10 9.4 ± 0.7a 23.9 ± 0.2a 1.95 ± 0.04a SRIAF, Shizuoka, Japan Murakami, (2020)
- 33. Figure 2. Fruit appearance and sections of fruits developed through artificial pollination, pistil removal with CPPU treatment, pistil removal, and natural pollination with CPPU treatment in three kiwifruit cultivars. A, ‘Rainbow Red’, B,‘Kaimitsu’, and C, ‘Hayward’. 30
- 34. Table 5: Effect of GA3 and Streptomycin (SM) application on fruit weight, fruit number per tree, yield and firmness in ‘Balady’ mandarin. Means followed by the same letter are not significantly different at 5% level by DMRT 31 Shereif et al., (2017) Agriculture Research Centre, Egypt Treatments Fruit Weight (g) Fruit No./tree Yield (kg/tree) Seed number/fruit 2015 2016 2015 2016 2015 2016 2015 2016 Control 140.18b 138.93b 604.00e 617.00e 84.60d 85.73c 17 17 GA3 (25 ppm) 146.94a 150.73a 615.00d 611.00e 90.38b 92.09b 10 9 SM (250 ppm) 128.49e 132.07d 674.67b 660.00c 86.69cd 87.17c 9 8 SM (500 ppm) 131.63de 133.49cd 647.00c 643.67d 85.16d 85.93c 8 7 SM (250) + GA3 (25) 134.07cd 137.49bc 671.67b 670.67b 89.88bc 92.22b 7 6 SM (500) + GA3 (25) 137.47bc 138.32bc 701.67a 708.33a 96.46a 97.98a 4 3
- 35. Hybridization ☻Many seedless grape varieties obtained by conventional crossbreeding crossing between seeded and seedless cultivars. 2n (seeded) x 2n (seedless) 2n (seedless) e.g. ☻Autumn Royal (Autumn Black x Fresno C74-1) 32
- 36. Cross breeding between seedless cultivars and seeded cultivars 2n (seedless) x 2n (seeded) 2n (seedless) Cross Female characteristics Male characteristics Flame Seedless x Beichun Vitis vinifera; stenospermic; diploid V. vinifera x Vitis amurensis hybrid; seeded, diploid; Blush Seedless x Shuangyou V. vinifera; stenospermic; diploid V. amurensis, seeded, diploid; Pink Seedless x Beichun V. vinifera; stenospermic; diploid V. vinifera x V. amurensis hybrid; seeded, diploid. DA7 x Shuangyou V. vinifera; hybrid of Delight x Emerald Seedless, stenospermic, diploid V. amurensis; seeded, diploid. 33
- 37. Changing ploidy level ☻ It was first demonstrated in Japan that by developing a triploid water melon (2n= 33) by crossing tetraploids x diploid varieties, seedlessness could be achieved. ☻ Naturally available seedless guava varieties are due to auto polyploidy (triploid) and not due to parthenocarpic fruit development. 4n (seed parent) 2n (pollen parent) + = Sterile 3n plant Diploid egg Haploid pollen 34 2n n
- 38. Polyploidy Breeding • Triploid breeding – 4 ways of triploid breeding is followed 1. 2. 3. Hybridization between different ploidy cultivars. 2n (diploid) x 4n (tetraploid) 3n (seedless) 35
- 39. 4. 4n (tetraploid) x 2n (diploid) 3n (seedless) eg: ☻Jingyou (4X) x Muscat Hamburg (2X) ☻Fenghou (4X) x Agate early (2X) ☻Delaware (4X x) Muscat Hamburg (2X) eg: Tahiti lime 36
- 40. Spontaneous Triploids • It may arise by the production of (2x) egg cell by diploid seed parent or 2x pollen and their union with a monoploid(x) gamete. • Commonly seen in Citrus • Eg: Winola – Wilking x Minneola – spontaneous triploid hybrid • Tahiti lime – commercially exploited among naturally occuring triploids 37 Mandarin
- 41. 38 Triploid banana Musa acuminata × Musa balbisiana = Musa Paradisiaca (Asian banana) (Asian banana) (Hybrid banana) AA BB AAB or ABB etc. (Fertile) (Fertile) (Sterile) A – One haploid set of chromosomes from diploid M. Acuminata B – One haploid set of chromosomes from diploid M. balbisiana The black spots in polyploidy bananas are aborted ovules, which would have become seeds
- 42. ☻ The triploid banana fruit is vegetatively parthenocarpic. ☻ The graphs of growth in volume of seeded banana fruits are sigmoid in shape. Those of parthenocarpic fruits are variable but are not sigmoid and the shapes are related to specific origins. ☻ Growth rates are related to certain ovule behaviours, to seed content of the fruit, and to ploidy. ☻ NAA induces parthenocarpy in seeded bananas and stimulates it in weakly parthenocarpic types. ☻ By contrast, coumarin, a hormone inhibitor, inhibits it in strongly parthenocarpic forms. 39 Triploid banana
- 43. Mutation Breeding •In the modern existing techniques irradiation is an effective method for introducing nearly complete to complete seedlessness in fruits. •Many spontaneous mutants characterized by better fruit colour and seedlessness have been found particularly in apple and citrus. •Mutagens : fast neutrons, x rays and gamma rays. •Gamma irradiation mutagenesis is a technique commonly used to induce seedlessness in citrus 40
- 44. Materials And Methods • The gamma-irradiated clones were obtained when buds exposed to 2.75 - 6.5 krad of 60Co gamma irradiation at 13 Gy/min,. • After irradiation treatment, budwoods were immediately grafted on to “Troyer citrange” rootstock • The number of surviving budwoods was measured three months after grafting and the survival rate of budwood was recorded as 60.34% at mV1 stage. • In order to stabilize the mutation, buds in the middle of the first shoots of mV1 plants were extracted, grafted onto “Troyer” citrange rootstock • In the following year, this process was repeated again at mV2 stage to increase mutant clonal population 41 Effects of Gamma-Irradiation Mutagenesis for Induction of Seedlessness, on the Quality of Mandarin Fruit Goldenberg et al., (2014) Agricultural Research Organization, Israel
- 45. Figure 3: Photographs of eight different natural seeded mandarin varieties, compared with their corresponding gamma-irradiated low-seeded mutants. 42 Goldenberg et al., (2014) Agricultural Research Organization, Israel
- 46. Figure 4: Effects of gamma-irradiation mutagenesis on seed number of eight different mandarin varieties. Data are means ± SE of 10 fruits, and asterisks indicate significant difference at P ≤ 0.05 between fruits from gamma-irradiated and from control trees. 43 Goldenberg et al., (2014) Agricultural Research Organization, Israel
- 47. Transverse section of fruit of Pamelo Nambangan (A) and mutant plant (B) 44 Mariana et al., (2018) Bud woods of ‘Pamelo Nambangan’ irradiated with dosage of 20, 40 and 60 Gy using Cobalt-60 source. The buds were grafted on 6 months-old rootstocks of Japanese Citroen ICSFRI, Indonasia • A mutant plant derived from 20 Gy irradiation. • Pamelo Nambangan has more than 40 seeds/fruit and the mutant has less than 10 seeds/fruit on average. • In the seedless mutant, it was observed that the mutant has embryo sac abortion leading to seedless fruit and low pollen viability (7.7%). • The mutant has been released in 2016 as a new seedless pummelo variety name of ‘Pamindo Agrihorti’. Seedless Fruit Pummelo induced by Gamma Ray irradiation: Fruit morphological characters and stability evaluation
- 48. Embryo Rescue • This technique was first documented in 18th century by Charles Bonnet. • The rescue of hybrid embyos which resulted from interspecific or other wide crosess, where pollination and fertilization occurs normally but endosperm fails to develop. • Immature embryos are exiced from seeds which usually reached heart shaped zygotic embryos. • Embyos are cultured on suitable media by invitro culture. 45
- 49. Breeding New Seedless Grapes using in Ovule Embryo Rescue And Marker-assisted Selection 46 Zhiqian et al., (2015) Xinjiang Development and Research Center of Grapes and Melons
- 50. Table 6: Effect of female parent characteristics on embryo rescue 47 Zhiqian et al., (2015) Crosses No. of ovules cultured No. of embryos developed No. of plantlets Percentage of embryos developed (%) Percentage of plantlets (%) DR1 × Monukka 108 32 22 29.6 20.4 DR2 × Monukka 75 20 11 26.7 14.7 DR3 × Monukka 109 39 24 35.8 22.0 Blush Seedless × Thompson Seedless 45 3 1 6.7 2.2 Blush Seedless × Flame Seedless 17 1 0 5.9 0 DRs was female parent and crossed with various seedless cultivars Xinjiang Development and Research Center of Grapes and Melons
- 51. Figure 5: Ovules of seedless and seed-trace parents. (A) DR1 ovule with no endosperm; (B) DR1 ovule with endosperm (endosperm marked with arrow) (C) Blush Seedless ovule with no endosperm. 48
- 52. Endosperm (isolated at proper stage) Cultured on suitable media Callus followed by embyogenesis or shoot bud differentiation Complete triploid (seedless) plantlet is obtained The endosperm is a unique tissue. Being the fusion product of three haploid nuclei; two polar nuclei and one pollen nucleus, in most angiosperms, is triploid. 49 Endosperm Culture
- 53. ☻It is useful breeding strategy for citrus, a vegetatively propagated perennial crop, for which seediness is undesirable and unnecessary. ☻It can also overcome barriers to sexual hybridization resulting from apomixis and embryo abortion. ☻It was done two months after flower anthesis. ☻Endosperm cultured in MT medium + 2 ppm 2, 4-D + 5 ppm Benzyl adenine + 1000 ppm casein hydrolysate . Eg: Embryogenesis in endosperm of sweet tangor (C. reticulate x C. sinensis) was also induced and triploid plants were regenerated (Mooney et al., 1996). 50
- 54. • Triploid papaya plants were obtained by immature endosperm culture • Young fruits [10–13 days after pollination (DAP)] of non transgenic papayas Carica papaya L. ‘Sunrise’ were collected. • The excised endosperm, with or without embryo, was cultured in the callus induction medium. • Shoot buds were produced when the callus was sub cultured (BA or TDZ + NAA). • Over 75% of the endosperm-derived plants were triploid with chromosome number 2n = 3x = 27. Comparison of the number of chloroplasts in the guard cells and stoma density for diploid and endosperm derived plant of Carica papaya. a,c Epidermal peels from the diploid parent plant. b,d Epidermal peels from the endosperm-derived plant. 51 Sun et al., (2010)
- 55. •Due to induction of male sterility. •Seed coat destruction by suicide gene expression. •Induction of female sterility- destruction of ovules or stigma by suicide genes. •Increased expression or sensitivity to GA and auxins in ovary. 52 Biotechnological Approches
- 56. Biotechnological Approches • They used a chimeric gene derived by using genetic information from bacterium Pseudomonas syringae pv. savastanoi and the plant Antirrhinum majus. Antirrhinum majus Pseudomonas syringae pv. savastanoi DefH9-iaaM coding region Regulated by the promoter and regulatory sequences Increase auxin biosynthesis in transgenic plant cells and organs expressed specifically in the ovules It codes for tryptophan mono-oxygenase oxidation Indolacetamide (chemically or enzymatically) by plant hydrolases indole-3-acetic acid (IAA) 53
- 57. Genetic Engineering of Parthenocarpic Fruit Development in Strawberry Objective To develop a seedless fruit in strawberry and raspberry by using defH9-iaaM auxin- synthesizing gene. Mezzetti et al., (2004) 54 University of Verona
- 58. MATERIALS AND METHODS Regeneration and Transformation • In vitro proliferating shoots of strawberry and raspberry were used for the regeneration and transformation experiments. • Cultures initiated from shoot-tips were sub-cultured monthly on MS medium supplemented with 3 % (w/v) sucrose, BA 2.2 µm, adjusted to pH 5.7. • Media were solidified with 0.7 % Agar before autoclaving for 20 min at 121 ℃. • Proliferating explants were maintained at 25+1 ℃ under 16/24 hrs photoperiod provided by cool white fluorescent lamps. 55
- 59. • Leaves excised from 30-day old proliferating cultures were plated on MS media supplemented with 3% sucrose, agar 0.7%. • supplemented with two combinations of plant growth regulator (PGR): A) 4.5 µM TDZ and 0.8 µM BSAA or B) 4.5 µM TDZ and 0.9 µM 2.4-D. • Transformation was done by using A. tumefacies non-oncogenic strain GV 3101 harbouring the parthenocarpic gene DefH9-iaaM by using two disarmed Ti-binary vector and the nptII gene as a selectable marker. • Transgenic clones able to root in the presence of kanamycin were analysed by Southern blot. 56
- 60. Table 7: Plant fecundity and fruit yield of transgenic with respect to control Means within a column followed by the same letter are not significantly different (Duncan’s multiple range test, P<0.05) 57 Mezzetti et al., (2004) Lines Inflorescence per plant/cane Fruit per inflorescence Fruit Production g/plant – cane Fragaria vesca – greenhouse Control 8.43 ± 0.79 b 2,41 ± 0.07 b 15.55 ± 1.28 c DefH9-iaaM 1 10.71 ± 0.68 ab 3.44 ± 0.09 a 32.94 ± 1.62 b DefH9-iaaM 2 11.94 ± 0.65 a 3.53 ± 0.09 a 41.32 ± 2.64 a Fragaria x ananassa – greenhouse Control 4.12 ± 0.66 b 4.27 ± 0.33 b 135.13 ± 12.48 b DefH9-iaaM 1 6.15 ± 0.47 a 5.03 ± 0.35 a 383.36 ± 36.56 a Rubus idaeus – open field Control 9.43 ± 0.37 b 9.50 ± 0.52 b 195.76 ± 17.29 b DefH9-iaaM 1 11.50 ± 0.45 a 13.96 ± 0.56 a 408.53 ± 34.94 a University of Verona
- 61. Figure 7. Strawberry and raspberry fruits from control and DefH9-iaaM plants Mezzetti et al., (2004) 58
- 62. Changes in physico-chemical characteristics in the fruits due to induction of seedlessness • Size : In mango seedless fruits had smaller size than seeded fruits but had good quality and matured earlier than seeded fruits. In grapes also the size of seedless berries was smaller than the seeded ones of the same variety. • Form: Parthenocarpic fruits (induced by GA) in apple were usually more elongated than the normal seeded fruits. • Composition and quality: In most grape varieties, seedless fruits are sweeter than the seeded fruits of the same variety. • Maturity: Seedless fruits ripen later than seeded fruits which may be attributed to ethylene produced by seeds. 59
- 63. Significance of Seedlessness • Parthenocarpic fruits are required by food preservationist for making jams, jellies, sauces, fruit drinks etc. because the edible fleshy part of fruit may increased by the absence of seeds. • Helps the growers to keep the insect and pest away because there no need for pollinating agent. • So it improves the crop yield without using pesticides. , • Preferred by consumers. • The absence of seeds is usually appreciated by consumers and producers because it increases fruit quality and fruit shelf-life. 60
- 64. Significance of seedlessness •Year round production •Gustatory advantage •Processing •Seed cavity can be filled with fruit tissue •Improve shelf life •Fruit quality •Organic acids and flavonoids •Carbohydrates, carotenoids and essential oil. 61
- 65. Limitation of seedlessness fruits •Seedless fruit is cannot be used to produce new progeny of plant. •Lack of stability and ununiformity in the expression of parthenocarpy •Small sized fruits •Malformed fruits •Hampers the production of commercial seeds 62
- 66. Future trust 63 •High level and stable parthenocarpy •Combining several parthenocarpy genes •Developing parthenocarpy in high value crops •Combining parthenocarpy with male sterility
- 67. Conclusions • Seedless fruits are a desirable commodity for consumers. • Evidence that seedless forms of Vitis vinifera grapes have been prized for many centuries as dried fruit. • Seedlessness is a boon to the processing industry and to a greater level to the consumers they can reduce the hardship regarding a crucial step in the fruit processing. • Seedlessness fruit production is a age old practice, several technologies have been evolved but relevant one are discussed here, in that foremost is the plant growth regulator application among which gibberellin was found to be promising in certain major fruit crops. Further, technologies which have greatly improved seedless fruit production are, suicide gene and mutation. • Nowadays trend is shifting, and urban culture is growing, and people are very busy and in this context, Seedless fruit: Fruits of future i.e. seedlessness is having great importance in the fruit production. 64