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Role of tissue culture techniques in overcoming major breeding constraints in fruit crops

•
•
M
MANDEEP KAUR

This document discusses how tissue culture techniques can help overcome major constraints in fruit crop breeding. It covers techniques like embryo rescue, haploid production, somatic hybridization, somaclonal variation, cryopreservation, and in vitro germplasm storage. Embryo rescue allows the development of interspecific and intergeneric hybrids by rescuing immature or weak embryos. Haploid production through anther or pollen culture can generate pure lines more quickly. Somatic hybridization combines genomes from incompatible parents to create novel hybrids. Somaclonal variation and in vitro mutagenesis are used to select stress-tolerant variants. Cryopreservation and in vitro storage help conserve genetic resources long-term. Overall, integrating these bi

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M K Saini 1 Presented by: Mandeep Kaur L-2018-A-35-D PhD Fruit Science Role of tissue culture techniques in overcoming major breeding constraints in fruit crops Presented to: Dr H S Dhaliwal Dr Monika Gupta Dr Rachna Arora
M K Saini CONTENT  Introduction to Fruit Breeding  Fruit Breeding Constraints  Tissue Culture Techniques a. Embryo Rescue b. Production of Haploid Plants c. Somatic Hybridization d. Somaclonal Variations e. Cryopreservation & In- vitro Germplasm Storage f. Quality Improvement  Conclusion
M K Saini Fruit Breeding • Breeding objectives for rootstock: – Resistant to biotic and abiotic stress – Dwarf growth stature – without affecting productivity of scion. – Wide geographical adaptability – Easily propagated through asexual means • Breeding objectives for scion cultivars: – High productivity – Resistant to biotic and abiotic stress – Dwarf growth stature – Regular, precocious and prolific bearer per unit canopy area – Longer shelf life and transport quality – Good quality fruits:  Appearance –> Size, shape, color  Eating quality –> Seedlessness (few or soft seeds), flesh color, texture, flavor
M K Saini Fruit Breeding Constraints • Long juvenile phase • Heterozygous nature • Wide/distant hybridization – Interspecific & Intergeneric crosses. • Sexual incompatibilities in both self and cross combination • Susceptibility to biotic & abiotic stress • Polyploidy – Banana, Strawberry • Polyembryony – Citrus, Mango • Parthenocarpy and seedlessness – Banana, Pineapple • Loss of natural germplasm • Lack of knowledge on inheritance pattern • Excessive fruit drop – Mango, Citrus • Presence of single seed in single fruit – Mango, Litchi • Seed dormancy • Low seed viability
M K Saini 1. Embryo Rescue
M K Saini 1. Embryo Rescue • Post - zygotic embryo abortion – Major breeding constraint. • Embryo rescue refers to in vitro technology whose purpose is to promote the development of an immature or weak embryo into a viable plant. • Embryo rescue procedures – Ovule culture, Ovary culture, Embryo culture. • It has been successfully applied in grapes, citrus, mango, stone fruits. • Grapes variety developed by IARI, New Delhi using embryo rescue :- • Pusa Purple Seedless: Cross between ‘Pearl of Casaba’ and ‘Beauty Seedless’, raised through embryo rescue. • It is an extra-early berry ripening variety (in the 3rd week of May) under sub-tropical region.
M K Saini Applications of embryo rescue techniques 1. Shortening the breeding cycle 2. Developing interspecific and intergeneric hybrids 3. Seedlessness 4. Overcoming embryo abortion 5. Determining of seed viability 6. Overcoming dormancy – Malus and Prunus spp.
M K Saini In vitro efficiency of embryo rescue of interspecific hybrids of sweet cherry and Chinese cherry cultivars Effects of different low temperature treatments & GA₃ on cherry embryo germination through in vitro rescue. Wu et al (2021) China  Chaoyang-1 (CY-1) – Sweet cherry  Duangbing (DB) – Chinese cherry • Failure to obtain hybrid cherry seedlings during outbreeding.
M K Saini In vitro embryo rescue for the production of hypotetraploids after cross between hypotetraploid and tetraploid grape cultivars Effect of the time of ovule culture and basal medium on development of the embryos and in vitro growth of embryos excised after ovule culture Kim et al (2020) Korea • Hypotetraploid (2n = 4x-1 = 75) - To develop seedless grapes with large berries. • (4x-1=75) => Hanareum => female parent • (4x=76) => Honey Black and Kyoho => male parents
M K Saini Effect of different sampling times on embryo recovery rate in different seedless grapes crosses Li et al (2020) China Plant Development (%) Embryo Recovery (%) • Stenospermocarpy leads to embryo abortion during development.
M K Saini Plant material Remarks Reference Vitis vinifera L. Ovule culture of seedless grapes (Vitis vinifera L.) Singh et al (1991) Peach Ovule rescue in peaches: incubation period Raseira and Einhardt (2010) Citrus Regenerating triploid plants by embryo rescue technique Liu et al (2010), Aleza et al (2010) Grape Embryo rescue of seedless grape with disease and cold resistance Niu et al (2012) Musa species Embryo culture and embryo rescue studies in wild Musa spp. (Musa ornata) Dayarani et al (2014) Citrus Production of common sour orange × Carrizo citrange hybrids using embryo rescue Kurt and Ulger (2014) Grapevine Immature embryo rescue and in vitro evaluation of intraspecific hybrids from Brazilian seedless grapevine ‘Superior × Thompson’ clones Menezes et al (2014) Embryo rescue in fruit crop improvement
M K Saini 12 2. In Vitro Production of Haploids
M K Saini 2. In Vitro Production of Haploids • Haploid plants are obtained from pollen grains by culturing them. • Two methods - Anther culture & pollen culture. • Double haploid - Homozygous pure plant. • Applications of haploid plants: – Production of genetically pureline by selfing is impractical because of their long generation cycle & frequent self incompatibility. – To solve of problem of high heterozygosity – No segregation in meiosis. – Shorten the time for generation of homozygous lines. – QTL mapping. – Genome sequencing. – DH: To study heterozygous maternal parent. Haploid 1 n Diploid 2 n Colchicine
M K Saini In vitro regeneration of haploids using isolated microspore culture of banana cv. Bimkol • Musa balbisiana cv. Bimkol => BB genomic group (2n= 2x= 22). • Long crop cycle (740 days) => Breeding problem. • 30 days old immature male flowers => Explants. • 58.33 % haploid plants • 41.66 % diploid plants Gogoi et al (2020) India Haploid plant Diploid (Control)
M K Saini 3. Somatic Hybridization
M K Saini 3. Somatic Hybridization • Somatic hybridization is an alternative to sexual reproduction in order to combine the genomes from otherwise incompatible parents. • It involves fusing protoplasts of two different genomes followed by the selection of desired somatic hybrid cells and subsequent regeneration of hybrid plant. • It involves four steps:  Protoplast isolation  Protoplast fusion  Selection of hybrid cells  Regeneration of hybrid plants • Two types of hybrids - symmetric hybrid and asymmetric hybrids
M K Saini Applications of somatic hybridization • Production of interspecific and intergeneric hybrids – by combining nuclear and cytoplasmic genomes belonging to different species & genus. • Production of fertile diploids and polypoids – by crossing sexually sterile haploids, triploids and aneuploids • Production of heterozygous lines in the asexually propagated species • Transfer of cytoplasmic information (mtDNA) for the production of male sterile plants and seedless triploids. • To create novel germplasm as a source of elite breeding parents. • To create novel hybrids with increased yield and resistance to biotic and abiotic stress.
M K Saini Triploid Mandarin: Sugar Belle X Nova + Succari Triploid Lime: ‘Todo del Ano’ lemon X ‘Mexican’ lime + ‘Valencia’ Triploid Lemon: ‘Todo del Ano’ lemon X ‘Hamlin’ + Femminello’ Fig: Seedless fruits from triploid hybrids produced by somatic hybridization Grosser and Gmitter (2011) USA
M K Saini Response of four allotetraploid somatic hybrids to Citrus tristeza virus induced infections Abbate et al (2018) Italy Fig: Detection of mild (CTV-DS1) and severe (CTV-DS2) strains in the investigated genotypes by using DAS-ELISA and qRT-PCR assays. Biological barriers - sexual incompatibility, polyembryony, male/female sterility, long juvenility and different bloom phases
M K Saini Production of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars • Polyembryony: Problem to obtain sexual hybrids • Protoplast fusion - Satsuma mandarin cv. Guoqing No. 1 (G1) and three seedy sweet oranges i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. • mtDNA of G1 SM introduced into SO cultivars for potential seedlessness. Xiao et al (2014) China
M K Saini Somatic hybridization in fruit crop improvement Somatic hybrids Characters References Musa acuminata cv. Mas + M. silk cv. Guoshanxiang Disease resistance (Fusarium) Xiao et al (2009) Citrus reticulata cv. Red Tangerine + Poncirus trifoliata Tolerant to CTV (citrus tristeza virus) and CEV ( citrus exocortis virus ) Guo et al (2002) Citrus reticulata + C. limon Carotenoid compounds Bassene et al (2009) Murcott tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) Seedless Cai et al (2010) Citrus sinensis Osbeck cv. Yoshida navel orange + Citrus unshiu Marc cv. Okitsu satsuma mandarin Seedless An et al (2008) Bingtang orange (Citrus sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge) Asiatic citrus canker-tolerant and ornamental citrus breeding Cai et al (2010)
M K Saini 4. Somaclonal variations & In vitro mutagenesis
M K Saini 4.Somaclonal variations & In vitro mutagenesis • Somaclonal variation is defined as genetic variation observed among progeny of plants regenerated from somatic cells cultured in vitro. • Somaclonal variants in vitro selected with or without induced mutagenesis to obtain tolerant plants to a variety of stresses. Grosser and Gmitter (2011) USA
M K Saini Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS • Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 hour. • The survival plants were tested by in vitro and in vivo inoculation with Xcc. • One somaclone (DG-2) was identified to be resistant to the canker disease. Ge et al (2015) China The symptoms of wild type plants on 3 dpi (j) and on 7 dpi (k); No symptoms of tolerant somaclone on 3 dpi (l) and on 7 dpi (m)
M K Saini In vitro mutagenesis for improvement of Banana • In vitro mutagenesis was applied to banana cv. “Giant Cavendish” to develop dwarf, early maturing mutants with increased fruit yield. • Multiple shoots of banana were gamma ray irradiated (10 Gy). • Later, irradiated plants and control plants were multiplied by tissue culture. Ganapathi and Badigannavar (2020) BARC, Mumbai TBM-2
M K Saini 5. Cryopreservation & in vitro germplasm storage
M K Saini 5. Cryopreservation & in vitro germplasm storage • Genetic resources are not only pre-requsite but key ingredient for genetic improvement. • Loss of genetic variability –> Genetic erosion. • Field gene bank –> Damage by pests, pathogens & climatic disorders. • In vitro germplasm conservation is the only solution. • Cryopreservation – – Storage at ultra cool temperature (-196 ᵒC). – Plants cells, Shoot tips, Somatic & Zygotic embryos. – 4 Steps –> Freezing, Storage, Thawing & Reculturing. • In vitro germplasm storage – INIBAP –> Banana – NBPGR –> Pomegranate, Banana, Phalsa, Bael & Jackfruit.
M K Saini Conclusion • Biotechnology has brought great challenges, opportunities and prospects for conventional breeding. • However, biotechnology as transgenic breeding or genetic manipulation cannot replace conventional breeding but is and only is a supplementary addition to conventional breeding. • Therefore, integration of biotechnology into conventional breeding programs will be an optimistic strategy for fruit crop improvement in the future.
M K Saini

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Role of tissue culture techniques in overcoming major breeding constraints in fruit crops

  • 1. M K Saini 1 Presented by: Mandeep Kaur L-2018-A-35-D PhD Fruit Science Role of tissue culture techniques in overcoming major breeding constraints in fruit crops Presented to: Dr H S Dhaliwal Dr Monika Gupta Dr Rachna Arora
  • 2. M K Saini CONTENT  Introduction to Fruit Breeding  Fruit Breeding Constraints  Tissue Culture Techniques a. Embryo Rescue b. Production of Haploid Plants c. Somatic Hybridization d. Somaclonal Variations e. Cryopreservation & In- vitro Germplasm Storage f. Quality Improvement  Conclusion
  • 3. M K Saini Fruit Breeding • Breeding objectives for rootstock: – Resistant to biotic and abiotic stress – Dwarf growth stature – without affecting productivity of scion. – Wide geographical adaptability – Easily propagated through asexual means • Breeding objectives for scion cultivars: – High productivity – Resistant to biotic and abiotic stress – Dwarf growth stature – Regular, precocious and prolific bearer per unit canopy area – Longer shelf life and transport quality – Good quality fruits:  Appearance –> Size, shape, color  Eating quality –> Seedlessness (few or soft seeds), flesh color, texture, flavor
  • 4. M K Saini Fruit Breeding Constraints • Long juvenile phase • Heterozygous nature • Wide/distant hybridization – Interspecific & Intergeneric crosses. • Sexual incompatibilities in both self and cross combination • Susceptibility to biotic & abiotic stress • Polyploidy – Banana, Strawberry • Polyembryony – Citrus, Mango • Parthenocarpy and seedlessness – Banana, Pineapple • Loss of natural germplasm • Lack of knowledge on inheritance pattern • Excessive fruit drop – Mango, Citrus • Presence of single seed in single fruit – Mango, Litchi • Seed dormancy • Low seed viability
  • 5. M K Saini 1. Embryo Rescue
  • 6. M K Saini 1. Embryo Rescue • Post - zygotic embryo abortion – Major breeding constraint. • Embryo rescue refers to in vitro technology whose purpose is to promote the development of an immature or weak embryo into a viable plant. • Embryo rescue procedures – Ovule culture, Ovary culture, Embryo culture. • It has been successfully applied in grapes, citrus, mango, stone fruits. • Grapes variety developed by IARI, New Delhi using embryo rescue :- • Pusa Purple Seedless: Cross between ‘Pearl of Casaba’ and ‘Beauty Seedless’, raised through embryo rescue. • It is an extra-early berry ripening variety (in the 3rd week of May) under sub-tropical region.
  • 7. M K Saini Applications of embryo rescue techniques 1. Shortening the breeding cycle 2. Developing interspecific and intergeneric hybrids 3. Seedlessness 4. Overcoming embryo abortion 5. Determining of seed viability 6. Overcoming dormancy – Malus and Prunus spp.
  • 8. M K Saini In vitro efficiency of embryo rescue of interspecific hybrids of sweet cherry and Chinese cherry cultivars Effects of different low temperature treatments & GA₃ on cherry embryo germination through in vitro rescue. Wu et al (2021) China  Chaoyang-1 (CY-1) – Sweet cherry  Duangbing (DB) – Chinese cherry • Failure to obtain hybrid cherry seedlings during outbreeding.
  • 9. M K Saini In vitro embryo rescue for the production of hypotetraploids after cross between hypotetraploid and tetraploid grape cultivars Effect of the time of ovule culture and basal medium on development of the embryos and in vitro growth of embryos excised after ovule culture Kim et al (2020) Korea • Hypotetraploid (2n = 4x-1 = 75) - To develop seedless grapes with large berries. • (4x-1=75) => Hanareum => female parent • (4x=76) => Honey Black and Kyoho => male parents
  • 10. M K Saini Effect of different sampling times on embryo recovery rate in different seedless grapes crosses Li et al (2020) China Plant Development (%) Embryo Recovery (%) • Stenospermocarpy leads to embryo abortion during development.
  • 11. M K Saini Plant material Remarks Reference Vitis vinifera L. Ovule culture of seedless grapes (Vitis vinifera L.) Singh et al (1991) Peach Ovule rescue in peaches: incubation period Raseira and Einhardt (2010) Citrus Regenerating triploid plants by embryo rescue technique Liu et al (2010), Aleza et al (2010) Grape Embryo rescue of seedless grape with disease and cold resistance Niu et al (2012) Musa species Embryo culture and embryo rescue studies in wild Musa spp. (Musa ornata) Dayarani et al (2014) Citrus Production of common sour orange × Carrizo citrange hybrids using embryo rescue Kurt and Ulger (2014) Grapevine Immature embryo rescue and in vitro evaluation of intraspecific hybrids from Brazilian seedless grapevine ‘Superior × Thompson’ clones Menezes et al (2014) Embryo rescue in fruit crop improvement
  • 12. M K Saini 12 2. In Vitro Production of Haploids
  • 13. M K Saini 2. In Vitro Production of Haploids • Haploid plants are obtained from pollen grains by culturing them. • Two methods - Anther culture & pollen culture. • Double haploid - Homozygous pure plant. • Applications of haploid plants: – Production of genetically pureline by selfing is impractical because of their long generation cycle & frequent self incompatibility. – To solve of problem of high heterozygosity – No segregation in meiosis. – Shorten the time for generation of homozygous lines. – QTL mapping. – Genome sequencing. – DH: To study heterozygous maternal parent. Haploid 1 n Diploid 2 n Colchicine
  • 14. M K Saini In vitro regeneration of haploids using isolated microspore culture of banana cv. Bimkol • Musa balbisiana cv. Bimkol => BB genomic group (2n= 2x= 22). • Long crop cycle (740 days) => Breeding problem. • 30 days old immature male flowers => Explants. • 58.33 % haploid plants • 41.66 % diploid plants Gogoi et al (2020) India Haploid plant Diploid (Control)
  • 15. M K Saini 3. Somatic Hybridization
  • 16. M K Saini 3. Somatic Hybridization • Somatic hybridization is an alternative to sexual reproduction in order to combine the genomes from otherwise incompatible parents. • It involves fusing protoplasts of two different genomes followed by the selection of desired somatic hybrid cells and subsequent regeneration of hybrid plant. • It involves four steps:  Protoplast isolation  Protoplast fusion  Selection of hybrid cells  Regeneration of hybrid plants • Two types of hybrids - symmetric hybrid and asymmetric hybrids
  • 17. M K Saini Applications of somatic hybridization • Production of interspecific and intergeneric hybrids – by combining nuclear and cytoplasmic genomes belonging to different species & genus. • Production of fertile diploids and polypoids – by crossing sexually sterile haploids, triploids and aneuploids • Production of heterozygous lines in the asexually propagated species • Transfer of cytoplasmic information (mtDNA) for the production of male sterile plants and seedless triploids. • To create novel germplasm as a source of elite breeding parents. • To create novel hybrids with increased yield and resistance to biotic and abiotic stress.
  • 18. M K Saini Triploid Mandarin: Sugar Belle X Nova + Succari Triploid Lime: ‘Todo del Ano’ lemon X ‘Mexican’ lime + ‘Valencia’ Triploid Lemon: ‘Todo del Ano’ lemon X ‘Hamlin’ + Femminello’ Fig: Seedless fruits from triploid hybrids produced by somatic hybridization Grosser and Gmitter (2011) USA
  • 19. M K Saini Response of four allotetraploid somatic hybrids to Citrus tristeza virus induced infections Abbate et al (2018) Italy Fig: Detection of mild (CTV-DS1) and severe (CTV-DS2) strains in the investigated genotypes by using DAS-ELISA and qRT-PCR assays. Biological barriers - sexual incompatibility, polyembryony, male/female sterility, long juvenility and different bloom phases
  • 20. M K Saini Production of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars • Polyembryony: Problem to obtain sexual hybrids • Protoplast fusion - Satsuma mandarin cv. Guoqing No. 1 (G1) and three seedy sweet oranges i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. • mtDNA of G1 SM introduced into SO cultivars for potential seedlessness. Xiao et al (2014) China
  • 21. M K Saini Somatic hybridization in fruit crop improvement Somatic hybrids Characters References Musa acuminata cv. Mas + M. silk cv. Guoshanxiang Disease resistance (Fusarium) Xiao et al (2009) Citrus reticulata cv. Red Tangerine + Poncirus trifoliata Tolerant to CTV (citrus tristeza virus) and CEV ( citrus exocortis virus ) Guo et al (2002) Citrus reticulata + C. limon Carotenoid compounds Bassene et al (2009) Murcott tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) Seedless Cai et al (2010) Citrus sinensis Osbeck cv. Yoshida navel orange + Citrus unshiu Marc cv. Okitsu satsuma mandarin Seedless An et al (2008) Bingtang orange (Citrus sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge) Asiatic citrus canker-tolerant and ornamental citrus breeding Cai et al (2010)
  • 22. M K Saini 4. Somaclonal variations & In vitro mutagenesis
  • 23. M K Saini 4.Somaclonal variations & In vitro mutagenesis • Somaclonal variation is defined as genetic variation observed among progeny of plants regenerated from somatic cells cultured in vitro. • Somaclonal variants in vitro selected with or without induced mutagenesis to obtain tolerant plants to a variety of stresses. Grosser and Gmitter (2011) USA
  • 24. M K Saini Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS • Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 hour. • The survival plants were tested by in vitro and in vivo inoculation with Xcc. • One somaclone (DG-2) was identified to be resistant to the canker disease. Ge et al (2015) China The symptoms of wild type plants on 3 dpi (j) and on 7 dpi (k); No symptoms of tolerant somaclone on 3 dpi (l) and on 7 dpi (m)
  • 25. M K Saini In vitro mutagenesis for improvement of Banana • In vitro mutagenesis was applied to banana cv. “Giant Cavendish” to develop dwarf, early maturing mutants with increased fruit yield. • Multiple shoots of banana were gamma ray irradiated (10 Gy). • Later, irradiated plants and control plants were multiplied by tissue culture. Ganapathi and Badigannavar (2020) BARC, Mumbai TBM-2
  • 26. M K Saini 5. Cryopreservation & in vitro germplasm storage
  • 27. M K Saini 5. Cryopreservation & in vitro germplasm storage • Genetic resources are not only pre-requsite but key ingredient for genetic improvement. • Loss of genetic variability –> Genetic erosion. • Field gene bank –> Damage by pests, pathogens & climatic disorders. • In vitro germplasm conservation is the only solution. • Cryopreservation – – Storage at ultra cool temperature (-196 ᵒC). – Plants cells, Shoot tips, Somatic & Zygotic embryos. – 4 Steps –> Freezing, Storage, Thawing & Reculturing. • In vitro germplasm storage – INIBAP –> Banana – NBPGR –> Pomegranate, Banana, Phalsa, Bael & Jackfruit.
  • 28. M K Saini Conclusion • Biotechnology has brought great challenges, opportunities and prospects for conventional breeding. • However, biotechnology as transgenic breeding or genetic manipulation cannot replace conventional breeding but is and only is a supplementary addition to conventional breeding. • Therefore, integration of biotechnology into conventional breeding programs will be an optimistic strategy for fruit crop improvement in the future.

Editor's Notes

  1. Young fruit stored at 4℃ for 35 days remained the best choice. Cold treatment affected the germination process in cherry by breaking the dormancy and accelerating the germination onset. This could be related to the poor development of embryos, which differentiate
  2. “Symmetric hybrid” is a combination of diploid nuclear genomes and two maternal cytoplasmic genomes. “Asymmetric hybrids” arises due to a combination of genomes of the parents or combination of the genome of one parent and the cytoplasm of another; these are also known as cybrids.
  3. Protoplast fusion was adopted to transfer CMS from Satsuma mandarin cv. Guoqing No. 1 (G1) to three seedy sweet oranges i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’.
  4. Tropical fruit crops such as banana are propagated vegetatively and creating genetic variability is highly limited due to heterozygosity, polyploidy and complex genome. Mutation induction is an important tool to create variability for fruit yield and quality traits.