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Genetic Transformation in Fruit Crops

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M
MANDEEP KAUR

Genetic transformation is a technique used to directly manipulate an organism's genome. It has several applications for fruit crop improvement, including shortening juvenile phase, increasing productivity, improving biotic/abiotic stress tolerance, and enhancing quality. Key methods are Agrobacterium-mediated transformation and particle bombardment. Studies show overexpression of flowering genes reduced juvenile phase in apple, while auxin-related genes increased grape fecundity. Disease resistance genes like Xa21 and NPR1 enhanced resistance to citrus canker and diseases in strawberry. Abiotic stress tolerance was achieved in strawberry and apple through expression of osmotin and Myb4 genes. Quality was improved by modulating pigmentation and shelf life genes in apple and

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M K Saini 1 Presented by: Mandeep Kaur L-2018-A-35-D PhD Fruit Science Genetic Transformation in Fruit Crops Presented to: Dr H S Dhaliwal Dr Monika Gupta Dr Rachna Arora
M K Saini Content – Genetic Transformation – – Shortening of Juvenile Phase – Increasing Productivity – Biotic Stress Tolerance – Abiotic Stress Tolerance – Quality Improvement
M K Saini Genetic Transformation PROCEDURE FOR GENETIC TRANSFORMATION Isolation of the gene(s) of interest Insertion of the gene(s) into a transfer vector Plant transformation Selection of the modified plant cells Regeneration into whole plants via tissue culture Verification of transformation and characterization of the inserted DNA fragment (Transient transformation and Stable transformation) Testing of the plant performance Safety assessment Genetic transformation also called genetic modification is the direct manipulation of an organism's genome using rDNA technology (Hota et al 2020).
M K Saini Direct Method Biolistic or Particle gun Electroporation Microinjection & Macroinjection PEG Indirect/Biological Method Agrobacterium tumefaciens Agrobacterium rhizogenes METHODS OF GENE TRANSFER
M K Saini Genetic Transformation in Fruit Crops b. Increasing Productivity c. Biotic Stress Tolerance a. Shortening of Juvenile Phase d. Abiotic Stress Tolerance e. Quality Improvement
M K Saini a. Shortening of Juvenile Phase
M K Saini FasTrack Breeding (Scorza et al (2012) West Virginia, USA STEP 1 STEP 2 Flowering within 6 months STEP 3 Breeding system that uses genetically engineered tree carrying early flowering gene. The variety released for commercial use is not a genetically modified plant.
M K Saini Overexpression of BpMADS4 gene from silver birch induces early-flowering in apple • Objective => To shorten the juvenile stage. • Proliferating axillary shoot cultures of apple cv. Pinova. • BpMADS4 gene from silver birch (Betula pendula Roth.) • Floral initiation within 3–4 months. Flower buds (13 weeks) 5–7 days later, Flowers were obvious After 10–14 days, Complete flower opened Morphologically similar to normal Flachowsky et al (2007) Finland
M K Saini b. Increasing Productivity
M K Saini Ovule-specific auxin-synthesizing (DefH9-iaaM) transgene enhances grape fecundity • Grapevine fecundity (fertility) is determined by its genetic makeup. • DefH9 coding region from Antirrhinum majus. • iaaM from Pseudomonas savastonoi. • Inflorescences per shoot - Doubled in transgenics of Thompson Seedless. • Berry number per bunch - Increased in both transgenic cultivars. Costantini et al (2007) Italy SILCORA THOMPSON SEEDLESS Control Transgenic Transgenic Control
M K Saini c. Biotic Stress Tolerance
M K Saini Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice • Disease susceptibility, inter- & intraspecific incompatibility => Breeding obstacles. • ‘W. Murcott’ mandarin - Direct DNA uptake using a protoplast transformation system. • DNA construct - cDNA of Xa21, a Xanthomonas resistance gene from rice. Omar et al (2018) USA qRT-PCR of W. Murcott Transgenic lines Resistant phenotypes of transgenic lines
M K Saini The Arabidopsis NPR1 gene confers broad- spectrum disease resistance in strawberry • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • AtNPR1 gene provides System Acquired Resistance. • Diploid strawberry (F. vesca) accession Hawaii 4 - Agrobacterium strain GV3101. • Overexpression of AtNPR1 increased resistance to 3 diseases. Fig 1: Resistance to Anthracnose rot in TL-16 Silva et al (2015) USA Fig 2: Powdery mildew in TL-14 & TL-53 Fig 3: Angular leaf spot in TL-61
M K Saini Production of transgenic kiwifruit plants harbouring the SbtCry1Ac gene • Loss of valuable kiwifruit germplasm & Auto-hexaploid nature => Breeding problem. • Objective: Transgenic kiwifruit against fruit-piercing moth (Oraesia excavata) • Kiwifruit (cv. Hongyang) leaf tissues - SbtCry1Ac gene. • Expression of insecticidal btCrylAc gene in tissues of transgenic kiwifruit. Zhang et al (2015) China Fig 1: Column 1. Non-transgenic plant Column 2. Transgenic plant Fig 2: Column 1. Non-transgenic plant Column 2. Transgenic plant Leaf damage rate Average weight of surviving larvae
M K Saini Transgenic PRSV Resistant Papaya cv. ‘SunUp’ • Monotypic Genus Carica => No resistance gene for PRSV. • Vasconcella cauliflora (Carica cauliflora) => Resistant to PRSV. • Intergeneric incompatibility => Breeding problem. • CP gene of PRSV strain • Line 55-1 => excellent resistance against PRSV. • Line 55-1 X non-transgenic - Sunset => ‘SunUp’ • SunUp X non-transgenic - Kapoho => ‘Rainbow’ Gonsalves et al (1998) Hawaii Transgenic Control-NT SunUp Rainbow
M K Saini Transgenic PPV resistant plum cv. ‘HoneySweet’ • Allo-hexaploid nature of European plum => Breeding problem. • Gene for PPV coat protein inserted into plant genome. • Bluebyrd plum seedling => A. tumefaciens EHA 101. • Field evaluation - 12 years in aphid vectored conditions. • Later patented as ‘HoneySweet’. Scorza et al (2016) US Kearneysville
M K Saini d. Abiotic Stress Tolerance
M K Saini Overexpression of tobacco osmotin gene leads to salt stress tolerance in strawberry plants • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • Strawberry plants => A. tumefaciens (GV2260) harbouring Osmotin gene. • Expression of osmotin gene increased in transgenic lines TL3, TL5, TL9. Husaini and Abdin (2008) India Proline content at 1 week and 2 week exposure to NaCl Chlorophyll content upon exposure to NaCl
M K Saini Osmyb4 expression improves adaptive responses to drought & cold stress in transgenic apples • Long juvenile period => Breeding problem • In vitro cultured apple shoots cv. Greensleeves transformed via A. tumefaciens strain EHA105. • Ectopic expression of the Myb4 transcription factor improved tolerance. Pasquali et al (2008) Italy RT-PCR analysis of Osmyb4 gene expression • WT - Wild type • HE - High transgene expression • LE - Low transgene expression
M K Saini Relative water content & ion leakage in apple leaves during the 15 days of drought treatment Glucose and proline contents of wild-type (WT) and high expression (HE) apple plants during cold treatment 4ᵒC for 1, 2 and 9 days.
M K Saini e. Quality Improvement
M K Saini MYB transcription factor (MdMYB10) increases red colouration in transgenic apple fruit • Long juvenile period => Breeding problem. • Objective => To increase red coloration in cortex & skin of fruit. • Leaf pieces of Malus domestica cv. Red chief - MdMYB10 cDNA. • Differences in expression of MdMYB10 gene that determine cortex colour. Espley et al (2007) New Zealand RT-PCR analysis of MdMYB10 cDNA in ‘Red Field’ & Control (Pacific Roseᵀᴹ) Red Field Pacific Roseᵀᴹ
M K Saini • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • Objective => To increase postharvest shelf life. • Strawberry plants => FaPG1-cDNA fragment in antisense orientation. • Antisense down-regulation of FaPG1 in AntiPG lines reduced fruit softening. • Total Polygalacturonase activity reduced in 3 transgenic lines. Antisense down-regulation of the FaPG1 reduces fruit softening in Strawberry cv. Chandler Quesada et al (2009) Spain Control & Transgenic (AntiPG) harvested at the full ripened stage. Total PG activity in ripened fruits from control and Transgenic lines (APG).
M K Saini Conclusion & Future Prospects • Biotechnology has brought great opportunities and prospects for overcoming problems of conventional breeding. • However, biotechnology as transgenic breeding or genetic manipulation cannot replace conventional breeding but it 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

More Related Content

Genetic Transformation in Fruit Crops

  • 1. M K Saini 1 Presented by: Mandeep Kaur L-2018-A-35-D PhD Fruit Science Genetic Transformation in Fruit Crops Presented to: Dr H S Dhaliwal Dr Monika Gupta Dr Rachna Arora
  • 2. M K Saini Content – Genetic Transformation – – Shortening of Juvenile Phase – Increasing Productivity – Biotic Stress Tolerance – Abiotic Stress Tolerance – Quality Improvement
  • 3. M K Saini Genetic Transformation PROCEDURE FOR GENETIC TRANSFORMATION Isolation of the gene(s) of interest Insertion of the gene(s) into a transfer vector Plant transformation Selection of the modified plant cells Regeneration into whole plants via tissue culture Verification of transformation and characterization of the inserted DNA fragment (Transient transformation and Stable transformation) Testing of the plant performance Safety assessment Genetic transformation also called genetic modification is the direct manipulation of an organism's genome using rDNA technology (Hota et al 2020).
  • 4. M K Saini Direct Method Biolistic or Particle gun Electroporation Microinjection & Macroinjection PEG Indirect/Biological Method Agrobacterium tumefaciens Agrobacterium rhizogenes METHODS OF GENE TRANSFER
  • 5. M K Saini Genetic Transformation in Fruit Crops b. Increasing Productivity c. Biotic Stress Tolerance a. Shortening of Juvenile Phase d. Abiotic Stress Tolerance e. Quality Improvement
  • 6. M K Saini a. Shortening of Juvenile Phase
  • 7. M K Saini FasTrack Breeding (Scorza et al (2012) West Virginia, USA STEP 1 STEP 2 Flowering within 6 months STEP 3 Breeding system that uses genetically engineered tree carrying early flowering gene. The variety released for commercial use is not a genetically modified plant.
  • 8. M K Saini Overexpression of BpMADS4 gene from silver birch induces early-flowering in apple • Objective => To shorten the juvenile stage. • Proliferating axillary shoot cultures of apple cv. Pinova. • BpMADS4 gene from silver birch (Betula pendula Roth.) • Floral initiation within 3–4 months. Flower buds (13 weeks) 5–7 days later, Flowers were obvious After 10–14 days, Complete flower opened Morphologically similar to normal Flachowsky et al (2007) Finland
  • 9. M K Saini b. Increasing Productivity
  • 10. M K Saini Ovule-specific auxin-synthesizing (DefH9-iaaM) transgene enhances grape fecundity • Grapevine fecundity (fertility) is determined by its genetic makeup. • DefH9 coding region from Antirrhinum majus. • iaaM from Pseudomonas savastonoi. • Inflorescences per shoot - Doubled in transgenics of Thompson Seedless. • Berry number per bunch - Increased in both transgenic cultivars. Costantini et al (2007) Italy SILCORA THOMPSON SEEDLESS Control Transgenic Transgenic Control
  • 11. M K Saini c. Biotic Stress Tolerance
  • 12. M K Saini Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice • Disease susceptibility, inter- & intraspecific incompatibility => Breeding obstacles. • ‘W. Murcott’ mandarin - Direct DNA uptake using a protoplast transformation system. • DNA construct - cDNA of Xa21, a Xanthomonas resistance gene from rice. Omar et al (2018) USA qRT-PCR of W. Murcott Transgenic lines Resistant phenotypes of transgenic lines
  • 13. M K Saini The Arabidopsis NPR1 gene confers broad- spectrum disease resistance in strawberry • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • AtNPR1 gene provides System Acquired Resistance. • Diploid strawberry (F. vesca) accession Hawaii 4 - Agrobacterium strain GV3101. • Overexpression of AtNPR1 increased resistance to 3 diseases. Fig 1: Resistance to Anthracnose rot in TL-16 Silva et al (2015) USA Fig 2: Powdery mildew in TL-14 & TL-53 Fig 3: Angular leaf spot in TL-61
  • 14. M K Saini Production of transgenic kiwifruit plants harbouring the SbtCry1Ac gene • Loss of valuable kiwifruit germplasm & Auto-hexaploid nature => Breeding problem. • Objective: Transgenic kiwifruit against fruit-piercing moth (Oraesia excavata) • Kiwifruit (cv. Hongyang) leaf tissues - SbtCry1Ac gene. • Expression of insecticidal btCrylAc gene in tissues of transgenic kiwifruit. Zhang et al (2015) China Fig 1: Column 1. Non-transgenic plant Column 2. Transgenic plant Fig 2: Column 1. Non-transgenic plant Column 2. Transgenic plant Leaf damage rate Average weight of surviving larvae
  • 15. M K Saini Transgenic PRSV Resistant Papaya cv. ‘SunUp’ • Monotypic Genus Carica => No resistance gene for PRSV. • Vasconcella cauliflora (Carica cauliflora) => Resistant to PRSV. • Intergeneric incompatibility => Breeding problem. • CP gene of PRSV strain • Line 55-1 => excellent resistance against PRSV. • Line 55-1 X non-transgenic - Sunset => ‘SunUp’ • SunUp X non-transgenic - Kapoho => ‘Rainbow’ Gonsalves et al (1998) Hawaii Transgenic Control-NT SunUp Rainbow
  • 16. M K Saini Transgenic PPV resistant plum cv. ‘HoneySweet’ • Allo-hexaploid nature of European plum => Breeding problem. • Gene for PPV coat protein inserted into plant genome. • Bluebyrd plum seedling => A. tumefaciens EHA 101. • Field evaluation - 12 years in aphid vectored conditions. • Later patented as ‘HoneySweet’. Scorza et al (2016) US Kearneysville
  • 17. M K Saini d. Abiotic Stress Tolerance
  • 18. M K Saini Overexpression of tobacco osmotin gene leads to salt stress tolerance in strawberry plants • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • Strawberry plants => A. tumefaciens (GV2260) harbouring Osmotin gene. • Expression of osmotin gene increased in transgenic lines TL3, TL5, TL9. Husaini and Abdin (2008) India Proline content at 1 week and 2 week exposure to NaCl Chlorophyll content upon exposure to NaCl
  • 19. M K Saini Osmyb4 expression improves adaptive responses to drought & cold stress in transgenic apples • Long juvenile period => Breeding problem • In vitro cultured apple shoots cv. Greensleeves transformed via A. tumefaciens strain EHA105. • Ectopic expression of the Myb4 transcription factor improved tolerance. Pasquali et al (2008) Italy RT-PCR analysis of Osmyb4 gene expression • WT - Wild type • HE - High transgene expression • LE - Low transgene expression
  • 20. M K Saini Relative water content & ion leakage in apple leaves during the 15 days of drought treatment Glucose and proline contents of wild-type (WT) and high expression (HE) apple plants during cold treatment 4ᵒC for 1, 2 and 9 days.
  • 21. M K Saini e. Quality Improvement
  • 22. M K Saini MYB transcription factor (MdMYB10) increases red colouration in transgenic apple fruit • Long juvenile period => Breeding problem. • Objective => To increase red coloration in cortex & skin of fruit. • Leaf pieces of Malus domestica cv. Red chief - MdMYB10 cDNA. • Differences in expression of MdMYB10 gene that determine cortex colour. Espley et al (2007) New Zealand RT-PCR analysis of MdMYB10 cDNA in ‘Red Field’ & Control (Pacific Roseᵀᴹ) Red Field Pacific Roseᵀᴹ
  • 23. M K Saini • Allo-octaploid nature of cultivated strawberry => Breeding constraint. • Objective => To increase postharvest shelf life. • Strawberry plants => FaPG1-cDNA fragment in antisense orientation. • Antisense down-regulation of FaPG1 in AntiPG lines reduced fruit softening. • Total Polygalacturonase activity reduced in 3 transgenic lines. Antisense down-regulation of the FaPG1 reduces fruit softening in Strawberry cv. Chandler Quesada et al (2009) Spain Control & Transgenic (AntiPG) harvested at the full ripened stage. Total PG activity in ripened fruits from control and Transgenic lines (APG).
  • 24. M K Saini Conclusion & Future Prospects • Biotechnology has brought great opportunities and prospects for overcoming problems of conventional breeding. • However, biotechnology as transgenic breeding or genetic manipulation cannot replace conventional breeding but it 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. Conventional breeding in octoploid strawberry is laborious, time-consuming, and expensive. The coding region of AtNPR1 was amplified from cDNA using PCR, cloned into pK7WG2D,1 and then transformed into the diploid strawberry F. vesca L. using the Agrobacterium strain GV3101. Transgenic manipulation of strawberry’s innate defense signaling pathways may offer an alternative approach to generate resistance or tolerance to various pathogens.
  2. CP gene of a mild mutant of a PRSV strain. High velocity micro-projectiles gun.