1. DOUBLED HAPLOIDS IN HOT AND SWEET PEPPERS: SUCCESS AND CHALLENGES Piyush Kumar Gupta Verdenta Hybrid Seeds Pvt. Ltd., Gurgaon, Haryana 122001 ASRT 2014, Sep 9-10, 2014, Bangalore, India

2. Content Outline  What is a Double Haploid (DH)?  How it reduces time?  Why Double Haploids?  Doubled haploids in Hot and Sweet Peppers ▫ Material and Methods ▫ Equipments ▫ Different stages of DH Pepper ▫ Challenges ▫ Major factors influencing DHs production ▫ Highlights  Cost and Financials  Doubled Haploids in Tomato

3. What is a Double Haploid?  A doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. Haploids (n) Double Haploids (2n) Cochicine treatment

4. How it reduces time? DH selection via molecular markers 100% DH AABB 6.25% homozyg ous 25% DH AABB Testing and Cultivar Release 3-4 2 1 1 2 3 4 5 6 7 8 9-10 Seasons

5.  Microspore and anther culture followed by colchicine helps rapid homozygosity following a cross ▫ Fast track stabilization of traits – major & minor genes. ▫ Using molecular markers helps select appropriate DH plant ▫ Inbred ready in 3rd season after F1 pollen used for DH Why Double Haploids? Cont..

6.  Quick development of recombinant inbred lines for mapping & genetics studies ▫ Molecular marker development, association mapping etc  Proven technology in several crops- Wheat, Corn, Rice, Pepper etc  Impact on Business ▫ Speed to deliver products - Prototype hybrids can be made in 3rd season itself vs 6 seasons in conventional breeding. 50% faster delivery  Quick return on investment Cont..

7.  Asian hot peppers ▫Light Green, Bydagi, varieties/breeding lines ▫European Sweet pepper, control (Light Green blocky)  Growth of anther donor plants of Hot and Sweet Peppers ▫In phytotron chamber 16-h illumination at 70 µmol m -2 s -1 with day/night temperatures of 25/20ºC day/night  Key Factors ▫Late-unicellular microspores ▫Heat stress of anthers (35ºC for 8 days, dark) ▫In vitro doubling of haploid regenerants with 400 mg/l colchicine ▫Acclimatization of regenerants step by step Material and Methods

8. Equipments required for DH lab  Autoclave (Small & Large)  Water Purifier distillation system  Gyratory Shaker  Refrigerator  Weighing balance  Magnetic Stirrer  pH Meter  BOD Incubator  Laminar Flow (6x2x2)  Microscope & Haemocytometer  Media Preparator  Media Pourer  Dish washer Media PreparatorMedia Pourer

9. Different stages of DH Pepper

10. Challenges faced  Recalcitrant Genotypes ▫ High genetic variability ▫ Significant genotype difference for the efficiency of DH production  No reproducible protocol available  Unusual regeneration ▫ Without shoot ▫ Without roots  Produce 2-4 plants per 100 anthers plated

11. Major factors influencing DHs production  Donor plant growth condition ▫ Green House Vs Growth Chamber Temperature Moisture Light Nutrition  In vitro and in vivo culture condition ▫ Pretreatment of the buds ▫ Media composition ▫ Optimal temperature and light regimes

12. Highlights  DH response of Recalcitrant genotype can be increased by crossing with high responsive lines  Need to study genetics of response to DH. (Gene was reported for such response in Cotton)  Anther donor plants in growth chambers reproduce 2 times more DH plants than Green house conditions. ▫ Reduce contamination (Thrips) ▫ Easy to target appropriate anther stage ▫ Less influence of uncontrolled environmental conditions  MS Media with 2,4D (10 -7 M- 10 -8 M) shows better response than without 2,4D

13. Costs & Financials 13

14. Capital Investment: Facilities and Costs  Assumptions: ▫ Annual volume is 2000 DH lines 10 projects and 200 DH Lines per project ▫ 2 growth chamber size 12x10 feet ▫ 1 greenhouse size 30x15 feet  $ 1 = 60  Employees: 1 FTE, 1 Technician, 2 Daily paid  Scaling up ▫ Double the DH plants will add only 50% cost in capital.

15. Cost per DH*: INDIA can be half of that in NAFTA  All pricing is based on the assumption of a 8% induction rate and a success rate of 4%  Annual volume is 2000 DH lines (10 projects and 200 DH Lines per project).  Scaling up: Double the DH plants will reduce per DH plant cost by 40%.

16. Financial Summary Pay back period – 3.7 years IRR – 62%

17. DH in Tomato

18.  Little progress in the tomato DH  No reliable and standardized methods available so far  Tomato is extremely recalcitrant  in vitro developmental pathways ▫Anther culture ▫ Microspores or microsporocytes  direct embryogenesis  callus formation ▫Ovary culture ▫Wide hybridization using Solanum sisymbriifolium Lam.  Patents ▫Tomato anther culture: US 4835339 A ▫http://www.google.com/patents/US4835339http://www.google.com/patents/US4835339 ▫Key claim 1 anthers are dissected from an inflorescence of a hybrid plant of L. esculentum (×) L. pennellii.

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