#Alliance4Science @BiovIntCIAT_eng @BiovIntCIAT_esp COAXIM, an innovative protocol for mass clonal propagation of coconuts: Merits of the Technology, Potency and Progress Bart Panis Hannes Wilms COAXIM (COconut AXIllary Meristems) Starting point of our involvement in coconut research; Project “Developing cryopreservation protocols for sub-tropical crops and establishing cryo-genebank at RDA with Bioversity International” Kick off meeting in Legazpi (Nov 2016) Which clonal in vitro multiplication methods are out there for plants ? 1. Somatic embryogenesis 2. Formation of adventitious (de novo) meristems 3. Formation of axillary meristems … and for coconuts ? 1. Somatic embryogenesis protocol for coconut is developed since >30 years (and has pros and cons), not really commercialized yet 2. Adventitious shoot formation in monocots is rare 3. Coconut palms do not form axillary shoots (branches) in nature. In vitro axillary shoot formation not reported yet Clonal in vitro multiplication GMO, gene editing, gene manipulation Making identical copies from field plants (Fruit trees, potato, cassava, strawberries,…..) Starting materials: shoots from in vitro germinated embryos Starting materials: shoots from in vitro germinated embryos Coconuts from Belgian supermarket used for embryo culture: problematic ! • Contamination variable but often very high (40 - 50%) • Shoot formation variable but often very low (average 20%) • Origin and age of material unknown Solution: Work with embryos excised at PCA and send to Belgium; In 2017 and 2018, 3600 embryos were received Markham valley tall (MVT), Equatiorial green dwarf (EGD), Laguna tall (LAGT), Catigan dwarf (CATD), Malayan yellow dwarf (MYD), West African tall (WAT), Rennell Island Tall (RIT) Optimization of germination of coconut embryos received from PCA • Sterilization procedure • Haustorium removal • Macro and micro nutrient composition • Vitamins • PGR • Sugar concentration • Active charcoal • cultivar 96% of embryos germinate and grow out to normal plants on Y60 AC Multiplication of single shoots How 1. Activation of axillary meristems by breaking dormancy and apical dominance 2. Activation through in vitro flower induction and inversion Different Plant Growth Regulators were tried • BA (benzyladenine) • PBZ (Paclobutrazol) • Strigolacton inhibitor ( TIS108) • TDZ (thidiazuron) No multiplication ! Proliferation! Treatment Plantlets initiated Proliferating plantlets % proliferation Thidiazuron only 144 0 0 Cut only 144 0 0 Thidiazuron+Cut 141 36 25.5 + 1 0 Multiplication? 1 1 Regeneration ? Applicable to other cvs? ➔ Induction of scalps is possible with all cultivars Markham valley tall (MVT), Equatiorial green dwarf (EGD), Laguna tall (LAGT), Catigan dwarf (CATD), Malayan yellow dwarf (MYD) and West African tall (WAT) 12 Cultivar Plantlets Initiated Meristematic proliferation (Percentage ± StD) Died after initiation (Percentage ± StD) Meristematic proliferation (Percentage ± StD) Dwarf CATD 72 22 (30.6±14.6%)a 8 (11.1±6.3%)a 58 (26.9±12.0%)aEGD 72 21 (29.2±14.4%)a 10 (13.8±13.4%)a MYD 72 15 (20.8±8.3%)a 5 (6.9±2.4%)a Tall LAGT 72 14 (19.4±2.4%)a 19 (26.4±15.8%)a 58 (27.1±12.0%)aMVT 72 24 (33.3±7.2%)a 10 (13.8±6.4%)a WAT 70 20 (28.6±19.1%)a 6 (8.6±8.3%)a Total 430 116 (27.1±11.6%) 58 (13.5±10.5%) Technique is repeatable in other laboratories solely based on the publication! Pictures by Dr. Rajesh M.K., Principal Scientist (Biotechnology), Division of Crop Improvement, ICAR- Central Plantation Crop Research Institute (CPCRI), Kerala, India First axillary in vitro shoot multiplication protocol for coconut palms ➢50% increase /month ➢Shoot to shoot in 11 months 1 6 What are next steps, mainly to “economize” the protocol • Optimizing shoot formation, avoid flower- like structures • Avoid blackening • Optimize (speed up) shoot growth and rooting • Increase acclimation success • Grow regenerants out in the field to screen for SV Advantages and strengths of our method • Clonal propagation of coconut plants with identical characteristics to create the opportunity to propagate and plant solely elite trees (for example hybrids) • Large-scale propagation of coconut palms to better respond to increased market demand • Compared to the already existing clonal propagation for coconuts, somatic embryogenesis, the present protocol does not involve a callus phase. In theory this could result in a lower chance for somaclonal variation ? • The propagation protocol is user-friendly and can be applied in all tissue culture laboratories • Protocol can be applied to all cvs (talls as well as dwarfs) • Provides clonal material for cryopreservation (long term preservation of genetic resources) but also for medium term conservation Patent procedure Months after first priority priority dates 25.01.2019 MO 17.06.2019 End priority & entry PCT phase 25.01.2020 M12 PCT search & publication of patent application 25.07.2020 M18 Demand: entry of arguments and amended claims 25.11.2020 M22 Leaving the PCT & national and regional entry 25.07.2021 M30 -M31 • 2 Inventors: Bart Panis (Alliance), Hannes Wilms (KU Leuven) • 3 Patent owners: Alliance, KU Leuven, RDA • Protected in 7 countries Principles that will guide the exploitation of the invention • The Alliance of Bioversity and CIAT, RDA and KU Leuven will make the invention available for use, free of charge, for non-commercial research and development. • We will develop licenses with organizations that want to exploit the invention for commercial purposes in specific geographic areas for limited periods of time. • Each license will be negotiated on a case by case basis, in accordance with the following principles: – Licensees must ensure quality control along the process and of the resulting plants – Resulting planting material must reach smallholder farmers at an affordable price. If not, the license will be revoked. – The Alliance will require licensees to make payments based on their sales of planting materials to the Benefit Sharing Fund of the International Treaty on Plant Genetic Resources for Food and Agriculture. – Other forms of benefit-sharing such as research partnerships, technology transfer and information sharing will be explored with research partners in coconut producing countries and with the International Coconut Genetic Resources Network. This licensing strategy is compliant with the CGIAR Principles for the Management of Intellectual Assets. More info • Contact: b.panis@cgiar.org or hannes.wilms@kuleuven.be • “Development of the first axillary in vitro shoot multiplication protocol for coconut palms” by Hannes Wilms, Dries De Bièvre, Kevin Longin, Rony Swennen, Juhee Rhee and Bart Panis, published in “Scientific Reports” • Patent application (CLONAL PROPAGATION OF COCONUT TREE, EP19180499.6) mailto:b.panis@cgiar.org mailto:hannes.wilms@kuleuven.be Funders: This project was funded by DGD (Directorate-general Development Cooperation and Humanitarian Aid, Belgium) This project was carried out with the support of “Research Program for Agricultural Science & Technology Development (Project No.PJ012225)” of International Cooperative Research Project in National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea. Dries De Bièvre, Kevin Longin (KU Leuven) Rony Swennen (KU Leuven, IITA) Juhee Rhee (RDA) Ramon Rivera, Cristeta Cueto (PCA) Yu-Chun Liao (Patty) Alliance Acknowledgements Philippine coconut authority Thank you! Slide 1: COAXIM, an innovative protocol for mass clonal propagation of coconuts: Merits of the Technology, Potency and Progress Slide 2: COAXIM (COconut AXIllary Meristems) Slide 3: Starting point of our involvement in coconut research; Project “Developing cryopreservation protocols for sub-tropical crops and establishing cryo-genebank at RDA with Bioversity International” Slide 4 Slide 5: Starting materials: shoots from in vitro germinated embryos Slide 6: Starting materials: shoots from in vitro germinated embryos Slide 7: Optimization of germination of coconut embryos received from PCA Slide 8: Multiplication of single shoots Slide 9: Proliferation! Slide 10: Multiplication? Slide 11: Regeneration ? Slide 12: Applicable to other cvs? Slide 13 Slide 14: Technique is repeatable in other laboratories solely based on the publication! Slide 15: First axillary in vitro shoot multiplication protocol for coconut palms Slide 16: What are next steps, mainly to “economize” the protocol Slide 17: Advantages and strengths of our method Slide 18: Patent procedure Slide 19: Principles that will guide the exploitation of the invention Slide 20: More info Slide 21: Acknowledgements Slide 22: Thank you!