Coconut Germplasm Conservation: Production Problems, Cryopreservation, and Tissue Culture

Coconut Germplasm Conservation

Funded by ACIAR, focusing on coconut livelihoods in the Pacific.

The Tree of Life: Uses of Coconut

• Grown in tropical and subtropical regions.

Coconut Husk and Shell

• Husk: Used as a pot for plants.

• Shell: Used to create bowls, utensils, handicrafts, and musical instruments; also used in exfoliating products.

• Husk and Shell: Used as fuel, to buff floors, and as mosquito repellent.

Coconut Meat, Oil, and Milk

• Coconut Meat

• Coconut Oil

• Coconut Milk

• Nectar

• Copra

• Coconut Sap: Can be turned into palm wine, candy, syrup, coconut sugar, or palm sugar.

Coconut Leaves

• Used for brooms, baskets, mats, cooking skewers, kindling, and roofing.

Coconut Trunk

• Used for furniture, houses, drums, and canoes.

Coconut Roots

• Used for dye and mouthwash; frayed pieces can be used as a toothbrush.

Coconut Water

• Consumed as a refreshing drink and for hydration in athletes and during illness; also used for skin and hair care.

Coir

• A natural elastic fiber extracted from coconut husks; used for floor mats, brushes, ropes, strings, mattress stuffing, and caulking for boats and fishing nets.

Coconut Products

• Coconut Sugar

• Coconut Milk

• Coconut Water

• Virgin Coconut Oil

• Auto Parts

• Coconut Diesel

• Coco- & Oleo Chemicals

• Macapuno or Kopyor coconut, other products

Comprehensive List of Coconut Products

1. Oleo chemicals

2. Glycerine

3. Fresh coconuts

4. Matured coconuts

5. Coconut seedlings

6. Bukayo

7. Coco Cream Powder

8. Coconut Milk

9. Frozen coco meat

10. Kopyor/Makapuno

11. Coconut vinegar

12. Nata de coco

13. Ubod

14. Coco acid oil

15. Alkanolamide

16. Paring oil

17. Coco coir waste

18. Coco coir fiber

19. Coconut water

20. Coco husk

21. Coco husk chips

22. Coco chips

23. Coco lumber

24. Coconut shell

25. Coconut shell charcoal powder

26. Toilet/Bath soaps

27. Husk nuts

28. Laundry soap

29. Shortening

30. Coco furniture

31. Coco cutlery

32. Coir bullet proof vest

33. Coco Jam

34. Spec Creamed Coconut

35. Coco Hydro Water

36. Coco Soy Sauce

37. Coco Fiber Dust

38. Coco Shell Powder

39. Coco Shampoo

40. Coco Wood Pallet

41. Margarine

42. Coconut Flour

43. Coconut Milk Powder

44. Coconut Liquor

45. Coco Handicrafts

46. Grated Coconut Meat

47. Coconut Honey

48. Coir Net

49. Soap Chips

50. Virgin Coconut Oil

51. Coconut sugar

52. Neera fresh

53. Neera products

54. Coco Culture

55. Coconut flour products

56. Coco Hostorium juice

57. Coco Mats

58. Coco Belt

59. Coco Vest

60. Coconut wines

61. Coir Twine

62. Coir Pads & Liner

63. Coir Doormats

64. Coco Husk Cubes

65. Hydrogenated C/Oil

66. Coconut Syrup

67. Charcoal briquette

68. Coconut shell oil

69. Coconut water blends

70. Coconut milk blends

71. Bio Fuel products

72. Coco pith products

73. Coconut sugar

74. Coco sugar 3-1 Coffee

75. Coco artifacts

76. Coco Art & Craft

77. Coco Fibre Shoes

78. Coconut yogurt

79. Coconut Arak

80. Coconut Vodka

81. Coco spirits

82. Coco Fabric

83. Coir portraits

84. MCT oil

85. Laurin MCT Boost

86. Laurin MCT Brain

87. Insect Repellent

88. Lip Balm

89. Charcoal T/paste

90. Infused oil

91. Roller Perfume

92. VCO capsules

93. VCO by products

94. Coco veneer

95. Coco wood panel

96. Organic fertilizer

• Many more products are being developed each year.

Traditional Uses of Coconut

Recent Research-Based Evidence

• Coconut meat contains large amounts of medium-chain fatty acids (MCFAs), which are easier for the human body to digest.

• Treatment of obesity, dyslipidemia, elevated low-density lipoprotein, insulin resistance, and hypertension, which are the risk factors for cardiovascular disease, type 2 diabetes, and Alzheimer’s disease.

• Virgin coconut oil has been reported to treat or recover patients from COVID-19, which are very positive effects of the coconut.

Market Demand

• Demand for coconut products has grown worldwide, up to 500% in the last decade.

Coconut Production Problems

• Palm senility – old age

• Immediate replanting of about 1 billion palms is needed (International Coconut Community).

Pests and Diseases

• Coconut Rhinoceros Beetle (CRB) - Oryctes rhinoceros

• Palm Weevil

  • Rhynchophorus palmarum

  • Rhynchophorus ferrugineus

• Lethal yellowing - Phytoplasma

  • Vector: Plant hoppers

• Cadang-Cadang disease - Viroid

• Bud rot - Phytophthora palmivora

  • Sporangia

• Red ring - Nematode

  • Bursaphelenchus cocophilus (Cobb)

  • Vector: Weevil

Other Threats

• Natural disasters and human activities such as cyclones, droughts, urban development, etc.

• Necessary to conserve coconut genetic diversity.

Problems with Field Gene Banks

• Susceptibility to pests, diseases, and natural/human induced calamities.

• Limited amounts of genetic diversity conserved.

• Vulnerability to changes in management practices.

• High maintenance costs.

• Duplication still required.

Cryopreservation

Why is Cryopreservation Important?

Cryopreservation is a process where tissues susceptible to damage caused by dehydration are preserved by cooling to -196^{\circ}C using Liquid N_2. Proven difficult to undertake.

Advantages of Cryopreservation for Conservation

• Long-term conservation for recalcitrant, sterile, or clonal species.

• Long-term safety of stored material.

• Limited maintenance/monitoring required once stored.

• Facilitates germplasm exchange.

• Rapid multiplication of desirable pathogen-free material.

Disadvantages of Cryopreservation

• Relatively high technology level required.

• High initial costs.

• Need to develop/adapt protocol for most species.

Cryopreservation for Germplasm Conservation

• Must end with ‘Plants in Soil’

• Can be applied to:

  • Embryos

  • Plumules

  • Shoot tips

  • Callus (Pollen)

Successful Cryopreservation Approach

• Low Cost

• Easy to Undertake

• Simple Equipment

• High Recovery

• Scale up Possible

Cryopreservation Decision Tree

• If the plant produces large numbers of quality, orthodox seeds, conserve seeds dry in a seed store at -20^{\circ}C.

• If it produces recalcitrant seeds but seeds are of quality and high in number:

  • Can those seeds yield embryos that can undergo partial or full dehydration?

    • If yes, use embryo cryopreservation by physical dehydration.

    • If not, consider plumule cryopreservation.

• If it produces few or no seeds but produces high numbers of vegetative shoot tips:

  • Cryopreservation of shoot tips by droplet vitrification.

Key Features of Successful Cryopreservation

1. Low cost

2. Ease of undertaking

3. Simple equipment and skills required

4. Quality and quantity and ease of plant recovery

5. Scale up possibilities

6. No genetic fidelity shifts

7. High seedling recovery rate (60%)

8. Tested on +15 varieties (T & D)

9. Simple, easy, scale up possible

Physical Dehydration of Embryos

1. Embryo isolation

2. Pre-treat, Dehydrate

3. Rapid Freezing

4. Rapid Warming \text{40}^{\circ}C 30 minutes

5. Recovery: Y3 Vitamins AC Sucrose

Desiccation and Cryopreservation Results

• Desiccated, Cryopreserved, Germinated, Soil (for 8 hours to 20% water content)

• Germination and Normal Seedlings observed

Rapid, Physical Desiccation

• To 20% water content before freezing at -196^{\circ}C

• Initially 8 hours, now 5 hours with flash drying

• Using Electric Fan and Silica gel

New Cryo Apparatus for Coconut Zygotic Embryos

• Amir et al. unpublished

Why Reduce Hours of Dehydration?

• Amir et al. unpublished

• Relationship between duration of dehydration, moisture content, and germination percentage.

Cryopreservation Summary

• A cryopreservation protocol based on rapid, physical dehydration of zygotic embryos has been developed & tested against > 15 varieties.

• No genetic fidelity shifts detected

• BEFORE: No (or very few) field-growing plants produced following Cryopreservation work

• AFTER: 20 to 60% coconut seedlings were produced from cryopreserved embryos and ready for field planting

• > 15 Varieties (T and D) tested

• No abnormalities observed for morphological, karyotype and molecular levels

Analysis

• Morphological Analysis

• CRYOPRESERVED vs CONTROL

Comparison of Embryo and Shoot Tip Cryopreservation

Response of Explants After Cryopreservation

Need for Coconut Tissue Culture

• Expensive and time-consuming

• Conventional breeding approach

• Needs to replant >1 billion coconut palms (International Coconut Community, 2020)

Objectives of Coconut Tissue Culture

• Mass production of high yielding, market-preferred, elite, and disease-resistant varieties.

• Production of high quality and early bearing plantlets.

• Supply of seedlings for large scale replanting of coconut palms worldwide.

Cloning Pathways

• Somatic Embryogenesis

  • Somatic embryogenesis is the developmental process by which somatic cells undergo restructuring to generate embryogenic cells. These cells then go through a series of morphological and biochemical changes that result in the formation of a somatic or non-zygotic embryo capable of regenerating plants.

• Direct Organogenesis

  • The production of direct buds or shoots from tissue with no intervening callus stage

Clonal Propagation By Direct Organogenesis

• Meristem explant

• Shoot induction (1 month)

• Selection of explant

• Shoot elongation (2 months)

• Root induction (2 months)

• Acclimatization of plantlets (2 months)

Coconut Cloning Pathway

• Plumula tissue

• Embryogenic callus

• Globular somatic embryos

• Callus formation

• Explant

• Somatic embryos

• Converted embryos

• Plantlets

• Coleoptile stage

• Immature inflorescence

Acclimatization Process

Thanks to Coconuts Research Team

Contact: Dr Sundar Kalaipandian (s.Kalaipandian@uq.edu.au)

Thanks for listening. Any Questions?