Protocol

Coconut (Cocos nucifera L.)

Coconut Embryo Culture Protocol for Germplasm Collection

Scientific Name: cocos nucifera Linn.

Family: Palmaceae

Origin

Comparatively little is known about the origin and early distribution of the coconut palm, probably because it was so widely spread throughout the tropical areas of the world so many years ago. It has variously been thought to be native to the Malay Archipelago, the South Pacific and tropical America.

Distribution

The coconut is widespread throughout the tropics, typically being found along sandy shorelines. It has been spread largely by man but also by natural means. The fruit can float for long distance and still germinate to form new trees after being washed ashore. Commercial plantings are confined to the tropical lowlands, but it will also fruit in a few warmer subtropical areas.

Uses

The meat of immature coconuts can be eaten with a spoon or be scooped out and made into ice cream. Coconut milk, abundant in unripe nuts, is a refreshing and nutritious drink. The most important economic product is obtained by drying the meat into copra which is pressed to produce coconut oil, primarily used in making soap. Coconut oil is also used for cooking and making margarine. The husk fiber is combed out and sold as coir, a material for making rope and coconut matting. The trunks may be used for building timbers and the leaves used for house thatching. The coconut palm has little commercial importance in Florida but is highly valued as an ornamental. It gives a tropical effect to the Florida landscape and provides fruit for home use.

Field collection kit

Materials and tools for collection of embryos (2000 nos.) in aseptic condition from the collection site are listed below

SI. No

Name of the items

Quantity

BOX-1 (75x60x1 Ocm)

1

Portable folding hood and folding chair

1 each

BOX-2 ( 100x70x60 cm)

2

Pressure cooker (7 I capacity)

1 no.

3

Absolute alcohol

3 l

4

Sodium hypochlorite Solution

2 l

5

Chlorine kit: Conical flask (1 I) separating funnel (500 ml), reagent bottle (500 ml), potssium permanganate (500 g) and Hcl (500 ml)

1 set

6

Test tube (20 cm length and 3 cm diameter)

20 nos.

7

Steel forceps (24 cm)

4 nos.

8

Stainless steel cork borer (18 cm length and 2 cm diameter)

2 nos.

9

Small knife (17.5 cm length)

1 no.

10

Big knife (40 cm length)

1 no.

11

Beakers of capacity 50 ml, 100 ml and 500 ml (preferably polypropylene)

1 each

12

Self sticking labels

3000 nos.

13

Scissors

2 nos.

14

Parafilm

1 roll

15

Bandage cloth (30 x 45 cm)

500 pieces

16

Plastic tray (45 x 30 cm)

500 pieces

17

Rubber bands

500 nos.

18

Non absorbent cotton

3 bundles

19

Sterile disposable gloves

200 nos.

20

Reagent bottles (500 ml and 250 ml)

1 each

21

Measuring cylinders (100 ml)

2 nos.

22

Aluminum foil

3 rolls

23

Polythene bags (45 x 30 cm)

100 nos.

24

Coconut dehusker

1 no.

25

Scalpel

2 nos.

26

Wash bottle

1 no.

27

Conical flasks (50 ml, 100 ml, 500 ml)

5 each

28

Cello tape (5 cm wide)

1 roll

29

Market pens (4 assorted colors)

1 set

BOX -3

30

Carry bag (60 x30x30 cm)

1 no.

31

Sterile vials containing either sterile water or minimal growth medium

2100 nos.

A folding type portable inoculation hood made out of plexiglass is used for inoculation of embryos in the field.

Extraction of embryos

Use mature nuts (11 to 12 months after fertilization) for seed nut collection

To extract embryos, first remove the husk of the harvested fruit and split open the nut. Embryo is located under the 'large eye' of the coconut embedded in the solid endosperm.

After locating this 'eye', scoop out the embryo along with a portion of the endosperm by means of a cork borer.

The extracted endosperm cylinders are collected in a beaker containing distilled water. Once all the plugs are collected from the split opened nuts, the embryo is extracted from the endosperm plug using scalpel or small knife. Extracted embryos alone are collected in a beaker containing distilled water.

Surface sterilization:

Surface sterilization of inoculation hood:

Immediately after the extraction of embryos the folding hood should be kept ready for surface sterilization of embryos.

Both inside and outside of the hood must be wiped thoroughly with alcohol for disinfection.

Disposable gloves must be used while working with bleach to reduce the contamination.

Before surface sterilization wash the embryos with absolute alcohol quickly to remove the fat globules on the surface of the embryos.

Inoculation of embryos:

After proper washing, inoculate each embryo directly into a small screw cap vial (5 ml capacity) containing 1.5 to 2 ml of sterile water. The sterilization of embryos and inoculation are carried out inside inoculation hood

An alternative to sterile water for in vitro active storage of embryos is half-strength Y3 medium (without sucrose) supplemented with charcoal. In either of the case the embryos could be stored for two months. Compared to nutrient medium, chance of contamination of cultures is leis with sterile water.

Inoculation of embryos in separate vials will reduce the loss of cultures due to contamination. After proper sealing of the cap (airtight) with parafilm, group the vials according to the accession and label them properly

Practical Utility:

This protocol could be used to bring the embryos from field condition to lab condition in an aseptic manner.

Coconut (Cocos nucifera L.) Coconut Embryo Culture Protocol for Germplasm Collection Scientific Name: cocos nucifera Linn.
Family: Palmaceae
Origin
Comparatively little is known about the origin and early distribution of the coconut palm, probably because it was so widely spread throughout the tropical areas of the world so many years ago. It has variously been thought to be native to the Malay Archipelago, the South Pacific and tropical America.
Distribution
The coconut is widespread throughout the tropics, typically being found along sandy shorelines. It has been spread largely by man but also by natural means. The fruit can float for long distance and still germinate to form new trees after being washed ashore. Commercial plantings are confined to the tropical lowlands, but it will also fruit in a few warmer subtropical areas.
Uses
The meat of immature coconuts can be eaten with a spoon or be scooped out and made into ice cream. Coconut milk, abundant in unripe nuts, is a refreshing and nutritious drink. The most important economic product is obtained by drying the meat into copra which is pressed to produce coconut oil, primarily used in making soap. Coconut oil is also used for cooking and making margarine. The husk fiber is combed out and sold as coir, a material for making rope and coconut matting. The trunks may be used for building timbers and the leaves used for house thatching. The coconut palm has little commercial importance in Florida but is highly valued as an ornamental. It gives a tropical effect to the Florida landscape and provides fruit for home use.
Field collection kit
Materials and tools for collection of embryos (2000 nos.) in aseptic condition from the collection site are listed below
	SI. No	Name of the items	Quantity
		BOX-1 (75x60x1 Ocm)
	1	Portable folding hood and folding chair	1 each
		BOX-2 ( 100x70x60 cm)
	2	Pressure cooker (7 I capacity)	1 no.
	3	Absolute alcohol	3 l
	4	Sodium hypochlorite Solution	2 l
	5	Chlorine kit: Conical flask (1 I) separating funnel (500 ml), reagent bottle (500 ml), potssium permanganate (500 g) and Hcl (500 ml)	1 set
	6	Test tube (20 cm length and 3 cm diameter)	20 nos.
	7	Steel forceps (24 cm)	4 nos.
	8	Stainless steel cork borer (18 cm length and 2 cm diameter)	2 nos.
	9	Small knife (17.5 cm length)	1 no.
	10	Big knife (40 cm length)	1 no.
	11	Beakers of capacity 50 ml, 100 ml and 500 ml (preferably polypropylene)	1 each
	12	Self sticking labels	3000 nos.
	13	Scissors	2 nos.
	14	Parafilm	1 roll
	15	Bandage cloth (30 x 45 cm)	500 pieces
	16	Plastic tray (45 x 30 cm)	500 pieces
	17	Rubber bands	500 nos.
	18	Non absorbent cotton	3 bundles
	19	Sterile disposable gloves	200 nos.
	20	Reagent bottles (500 ml and 250 ml)	1 each
	21	Measuring cylinders (100 ml)	2 nos.
	22	Aluminum foil	3 rolls
	23	Polythene bags (45 x 30 cm)	100 nos.
	24	Coconut dehusker	1 no.
	25	Scalpel	2 nos.
	26	Wash bottle	1 no.
	27	Conical flasks (50 ml, 100 ml, 500 ml)	5 each
	28	Cello tape (5 cm wide)	1 roll
	29	Market pens (4 assorted colors)	1 set
		BOX -3
	30	Carry bag (60 x30x30 cm)	1 no.
	31	Sterile vials containing either sterile water or minimal growth medium	2100 nos.

A folding type portable inoculation hood made out of plexiglass is used for inoculation of embryos in the field.
Extraction of embryos
Use mature nuts (11 to 12 months after fertilization) for seed nut collection To extract embryos, first remove the husk of the harvested fruit and split open the nut. Embryo is located under the 'large eye' of the coconut embedded in the solid endosperm. After locating this 'eye', scoop out the embryo along with a portion of the endosperm by means of a cork borer. The extracted endosperm cylinders are collected in a beaker containing distilled water. Once all the plugs are collected from the split opened nuts, the embryo is extracted from the endosperm plug using scalpel or small knife. Extracted embryos alone are collected in a beaker containing distilled water.
Surface sterilization:
Surface sterilization of inoculation hood:
Immediately after the extraction of embryos the folding hood should be kept ready for surface sterilization of embryos. Both inside and outside of the hood must be wiped thoroughly with alcohol for disinfection. Disposable gloves must be used while working with bleach to reduce the contamination. Before surface sterilization wash the embryos with absolute alcohol quickly to remove the fat globules on the surface of the embryos.
Inoculation of embryos:
After proper washing, inoculate each embryo directly into a small screw cap vial (5 ml capacity) containing 1.5 to 2 ml of sterile water. The sterilization of embryos and inoculation are carried out inside inoculation hood An alternative to sterile water for in vitro active storage of embryos is half-strength Y3 medium (without sucrose) supplemented with charcoal. In either of the case the embryos could be stored for two months. Compared to nutrient medium, chance of contamination of cultures is leis with sterile water. Inoculation of embryos in separate vials will reduce the loss of cultures due to contamination. After proper sealing of the cap (airtight) with parafilm, group the vials according to the accession and label them properly
Practical Utility:
This protocol could be used to bring the embryos from field condition to lab condition in an aseptic manner.