PLANT TISSUE CULTURE OF NAIPIER GRASS

INTRODUCTION :-

Napier grass (Pennisetum purpureum), also known as elephant grass, is a tall perennial grass widely cultivated as a high-yielding fodder crop for livestock. It is valued for its fast growth, high biomass production, palatability, and nutritional quality. Traditionally, Napier grass is propagated through stem cuttings and root splits. However, these conventional methods are slow, prone to disease transmission, and require large planting materials.To overcome these limitations, plant tissue culture has emerged as an efficient technique for the rapid, large-scale, and disease-free propagation of Napier grass. Through micropropagation, explants such as shoot tips, nodal segments, or meristems are cultured under sterile conditions on nutrient media enriched with growth regulators.

EXPLANT CAN USE :-

Nodal segments

Shoot tips / apical meristems

Leaf bases or immature inflorescences

STAGES :-

Preparation of Explant

Initiation

Shoot Multiplication

Rooting

Hardening/Acclimatization

MATERIALS :-

Beaker	Surgical Blade
Conical flask	Autoclave Bag
Petri Plate	Scissors
Cotton Plug	Tissue Paper
Test Tube	Parrafilm Tape
Test Tube Stand	Distilled Water
Forceps	Sterile Distilled Water
Spatula	Culture Bottle
Measuring Cylinder	Scalpel

CHEMICALS FOR STERILIZATION :-

Liquid Soap

Savlon ( Dettol )

Ethanol

Sodium Hypochlorite

Antibiotics

CHEMICALS FOR MS MEDIA :-

Macronutrients

Micronutrients

Vitamins

Iron Source

Mesoinositol

Sucrose

Agar

Hormones

HCl / NaOH

PREPARATION OF MACRONUTRIENTS :-

Macronutrients	Original Concentration mg/l	Amount of salt after multiplying original concentration by 20X, Solution Volume (250ml)
NH4NO3	1650	8250mg
KNO3	1900	9500mg
KH2PO4	170	850mg
MgSO4.7H2O	370	1850mg
CaCl2.2H2O	440	2200mg

PREPARATION OF MICRONUTRIENTS :-

Micronutrients	Original Concentration mg/l	Amount of salt after multiplying original concentration by 100X, Solution Volume (250ml)
H3BO3	6.2	155mg
MnSO4.4H2O	22.2	557.5mg
ZnSO4.7H2O	8.6	215mg
Na2MoO4.2H2O	0.25	6.26mg
CoCl2.6H2O	0.025	0.625mg
CuSO4.5H2O	0.025	0.625mg
Kl	0.83	20.75mg

PREPARATION OF VITAMINS :-

Vitamins	Original Concentration mg/l	Original Concentration multiplying by 1000X, Final solution volume (50ml)
Thiamine HCl	0.1	5mg
Pyridoxin HCl	0.5	25mg
Nicotinic	0.5	25mg
Glycin	2.0	100mg

PREPARATION OF IRON SOURCE :-

Iron Source	Original Concentration mg/l	Original Concentration multiplying by 400X, Final solution volume (50ml)
FeSO4.7H2O	27.8	2780mg
Na2EDTA.2H2O	37.3	3730mg

PREPARATION OF MS MEDIA :-

Materials	Final Volume ( 250ml )
Macronutrients	12.5ml
Micronutrients	0.625ml
Vitamins	0.625ml
Iron Source	0.250ml
Mesoinositol	25mg
Sucrose	7.5gm
Agar	2gm
Distilled Water	100ml

MESOINOSITOL :-

When we prepare 1 litre ms media then we will take 100mg mesoinositol.

SUCROSE (3%):-

When we prepare 1 litre ms media then we will take 30gm sucrose.

PREPARATION OF HORMONES:-

BAP Stock:– 100mg BAP in conical flask (250ml) dissolve with ethanol or 1M NaOH solution and BAP powder dissolve completely in solution and make final volume 100ml solution with Distilled water.

IBA Stock:- 100mg of IBA in conical flask (250) dissolve with ethanol or 1M NaOH solution and IBA powder dissolve completely in solution and make final volume to 100ml with Distilled water.

pH CHECK:- Prepare solution media pH range is 5.8 – 5.9 by using HCl or 1M NaOH.

Agar:- Take 8 gm agar powder and add in media ,mix with glass rod then in microwave the media for 14 minutes and agar dissolve completely in media.

STERILIZATION PROCESS :-

Autoclave the used media to sterilize it before disposal. Discard the used media from the culture bottle. Soak the empty culture bottle in chromic acid (for cleaning or decontamination) for 24 hours. After 24 hours, remove the culture bottle from the chromic acid and wash it with liquid soap. Then dry it in a hot air oven at 180°C for 12 hours. Then we have wrapped glassware in autoclave bag then put in autoclave with distilled water and culture media.

Autoclave work at high pressure and temperature ( 15 psi, 121°C) for 15 to 20 minutes and kill the microorganisms. After sterilization removed glassware from sterilizer. And placed in laminar air flow then when we use it, we will turn on UV – ray light for 1 hour.

METHODOLOGY :-

11 June 2025

1.Select mother plants ( Shoot tips, Nodal part ) that are young shoot tips disease-free, and have high-yield and desirable traits.

2. Cut the mother plant in desirable shape and placed dry tray.

3. Wash the cut explant in running water for 15 minutes.

4.Handle the plant material with sterile gloves.

5. Then prepare total volume 300ml soap solution (30ml soap liquid and 270ml distilled water).

6. Wear glove hand and wash the plant material for 5 minutes.

7.Then remove the soap solution and placed in glass beaker.

8. Cut the material desirable size with the help of knife.

9. Then soak the material in savlon solution ( 3ml savlon and 300 ml distilled water) for 15 minutes.

10. Then wash the plant material from sterile distilled water 3 to 5 time in bottle beaker.

11. Prepare 4% sodium hypochlorite solution and taking sodium hypochlorite solution into a laminar air flow and soak the plant material in sodium hypochlorite solution for 20 minutes( for 1 jar 10ml sodium hypochlorite and 250ml sterile distilled water).

12. After 20 minutes then remove plant material from sodium hypochlorite solution and wash the plant material 3 to 5 times in sterile distilled water.

13. Then cut the plant material required culture bottle size with the help of knife on sterilized brown paper.

14. Prepare antibiotic solution using Cefotaxime antibiotic( 1ml antibiotic and 200ml sterile distilled water) and soak the plant material in this solution for 30 minutes. Using only fresh antibiotic solution.

15. Then inoculate the plant material in culture medium( bottle beaker) and remove laminar air flow placed inoculate culture bottle at 24°C with photo periods of 6 to 7 hours light and 8 hours darkness for tissue culture sterile environment.

RESULT :-

After two to three days, the inoculated naipier grass explants showed no growth and were contaminated.

2 July 2025

I took a one-month-old plant, performed surface sterilization, and inoculated it onto MS medium.

5 August 2025

After discussing with Dixit Sir, I grew Napier grass in a hydroponic setup and treated it with fungicide and UV light.

RESULT :-

Contamination appeared within 3 to 4 days after inoculation.

8 August 2025

I took the shoot tips of Napier grass and kept them in running water with UV light for 15 minutes. After that, I performed surface sterilization and inoculated it onto MS medium.