Plant Tissue Culture Of Moringa

Plant Tissue Culture Of Moringa / Drumstick (Moringa Oleifera )

Objective :

  1. To study the effect of different concentration of hormones on germination under

     in vitro condition.

  2. To know the ideal concentration and constitution of germination media.

   3. To know the contamination percentage and develop protocols to avoid it.

Materials and Methods:

1. Preparation of Stock Solution (M S Media) and Hormones combination

Stock Classification

Stock	Type
Stock I	Macronutrients
Stock II	Macronutrient
Stock III	Micronutrient (Iron)
Stock IV	Micronutrients
Stock V	Vitamins

1. Stock I – Macronutrients

Sr. No.	Chemical	Full Name	1000 ml (g)	250 ml (g)	100 ml (g)
1	NH₄NO₃	Ammonium Nitrate	85.5	21.375	8.55
2	KNO₃	Potassium Nitrate	95.0	23.75	9.5
3	MgSO₄·7H₂O	Magnesium Sulphate Heptahydrate	18.5	4.625	1.85
4	KH₂PO₄	Potassium Dihydrogen Phosphate	8.5	2.125	0.85

2. Stock II – Macronutrient

Chemical	Full Name	1000 ml (g)	250 ml (g)	100 ml
CaCl₂·2H₂O	Calcium Chloride Dihydrate	22	5.5	2.2

3. Stock III – Micronutrient (Iron)

Chemical	Full Name	1000 ml (g)	250 ml (g)	100 ml (g)
FeSO₄·7H₂O	Ferrous Sulphate Heptahydrate	1.39	0.3475	0.139
Na₂EDTA·2H₂O	Disodium EDTA Dihydrate	1.863	0.46575	0.1863

4. Stock IV – Micronutrients

Chemical	Full Name	1000 ml (g)	250 ml (g)	100 ml (g)
MnSO₄·4H₂O	Manganese Sulphate Tetrahydrate	2.23	0.5575	0.223
ZnSO₄·7H₂O	Zinc Sulphate Heptahydrate	0.86	0.215	0.086
H₃BO₃	Boric Acid	0.62	0.155	0.062
KI	Potassium Iodide	0.083	0.02075	0.0083
Na₂MoO₄·2H₂O	Sodium Molybdate Dihydrate	0.025	0.00625	0.0025
CuSO₄·5H₂O	Copper Sulphate Pentahydrate	0.0025	0.000625	0.00025
CoCl₂·6H₂O	Cobalt Chloride Hexahydrate	0.0025	0.000625	0.00025

5. Stock V – Vitamins 

Chemical	Full Name	1000 ml (g)	250 ml (g)	100 ml (g)
Glycine	Glycine (Amino acid)	0.2	0.05	0.02
Nicotinic acid	Vitamin B3	0.05	0.0125	0.005
Pyridoxine HCl	Vitamin B6	0.05	0.0125	0.005
Thiamine HCl	Vitamin B1	0.01	0.0025	0.001

 MS Media Preparation Table

Component	Category	250 ml Requirement	100 ml Requirement
Stock I	Macronutrients	5 ml	2 ml
Stock II	Macronutrient (Ca)	5 ml	2 ml
Stock III	Iron source	2.5 ml	1 ml
Stock IV	Micronutrients	2.5 ml	1 ml
Stock V	Vitamins	2.5 ml	1 ml
Sucrose	Carbon source	7.5 g	3 g
Inositol	Organic supplement	25 mg (0.025 g)	10 mg (0.01 g)
Agar	Solidifying agent	2 g	0.75 g
Distilled Water	—	Make up to 250 ml	Make up to 100 ml

Role of Hormones in MS Media (Micropropagation)

In plant tissue culture, plant growth regulators (hormones) control the direction of growth. They determine whether the explant will develop into shoots, roots, or callus.

 1. BAP (BA – 6-Benzylaminopurine)

 Type: Cytokinin

 Role:

• Promotes shoot formation 
• Stimulates axillary bud growth 
• Induces multiple shoot production (multiplication stage)

2. NAA (Naphthalene Acetic Acid)

 Type: Auxin

 Role:

• Promotes root formation 
• Enhances cell elongation 
• Supports callus formation at low concentrations 

3. 2,4-D (2,4-Dichlorophenoxyacetic Acid)

Type: Strong Auxin

 Role:

• Induces callus formation
• Promotes cell division 
• Initially suppresses organ formation 

Combination	Full Form	Role
MS (without hormone)	Murashige and Skoog Medium	Control (natural growth)
BAP	6-Benzylaminopurine	Shoot formation
NAA	Naphthalene Acetic Acid	Root formation
2,4-D	2,4-Dichlorophenoxyacetic Acid	Callus formation
BAP + NAA	6-Benzylaminopurine + Naphthalene Acetic Acid	Balanced shoot and root development
BAP + 2,4-D	6-Benzylaminopurine + 2,4-Dichlorophenoxyacetic Acid	Shoot and callus formation
NAA + 2,4-D	Naphthalene Acetic Acid + 2,4-Dichlorophenoxyacetic Acid	Root and callus formation

protocol for Preparation of 250 ml MS Medium

1. The required volumes of stock solutions were calculated and added into a 250 ml beaker as follows:
   • Stock I → 5 ml 
   • Stock II → 5 ml 
   • Stock III → 2.5 ml 
   • Stock IV → 2.5 ml 
   • Stock V → 2.5 ml 
2. Approximately 150–200 ml distilled water was added. 
3. Sucrose (7.5 g) and inositol (0.025 g) were added and stirred until completely dissolved. 
4. Required concentration of plant growth regulators (hormones) was added as per treatment. 
5. The final volume was made up to 250 ml using distilled water. 
6. The pH of the medium was adjusted to 5.7–5.9 using (To adjust Ph NaOH or HCl.)
7. Agar 2.0 g added and mixed thoroughly. 
8. The medium was heated in a microwave oven until agar was completely dissolved. 
9. The medium was sterilized by autoclaving at 121°C and 15 psi for 15–20 minutes. 
10. After autoclaving, the medium was allowed to cool to about 45–50°C. 
11. Heat-sensitive components such as antibiotics (e.g., Cefotaxime sodium) were added aseptically and mixed gently. 
12. The medium was poured into sterile culture tubes / bottles under laminar airflow conditions and allowed to solidify.

Explant Sterilization & Inoculation Protocol 

 Explant Source

• Nodal segments (21 days old) from greenhouse plants 
• Tender explants from field/outside plants 

     Note: plants were sprayed with fungicide (Bavistin) one day prior to explant collection.

 Why are 21-day-old explants used?

 21-day-old nodal explants are used because the tissue is young, active, and highly responsive.

 Reasons

• High meristematic activity: Cells divide rapidly, leading to faster shoot formation. 
• Low contamination: Younger tissues have fewer microbes, making sterilization easier. 
• Better hormone response: Explants respond quickly to hormones like BAP, NAA, and 2,4-D. 
• Less lignification: Soft tissues allow better absorption of nutrients and hormones. 
• High survival rate: Young explants adapt well and show better growth in culture.

 Surface Sterilization Procedure

1. Nodal segments were excised from actively growing plants and cut into small pieces

            (2-3 cm). 

2. Explants were rinsed with running tap water to remove dust and debris. 
3. Surface cleaning was done using 2–3 drops of Labolene + Bavistin (0.2 g/L) for 10 minutes. 
4. Explants were washed thoroughly with distilled water (3–4 times). 
5. Explants were surface sterilized using 70% ethanol for 30 seconds to 1 minute.
6. Then treated with Sodium hypochlorite solution (1–2%) for 5–10 minutes.

           (Few drops of Tween-20 can be added for better sterilization)

7. Explants were rinsed 3–4 times with sterile distilled water to remove chemical residues. 
8. Surface-dried using sterile blotting paper. 

Inoculation

9. Explants were inoculated aseptically into MS medium under laminar air flow. 
10. Cultures were incubated at 25 ± 2°C for 5 weeks. 

 Subculture

• First subculture → after 14 days 
• Second subculture → after 6 days 
• Same hormone concentration maintained 
• Total duration → 5 weeks 

 Hardening (Acclimatization)

• Initiated after sufficient shoot/root development 
• Duration → 7–10 weeks 
• Plantlets transferred gradually to greenhouse conditions

7 April 2026

On 7 April, a discussion was held with Dixit Sir and Yashwant Sir, and it was decided to conduct a plant tissue culture trial of Moringa using nodal and seed explants.

 Experimental Trial Design (Nodal vs Seed Explants)

 Table: Hormone Treatment with Two Explant Types

Sr. No.	Treatment (MS Media +Hormone)	Hormone Conc.	Nodal Explants	Seed Explants	Total Explants
1	MS (Control – no hormone)	—	5	5	10
2	MS + BAP	0.5 mg/L	5	5	10
3	MS + NAA	0.5 mg/L	5	5	10
4	MS + 2,4-D	0.5 mg/L	5	5	10
5	MS + BAP + NAA	0.5 + 0.5 mg/L	5	5	10
6	MS + BAP + 2,4-D	0.5 + 0.5 mg/L	5	5	10