Moringa oleifera

“Micro-propagation through organ/ tissue/cell culture in drumstick (Moringa oleifera Lam.)” Abstract: Moringa is an old-world dry tropical plant genus with great food, horticultural, industrial, and pharmaceutical potential. The present study is conducted at laboratory of Plant Tissue Culture, Department of Olericulture, College of Horticulture, Kerala Agricultural University, Thrissur. The KAU has recently released the “Anupama” a high yielding cultivar of Drumstick. To obtain plantlets of drumstick tree (Moringa oleifera Lam.) with identical genetic make up for propagation or field studies and to make availability of diseases free seedling to farmers for commercial cultivation, in vitro cultures of drumstick tree are required to produce. For multiple root-shoot induction, the young stem explants from green house and from field of drumstick tree plantlet were cultured in Murashige and Skoog (MS) agar medium supplemented with the 6 combination different hormones of 6-benzyl adenine purine (BA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1-napthalene acetic acid (NAA) with the concentration of 0.5ml/Lit, respectively and maintained at 25 ± 2°C for 5 weeks. The subculture of explants is done at 14 days interval and second after at 6 days interval. The hormonal combinations like B A (0.5ml/lit) + NA (0.5ml/lit) and B A (0.5ml/lit) + 2, 4-D NA (0.5ml/lit) were shown very good (+++) quality of multiple shoot and root in micropropagation in an experiment. On average basis, the contamination percentage in out side explants was 57.1 % and 34.3 % in test tubes where explants used from green house with pretreatment of fungicide (Bavistin) before one day of taking explants. Proper pretreatment of explants with fungicide (Bavistin) with standard concentration and aseptic handling (treatment with alcohol (50 % and 70%) and Mercuric chloride) is effective to check the contamination and for efficient in -vitro propagation. Tissue culture protocol reported in this study is an alternative mean of propagation of drumstick plantlets.

Key words: Moringa, Genetic Make-up, in vitro, Hormone, contamination, Protocol

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“Micro-propagation through organ/ tissue/cell culture in drumstick (Moringa oleifera Lam.)” Introduction: Dumstick tree or horseradish tree (Moringa oleifera) is one of the commonest trees in India. It is distributed in the wild in the sub-Himalayan tract and cultivated widely throughout India. Moringa oleifera, the word Moringa probably came from dravidian language Tamil and commonly referred to as "Shojne" in Bengali, "Munagakaya" in Telugu, "Shenano" in Rajasthani, "Shevaga" in Marathi, "Nuggekai" in Kannada, "Moringa" (from Tamil: Murungakai, Malayalam: Muringa, Konkani: Mashinga sanga, and Malunggáy in Filipino, is the most widely cultivated species of the genus Moringa, which is the only genus in the family Moringaceae. It is an exceptionally nutritious vegetable tree with a variety of potential uses. The tree itself is rather slender, with drooping branches that grow to approximately 10 m in height. In cultivation, it is often cut back annually to 1 meter or less and allowed to regrow so that pods and leaves remain within arm's reach. The immature green pods called “drumstick” are probably the most valued and widely used part of the tree. They are commonly consumed in India and are generally prepared in a similar fashion to green beans and have a slight asparagus taste. The seeds are sometimes removed from more mature pods and eaten like peas or roasted like nuts. The flowers are edible when cooked, and are said to taste like mushrooms. The roots are shredded and used as a condiment in the same way as horseradish.

Photo 1: Drumstick (Moringa oleifera) leaves with flowers and drumstick

The leaves are highly nutritious, being a significant source of beta-carotene, Vitamin C, protein, iron, and potassium. The leaves are cooked and used like spinach. Its leaves are commonly dried and crushed into a powder, and used in soups and sauces. Murungakai, as it is locally known in Tamil Nadu and Kerala, is used in Siddha medicine. The tree is a good source for calcium and phosphorus. In Siddha medicines, these drumstick seeds are used as a sexual virility drug for treating erectile dysfunction in men and also in women for prolonging sexual activity. Moringa leaves and pods are helpful in increasing breast milk in the breastfeeding months. One tablespoon of leaf powder provides 14% of the protein, 40% of the calcium, 23% of the iron and most of the vitamin A needs of a child aged one to three. Six tablespoons of leaf

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powder will provide nearly all of the woman's daily iron and calcium needs during pregnancy and breastfeeding. The Moringa seeds yield 38–40% edible oil (called ben oil from the high concentration of behenic acid contained in the oil). The refined oil is clear and odorless and resists rancidity at least as well as any other botanical oil. The seed cake remaining after oil extraction may be used as a fertilizer or as a flocculent to purify water. The bark, sap, roots, leaves, seeds, oil, and flowers are used in traditional in several countries. In Jamaica, the sap is used for a blue dye. The flowers are also cooked and relished as a delicacy in West Bengal and Bangladesh, especially during early spring. There it is called shojne ful and is usually cooked with green peas and potato. Cultivation: Drumstick is propagated by planting 1–2 m August. The plant starts bearing pods 6–8 commences after the second year, continuing by seeds, which are planted an inch below the well-draining soil.

long limb cuttings, preferably from June to months after planting, but regular bearing for several years. It can also be propagated surface and can be germinated year-round in

As with all plants, optimum cultivation depends on producing the right environment for the plant to thrive. Drumstick is a sun and heat-loving plant, and thus does not tolerate freeze or frost. To obtain plantlets of drumstick tree (Moringa oleifera Lam.) with identical genetic make up for propagation or field studies, in vitro cultures of drumstick tree are required to produce. Different varieties of Drumsticks: Though the origin of drumsticks is India, due to its medicinal uses it has reached other countries also. Different varieties and types of drumsticks are found in specific part of India. But no research has been done in different agricultural institute and universities. The universities in south India are ahead in these researches. With the research and considering the geographical conditions, there are following varieties of Drumsticks available Table No. 1: Varieties of Drumsticks Coimbator-1 P.K.M. -1 Jafna Coimbator-2 P.K.M. -2 K.M.1 Durga G.K.V.K. -1 Chavalavacheri Chem Moringa G.K.V.K. -2 Yalpanam Moringa G.K.V.K. -3 Shabanam Anupama: A high yielding Drumstick variety recently released by Kerala Agriculture University, Thrissur Objectives of study: To obtain plantlets of drumstick tree (Moringa oleifera Lam.) with identical genetic make up for propagation or field studies, in vitro cultures of drumstick tree are required to produce. Keeping in view all above facts, the present research study entitled “Micro-propagation

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through organ/ tissue/cell culture in drumstick (Moringa oleifera Lam.)” is under taken with the following objectives: 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. Review of Literature: Eltayb and Mutasim (2010) conducted the in vitro morphogenesis study and found that adventitious shoot could be induced directly from stem node. Shoot development was induced in MS media supplemented with Benzyladenine (BA), kinetin (6-furfuryl amino purine) (Kin) or N-6, 2, iso pentile adenine (2ip). Both types and concentrations of cytokinins significantly influenced shoot proliferation. BA proved to be more effective than other cytokinins. The best response, however was obtained when 0.1 mg L-1 BA was used. For callus induction it was found that 2, 4-Dichloro-Phenoxyacetic acid (2, 4-D) had a significant effect on the callus induction, callus index and callus physical appearance. The highest frequencies of callus growth index (7.8) were observed on leaves explants cultured on MS medium supplemented with 0.1 mg/L 2, 4-D. in vitro produced callus were proliferated on MS free hormone medium. Riyathong et al. (2010) carried out experiment on Shoot Multiplication and Plant Regeneration from In Vitro Cultures of Drumstick Tree (Moringa oleifera Lam.) and observed that for multiple shoot induction, the stem explants of drumstick tree plantlet were cultured on Murashige and Skoog (MS) agar medium supplemented with different concentrations of 6-benzyl adenine purine (BA) and maintained at 25 ± 2°C for 5 weeks. Multiple shoot induction was present in the stem explants that were cultured on the MS medium supplemented with BA at the concentration range of 1.0 - 4.0 mg/l. The MS medium containing BA at 2.0 mg/l was found to produce 100% shoot formation with the highest average number of 10.8 shoots per explant. For plant regeneration, the shoot explants were cultured on MS agar medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) at 0.5 mg/l for callus production. Friable callus were transferred to MS agar medium supplemented with different concentrations of 1-napthalene acetic acid (NAA) or BA for 4 weeks. Callus grown in MS medium supplemented with BA (0 – 2.0 mg/l) did not produce either shoots or roots, but showed green spots on the surface of callus. Callus grown in MS medium containing NAA 0.5 mg/l produced both shoots and roots. While, only root production were found in the medium supplemented with NAA 1.0 mg/l. Moreover, when NAA concentration was raised to 2.0 mg/l green spots appeared on the surface of callus without further developing into the whole plants Katherine et al. (2004) studied the development of tissue culture methods for the rescue and propagation of endangered moringa spp. germplasm and found that many of the thirteen known Moringa species are in danger of extinction, one species, M. oleifera Lam., is now widely cultivated. M. oleifera was therefore utilized to develop micro propagation techniques that may be applicable to the more endangered members of this genus. Immature seeds were the most responsive tissue source, and greatest success was achieved using membrane rafts and a liquid growth medium. The success rate was 73%, but the multiplication rate averaged

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only 4.7 shoots per culture. Most vigorous plantlet development through the transplant stage was achieved using a commercial plant preservative formulation of isothiazolones following shoot proliferation. Although there was no evidence of contamination, treatment with this microbiocide prevented early tissue senescence and it increased culture survivability.

Materials and Methods: 1. Preparation of Stock Solution (M S Media) and Hormones combination A. Stock I (50 X) (Table 2) Element NH4 NO3 KNO3 MgSO4.7 H2O KH2 (PO4)

B. Stock II (50 X) (Table 3) Element Ca Cl2.2H2O

C. Stock III (50X) (Table 4) Element

Require for 1000 Stock for 1000 ml ml medium (mg/l) (mg/l) 1650 82500 1900 95000 370 18500 170 8500

Stock for 1000ml (g/l) 85.5 95.0 18.5 8.5

Require for 1000 Stock for 1000 ml ml medium (mg/l) (mg/l) 440 22000

Stock for 1000ml (g/l) 22.0

Require for 1000 Stock for 1000 ml ml medium (mg/l) (mg/l) 27.8 1390 37.26 1863

Stock for 1000ml (g/l) 1.39 1.863

D. Stock IV (100X) (Table 5) Element Require for 1000 Stock for 1000 ml ml medium (mg/l) (mg/l) 22.3 2230 MnSO4. 4H2O 8.6 860 ZnSO4.7H2O 6.2 620 H3 BO3 0.83 83 KI 0.250 25 Na2M0 04.2H20 0.025 2.5 CuSo4.5H2O 0.025 2.5 COCl2.6 H20

Stock for 1000ml (g/l) 2.23 0.86 0.62 0.083 0.025 0.0025 0.0025

FeSO4.7H2O Na2EDTA.2 H2O

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E. Stock V (100X) (Table 6) Element

Require for 1000 Stock for 1000 ml ml medium (mg/l) (mg/l) 2 200 0.5 50 0.5 50 0.1 10

Glycine Nicotine acid Pyridocrine HCL Thiamine HCL

Stock for 1000ml (g/l) 0.2 0.05 0.05 0.01

Note: Inositol-100mg/l added at the time of media preparation. Table 7: Stock Used in ml for preparation M S Media Composition Sr. No.

1. 2. 3. 4. 5.

Stock

Full MS (per liter)

Half MS (per liter)

I II III IV V

20 20 10 10 10

10 10 5 5 10

Half MS Media for 100ml 1 1 0.5 0.5 1

2. Hormones Used The following hormone combinations are made for optimization of media and effective Micro-propagation. Table 8: Hormone combinations Sr. No.

1 2 3 4 5 6 7

Hormone used in M S Media

M S Media (without any Hormone) B A (0.5ml/lit) N A (0.5ml/lit) 2, 4-D (0.5ml/lit) B A (0.5ml/lit) + NA (0.5ml/lit) B A (0.5ml/lit) + 2, 4-D NA (0.5ml/lit) N A (0.5ml/lit) + 2, 4-D NA (0.5ml/lit)

Explants from Green House 5 5 5 5 5 5 5

Explants from outside 5 5 5 5 5 5 5

Preparation of stock (Hormones): Weigh 0.1g of hormone and dissolve it in minimum dissolve solution (NaoH) and make up to 100 ml in a standard flask with distilled water. Note: Hormone 2, 4-D stock is prepared by taking 0.1g of 2, 4-D and dissolved first with absolute alcohol and then added distilled water to make final volume 100ml.

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*Carbon source used in the media: Sucrose (30g/l) * Solidifying agaent: Agar Agar (7.5g/l) * pH= 5.7-6.0 3. Protocol for Preparing 1 liter MS Medium 1. Required volume of each stock solution were calculated by using formula (Formula A) and obtained into a 1000 ml beaker. 2. 30 g/l sucrose and 0.1g/l inositol were added and stirred until fully dissolved. 3. Required volume of each hormone solution is added. 4. The volume is then make up to 1000ml with distilled water and pH is adjusted to 5.7 to 6.0 with NaoH. 5. To this 7.5g/l Agar is added and stirred for complete mixing. 6. Medium was heated up in microwave oven to dissolve the agar. 7. The medium was autoclaved for 15-20 min at 121degree Celsius temperature. 8. After autoclaving medium was kept for one day to check the contamination. 9. If there is no any contamination then such medium is heated with wire-mess on low flame of gas. 10. After cooling the add the 1μ/ml antibiotic like Cefotoxine Sodium injection IP to medium and shake properly to mix it. 11. Then, medium was distributed approximately 20ml to each sterile test tube in \ laminar air flow and allow to solidify followed by exposing to UV light for 20 minutes and stored for use.

4. Explants: Nodal Segment of 21 days old from green house and tender from field/ outside. Note: The source plant of explants from green house was sprayed with fungicide bavistin one day before taking the explants. Protocol for explants Inoculation 1. Veins cut from actively growing plants (From green house or open field) of Moringa/ drumstick and into small nodal segments. 2. Then wipe with 70% alcohol (3ml alcohol + 7ml distilled water for 20 explants). 3. Top of the nodal segment will be round and bottom slant. 4. Washed with 2 drops of pril solution and 0.2 g/lit Bavistin (fungicide) for 10 minutes. 5. Explants thoroughly washed with distilled water for 3-4 times. 6. Treat with 50 % alcohol for 5 minutes. 7. Treat with 0.05 % HgCl2 (Mercuric Chloride) for 8-10 minutes Note: HgCl2 stock- 0.05g HgCl2 (Mercuric Chloride) dissolved in 100 ml distilled water and keep in standard flask. 8. Wash with sterile water and kept in sterile blotting paper for draining water. 9. Inoculate the explants into medium at 25 ± 2°C for 5 weeks 5. Subculture: The subculture of explants is done at 14 days interval and second subculture done after 6 days interval with same concentration of hormones up to 5 weeks.

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6. Hardening: The hardening, or acclimatization, process begins while the plantlets are still in vitro, i.e., growing in the culture vessels. This will be Final phase which takes 7–10 weeks. The hardened-off plants should be quickly prepared for the greenhouse nursery.

7. Calculations: A. Calculation for MS Media preparation M1 V1 = M2 V2 Where, M1= concentration of stock M2= Concentration of MS medium required V1= Volume will be taken from Stock (X) V2= Required volume of medium B. Calculation of Contamination percentage

Number of Test tube Having fungal contamination Contamination Percentage = ------------------------------------- X 100 Total Number of test tubes

Results: The result of experiments collected and presented in terms of shoot quality and characteristics of explants growth through visual scoring also contamination percentage is calculated from different source of explants used in experiment. Table 9: Effect of different hormone concentrations on multiple shoot inductions of Moringa olifera from node explants from Green house after 5 weeks of culture and its scoring Sr. No.


Shoot quality*

Characteristic of explants growth**

1 2 3 4 5 6


++ + + +++ +++

C S R R SR SR

++

R

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Table 10: Effect of different hormone concentrations on multiple shoot inductions of Moringa olifera from node explants from outside after 5 weeks of culture and its scoring Sr. No.

1 2 3 4 5 6 7


Shoot Characteristic quality* of explants growth**


-

C

+ + ++ +++

S R R SR SR

+

R

Notes: 1) * +++ ~ very good, ++ = good, + = moderately good and - = poor. 2) **S: Shoot formation, R: Root formation, C: Callus formation

Table 11: Contamination percentage in micro propagation (Source of explants = Green house) Sr. No.

1 2 3 4 5 6 7


Explants from Green House


5 5 5 5 5 5 5

Test tube contaminated by fungal growth 2 2 1 0 3 2 2

contamination %

40 40 20 0 60 40 40

Table12: Contamination percentage in micro propagation (Source of explants = open field or out side) Sr. No.

1 2 3 4 5 6 7


Explants from open field or out side


5 5 5 5 5 5 5

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Test tube contaminated by fungal growth 4 3 2 2 4 3 2

contamination %

80 60 40 40 80 60 40

Discussion: The results obtained in above experiment are discussed with following Photos: Effect of Hormones and its combinations: In general the hormones like Cytokinines (B A) and Auxins (NA, 2, 4-D) are used to develop multiple shoots and roots, respectively in growth media. The same pattern was observed in the experiment. But, the quality of growth was different with each hormone and its combinations. The hormonal combinations like B A (0.5ml/lit) + NA (0.5ml/lit) and B A (0.5ml/lit) + 2, 4D NA (0.5ml/lit) were shown very good (+++) quality of multiple shoot and root in micropropagation in above experiment.

Photo 2: The raised test tube in tray showing the multiple shoot and root formation of B A (0.5ml/lit) + NA (0.5ml/lit) hormonal combinations

The similar results of above quality through B A (0.5ml/lit) + NA (0.5ml/lit) and B A (0.5ml/lit) + 2, 4-D NA (0.5ml/lit) hormonal combinations were previously reported by Eltayb and Mutasim (2010) and Riyathong et al. (2010) with different concentration.

Contamination and Source of Explants: In general, the contamination was more in test tubes where explants used from outside or from open field as compared to explants used from green house.

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On average basis, the contamination percentage in out side explants was 57.1 % and 34.3 % in test tubes where explants used from green house with pretreatment of fungicide (Bavistin) before one day of taking explants.

Explants from green house

Explants from outside

Photo 3: Contamination in test tube green colour rubber band showing the explants used from green house and back side explants from outside or open field. The is more in explants source open field or outside. Therefore, proper pretreatment of explants with fungicide (Bavistin) with standard concentration and aseptic handling (treatment with alcohol (50 % and 70%) and Mercuric chloride) is required to check the contamination and for effective in -vitro propagation. Conclusion: Tissue culture protocol reported in this study is an alternative mean of propagation of drumstick plantlets with uniform genotypes for breeding selection, field experiments and to make availability of disease free seedlings to farmers for commercial cultivation and to achieve greater profits.

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Reference: Eltayb A. and Mutasim M. Khalafalla (2010) In vitro Morphogenesis Studies on Moringa olifera L. an Important Medicinal Tree. Int J Med Res. 1(2): 85-89. Katherine K. S. and Jed W. F. (2004) Development of Tissue Culture Methods For The Rescue and Propagation of Endangered Moringa Spp. Germplasm Eco. Bot. 58 (Supplement) Pp. S116-S124. Murashige, T. and Skoog, F., (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15, 473-497. Thidarat R. , Srisulak D. , Jiraporn P. and Lalida S.(2010) The 8th International Symposium on Bio-control and Biotechnology Wekipedia: http://en.wikipedia.org/wiki/Drumstick_(vegetable)

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Moringa oleifera

Moringa oleifera

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