Regeneration of Aegle marmelos (l.) Through Enhanced Axillary Branching from Cotyledenory Node

Asha Gupta1*, Tessy Thomas1, Shagufta Khan2 

1Department of Botany, Govt. P.G. College, Bhopal (MP)- 462022, India

3GrowTips Biotech, Saket Nagar, Bhopal (MP)- 462024, India

Received: 22-Feb-2018 , Accepted: 10-Apr-2018

Keywords: Axillary proliferation, Medicinal tree, Cotyledonary nodes, Micropropagation



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A complete protocol is standardized for in vitro micropropogation of Aegle marmelos for the first time using cotyledonary nodes derived through invitro raised seedlings. Higher percentage of direct multiple shoots were regenerated from cotyledonary nodal segments through forced axillary branching. The cotyledonary nodes of invitro raised seedlings were used as explants for shoot formation on MS Medium supplemented with Cytokinins (BAP) and Auxins (NAA), either alone or in combinations. Maximum (80%) shoots having shoot length of 2-3 cm were achieved on MS medium fortified with BAP (1.0 mg/l) and NAA (0.5mg/l). By repeating sub culturing of the cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 20-25 shoots were obtained. Shoots formed in vitro were best rooted on MS medium supplemented with 1.0-2.0 mg/l Napthalene acetic acid(NAA).

1 Introduction

Aegle marmelos (L.) Corr. belongs to Family Rutaceae is a spiny species medicinal tree mainly distributed wildly throughout India. Commonly known as Bael fruit is in high demand for its religious and pharma-ceutical purposes1. Every parts of the tree have tremendous nutritive and medicinal properties are used in ayurvedic preparation for various ailments. Several workers investigated different parts of A. marmelos has reported the plant contains coumarins, alkaloids, triterpenes, sterols and essential oils2.

There are many investigations about antibacterial and antifungal importance of essential oils has reported. The bael fruits contains marmelosin, which is laxative, diuretic, being used in many Indian drugs3. Has a wide therapeutic value in the treatment of diabetes, anaemia, fractures, healing of wound, swollen Joints, high blood pressure, Jundice, diarrhoea, troubles during pregnancy and typhoid. Fruits and roots also shows antiamoebic and hypoglycemic activities4. The leaves contain alkaloid aegeline is a potent antiasthamatic agent5. Green leaves shows anti-inflamatory, antipyretic and analgesic properties, leaves juices are used for the treatment of bronchitis, and the decoction of root barks has also been used as anti-malarial drug6. It is reported that the leaves also helps in controlling pollution by absorbing foul gases from the atmosphere and keep it clean and salubrious and can be grown under various wasteland suitably. Due to heavy unrestricted exploitation and demand in ayurvedics and pharmaceuticals industries the natural occurrence of this important medicinal plant has been markedly depleted. Thereby it is included in the red listed medicinal tree7. However, its commercial orcharding is not expanding at a faster pace due to severe shortage of planting material.

Conventionally the plant propagation of Aegle marmelos (In arching, budding and soft wood grafting) is season bound and slow. The seeds are mostly very small in number and have poor potential to germination. In vitro micropropagation technology can be beneficially employed in mass multiplication of elite bael plant varieties8. To accomplish in this contest an attempt was made to standardize an efficient protocol for tissue culture so as to conserve the species in vitro. Although there are many techniques have been reported forpropagation of Aegle marmelos by using in vitro raisedseedling explants, i.e. from roots9, from hypocotyl10 cotyledonary node11 and excised leaf explants12 have more been concentrated on in vitro grown seedling explants may be due to more efficiency to regenerate through juvenile tissues.

Therefore the present investigation has been carried out to establish the most appropriate protocol for invitro regeneration and mass propagation of A. marmelos of M.P. region.

2 Materials and Methods

2.1 Collection of Plant material

Unripe fruits of A. marmelos were collected from an elite tree growing in the Sehore district area. Immature white seeds were isolated from the fruits and used as explants material for the present experiments.

After removing the mucilaginous coat the seeds, were washed three to four times with plain water and then with liquid soap solution followed by washing with tap water. Further surface sterilization treatment was conducted in a laminar air flow chamber.  Seeds were dipped into 0.1% (w/v) freshly prepared mercuric chloride solution for 5 minutes, and then washed with 4-5 times in sterile double distilled water and are then inoculated aseptically in different composition medias.

To obtain the in vitro seed germination surface-sterilized seeds Aegle marmelosof were inoculated aseptically on basal media namely MS13 (Murashige and Skoog, 1962).  Seeds were also inoculated on the concentration of half strength MS media containing 3% sucrose and gelled with 0.8% agar having pH 5.7 in culture bottles. The cultures incubated at 24-25oc under dark for 10 days and under fluorescent light with a 16/8 light/dark photoperiod.

 2.2 Shoot Regeneration Experiment

Cotyledonary nodes (CNs), excised from 1-2 cm length in vitro raised seedlings and were taken for shoot development experiment on MS medium supplemented with varied concentration of cytokinins like BAP (0.5-2.0 mg/l), and Kinetin (0.5mg/l-1.0 mg/l) alone and with combination. The pH of all medium was adjusted to 5.8 before autoclaving. The cultures were incubated in a culture room at 25 ± 2oc under sixteen hours photo period provided by cool white fluorescent tubes (Phillips India). After shoot initiation from CNs of seedlings, the initiated cultures were sub culture in the fresh medium for profuse multiplication of shoots on different higher concentration of cytokinin in combination with NAA and additive Adenine sulphate. The growth responses of explants were studied at weekly interval. The parameters were taken as the number of shoots initiated and multiplied, the height of regenerated and multiplied shoots and the callus developed.

2.3 Root Induction and Root Growth

Multiplied shoots developed in the presence of different growth regulators (cytokinins) and additives generally lack roots.To obtain full plants the shoots must be transferred to a rooting medium, which is different from the shoot multiplication medium, especially in its hormonal composition. The shoot were sub cultured for further growth and after attainment to a height of 2 cm the shoots were transferred to rooting media and number of days taken for root initiation was observed and recorded in the medium containing different concentration of auxins like IAA, NAA, IBA and Activated charcoal (AC). The growth parameter was observed as percentage of root initiate, length in cms and number of roots formed.

2.4 Data scoring

Data were recorded on percentage of response, shoot number, shoot length after 4 weeks of culture on MS basal medium with or without addition of plant growth regulators (PGRS). Similarly, data were also recorded on percentage of root induction, root numbers, and root length after 4 weeks of culture in the root induction medium. Data was analyzed statistically using analysis of variance (ANOVA) for a completely randomized design. Ten replicates were used for every treatment and repeated thrice.

3 Results and Discussion

In vitro micropropagation of woody tree species shows some difficulties due to slow growth, formation of phenolic exudates in the culture medium, long complex life cycles and great genetic variations.

So it is essential to standardize an efficient regeneration protocol to establishment of given species under in vitro condition. Standardization begins with use of explants and various media and different hormonal concentration to different explants. Various previous studies reported that cytokine: auxin ratio is the deciding factor in the establishment of efficient reproducible protocol, Higher cytokine and low auxin concentration helps to multiple shoot regeneration.

The method of in vitro seed germination and use of seedling explants could be exploited for the microprapogation of woody species to preserve intrinsic genetic variability and also prove useful for obtaining sterile explants as juvenile tissues shows better regenerability potential in the technique.

Initiation and Adventitious shoot Regeneration

In all the subsequent experiments, MS medium has been frequently used for the in vitro micropropagation of a large number of plants14,15. In the present study isolated cotyledonary nodes (CNs), from in vitro raised seedling shows shoot induction in every medium tried for shoot development experiment. Incorporation of a cytokinin in the medium greatly affected the induction of axillary shoot proliferation from cotyledonary nodes.

Higher cytokine with the combination of low auxins concentration helps to multiple shoot proliferation. Shoot formation was stimulated by the media with various concentrations cytokinin (BAP) either alone with combination with different concentrations of Auxin (NAA). Maximum shoots per CNs, was achieved on the frequency or regeneration (70%) and the average mean 6.8 ± 0.10  shoots  per CN, having 6.4 ± 0.13 of length developed from explants were maximum on MS medium containing BAP (1.0 mg/l) and NAA (0.5mg/l) within 6 weeks (Table 1).

Further subculture of shoots on the fresh medium could not enhance shoot elongation. These results are similar to earlier reports where cytokinins such as BAP, zeatin, and kinetin have been shown to stimulate shoot proliferation while inhibiting shoot elongation16,17. To improve shoot multiplication and elongation an additive AS was incorporated in combination with 1.0 mg/l BAP and 0.5 NAA. Incorporation of 20 mg/l AS induced a significant increase (80%) of regeneration response having the maximum mean number of 19.9±0.76 of mean length of 8.5± 0.06 elongated shoots within 40 to 45 days culture (Table 2). Multiple shoots obtained were regenerated into 2-3 clumps after each subcultured in the fresh multiplication media having additional substance (AS- 20mg/l)  with the formation of about 20-25 number of shoots.  Multiple shoot formation from the young shoot explants of Clitoria ternatea on 0.5mg/l with the addition of lower concentration of NAA or IAA has also been reported by Kalamni and Michael Gomez (2002)18.

The result shows the addition of small amount of AS were effective in Aegle marmelos production and elongation. For the root induction on regenerated shoots addition of auxins  in the medium was essential. Maximum percentage of root initiated occurred within 18-20 days culture on both MS half and full strength medium supplemented with 2.0 mg/l of NAA (Auxin). However 50 to 60 percent shoots were rooted on medium contains higher concentration of NAA. (Table 3; Fig. 1). While IAA widely used as rooting hormones also shows good results as compared to IBA. As IBA auxin has proved the mostly used growth inducing hormone for the woody plants both under in vivo and in vitro conditions19.

4 Conclusion

In vitro micropropogation contribute the best and efficient of many important and  economically important plants as the rate of conventional method are very low in woody plants.

The present studies have been successful in micropropagation protocol by using CN segment from axenic seedlings. The fact may be attributed that the use of juvenile tissues to increase enzymatic activity at the cotyledonary node zone as also observed in case of bean20. Successful shoot regeneration from cotyledon node segments of A. marmelos has been reported earlier21- 24. The regeneration system can be adopted for mass production due to readily available immature seed embryos maybe a starting point for the development of genetic transformation technologies in this important medicinal tree species. 

5 Conflict of interests


6 Authors contributions

AG and TT design and carried out research work and SK drafted the manuscript.

7 References

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