Abstract: A protocol has been developed for regeneration of plantlets from leaf primordial of Aegle marmelos. Aegle marmelos is a middle sized slender aromatic armed tree native to India and distributed South-east Asia. The in vitro plant regeneration studies indicated that the following combinations of hormones with MS media containing Kn (1.5 mg L-1) + 0.5 mg L-1 2, 4-D with NAA (1.0 mg L-1) is most suitable for organogenic calli formation and shoot induction. These calli later developed shoots when transferred to MS medium containing NAA (1.5 mg L-1) with (0.5 mg L-1) IBA for root initiation. Rooted plants survived well under acclimatization.
INTRODUCTION
Aegle marmelos, commonly known as bael, is a spiny tree belonging to the family Rutaceae. The leaves, roots, bark, seeds and fruits of A. marmelos are edible. The medicinal properties of this plant have been described in the Ayurveda. It is indigenous to Indian subcontinent and mostly found in Tropical and subtropical region (Purohit and Vyas, 2005). It is medium sized tree having profuse dimorphic branched, alternate, trifoliate, deep green leaves; membranous leaflets, large, sweet scented, greenish white flowers; large, oblong or globose fruits (Purohit and Vyas, 2005). Almost all parts of the tree are used in preparing herbal medicine (Kala, 2006). The roots and bark are used in the treatment of diarrhea, fever (Mazumder et al., 2006) and to control pain in the abdomen (Kirtikar and Basu, 1935). The leaves are used in the treatment of diabetes (Narendhirakannan et al., 2005), snakebites (Purohit and Vyas, 2005). Fresh aqueous and alcoholic leaf extracts of A. marmelos are reported to have a cardio tonic effect, like digitalis and to decrease the requirement for circulatory stimulants (Nadkarni, 1976). Conventionally, grafting and layering are carried out to achieve this, but for large-scale propagation, they are not feasible methods. There is wide genetic variability in terms of quality, form and size of the fruit (Bhati et al., 1992). Also, seeds have short viability and are prone to insect and fungal attack (Purohit and Vyas, 2005). Although, vegetative propagation through root suckers is possible, the number of propagules produced through this technique is very limited. Alternatively, in vitro micropropagation techniques offer opportunities for multiplying disease-free planting material in a larger quantity within a short span of time. Nucleolus culture of Aegle has been attempted to develop plants with seedless fruits (Hossain et al., 1993) and in vitro axillary shoot proliferation is reported for rapid clonal propagation (Ajithkumar and Seeni, 1998; Arumugam and Rao, 1996). A tissue-to-plant regeneration system is thus an important pre-requisite for application of this technology for Aegle marmelos. Micro propagation using tissue culture is an alternative method used to multiply mainly vegetatively propagated crops. In this circumstance, the objective of this study was to develop an efficient method of plant regeneration system using leaf primodia explants of A. marmelos.
MATERIALS AND METHODS
Plant Material and Surface Sterilization
Aegle marmelos were freshly collected during (April 2010) from the
southeast coast of Tamil Nadu, India and kept under shade net (50%) house environment.
The specimen was certified by Botanical Survey of India (BSI) Coimbatore and
by the Herbaria of CAS in Marine Biology, Annamalai University, Parangipettai,
Tamil Nadu and India. The leaf primordia were used as explants material for
initiating the organogenic calli. The explants were surface sterilized with
70% (v/v) ethyl alcohol for 1-5 min followed by 0.1% HgCl2 for 3
min. The explants were then washed 4 times with sterile distilled water to remove
traces of HgCl2.
Culturing Tissues and Shoots Regeneration
The leaf primordial explants were cultured for plantlet regeneration in
250 mL conical flask and 25 mL Petri plates containing Murashige
and Skoog (1962) medium (MS) supplemented with 30 g L-1 sucrose,
8 g L-1 agar and various concentration of cytokinin and auxin. Four
growth regulator combinations, denoted as T1-T4 were tested [T1: 2,4-D (0.5
mg L-1); T2: 2,4-D (0.5 mg L-1) with NAA (0.5 mg L-1);
T3: Zeatin (1.5 mg L-1)+ 0.5 mg L-1 2,4-D with NAA (1.0
mg L-1); T4: Kn (1.5 mg L-1) + 0.5 mg L-1 2,4-D
with NAA (1.0 mg L-1)]. The pH was adjusted to 5.8 prior to the addition
of agar. Media were autoclaved at 15 lbs for 15 min. Culture was incubated at
25±2°C and relative humidity of 60±10%. An 8 h photoperiod
(16 h dark) with light intensity of 20-30 μmol/m2/sec was provided
by cool day light fluorescent tubes. The experimental chemicals were purchased
from (Hi-Media, Mumbai).
Each treatment consisted of 10 replicates. Cultures Once calli were initiated, globular-shaped yellowish green organogenic calli were selected and sub cultured in a fresh medium and incubated under dark conditions to promote further calli proliferation. Proliferated calli that were sub-cultured in fresh medium were transferred to light conditions and when the calli turned green in colour, The shoots (1-2 cm) were separated and cultured in various concentration of MS medium (above mentioned T1, T2, T3 and T4) to enhance further shoot growth.
Root Formation
Shoots that were 3-4 cm were separated and cultured in MS-based basal medium
[MS medium, sucrose (3% w/v) and solidified with agar (0.8% w/v)] added with
several growth regulator combinations to promote root initiation and growth.
Four media compositions, denoted as R1 – R4, were tested [R1: IBA (0.5
mg L-1); R2: IBA (0.5 mg L-1) with BAP (0.2 mg L-1)
+IAA (0.5 mg L-1); R3: IBA (1.0 mg L-1) + NAA (2.0 mg
L-1) with 0.5 mg L-1; R4: NAA (1.5 mg L-1)
with 0.5 mg L-1 IBA]. Each treatment consisted of 10 replicates.
Acclimatization of Plants
The rooted plantlets were transferred to a potting mixture of compost: sand
(1:1) in 5 cm diameter pots. They were kept inside a poly chamber for 3 weeks
and the poly cover was gradually removed. The plants were allowed to grow in
a plant house with application of a liquid fertilizer once a week.
RESULTS AND DISCUSSION
Calli formation was observed in leaf primordia explants after 7-8 weeks. This response was highly observed in medium T3: Zeatin (1.5 mg L-1) + 0.5 mg L-1 2, 4-D with NAA (1.0 mg L-1); T4: Kn (1.5 mg L-1) + 0.5 mg L-1 2, 4-D with NAA (1.0 mg L-1)]. They were compact yellowish green in color with an embryogenic nature. These calli proliferated rapidly when separated and sub cultured in a fresh T4 medium. Hence this system was selected for further studies. The results showed (Table 1) that, out of the several types of cytokinins tested, zeatin is suitable for callusing of Aegle at lower concentrations (2.0 mg L-1) but at higher levels (4.0 mg L-1), the response was very low. It was also shown that NAA and lower concentration of 2, 4-D was the suitable auxin here to initiate calli in the presence of zeatin (T3) as well as of Kn (T4). Creamish friable competent callus was achieved from nodal segments on MS medium augmented with 4.0 mg L-1, 2, 4-D within two weeks of inoculation. The callus produced large number of shoots when cultured on MS medium fortified with 2.0 mg L-1 BAP + 0.1 mg L-1 NAA within ten days of culture. In vitro raised shoots were rooted on half strength MS medium enriched with 1.0 mg L-1 IBA within fifteen days of culture (Ranjoy et al., 2009). Similar results were reported in several plants including, Ceropegia candelabrum (Beena and Martin, 2003), Ocimum sanctum L. (Singh and Sehgal, 1999), Gentiana sp. (Fiuk and Rybczynski, 2008), Azadirachta indica (Quraishi et al., 2004). Since the growth regulator 2, 4-D is promoting abnormal cell divisions that can induce mutations 2, 4-D was in this study for callus initiation at lower concentration, although Islam et al. (1992) reported 2, 4-D at 1-5 mg L-1 induced calli from young leaves of A.marmelos. Organogenic calli formation on a MS-based medium fortified with Zeatin or 6-furfurylamine (KN) (2.0 mg L-1) and NAA (0.5 mg L-1) under dark conditions. Such type of plant regeneration was also reported in several medicinal plant species including, Carica papaya (Islam et al., 2000), Phellodendron amurense Rupr (Azad et al., 2005), Amorphophallus albus (Hu and Li, 2008), Gentiana sp. (Fiuk and Rybczynski, 2008). Separated shoots continued to grow in liquid medium, free of hormones and produced roots at 30% efficiency in the presence of NAA (1.0 mg L-1). This protocol is suitable to produce number of plants from cotyledon, hypocotyls and immature leaves of A. marmelos (Prematilak et al., 2006).
| Table 1: | Effect of different concentration of auxin and cytokinin for callus induction and plantlet regeneration from leaf primordia explants of Aegle marmelos |
| Fig. 1: | In vitro plant regeneration of leaf primordial explants of Aegle marmelos (A) Callus induction, (B) Root and shoot induction on MS medium containing K. (1.5 mg L-1)+0.5 mg L-1 2,4-D with NAA (1.0 mg L-1) and (C) Plnatlet regeneration |
When the calli that were formed in the T3 and T4 media were allowed to be in the same medium without sub-culture for 6-8 weeks, they turned green and when transferred to light, produced shoot buds. Enlargement of shoot base occurred prior to root initiation. Root initiation efficiency was around and was observed in the presence of NAA (1.5 mg L-1) with 0.5 mg L-1 IBA after 3-4 weeks of culture. Many other workers reported similar results for root induction in various types of plants, namely Carica papaya (Islam et al., 2000), Gymnema sylvestre (Komalavalli and Rao, 2000), Tylophora indica (Faisal et al., 2007). Plants were successfully acclimatized under the given conditions and are now growing well in the plant house. It is a well known fact that the plants regenerated via a callus phase may carry features that are different to the mother plant (somaclonal variations). After another 3-5 weeks the plants were suitable to be transplanted in soil (Fig. 1A-C). The potted plantlets were covered by transparent polythene sheet to maintain high humidity and within 20 days new leaves were emerged from the plantlets that resumed new growth. After 40-45 days, the plants were transplanted in the field condition, where 80% plants were survived and grown satisfactory.
ACKNOWLEDGMENTS
The authors are gratefully acknowledge to the Dean, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period.