Standardization of Protocol for In Vitro Micropropagation of Morus alba L., An Important Economical and Medicinal Plant

An efficient regeneration protocol for Morus alba L. was developed using nodal explants on MS medium augmented with different concentrations and combinations of plant growth regulators. The highest frequency (80.0%) of bud breaks and shoot induction was observed on medium supplemented with 1.0 mg/l BAP along with 0.5 mg/l NAA with the formation of 5.1±0.1 number of shoots 30 days. MS medium fortified with the same combination of GR (1.0 mg/l BAP and NAA (0.5 mg/L) added with 10% Coconut water showed the highest percentage (80.0%) shoot multiplication, directly from the explants, without any callus formation. Elongated shoots were rooted best on MS medium with 20 mg/l AC producing maximum number of roots with average length 7.0±0.6 cm within 20 days. The plantlets were gradually acclimatized and successfully transferred to field condition with 90% survival rate after rooting. The standardized protocol reported in this study may help in large scale propagation of this plant species which is currently exploited from the nature.


Introduction
Phytochemicals extracted from different parts of plant is used for the treatment of various diseases, for supplementing nutrition in foods and cosmetic industries has a great potential in this Morus alba is an important economical and medicinal plant, commercially grown for the usage of leaves as a food of insect's silkworm (Bombyx mori L.) in the commercial sericulture technologies 2 . The species are also grown as fruit crop in the countries like Turkey and Greece for the production of mulberries fruits which were used for various local food recipes 3  Additionally, the saplings obtained through cuttings show inferior vigor when compared with micro propagated plants 8 . In vitro establishment of nodal segments collected from mature trees has been reported in different species of mulberry [9][10][11] but to the best of our knowledge, very less significant prior information in the contemporary literature is available on in vitro micropropagation or plant regeneration in this economically important tree species of M. alba.
Thus, traditional propagation methods through seeds and vegetative method is not sufficient and reliable hence, plant production through tissue culture method is required. To overcome these problems and to produce large number of plants of same genetic makeup, a simple and efficient micropropagation protocol is presented in this investigation.

Materials and Methods
Nodal segments, for axillary and apical meristems of 5-10 centimeter length of Morus alba were excised from the mother plants were selected as explants for inoculation.

Data analyses
Complete randomized design was used for all the experiments.
The data were analyzed by ANOVA (Duncan's multiple range test) using SPSS release 12.0. The data were transformed where necessary by using various formulae.

Axillary shoot initiation
Axillary shoots were observed emerging from nodal explants of mature tree on MS medium supplemented with BAP, KN and NAA (Table 1). Generally, the rate of bud break was increased by increasing the BAP level in combination with either concentration of auxin after different periods of time. The frequency of bud break was maximum in MS medium supplemented with BA (1.0 mg/l) and NAA (0.5 mg/l) after 30 days of culture with little browning of the medium and longest (5.1±0.1) shoot production. It was observed that shoot growth was not affected by the development of these calluses (Fig 1A &   B).

In vitro shoot multiplication
The axillary shoots were excised and cut into further nodal segments for multiple shoot induction ( Table 2). For this purpose, BAP alone or in combination with NAA and Coconut water were tested. Generally, the rate of shoot induction was low on individual cytokinins. Such shoot induction response was significantly improved by adding NAA with either cytokinin. BAP was superior over Kinetin to induce such responses when NAA was used along with Additive CW in MS medium. Multiple shoots produced by BAP were vigorous and green as compared to without CW supplemented media (Fig 1 C & D). The rate of

In vitro rhizogenesis
Well-developed in vitro shoots were used for rooting in MS full or half strength basal media or supplemented with 20% activated charcoal (Table 3). Rooting was observed after 20 days of culture and the data for further development of roots were recorded after 25 days. Significant difference was observed for root induction in MS half medium with the supplementation of AC and IBA.
However, rooting was better when activated charcoal was added in half strength MS medium. The rate of root induction was highest (80.0%) in half-strength MS medium supplemented with 20% activated charcoal after 20 days of culture. The number of primary as well as secondary roots was also highest on this medium with mean root length (Fig 1 E).