In Vitro Seed Germination and Induction of Enhanced Shoot Multiplication in Pterocarpus santalinus Linn.f: An Endemic Medicinal Plant of Seshachalam Hills, Tirumala

Headnote

Abstract: Pterocarpus santalinus belongs to fabaceae family and is one of the endemic and endangered medicinal plant species of seshachalam hills, Tirumala, Andhra Pradesh state, India. In the present study we have successfully demonstrated the in vitro regenartion of this plant through shoot bud culture. The seeds germinated sequentially on plastic trays and transferred polythene bags for 21 days served as explant source. Nodal segments from the seedlings were established on MS (Murashige and Skoog 1962), B5(Gamborg et.al 1969), and WPM(Woody Plant Medium) medium supplemented (84.4%,76.6%,80.2%) with BAP, NAA(α- Naphthalene acetic acid), and TDZ (Thidiazuron), in which BA 85% of nodes showed shoot bud elongation. Then nodal segments from the developed shoots were cultured on MS medium with several BA concentrations best shoot multiplication was obtained with 1.0mg BA+0.5mgNAA and 0.8%Agar. Successful in vitro rooting was induced form cut end of the micro shoots when placed on half strength MS+IBA (1500ppm). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure(1:1:1) and grown under green house conditions 85.4% survival rates.

Keywords: Pterocarpus santalinus, fabaceae, in vitro shoot regeneration.

1. Introduction:

Seven hills popularly known as Tirumala hills which lie between 79° 19' to 79° 23' East and 13° 37' to 13° 43' north latitude at the height of about 900m above MSL, in Chittoor District of Andhra Pradesh, India. Being a busy pilgrimage centre of Lord Balaji or Sri Venkateswara, this hill region is ever flocked with thousands of flouting population from all over India as well as from the rest of the world. Pterocarpus santalinus (Linn.f) is an endemic and an endangered species of seshachalam hills, Tirumala. Pterocarpus santalinus is having high medicinal properties due to which it is being exploited recklessly and which is already included in the endangered category of IUCN red list. Pterocarpus santalinus L.f belongs to the family papilionaceae/fabaceae, and popularly known as "Red sanders"(Naidu et al., 2001). These are used in treatment of headache, skin diseases, fever, boils, scorpion sting and to improve sight (Chopra RN et al.,1956). Ethno-botanical reports list several plants used for diabetes. Pterocarpus santalinus is one of such plants used for treatment of diabetes. Ethno-botanical reports indicates that P.santalinus is being used to treat diabetes mellitus and related symptoms along with use for many other curative properties including bilious affections, skin diseases as antihelmentic,aphrodisiac,alexiteric and also useful vomiting thirst, eye diseases, ulcers and disease of the blood (Chopra et al.,1956;Kirtikar and Latheef et al.,2008) Infusion of the fruit is used as an astringent tonic in chronic dysentery.

Drinking water in Pterocarpus santalinus wooden cups twice a day is used for the treatment of diabetes (Latheef et al., 2008). This species occurs exclusively in a well defined forest tract of Andhra Pradesh in southern India (Sanjappa, 2001).Earlier reports indicated that the red sanders wood contains of two red organic compounds called Santalin i.e., Santalic acid C3OH16O6 (OCH3)4 having a quinoid structure and derory santalin (C2OH16O6) a supposed derivative of napthaquinonoid.

The bark of the tree also yields a Kino similar to Pterocarpus santalinus (Anonymous 1969). Indiscriminate and illegal logging, low natural regeneration potential, narrow habitat specificity and micro climatic changes in the habitat resulted in the severe depletion of natural population of Pterocarpus santalinus (Madhava Chetty and Rao 1990; Sanjappa 2001). Red sanders are conventionally propagated through vegetative methods and seed is often very difficult because of a hard seed coat coupled with poor viability (Dayanad et al., 1988; Naidu et al 2001). Earlier studies using conventional vegetative propagation methods like semi hard woodcuttings, cleftgrafting and air layering were not successful in producing sufficient numbers of planting material for forestry programs (Kesava reddy et al., 1990).

Tissue culture has proved to be a promising technique for conservation and large scale multiplication of several woody species. However the members of Fabaceae have been difficult to culture in vitro owing to their recalcitrant nature . Attempts have already made in the recent past to micro propagate P.marsupium, another endangered species of the genus Pterocarpus santalinus using seedling explants (Chand and Singh 2004 a,b; Anis et al., 2005). Tissue culture of red sanders also began in the 1980s with more recent attempts to improve shoot sprouting using seedling explants. Anuradha et al 1999; Arockiasamy et al., 2000). Methanolic extract of Pterocarpus santalinus shown to exhibit antiinflammations and anticancer effects shown cytotoxic activity.

Many experimental studies in chemically induced diabetes have been conducted with whole extracts of bark or heard wood of P.santalinus, which have shown its efficacy in reducing blood sugar levels. The sandal wood extract has been found to increase antioxidant levels and also Consequently prolong subject viability.

2. Materials and Methods:

The mature weighed fruits of P.santalinus were obtained from the forest department laboratory 'BIOTRIM' (Biotechnology Centre for Tree Improvement) Government of Andhra Pradesh, Tirupati, India. The healthy seeds were isolated mechanically from the fruits with the help of a plant cutter. Each pod generally contained one seed but occasionally two. The visible seeds were washed thoroughly under running tap water for 30 to 40 minutes to remove any phenolic exudates. The seeds were then washed with 5%(v/v) Teepol-B-300 (Superior wetting agent) a liquid detergent, for 15 minutes at low speed on magnetic stirrer and kept in 1% (Bavistin Carbandazim powder) a broad spectrum fungicide for 10 minutes. The treated seeds were agitated in distilled water for 30 minutes to remove the inhibitors to germination. The seeds were surface sterilized with 70 %( v/v) ethanol for 30 sec. Subsequently surface disinfested with 0.05Hgcl2 for 5 to 10 minutes and raised four times with sterile distilled water. The seeds were placed aseptically into water agar medium (8mgL-1) with 3 %( w/v) Sucrose.

Culture Media and Condition:

MS (Murashige and Skoog) medium augmented with 3%(w/v) sucrose and 0.8(w/v) agar was in all the experiments. The pH of the medium was adjusted to 5.8 by 1N NaOH or 1N HCl before autoclaving at 121o C and 1.05 kg cm-2 pressures for 20 minutes. The medium was dispensed in20ml aliquots in 25x150 cm culture tubes. All the cultures were incubated in a culture room at an air temperature of 25° ±2°C with 65% relative humidity under a 16h photoperiod with irradiation of 50µEm-2 s-1 photo synthetically active radiation (PAR) provide by cool white fluorescent tubes.

Shoot Induction and Multiplication:

Cotyledonary nodes were placed vertically on to MS medium supplemented with various cytokines (BA,NAA,TDZ ) at different concentrations (1.0,2.0,3.0,4.0)either alone or in combination with NAA at three different concentrations(0.5,1.0,and1.5).To study the combined effect and stabilize the best combination . We tested various concentrations (0.1, 0.5, 1.0,) of in combination with optimal cytokinins and auxin concentration.

In Vitro Rooting in Micro Shoots:

The regenerated leafy shoots (3-4cm) grown on elongation with more than three or four leaves were harvested and rooting was attempted by a two step rooting procedure, Step one involved the pulse treatment of individual shoots with IBA at different concentrations. (1000ppm, 1500ppm, 2000ppm,2500ppm,). In step two the treated micro shoots were transferred on to half strength MS, Agar-gelled semi solid medium with 3% sucrose without IBA. After 4 weeks the data on percentage of rooting mean number of roots per shoots and root length were recorded.

Hardening and Acclimatization:

The in vitro raised micro shoots (>3-4cm) and root system were removed shoot cultures. Ex vitro root induction was performed by dipping the basal portion of micro shoots in double distilled water containing an auxin. (In dole -3-buteric acid) at various concentrations (1000ppm, 1500ppm, 2000ppm,2500ppm,) for different time duration (5 to 10 minutes) and transferred to thermo cups containing sterile soil rite. These were kept under diffuse light conditions. (16:8h photoperiod) covered with transferent polythene bags to ensure high humidity and maintained in culture room conditions (25±2°C), 16h photo period. These were irrigated after every 3 days with ½ strength MS salts solution (without vitamins) for two weeks one month following planting, the rooting percentage as well as the mean number of roots for each treatment was estimated. Relative humidity was progressively reduced during the incubation period. The poly bags were removed gradually over a period of 4 weeks in order to acclimatize plantlets primary hardened plantlets.

Statistitical Data Analysis:

The data for percentage of regeneration, number of shoots per explants and shoot length here recorded after 8 weeks. All the experiments were conducted with 10 replicates per treatment and repeated thrice.

3. Results and Discussion:

Establishment of Seed Regeneration:

The seeds were germinated under controlled conditions on MS basal and ½ strength MS media with or without GA3. A maximum of 90% of seed germination along with healthy growth was achieved on full strength MS +2.0µM GA3 (Table 1). The media lacking GA3 showed poor response (30% to 45%) for seed germination of GA3 from 1.0 to 5.0µM the germination percentage was reduced to 60% GA3 is known to breack dormancy of several types of seeds at critical concentrations. It stimulates seed germination via synthesis of α-analyse and other hydrolyses.

Shoot Induction and Multiplication:

Shoot bud elongation occurred in 85% nodal explants placed on MS medium supplemented with MS medium +BA1.0 mg/l-NAA 0.5mg/l and 0.8% agar. The explants preparation and double sterilization methods using 70 %( v/v) ethanol for 1 min and 0.1 %( w/v) aqueous mercuric chloride solution for 5 min yielded 85% of contamination free nodal explants in Pterocarpus santalinus. The response of various cytokinens (BA 1.0mg/l+NAA0.5mg/l+0.8 agar for shoot germination from cotyledonary node explants is depicted in (Table: 2) .The additions of plant growth regulators enhanced the multiplication rate and the number of shoots per explant. The percentage response varied with the type of growth regulator used and its concentration. All the concentrations of BA, NAA, TDZ (1.0µM, 0.5µM, and 1.0µM) alone resulted in direct shoot bud differentiation from the explant within 4 weeks of incubation. Among the three cytokinins tested. BA was found to be more efficient than others with the respect to initiation and subsequent proliferation of shoots (Table2), of the various concentrations of BA tested, 1.0µM proved to be most effective as in this medium an average of 8.80±0.76 shoots per explant were developed in 80% of cultures. Our results are in consistence earlier findings in woody tree species like Sterculia- urens. Dalbergia sissoo and Pterocarpus marsupium where BA was found to be more effective than other cytokinins for mass multiplication

The effect of auxins such as IAA, IBA, NAA (0.1, 0.5 and 1.0 µM) with optimal concentration of BA (1.0 µM) was also evaluated. Among various combinations used ,IBA (1.0 µM) + NAA (0.5 µM) was found to be most effective (Table:3), MS medium supplemented with IBA (1.0 µM) +NAA (0.5 µM) exhibited 85% , 80% shoot regeneration and induced maximum (6.53± 0.41 ; 11.20±0.81) shoots per explants with an average shoot length of (4.21 ±0.13cm; 7.40 ± 0.15 cm) increasing the concentration of NAA 1.0 µM regeneration frequency (76%) as well as the number of shoots per explants was reduced (10.6 ± 0.8 cm) moderatively. Higher concentration of NAA reduced the regeneration frequency. Our results substantiate with earlier findings in Oroxylum indicum Boswellia ovalifoliolata Pterocarpus santalinus and Balanites aegyptiaca (Anis et al.,) and Carria siamea. Among different combinations of IBA-IAA used the highest shoot regeneration frequency (72%) maximum shoot length were recorded on MS medium augmented with IBA (1.0 µM) +NAA (0.5 µM) followed by IBA+IAA combination. Where in the highest shoot regeneration frequency (70%) maximum number of shoots (8.20±0.56) per explant and the maximum shoot length (5.02±0.26) were recorded on MS supplemented with (IAA 0.5 µM + IBA 0.5 µM).

Ex vitro Root Induction:

Treatment of micro shoots with 1500ppm IBA for 10min resulted in best rooting response (85%) and induced maximum number (4.6 ±0.12) of roots per shoot after 4 weeks of transplantation to potting mixture Table 4. The roots produced were thick, long and well developed with secondary branching. Ex vitro rooting is reported to be a promising and attractive method; used reduced the micro propagation cost and also the time of established from lab to field. The main advantage of ex vitro rooting is that the chance of root damage is less rooting rates are often higher and quality is better. Our results are similar with earlier findings in other leguminous plants such as Pterocarpus marsupium (Chand and Sing 2004); Cassia angustifolia Pterocarpus santalinus (Prakash et al., 2006). Where lower concentration of IBA proved to the best for rizogenesis. Plantlets with fully expanded leaflets and well developed roots were successfully hardened offinside the culture room, and then transferred to earthen pots containing garden soil and vermiculate in a green house, where they grew normally (Fig .f ) with 85.4% survival rate. The plants were appeared normal morphologically and growth patterns.

Hardening and Acclimatization:

The most critical and important step of micro propagation studies is the transfer of regenerates from artificial to natural environment to ensure the maximum application of the technique. Treatment of micro shoots with 1500ppm IBA for 5minutes resulted in best rooting response (85%) and induced maximum number (4.6± 0.12) of roots per shoot after 4 weeks of transplantation to potting mixture.

4. Conclusion:

In conclusion, an efficient and effective protocol was developed for micro propagation of Pterocarpus santalinus a leguminous tree of great importance. This protocol provides a successful and rapid technique that can be used for the propagation and ex vitro conservation of this important species.

Acknowledgement

The authors thankful to the In vitro labs in tissue culture plants development private limited Tirupati, for providing Laboratory facilities.

References

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