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Micropropagation of Pongamia pinnata (L.). Pierre- a native Indian biodiesel tree from cotyledonary node.


Pongamia pinnata (L.) Pierre belongs to family Fabaceae. It's known by many names (Indian Beech, Pongam, Honge, Ponge, and Karanj among others). Pongamia is native to India. It is a fast growing, glabrous, deciduous, tree about 25 m tall, branches drooping; trunk diameter to 60 cm; bark smooth, gray. Leaves imparipinnate, shiny; young leaves pinkish red, mature leaves glossy, deep green; Flowers fragrant, white to pinkish, 1-seeded; seed thick, reniform [1]. The thick oil from the seeds is used for illumination, as a kerosene substitute, water paint binder, pesticide, and in soap and tanning industries and lubrication. The oil has been tried as fuel in diesel engines, showing a good thermal efficiency. Pongamia pinnata is also having immense potential as a bio-diesel plant [2], [3] and for real development in the biofuel sector the promotion of Pongamia is necessary. From scientific point of view Pongamia is far better than Jatropha. It also possesses valuable medicinal properties. The literatures related to different systems of medicine in India specially related to Ayurveda are full of miraculous therapeutic properties of Pongamia. It is cultivated for two purposes, one -as an ornamental in gardens and two -as a host plant for lac insects [4].

We describe here a protocol for micropropagation of P. pinnata through in vitro raised seedling derived cotyledonary nodes.

Materials and Methods

Plant material and explants source: Seeds of Pongamia were collected from ripe fruits. After removal of fruit coat, the seeds were washed thoroughly under tap water 4-5 times, followed by washing with distilled water 3-4 times. These were then treated with 1% (v/v) Rankleen liquid detergent (Ranbaxy Fine Chemicals Ltd., India) and thereafter rinsed thoroughly with distilled water thrice. These washed and cleaned seeds were surface sterilized in two steps. First these were rinsed with 70% ethanol for 3 minute and then treated in NaOCl Solution for 5 minutes with constant shaking followed by rinsing with sterile distilled water 4 times. The surface sterilized seeds were then inoculated in culture flaks with Murashige and Skoog (MS) medium [5] for germination. For multiple shoot induction, cotyledonary node (1-2 cm in length) obtained from 4 wk old seedlings.

Culture media and condition: MS medium supplemented with 3% (w/v) sucrose (HiMedia, India) and 0.8% w/v agar (Himedia, India) was used during the study. The pH of the medium was adjusted to 5.8 prior to autoclaving at 120[degrees] C and 104 kpa for 15 min. The cultures were incubated at 25 [+ or -] 2 [degrees]c under a 16-h photoperiod with 35[micro] mol [m.sup.-2[[s.sup.-1] photon flux density provided by cool white fluorescent tubes and incandescent bulbs.

Multiple shoot induction and proliferation: Seeds were inoculated on hormone free MS medium for germination. For multiple shoot induction, cotyledonary node (1-2 cm in length) obtained from 4 wk old seedlings were raised on MS medium supplemented with 8.8 [micro]M BAP and 0.53 [micro]M NAA. Once culture conditions for optimum shoot induction from explants were established, the shoot clumps produced in vitro were subcultured onto fresh medium or medium without auxin (NAA) with reduced callusing to further proliferation and elongation of shoots, as also reported by Sugla et al in 2007 [6] and Sujatha and Hazra also in 2007 [7]. The original explants were repeatedly sub cultured on shoot multiplication medium and subsequently on shoot elongation medium after each harvest of the elongated shoots. All cultures were maintained under similar condition as described earlier. The frequency of explants producing shoots, number of shoots per explants and shoot length were scored after 8 week of culture.

Root formation: Elongated shoots, 3-4 cm. in length, were excised after 12 week of culture and transferred individually to full strength MS medium supplemented with different concentration of Indole-3-butyric acid (IBA) for rooting. Observation on percent rooting, number of roots per shoot and root length were recorded after 4 weeks.

Acclimatization: Rooted shoots were taken out from the culture tubes; well- rooted plantlets were washed with sterilized distilled water and transferred in to vermiculite moistened with autoclaved Hoagland's solution. After 4 weeks, in vitro acclimatized plantlets were transferred to thermocol (Styrofoam) cups containing vermiculate and kept in mist chamber maintained between 28 and 30[degrees]C, where 90 sec misting at 10 min interval was given to attain RH between 85 and 95% for ex vitro acclimatization. The acclimatized plants were finally transferred to polybags bags containing FYM: sand :: 1:3 mixture.

Data analysis: Each treatment consisted of 10 explants and all experiments were repeated thrice. The results were analyzed statistically using SPSS ver. 10 (SPSS Inc., Chicago, IL, USA). The significance of differences among means was carried out using Duncan's multiple range test at P =0.00. The results are expressed as the means [+ or -] SE of three experiments.

Result and Discussion

The cotyledonary nodes, when inoculated on MS medium supplemented with 8.8 [micro]M BAP and 0.53 [micro]M NAA showed good shoot proliferation (Table 1). The frequency of regeneration and number of shoots from explants were maximum on MS medium supplemented with 8.8 [micro]M BAP which induce the highest shoot regeneration frequency (90 [+ or -] 5.7%), number of shoots per explant (2.8 [+ or -] 0.1) and shoot length (7.0 [+ or -] 0.2) (Fig.1 A-C). BAP is reported to have favored axillary shoot proliferation from cotyledonary nodes of several other tree species including Sterculia urens [8], Dalbergia sissoo [9], Achras sapota [10], and Punica granatu [11]. After induction on medium containing 8.8 [micro]M BAP, shoots along with the original explants, were sub-cultured on medium containing the same concentrations of BAP and this was repeated at a 4 week interval. Shoot elongation on these media was high until the fourth subculture after which it declined.

Each explant could be subcultured for upto a maximum of three times and produced an average of four to five shoots in 25-30 days. Therefore, from a single cotyledonary node, about 16-24 shoots were obtained within 60 days. Similar repeated culture of the mother explants has resulted in continuous harvest of shoots for a few generations in pomegranate [11] and Vanilla [12]. Addition of an auxin to the medium was essential to induce rooting in the regenerated shoots. Root initiation occurred within 10-15 day on full strength MS medium supplemented with a range of IBA concentrations (Fig.1 D). IBA has been widely used as a root-inducing hormone in difficult-to-root plants both under in vitro and in vivo conditions [13]. Analysis of variance showed that the root number was significantly influenced by the concentration of IBA used. The highest frequency of root formation (93 [+ or -] 3.3), number of roots (2.5 [+ or -] 0.1) and root length (4.3 [+ or -] 0.3) were developed on the medium containing 9.8 [micro]M IBA without intervening callus formation (Table 2). Plantlets with well-developed roots were successfully acclimatized (Fig.1 E) and eventually established in thermocol cups (Fig.1 F) and then polybags.



[1] Allen, O.N. and Allen E.K., 1981, "The Leguminosae. A Source Book of Characteristics, Uses and Nodulation. University of Wisconsin Press, Madison".

[2] Srinivasa, U. 1997, "Power to the people! Electricity from seeds! Pongam oil a dieselsubstitute. e.html."

[3] Vivek, Gupta, A.K., 2004, "Biodiesel production from Pongamia oil". J. Sci. Ind. Res. 63, pp. 39-47.

[4] Duke, J., 1983. Handbook of Energy Crops. Unpblished.

[5] Murashige, T., and Skoog, F., 1962. "A revised medium for rapid growth and bioassays with tobacco tissue culture," Physiol. Plant., 15, pp. 473.

[6] Sugla, T., Purkayastha, J., Singh, S.K., Sahoo, L., and Solleti, S.K., 2007, "Micropropagation of Pongamia pinnata through enhanced axillary branching," In Vitro Cell. Dev. Biol. Plant., 43 pp. 409-414.

[7] Sujatha, K., and Hazra, S., 2006, "In Vitro regeneration Pongamia pinnata Pierre," J. Plant Biotechnol., 33, pp. 263-270.

[8] Purohit, S.D., and Dave, A., 1996, "Micropropagation of Sterculia urens Roxb. An endangered tree species," Plant Cell Rep., 15, pp. 704-706.

[9] Pradhan, C., Kar, S., Pattnaik, S., and Chand, P.K. 1998, "Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes," Plant Cell Rep., 18, pp. 122-126.

[10] Purohit, S.D., and Singhvi, A., 1998, "Micropopagation of Achras sapota through enhanced axillary branching," Sci. Hort., 76, pp. 219-229.

[11] Naik, S.K., Pattnaik, S., and Chand, P.K., 2000, "High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Puncia granatum L.)," Sci. Hort., 85, pp. 261-270.

[12] Gopi, C., Vatsala, T.M., and Ponmurugan, P., 2006, "In vitro multiple shoot proliferation and plant regeneration of Vanilla planifolia Andr.-A commercial spicy orchid," J. Plant Biotech. 8(1), pp. 37-41.

[13] Minocha, S.C. 1987, "pH of the medium and the growth and metabolism of cells in culture," In: Bonga, J.M., and Durzan, D.J. (eds.,) Cell and Tissue Culture in Forestry. Martinus Nijhoff Pub., pp. 125-141.

Abbreviations: BAP, 6-Benzylaminopurine; IBA, Indole-3-butyric acid; NAA, Naphthalene acetic acid; MS, Murashige and Skoog's (1962) medium; NaOCl, Sodium hypochloride.

Vineeta Shrivastava and Tarun Kant *

Biotechnology Laboratory, FGTB Division, Arid Forest Research Institute, New Pali Road, Jodhpur 342005--India

* Corresponding author. Email:
Table 1: Effect of Cytokinin and auxin on shoot proliferation
from cotyledonary nodes of Pongamia pinnata grown on MS medium
after 4 week.

BAP ([micro]M) NAA ([micro]M) Regeneration

 0.0 0.0 30 [+ or -] 5.7 (c)
 0.0 0.53 13 [+ or -] 3.3 (cd)
 8.8 0.0 90 [+ or -] 5.7 (a)
 8.8 0.53 80 [+ or -] 5.7 (ab)

BAP ([micro]M) Number of Shoot length
 shoots/explant (cm)

 0.0 0.27 [+ or -] 0.0 (c) 1.8 [+ or -] 0.2 (c)
 0.0 0.13 [+ or -] 0.0 (cd) 1.5 [+ or -] 0.5 (c)
 8.8 2.8 [+ or -] 0.1 (a) 7.0 [+ or -] 0.2 (a)

Values represent [+ or -] SE. Means followed by the same
letter within columns are not significantly different
(P = 0.00) using Duncan's multiple range test

Table 2: Effect of various concentration of IBA on rooting of
proliferated shoots of pongamia pinnata cultured on full MS medium

 IBA ([micro]M) Rooting (%) Number of

 0.00 30 [+ or -] 5.7 (f) .33 [+ or -] 0.0 (e)
 4.9 73 [+ or -] 3.3 (bc) 1.1 [+ or -] 0.0 (c)
 9.8 93 [+ or -] 3.3 (a) 2.5 [+ or -] 0.1 (a)
 14.7 76 [+ or -] 3.3 (b) 1.5 [+ or -] 0.1 (b)
 19.6 66 [+ or -] 3.3 (bd) 1.0 [+ or -] 0.1 (d)
 24.6 50 [+ or -] 5.7 (e) .93 [+ or -] 0.0 (cd)

 IBA ([micro]M) Root length (cm)

 0.00 1.5 [+ or -] 0.1 (e)
 4.9 2.3 [+ or -] 0.1 (cd)
 9.8 4.3 [+ or -] 0.3 (a)
 14.7 3.2 [+ or -] 0.1 (b)
 19.6 2.1 [+ or -] 0.1 (de)
 24.6 2.9 [+ or -] 0.2 (c)

Values represent [+ or -] SE. Means followed by the same
letter within columns are not significantly different
(P = 0.00) using Duncan's multiple range test.
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Author:Shrivastava, Vineeta; Kant, Tarun
Publication:International Journal of Biotechnology & Biochemistry
Date:Oct 1, 2010
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