J. Plant Biochemistry & Biotechnology Vol.

16(2), 151-153, July 2007

Short Communication

A Rapid In Vitro Morphogenesis and Acclimatization Protocol for Balanites aegyptiaca (L) Del - a Medicinally Important Xerophytic Tree
Vinod Singh Gour, S K Sharma, C J S K Emmanuel and Tarun Kant*
Biotechnology Laboratory, Forest Genetics and Tree Breeding Division, Arid Forest Research Institute, Jodhpur 342 005, India The callus mediated regeneration system for Balanites aegyptiaca (L) Del is reported here. Different explants like apical buds, young thorns and cotyledon pieces from mature tree and root segments from in vitro raised seedlings were used for callus induction on MS medium supplemented with 2.23 M 2,4-Dichlorophenoxyacetic acid. Seven to eight weeks old calli were transferred on hormone free MS medium in order to get regeneration. Shoot morphogenesis was achieved only from cotyledon-derived callus. The shoots so produced rooted well, when cultured on B5 medium supplemented with 9.84 M Indole-3-butyric acid. Plantlets have been transferred to the field after two-phase hardening and are performing well. Key words: arid, diosgenin, hardening, hingota, in vitro rooting, shoot morphogenesis,

Balanites aegyptiaca (L) Del is a multipurpose xerophytic tree, belongs to family Balanitaceae and is commonly known as desert date (vernacular: hingota). It is welladapted to different agro-climatic regions characterized by arid and semi-arid climatic features (1). Diosgenin - a sapogenin, in its fruits and roots has the potential for use in the production of oral contraceptives. The plant is also well-known for its anti-leishmanial activity (2) and molluscicide properties (3). Callus based regeneration protocol for this plant will not only help in its mass propagation but also in development of a gene transfer protocol, preferably using Agrobacterium based system in future. This regeneration protocol will enable high frequency in vitro mass multiplication of quality planting material to help the people of drought prone area in their efforts towards combating desertification, reforestation and for erecting wind breaks. Very little work on in vitro morphogenesis has been carried out on this promising desert tree. Only regeneration through stem nodal segments from mature tree (4) and through root explants (5) has been reported. Present work is the first report on callus-based in vitro regeneration of Balanites aegyptiaca.
*Corresponding author. E-mail: tarunkant@gmail.com Abbreviations: 2,4-D: 2,4-Dichlorophenoxyacetic acid; IBA: Indole-3-butyric acid; FYM: farm yard manure; RH: relative humidity.

Mature fruits were collected from the visibly healthy mature trees growing in Arid Forest Research Institute campus and from areas in and around Jodhpur. The endocarp of fruit is very hard and woody and after depulping the seeds were mechanically released with a light hammer. Seeds and cotyledon pieces of about 1 cm 3 were first washed thoroughly with running tap water, then with 2% (v/v) Rankleen liquid detergent (Ranbaxy Fine Chemicals Ltd) and thereafter rinsed thoroughly with distilled water thrice. These explants were surface sterilized with 0.1% (w/v) mercuric chloride solution for 10 min, followed by three rinses with sterilized distilled water. The seeds were inoculated on semi-solid medium (0.8% w/v; pH 5.8). Which germinated within 1 to 2 weeks. Six to 8-week-old seedlings were used as source of root explants. The apical buds and young thorns of mature tree were surface sterilized with 0.1% (w/v) mercuric chloride solution for 3-5 min. Rest of the treatments were similar as that used for cotyledon explants. Gamborgs (B5) medium (6) and Murashige and Skoogs (MS) medium (7) were used in different experiments. MS and B5 media respectively had 3% and 2 % (w/v) sucrose. Both the media were supplemented with phytohormones (HiMedia Laboratories) and solidified with agar (0.8% w/ v). Before adding agar the pH of MS and B5 media was adjusted to 5.8 and 5.5, respectively, with the help of 1.0 N NaOH and/or 1.0 N HCl. The phytohormones used were

152 J Plant Biochem Biotech

2,4-D and IBA. Forty ml of the medium was dispensed per glass jam jar (350 ml) and conical flask (150 ml) while 20 ml per borosilicate culture tube (60 ml). Hoaglands solution (8) at pH 6.0 was initially used to water the plantlets. The glassware, forceps, scalpels, media and distilled water o were autoclaved for 20 min at 121 C and 15 psi pressure. The cultures were incubated for 16 h light (1400-lux light intensity using 40W fluorescent tubes) and 8 h dark photoperiod at a temperature of 26 + 2C. The cultures were regularly subcultured every 4 weeks on fresh medium. Observations were recorded at one-week interval. Explants (apical buds, young thorns, cotyledonary segments and seedling-derived roots) were inoculated on MS medium supplemented with 2.23 M 2,4-D for callus induction. Callus was multiplied by subculturing on the same medium and were then transferred on hormonefree MS medium for organogenesis. Calli were fixed in FAA solution [5 ml 5% formalin (v/v), 5 ml glacial acetic acid and 90 ml 50% ethanol] after shoot morphogenesis. Sections were dehydrated by passing through ethanolxylene series and then subjected to histowax infiltration. About 10-12 thick sections were cut with a hand rotary microtome (Yorko). The sections were then stained with saffranin, washed and finally mounted in DPX (9). Photographs were taken under phase contrast microscope (Nikon), attached to a 35 mm film camera (Nikon). The elongated micro-shoots were harvested and then placed on hormone-free as well as IBA supplemented B5 medium for root induction. Plantlets were gradually acclimatized to the ambient environmental conditions. Acclimatization of plantlets was carried out in two phases. Phase one involved transfer of plantlets into glass jam jars containing vermiculite. Here RH was reduced by gradual unscrewing of the caps of jars. During this phase the cultures were kept in growth chamber having conditions

as described earlier. In the second phase of acclimatization plantlets were transferred into thermocol cups containing vermiculite. After two weeks, the plantlets were transferred to soil: FYM (1:1) mixture in plastic plantation bags (polybags) of size 9x9x36 cm. This second phase was carried out entirely in mist chamber, maintained between 28 and 30C, where 90 sec misting at 10 min interval was given to attain RH between 85 and 95%. With the aim to study the callusing behavior various explants were cultured on MS medium with 2.23 M 2,4-D. Callus induction was observed within 1-4 weeks from all four types of explants inoculated on callus induction media (Table 1). The callus grew well and was compact to friable with creamish-white colour. The callus derived from apical bud grew faster than the remaining three explants on hormone-free MS medium. However, only the callus derived from cotyledons (Fig. 1A) turned green and compact which then produced shoot primordia (Fig. 1B). This may be due to the comparatively slow growth of callus derived from cotyledonary explants so that it got a better chance to undergo morphogenesis, as also reported by Lakshmisita et al (10) in rosewood and by Manickam et al (11) in Withania somnifera. Upon subsequent subculturings, cells within callus may establish polarity resulting in the formation of shoot primordia and subsequently shoot development. Histological preparation of regenerative callus showed differentiation of vascular strand (Fig. 1C), which confirmed regeneration of shoots from callus only. Out of four types of callus source tested in present investigation, 36.36% shoot morphogenesis was obtained from the calli derived from cotyledon segments. Calli derived from the apical buds, young thorns and root segments did not regenerate into shoots. The micro-shoots elongated on hormone-free MS medium after one subculture (Fig. 1D and E). Some root induction was observed when the shoots were excised

Table 1. Callus induction in various explants cultured on MS medium with 2.23 M 2,4-D Explants Apical Buds Cotyledon segments Young thorns Root segments Percentage of explants producing callus 95.8a 34.2b 87.8a 93.33a Duration (weeks) for callus induction 3 4 3 1 Callus growth rate High Moderate Moderate Moderate

Note - No. of each explant cultured was 15. Each experiment was repeated thrice. The percentage explants showing callusing marked by based on analysis of variance (ANOVA)

indicate significant differences from rest of the values marked by a at a 5% level

Short Communication

153

and transferred to hormone-free B5 medium. However, good rooting percentage was achieved within 4-5 weeks on B5 medium supplemented with 9.84 M IBA (Table 2; Fig. 1F) as also reported by Ndoye et al (4) in Balanites aegyptiaca. It was clearly observed that the number of roots per shoot was much higher on IBA induced rooting compared to that observed on medium lacking IBA (hormone-free).
Table 2. Effect of IBA on rooting of microshoots after 4 wk incubation IBA (M) 0 9.84 Rooting (%) 86.96 97.36

hardening percentage of 43%. It has been observed that hardening is a very important stage in successful micropropagation protocol. The phase-wise hardening as described in this paper was instrumental in obtaining hardy plantlets for successful field plantations. The hardened plants have now been planted in the experimental field at 2x2m spacing and are performing well.

Shoot length* (cm) 3.14 + 0.40 4.29 + 0.28

No. of Roots/shoot* 3.96 + 0.56 6.18 0+ 0.67

Root length* (cm) 1.04 + 0.17 1.56 + 0.13

Note - * the value represent mean+SE; no. of explants per treatment were 15 and experiments were repeated thrice.

The plantlets looked robust, and hardened well (Fig. 1G, H). It takes 8-10 weeks for complete hardening of plantlets. Successful hardening percentage varied from a maximum of 77% to as low as 33% with a mean

Acknowledgements
Authors are grateful to CSIR, New Delhi for financial assistance, to Arid Forest Research Institute, Jodhpur for providing research facilities, and to Dr R C Purohit and N Ravi for continued support.
Received 18 November, 2006; accepted 13 April, 2007.

References
1 2 3 4 5 Von-Maydell H-J, Eschborn: GTZ, (1983) 531. El-Tahir A, Ibrahim AM, Satti GMH, Theander TG, Kharazmi A & Khalid SA, Phytoth Res, 12 (1998) 576. Jain DC, Phytochemistry, 26 (1987) 2223. Ndoye M, Diallo I & Gassama/Dia YK, African J Biotechnol, 2 (2003) 421. Gour VS, Emmanuel CJSK & Kant T, In Proc IUFRO Int Conf on Multipurpose Trees in the tropics: Assessment, Growth and Management (VP Tewari, RL Srivastava, Editors) Scientific Publishers, India (2005) pp 701-704. Gamborg OL, Miller A & Ojima K, Expt Cell Res, 50 (1968) 151. Murashige T & Skoog F, Physiol Plant, 15 (1962) 473. Hoagland DR & Arnon DI , Calif Agric Exp Stn Circ , 347(1950) 1. Jensen WA , Botanical histochemistry: Principles and practice, WJ Freeman & Co, San Francisco, CA (1962). Lakshmisita G, Chattopadhay S & Tejawathi DH, Plant Cell Rep, 5 (1986) 266. Manickam VS, Elangomathavan R & Antonisamy R, Plant Cell Tiss Org Cult, 62 (2000) 181.

6 7 8 Fig. 1. (A) Callus induction in cotyledons, (B) shoot morphogenesis through callus, ( C ) cross section of callus (ca) through regenerating shoot showing distinct vasculature (vt) after histochemical staining (magnification: 200X), ( D) early stage shoot elongation, (E) late stage shoot elongation, (F) rooting in regenerated shoots, (G) plantlets transferred to vermiculite, and (H) plantlets on vermiculite in thermocol cups undergoing hardening. 9 10 11