In vitro multiplication of the semi-arid forest tree, Balanites aegyptiaca (L.) Del.

Procedures were developed for micropropagation of Balanites aegyptiaca using axillary bud explants obtained from mature trees. Cultures were established in Murashige and Skoog (MS) medium supplemented with 2.5 mg/l 6-benzylaminopurine (BAP) and 0.1 mg/l naphthalene acetic acid (NAA). The effects of kinetin on shoot growth and proliferation in vitro was also investigated. Results show that shoot multiplication required 2.5 mg/l of BAP. Shoot length was significantly affected by the presence of BAP or 6-furfurylaminopurine (Kin). Rooting of shoots in vitro was achieved on MS medium containing 20 mg/l of the auxin, indolebutyric acid (IBA). Rooted shoots acclimated and were successfully transferred into soil, with 48% of the plantlets surviving.


INTRODUCTION
Balanites aegyptiaca an evergreen tree is a multipurpose plant known for its many uses as fodder, charcoal, timber, fuelwood, antifeed etc (Von Maydell, 1984). It is widely distributed from Guinea through the Sahara into Egypt, and as far as Yemen, Iran to India. It is encountered on clay soils under rainfall of 500 mm (Giffard, 1974). It is sporadically *Corresponding author. E-mail: mndoye@ucad.sn. Tel: 5331084. Fax: (221) 824 63 18.
NAA: α-naphthalene acetic acid. BAP: 6benzylaminopurine. Kin: 6-furfurylaminopurine. MS: Murashige and Skoog medium. distributed on sandy soils under rainfall of 250 mm. The species, which occurs in arid zones, grows very slowly and has a slow fruit development. B. aegyptiaca is ecologically very flexible with excellent persistence. It is adaptable to a wide range of sites and climatic conditions (Von Maydell, 1984).
The tree is drought and fire resistant, and withstands up to 2 months flooding in areas near a river but it can not tolerate prolonged water logging (IPGRI, 1984). In Senegal, B. aegyptiaca was formerly protected. However, with the increased human pressures for its valuable timber and fodder, the tree is being continuously felled. Although the tree regenerates naturally by seed or after moderate coppice, the species is endangered because of the high rate of clearance (El Nour et al., 1991).
Micropropagation of tree species offers a rapid means of producing clonal planting stock for afforestation, woody biomass, and conservation of elite germplasm (Bonga and Durzan, 1982). The tissue culture of forest trees has shown promise in obtaining regenerants and clonal multiplication for domestication of wild populations, afforestation and improvement of economically important trees that have been cultivated for generations. Woody taxa are generally difficult to regenerate by in vitro techniques, but some success has been achieved in a few leguminous tree species (Dhawan, 1987). This paper describes preliminary observations on clonal multiplication of axillary buds of mature B. aegyptiaca tree, popularly known as "desert date".

Plant material and explant source
Vegetative shoots were collected from ten year old tree of Balanites aegyptiaca located in Hann forestry Park in Senegal (14°43′N; 17°26′W). Nodes (1.5 to 3 cm) were used as source of explant. Nodes segments bearing axillary buds were surface sterilized by quick dip in 90% ethanol followed by first, sodium hypochlorite solution for 5 min, and rinsed 5 times in sterilised distilled water, then in 0.1% HgCl 2 containing tween 20, finally 5 rinsed times in sterilised distilled water.

Culture medium and condition
The explants were placed on solid basal MS (Murashige and Skoog, 1962) medium supplemented with different concentrations and combinations of BAP: 0, 1, 2.5, 5 mg/l; kin: 0, 1, 2 and IAA 0, 0.1 mg/l for shoot proliferation and multiplication. The pH was adjusted to 5.6 using 0.1 N NaOH or 0.1 N HCl before autoclaving (110°C, 20 min). 20 ml of medium were dispensed into culture tubes (25 x 150 mm. The cultures were maintained at 25±2°C either under 16 h/day photoperiod from cool white fluorescent lamps (3000 lux). The shoots were maintained by regular subcultures at 4week intervals on fresh medium with the same compositions.

Induction of rooting and acclimatization
For root induction, excised microshoots (1-2 cm length) were transferred in liquid or solidified MS basal medium supplemented with different concentrations of NAA or IBA (5 and 20 mg/l). One excised shoot was placed in each tube (25 x 150 mm) containing 15 ml of the culture media. All the cultures were incubated at 25±2 0 C under 14 h photoperiod with cool, white fluorescent lamps. Rooted explants were planted in pots containing a sterile soil and kept in the greenhouse for acclimatization.

Observation of cultures and presentation of results
24 explants were used per treatment. The data pertaining to mean percentage of cultures regeneration, number and length of shoots/explant and mean percentage of rooting were statistically analysed by the Fisher's test.

RESULTS AND DISCUSSION
Among all the used media, the explants in MS medium were healthy and grew vigorously. 100% of segment explants produced shoots within 2 weeks and explants possessed 2.25 shoots having broad leaves. B5 (Gamborg et al., 1968) and SH media (Schenk and Hildebrandt, 1972) induce vitrifaction with an average of 97% of new shoots produced. B5 medium produced rosette plants. Cultured axillary buds started to grow within 5 to 10 days. Excised explants cultured on MS medium formed callus at the cut and along the margin which remained unorganised. Callus formation at the proximal ends of the node explants in the present study is in line with reports on Peganum harmala (Saini and Jaiwal, 2000) and Holostemma ada-kodien (Martin, 2000). Marks and Simpson (1994) suggested that this callus formation may be due to the action of accumulated auxin at the basal cut ends, which stimulates cell proliferation especially in the presence of cytokinins. According to Preece et al. (1991) the formation of callus at basal cut ends of nod explants on cytokinin enriched medium is frequent in species with strong apical dominance.
Addition of BAP (1 -5 mg/l) increased the number of shoots. Medium supplemented with 5 mg/l BAP induced a mean of 4.30 shoots per node explant (Table 1), while medium supplemented with Kin resulted in a reduced number of shoots although with longer internodes. Shoot multiplication required BAP, and Kin was found to be less effective than BAP. 2.5 mg/l BAP in combination with 0.1 mg/l NAA was the most effective combination for axillary bud multiplication, resulting in 3.13 shoots per node explant. This synergistic effect of BAP and auxin has been demonstrated in many plants including Santolina canescens (Casado et al., 2002), Bupleurum fruticosum (Fraternale et al., 2002), Acacia nilotica (Sane et al., 2001), and A. albida (Gassama et al., 1986).
Shoot length was significantly affected by the presence of BAP or Kin (Table 1). The highest BAP concentration (5 mg/l) reduced shoot length (2.16 cm) in cultures. 2.5 mg/l BAP was optimal for growth in this study. The overall number of shoots produced was significantly higher in the presence of 5 mg/l BAP. Other forestry species where BAP induced shoot multiplication are A. tortilis subsp. raddiana (Sane et al., 2000), A. Koa (Skolmen and Mapes, 1976) and Leucaena leucocephala ( Dhawan and Bhojwani, 1985). Kin promoted shoot elongation in B. aegyptiaca but led to decline of shoot multiplication. In Prosopis, however, Kin with IAA induced higher rate of shoot multiplication than BAP Arya, 1979, 1984).
Individual shoots were excised from 4 -6 week old cultures on multiplication medium and placed on the rooting medium. Root on induced shoots has been observed 10 days after rooting induction. Rooting of shoots was best achieved using high concentrations (10 to 20 mg/l) of auxin (Table 2). Shoots produced in vitro rooted well when treated with 20 mg/l of IBA or NAA. The number of roots per shoot is rather variable with NAA   (1991), the IBA hormone did not improve rooting significantly, which is contrary to our results. After 4 weeks, plantlets were transfered to pots and later established in soil. 48% of transferred plantlets survived with subsequent establishment. The results of this study show that axillary bud explants of B. aegyptiaca from mature mother plants can be readily established in MS medium containing 2,5 mg/l BAP to produce shoot multiplication. Multiple shoot regeneration has also been reported in Ziziphus mauritiana Lam. (Goyal and Arya, 1985;Mathur et al., 1995;Sudhersan et al., 2001). However, there are no published reports on the micropropagation of B. aegyptiaca. MS media without any growth hormones enhanced shoot growth and elongation,while media with BAP enhanced axillary branching explants furthering multiple shoot development of nods.