Thirty root cuttings were selected randomly from accessions of Cryptolepis sanguinolenta plants collected from the Ashanti, Eastern, Volta and Brong Ahafo regions of Ghana between July and August 2011. Plants used in the study were identified based on morphological characteristics of the species. Accession and voucher specimen details are as indicated in [7] .

Parent plants obtained from the root cuttings were sprayed with 0.3% systemic fungicide solution (Agriette 80 WP a.i. 800 g/kg Fosetyl-AL) about five days prior to the harvest of explants for in vitro culture. Explants were harvested usually about two weeks after cut back. They comprised of semi-hardwood single node cuttings, 1 - 1.5 cm long. Leaves were trimmed off the nodal cuttings and the explants washed with mild detergent (two drops of sunlight washing up liquid), run under water for five minutes and transferred to the laminar flow cabinet. Under the laminar cabinet, the explants were soaked in 20% bleach (a.i. 6% NaOCl) and gently agitated for 10 minutes. They were subsequently rinsed thoroughly to remove all traces of sterilant with three changes of sterile, triple filtered distilled water.

For shoot regeneration, cultures were maintained on MS (Murashige and Skoog) basic nutrient medium [8] supplemented with MS vitamins, 30 g/L sucrose and BA at 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/L in combination with 0.1 mg/L IBA or NAA. Control consisted of hormone free MS medium. For root induction and subsequent rooting, microshoots, not less than 1 cm were transferred onto full strength MS medium with vitamins (supplemented with 0.5 mg/L IBA or 0.1 mg/L IBA) or half strength MS medium with vitamins (supplemented with 0.01 mg/L IBA) or hormone freeHalf strength MS medium with vitamins. All media were solidified with 0.3% gelrite. The pH was adjusted to 5.7 ± 0.1 using 1 N NaOH and 1 N H₂SO₄. About 5 ml of media were dispensed into flat bottom polycarbonate culture tubes (25 mm × 95 mm) with polypropylene screw caps (Phyto Technology labs, US) and autoclaved at 121˚C and 120 kPa for 18 minutes.

With the aid of sterile, fine, stainless steel scalpel and forceps, the explants were trimmed to about 1 cm before inoculation. The bases of the explants were inserted vertically into the semi-solid medium. Microshoots not less than 1 cm were transferred onto rooting media after 8 weeks.

Each treatment involved 10 cultures and was replicated three times, in a completely randomized design. Data taken (number of days to shoot induction, percentage shoot induction, shoot length, number of nodes per shoot, number of days to root induction and percentage rooting) were analyzed using analysis of variance with GENSTAT (12^th edition). Significantly different means were separated using Duncan’s Multiple Range Test.

The auxin type played a very important role in shoot induction and proliferation. Generally, BA and IBA combinations were found to be more efficient in shoot regeneration and multiplication of nodal segments of Cryptolepis sanguinolenta than the BA and NAA combinations (Figures 1-3). Benzyladenine (BA) and NAA combinations produced lower percentage shoot inductions and higher mean number of days to shoot induction and lower percentage shoot induction than the control (Table 1).

The combination of BA and NAA was not only inefficient in breaking axillary shoot dormancy, but also had detrimental effects on the cultures. Cultures maintained on NAA supplemented media produced profuse callus, which retarded shoot formation and subsequent shoot growth (Figures 2(e)-(k), Figure 3(l), Figure 3(m)).

Benzyladenine (BA) and IBA combinations proved to be efficient for shoot multiplication in a large number of species belonging to the Asclepiadaceae family [3] [4] [9] - [11] . NAA and BA combinations however, have also been reported to be efficient in some plant species in the Asclepiadaceae family [12] [13] .

The number of nodes produced within a period of time gives an estimate of the multiplication rate while shoot height determines the feasibility of subculture. Although the highest mean shoot length (1.28 cm) was recorded in

Values represent means ± se. Any two means not followed by one or more letters in common within a column are significantly different at p < 0.05 according to Duncan’s Multiple Range Test.

cultures maintained on MS medium supplemented with 3.0 mg/L BA (in combination with 0.1 mg/L IBA), it did not differ significantly from cultures maintained on the other high BA concentrations (1.5, 2.0, 2.5 mg/L) and 0.1 mg/L IBA supplemented media and also the control (Table 1). However, it was significantly higher than mean shoot lengths recorded in the low BA concentrations (0.5 and 1.0 mg/L BA) and 0.1 mg/L IBA (Table 1). The efficacy of plant growth regulator is dependent on its concentration [14] - [17] .

High shoot length recorded in the control suggests the presence of endogenous auxins which may have induced shoot elongation [18] . In addition to increased shoot length, the number of nodes was significantly improved by BA, proving its ability to enhance axillary bud development in Cryptolepis sanguinolenta.

Some species may root readily even in the course of multiplication, while others may require the inclusion of auxin in the medium. According to Sathyanarayana and Varghese [19] , transfer onto auxin supplemented medium becomes necessary if rooting is not observed after two monthly passages on the basal nutrient medium. Other species may also root when simply transferred onto a hormone free medium [14] [15] .

Cultures transferred onto hormone free half strength medium, began to form callus at the base [first sign of rooting; Figure 4(b)] by the fourth week after transfer. However, no rooting was observed by the sixth week (Table 2; Figure 5(c)).

Cultures transferred onto full strength MS medium, supplemented with 0.5 mg/L IBA, exhibited the highest

level of stress. Leaves of cultures began to drop after 24 hours of transfer and within the first week, almost all the cultures shed off their leaves and began to show signs of scorching (Figure 5(a)). No signs of rooting were observed even after the six weeks of transfer (Table 2). Reducing IBA concentration to 0.1 mg/L reduced the extent of necrosis (Figure 5(b)).

Cultures transferred onto half strength MS medium supplemented with 0.01 mg/L IBA began to form callus at the base by the second week, and by the sixth week all cultures had formed callus with about 60% rooting (Table (2) & Figure 5(d)).

Rooting of cultures in half strength MS basal medium has been successful in a number of species belonging to the Asclepiadaceae family [11] [12] .

It was observed that roots developed by microshoots previously maintained on hormone supplemented media (BA in combination with IBA) were thicker, deep yellow in colour and had more lateral roots than roots developed by microshoots raised on hormone free medium (Figure 6).