*2.2.3 Objective 3: undertake the BAT species regeneration, and mass seedling propagation and test the in-situ rescue performance*

Mass regeneration of BAT species was done by rapid in-vitro micropropagation, ex-vitro plantlet growth, and in-situ re-establishment of the species in the wild. The BAT plant materials: vegetative portions and mature fruits, were collected from the Mt. Elgon region (Site 1) and transported to Makerere University Microbiology

#### *Integrated Conservation Approaches for Rescuing, Regeneration and Adaptive Management… DOI: http://dx.doi.org/10.5772/intechopen.106893*

Laboratory where the species were regenerated into mass seedlings using micropropagation. The plant part(s) were cut open or seeds extracted from pods for fruits and rinsed in distilled water three times. The parts/seeds were washed in 2%(v/V) Tween-20 detergent solution for 8 minutes, and later sterilized with 1%NaClO solution containing 2-drops of Tween-20 for 15 minutes. The plant part tissues/seeds were re-washed with sterile distilled water under aseptic conditions.

The extracted plant parts and seeds were regenerated into mass seedlings using tissue culture and in-vitro seed germination techniques; respectively [14], − where they were cultured on growth media and incubated under aseptic conditions. The growth media were prepared by mixing equal volumes of 3%Sucrose and 0.8%Agar into and diluting to 50% concentration while keeping the pH at 5.8. About 20 ml of the growth media will each be dispersed into sterilized 100 ml culture jars, where the plant tissues/seeds were inoculated. Non-absorbent cotton wrapped in cheesecloth was plugged into the culture jars, and the jars were autoclaved at 1.06kgcm−2 and 121°C for 15 minutes [14]. The inoculated tissues/seed samples were kept under aseptic conditions, at a temperature of 25 ± 2°C and 16/8 light–dark photoperiod supplied by white fluorescent lights under tissue culture rooms, until full tissue regeneration/ seed germination. At least 20 seeds and 50 plant tissues were regenerated during 1st micropropagation cycle and were exponentially increased for five successive seedling generations, so that mass BAT seedlings were rescued during the five micropropagation phases.

The newly germinated BAT seedlings, after 3–4 weeks from inoculation in the Tissue Culture Laboratory, were removed from the laboratory to artificially induced shoot and root formation. The regenerated shoot tips from the seedlings were cut using a sterile blade, and cultured in Murashige and Skoog (MS) medium supplemented with varying concentrations of BA/KA: 0.5, 1.0, 1.5 and 2 mg/l for 5–6 weeks [15]. The best resulting micro-shoots were incubated on a half-strength MS medium containing varying concentrations of indole-3-butyric acid (IBA)/naphthalene-1-acetic acid (NAA): 0.5, 1.0, 1.5 and 2 mgl−1 to induce root formation and growth [15]. After the mass micropropagation process, regeneration success was assessed as the percentage ratio of fully developed seedlings to the total number of seedlings cultured.

Seedling hardening: the most vigorous BAT plantlets were transplanted into PE-plastic pots (dimensions: 10 × 15 × 7cm), filled with soil growth media artificially sterilized by autoclaving and mixed peat-moss, perlite and soil in ratios of 1:1:1(v/v). During hardening, the potted plantlets were irrigated with distilled water for about 10 days under culture room conditions before being moved to greenhouse and field weather conditions for further in-situ re-establishment.
