Somaclonal variation induced in vitro during tissue culture can be a problem in clonal micropropagation of elite plants. This study investigated the extent of morphological and genetic similarity or dissimilarity between Melia volkensii in vitro plants (somatic seedlings) obtained via somatic embryogenesis and normal seedlings. Comparisons were made between in vitro plants regenerated directly from cotyledon explants, indirectly from zygotic embryos and normal seedlings of the same parent trees. Regeneration was achieved using half MS medium supplemented with 0.05 mg/l thidiazuron. Shoots were elongated in half MS with 0.1 mg/l BAP plus 0.01 mg/l IAA then rooted in half MS with 0.1 mg/l IBA and 0.1 mg/l NAA. Six morphometric and five meristic characters were used for the morphological characterization. PCR-RAPD markers were used for assessment of genetic similarity or distance. Multivariate analysis using principal coordinates, cluster analysis, analysis of similarities (Anosim) and similarity percentages analysis (SIMPER) revealed significant dissimilarities (p< 0.0001) in morphometric and meristic characters between the in vitro plants and normal seedlings. However, significant similarity (p<0.01) was observed in the RAPD-genic characters of the in vitro plants and normal seedlings. Out of six morphometric traits, taproot length, internode length and shoot height were the most important sources of dissimilarity, cumulatively accounting for 72.37% of overall morphometric dissimilarity. Number of lateral roots was the single most important source of meristic dissimilarity, with 77.02% contribution. Plants regenerated directly from cotyledons were more similar to the normal seedlings in morphological and RAPD-marker characters than those regenerated indirectly from zygotic embryos. This study paves the way for identification of trait-specific RAPD markers for further characterization through sequence-characterized amplified regions (SCARs).

Melia volkensii Gurke is a drought-tolerant tree native to East Africa's arid and semiarid lands (ASALs), with vast but underutilized potential for agroforestry and sustainable livelihoods in the ASALs. Its cultivation is limited by difficulties in propagation via conventional means. Full exploitation of the ability of thidiazuron (TDZ) to elicit regeneration in plant tissue cultures, as sole plant growth regulator (PGR), is hampered by high costs. This study tested the effectiveness of a low-cost agrochemical TDZ for in vitro propagation of M. volkensii. Zygotic embryos from mature seeds were cultured on Gamborg's B5 medium containing 0 to 4 mg/L of agrochemical TDZ from Kingtai Chemicals Co.,Ltd., China. Callus induction frequency was 96.67 to 100%. Significantly large callus fresh mass was produced at 0.05 mg/L TDZ concentration (ANOVA, P < 0.001). The effect of TDZ on embryogenicity was significant over certain ranges of concentrations (Anova, P < 0.001). Multiple somatic embryos developed within 14 days of subculture to hormone-free B5 medium. Somatic embryos developed into microshoots which elongated when transferred to 1/2 MS medium supplemented with 0.1 mg/L 6-benzylaminopurine plus 10% coconut water. The Kingtai-TDZ showed a high potency and suitability for use in M. volkensii tissue culture.

The study reports the first DNA-barcode and molecular phylogeny of the East African endemic tree species Melia volkensii using the standard two-locus plant barcoding genes (rbcL and matK). The two genes were amplified and the PCR products sequenced. Complete coding sequences were obtained for both genes. The edited and aligned sequences had lengths of 1371 bp for rbcL and 1524 bp for matK. These DNA sequences were deposited into the DNA Data Bank of Japan (DDBJ) with cross-listing in the European Molecular Biology Labaratory (EMBL) and GenBank databases. The deposited gene sequences were then subjected to separate nucleotide BLASTs in NCBI's GenBank database. Out of 100 Blast results in which the query (M. volkensii) had 96-100 percentage similarity in nucleotide sequence for the rbcL gene and 90-100% similarity for the matK gene, only 16 taxa had data for both rbcL and matK genes. These 16 taxa were used for the phylogenetic analysis and comprised of 6, 9 and 1 taxa respectively from the families Meliaceae, Simaroubaceae and Rutaceae. The barcode allowed adequate discrimination of the taxa into their respective generic and species clades. Availability of a barcode for M. volkensii will ease identification of the species, provide more robust phylogenetic reconstructions and allow for better tracking of its exotic dispersal.