Nelson Onzere Amugune

Morphological and RAPD-marker characterization of Melia volkensii (Gürke) in vitro plants regenerated via direct and indirect somatic embryogenesis.

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). Key words : Morphometric, meristic, PCR-RAPD, Melia volkensii, tissue culture, somaclonal variation.

Dominant allele phylogeny and constitutive subgenome haplotype inference in bananas using mitochondrial and nuclear markers

Abstract Cultivated bananas (Musa spp.) have undergone domestication patterns involving crosses of wild progenitors followed by long periods of clonal propagation. Majority of cultivated bananas are polyploids with different constitutive subgenomes and knowledge on phylogenies to their progenitors at the species and subspecies levels is essential. Here, the mitochondrial (NAD1) and nuclear (CENH3) markers were used to phylogenetically position cultivated banana genotypes to diploid progenitors. The CENH3 nuclear marker was used to identify a minimum representative haplotype number in polyploids and diploid bananas based on single nucleotide polymorphisms. The mitochondrial marker NAD1 was observed to be ideal in differentiating bananas of different genomic constitutions based on size of amplicons as well as sequence. The genotypes phylogenetically segregated based on the dominant genome; AAB genotypes grouped with AA and AAA, and the ABB together with BB. Both markers differentiated banana sections, but could not differentiate subspecies within the A genomic group. On the basis of CENH3 marker, a total of 13 haplotypes (five in both diploid and triploid, three in diploids, and rest unique to triploids) were identified from the genotypes tested. The presence of haplotypes, which were common in diploids and triploids, stipulate possibility of a shared ancestry in the genotypes involved in this study. Furthermore, the presence of multiple haplotypes in some diploid bananas indicates their being heterozygous. The haplotypes identified in this study are of importance because they can be used to check the level of homozygozity in breeding lines as well as to track segregation in progenies.

High-Frequency Regeneration of the Drought-Tolerant Tree Melia volkensii Gurke Using Low-Cost Agrochemical Thidiazuron.

Melia volkensii Gurke is a drought-tolerant tree native to East Africas 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 Gamborgs 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.

Expressed Centromere Specific Histone 3 (CENH3) Variants in Cultivated Triploid and Wild Diploid Bananas (Musa spp.)

Centromeres are specified by a centromere specific histone 3 (CENH3) protein, which exists in a complex environment, interacting with conserved proteins and rapidly evolving satellite DNA sequences. The interactions may become more challenging if multiple CENH3 versions are introduced into the zygote as this can affect post-zygotic mitosis and ultimately sexual reproduction. Here, we characterize CENH3 variant transcripts expressed in cultivated triploid and wild diploid progenitor bananas. We describe both splice- and allelic-[Single Nucleotide Polymorphisms (SNP)] variants and their effects on the predicted secondary structures of protein. Expressed CENH3 transcripts from six banana genotypes were characterized and clustered into three groups (MusaCENH-1A, MusaCENH-1B, and MusaCENH-2) based on similarity. The CENH3 groups differed with SNPs as well as presence of indels resulting from retained and/or skipped exons. The CENH3 transcripts from different banana genotypes were spliced in either 7/6, 5/4 or 6/5 exons/introns. The 7/6 and the 5/4 exon/intron structures were found in both diploids and triploids, however, 7/6 was most predominant. The 6/5 exon/introns structure was a result of failure of the 7/6 to splice correctly. The various transcripts obtained were predicted to encode highly variable N-terminal tails and a relatively conserved C-terminal histone fold domain (HFD). The SNPs were predicted in some cases to affect the secondary structure of protein by lengthening or shorting the affected domains. Sequencing of banana CENH3 transcripts predicts SNP variations that affect amino acid sequences and alternatively spliced transcripts. Most of these changes affect the N-terminal tail of CENH3.

Interaction of Fusarium oxysporum f. sp. vasinfectum and the fungal feeding nematode Aphelenchus avenae on Bt cotton.

The fungal feeding nematode Aphelenchus avenae (APH) feeds on different species of fungi including Fusarium oxysporum f. sp. vasinfectum (FOV) that causes wilt in cotton. The objective of this study was to assess the interactions FOV and APH on Bt cotton and its isogenic counterpart (isoline) under greenhouse conditions. The treatments consisted of three levels, where Bt cotton, isoline and HART 89M were inoculated with: (i) APH alone; (ii) FOV alone and (iii) APH+FOV. Vascular discoloration, plant height, number of nodes, number of bolls, fresh shoot and root weight were recorded 180 days after planting (dap). Foliar symptoms were recorded throughout the growing season, and ELISA was used to determine the presence of Bt protein in soil and roots at 180 dap. Whereas no Bt protein was detected in roots and soil of HART 89M and isoline, it was found in Bt cotton. The isoline was more susceptible to FOV and APH+FOV than Bt cotton and HART 89M. FOV and APH+FOV caused a reduction in plant height, number of nodes, number of bolls, fresh shoot and root weight but the decrease was greater in the FOV treatment. There was also a higher reduction of growth parameters in the FOV treatment than in APH. The number of nematodes in the APH+FOV treatment of Bt cotton and isoline were not significantly different. The isoline was more susceptible than HART 89M to FOV .