Summary• Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees.• For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants.• Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age.• Statistical analysis confirmed this rather counterintuitive finding that leaves of 'late regenerants' exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.
Cacao swollen shoot virus is a member of the family Caulimoviridae, genus Badnavirus and is naturally transmitted to Theobroma cacao (L.) by several mealybug species. CSSV populations in West African countries are highly variable and genetically structured into several different groups based on the diversity in the first part of ORF3 which encodes the movement protein. To unravel the extent of isolate diversity and address the problems of low titer and mixed viral sequences in samples, we used Illumina MiSeq and HiSeq technology. We were able to reconstruct de novo 20 new complete genomes from cacao samples collected in the Cocoa Research Institute of Ghana (CRIG) Museum and from the field samples collected in Côte d'Ivoire or Ghana. Based on the 20% threshold of nucleotide divergence in the reverse transcriptase/ribonuclease H (RT/RNase H) region which denotes species demarcation, we conclude there exist seven new species associated with the cacao swollen shoot disease. These new species along with the three already described leads to ten, the total number of the complex of viral species associated with the disease. A sample from Sri Lanka exhibiting similar leaf symptomology to West African CSSD-affected plants was also included in the study and the corresponding sequence represents the genome of a new virus named cacao bacilliform SriLanka virus (CBSLV).
By identifying antibiotics that eliminated Agrobacterium tumefaciens with the least phytotoxic effects, we were able to select appropriate A. tumefaciens strains for a more efficient transformation of seasonal Fragaria vesca and everbearing F. v. semperflorens. An efficient and reproducible method of shoot regeneration from leaf discs was developed with optimal shoot regeneration obtained on medium supplemented with 0.25 mg l -1 indole-3-butyric acid and 3 mg l -1 benzyladenine for both F. vesca forms. Petiole sections were also effective for F. v. semperflorens regeneration, and culture vessel capacity was found to influence shooting from both explant types and F. vesca forms. Of a range of antibiotics commonly used for Agrobacterium elimination, carbenicillin had the least inhibitory effect on shoot regeneration and was found to effectively control strain LBA4404. Both F. vesca forms showed a high sensitivity to kanamycin and, therefore, a selection regime ramped from 10 mg l -1 to 25 mg l -1 kanamycin over a period of 8 weeks was developed. Optimal transformation efficiency (15%) was achieved by the appropriate use of explant type and age, leaf-disc orientation, Agrobacterium density and inoculation time and phenolic compounds for bacterial virulence induction. Southern analysis confirmed the integration of the β-glucuronidase reporter gene into the genomic DNA of both F. vesca L. forms.
The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.
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