Establishment of Oil Palm Suspension Culture in the Sixfors Bioreactor

In vitro propagation is an important part of the oil palm industry’s approach towards clonal propagation of high-yielding materials. Oil palm suspension cultures have been established using the shake flask system which was developed for production of a reliable supply of regenerable plant tissues. However, this system is inefficient for fast large scale proliferation of embryogenic suspension cultures. Bioreactors have been used for the industrial production of microbial, animal and plant metabolites. However, it’s used was not well known in oil palm suspension culture. During the development of oil palm suspension culture in the sixfors multibioreactor, nutrients and extra cellular metabolites were monitored where kinetic parameters and nutrients to biomass conversion yield were calculated to better characterise the behaviour of oil palm suspension culture. It was observed that the amount of biomass of all the cell lines was at an average of 2-3 fold higher than the original inoculums weight with an incubation period of 30 to 60 days. The carbon source, which is sucrose, was hydrolysed to glucose and fructose in the first 10 days and both were completely utilised after the 25th day. The sugar to biomass conversion yield was low and the mainly linear growth showed that the growth of the cell was limited by the culture conditions. Nitrogen sources from the MS media remained in excess until the end of the growth period where only 30% of ammonia and 15% of nitrates were utilised which resulted in the cell being toxic and thus limiting cell growth. The growth was exponential in the first 10 days with a maximum specific growth rate of 0.07 day-1 which corresponded to a doubling time of 10 days. The cells then entered a period of linear growth until Day 25 to reach the maximum dry weight of 4 g/l, after which the cells began to die off causing the dry weight to fall to 2.8 g/l at Day 45. The pH profile was an indication of the nitrogen and sugar uptake by the cells. The pH decreased rapidly from 5.6 to 4.0 in the first 9 days and then increased gradually to 4.4 at the 25th day. At this point, the cell growth had stagnated, and the pH quickly increased to 5.5 before declining again to the end of the culture at Day 45. The initial pH decrease was partly due to the uptake of ammonium. After this, however, the great increase was due to the uptake of nitrate ions to the ammonium stored in the vacuoles of the cell

Analysis of oil palm clones, their suspension calli and regenerants via flow cytometry (FCM) and rDNA- fluorescence in situ hybridiSsation (rDNA-FISH)

Clonal propagation of palms with good traits is desirable for the oil palm industry. In this study, flow cytometry (FCM) and 18S-25S ribosomal DNA-fluorescence in situ hybridisation (rDNA-FISH) were utilised to analyse genetic variation in adult clonal palms, their respective suspension cultures and regenerant plantlets. The genome sizes estimated by FCM for the four adult clonal palms (using leaf samples from Frond-1) varied from 2C=2.59±0.19 pg to 2.91±0.14 pg and for their respective regenerants, the genome size varied from 2C=2.14±0.21 pg to 3.05±0.11 pg. The genome size of oil palm suspension cultures could not be analysed by FCM due to the low nuclei population, which was less than 1000. The rDNA-FISH analysis showed two hybridisation signals on interphase nuclei of suspension culture calli and regenerant plantlets, hence indicating the diploid ploidy level. Adult clonal palms with their suspension culture calli and regenerants therefore showed a similar ploidy level. However, the measurement of genome size was found to vary between the adult clonal palms and their regenerants

Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm

Somaclonal variation arises in plants and animals when differentiated somatic cells are induced into a pluripotent state, but the resulting clones differ from each other and from their parents. In agriculture, somaclonal variation has hindered the micropropagation of elite hybrids and genetically modified crops, but the mechanism responsible remains unknown. The oil palm fruit 'mantled' abnormality is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for oil production. Widely regarded as an epigenetic phenomenon, 'mantling' has defied explanation, but here we identify the MANTLED locus using epigenome-wide association studies of the African oil palm Elaeis guineensis. DNA hypomethylation of a LINE retrotransposon related to rice Karma, in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is associated with alternative splicing and premature termination. Dense methylation near the Karma splice site (termed the Good Karma epiallele) predicts normal fruit set, whereas hypomethylation (the Bad Karma epiallele) predicts homeotic transformation, parthenocarpy and marked loss of yield. Loss of Karma methylation and of small RNA in tissue culture contributes to the origin of mantled, while restoration in spontaneous revertants accounts for non-Mendelian inheritance. The ability to predict and cull mantling at the plantlet stage will facilitate the introduction of higher performing clones and optimize environmentally sensitive land resources