Plant DNA flow cytometry and estimation of nuclear genome size.

Principles of Plant Breeding

Global forest resources assessment 2000 : main report

Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.

Somaclonal variation in plants: causes and detection methods

In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful.

DNA Methylation Analysis: Choosing the Right Method

Jacq.) currently hampers the scaling-up of clonal plant production. In order to investigate the relationship between the “mantled” somaclonal variant and possible alterations in genomic DNA methylation rate, two complementary approaches have been used. HPLC quantification of relative amounts of 5-methyl-deoxycytidine has shown that global methylation in leaf DNA of abnormal regenerants is 0.5–2.5% lower than in their normal counterparts (20.8% vs 22%, respectively). When comparing nodular compact calli and fast growing calli, yielding respectively 5% and 100% of “mantled” plantlets, this decrease was up to 4.5% (from 23.2 to 18.7%). An alternative method, the SssI-methylase accepting assay, based on the enzymatic saturation of CG sites with methyl groups, gave convergent results. This work demonstrates that a correlation exists between DNA hypomethylation and the “mantled” somaclonal variation in oil palm.

Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis

An improved protocol is described for the large-scale micropropagation of an elite date palm (Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l-1 2,4-dichlorophenoxyacetic acid with 300 mg l-1 charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.

An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, Phoenix dactylifera L., cv. Deglet Nour

Flow cytometric analysis performed on two different crosses of dura×pisifera oil palm gave an accurate estimation of nuclear DNA content. The genome size of Elaeis guineensis was found to be 2C=3.76±0.09 pg and therefore ca. 3.4×109 bp. Embryogenic calli and plants showed the same ploidy level, but the measured 2C DNA values differed significantly. No variation in the ploidy level between three different types of calli originating from foliar explants, namely nodular compact callus, fast-growing callus and friable callus was observed. Since fast-growing callus (FGC), already identified as a source of `mantled' phenotype variants, did not show any difference in their ploidy level, these results are consistent with the hypothesis of an epigenetic origin for this type of somaclonal variant.

Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants.

Random amplified polymorphic DNA (RAPD) analysis using arbitrary 10-mer oligonucleotide primers was employed in order to investigate the genetic fidelity of somatic embryogenesis-derived regenerants of oil palm (#Elaeis guineensis# Jacq). Clonal palms bearing the "mantled" phenotype were identified in the field and the ability of RAPD markers to distinguish these variants from palms of the normal type was assessed. Of the 387 arbitrary primers used, 259 (67%) were successfully used to amplify oil palm DNA genomic fragments with consistently reproducible banding. Of these 387 primers, 73 (19%) primers enabled the identification of polymorphism between clones. No intraclonal variability and no differences between mother palms and regenerants could be identified using the total number of markers scored (8900). Twenty-four of these 73 primers were chosen for use in a larger experiment aimed at comparing, first, the mother palm genome with that of its clonal offspring and, second, true-to-type and variant regenerants. Thus, the regeneration protocol based on somatic embryogenesis set up for oil palm clonal propagation does not induce any gross genetic changes. The results obtained revealed however, that the RAPD approach is not suitable for the detection of the "mantled" variant phenotype. The use of RAPD markers for the detection of somaclonal variation in oil palm is discussed. (Resume d'auteur)

/pdf/suitability-of-rapd-analysis-for-the-detection-of-somaclonal-zo63ftka9n.pdf

Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq)

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.

/pdf/search-for-methylation-sensitive-amplification-polymorphisms-17l7tefhu2.pdf

Alain Rival

Papers

Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis

An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, Phoenix dactylifera L., cv. Deglet Nour

Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants.

Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq)

Search for methylation-sensitive amplification polymorphisms associated with the "mantled" variant phenotype in oil palm (Elaeis guineensis Jacq.)