Agromorphologic, genetic and methylation profiling of Dioscorea and Musa species multiplied under three micropropagation systems

Abstract

Plant in vitro vegetative propagation using classical semi-solid culture medium is limited due to the low degree of automation, suboptimal nutrient availability and induced physiological stress which often reduce its efficiency. Temporary Immersion System (TIS) emerged as an innovative approach to optimize and eliminate the drawbacks associated with the conventional system of micropropagation. In this study, both Dioscorea and Musa spp. were subjected to conventional semi-solid culture media, complete immersion in shaking liquid culture media and TIS using RITA bioreactor. In vitro grown plantlets were screened for possible vegetative changes using agro-morphological descriptors while genetic and methylation differences were assessed using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). In vitro results showed that the number of shoots produced in Musa spp. varied significantly (P≤0.001) with the type of culture system. The highest mean shoot produced was observed with TIS (28.40) and the least using semi-solid culture medium (1.13). For Dioscorea spp., there was no significant interaction between the hormone combination and the culture system. However, the lowest mean shoot value (1.55) was observed in the semi-solid culture medium. Genetic analysis via AFLP using 15 primer pair combinations revealed that the 3 culture systems maintained genetic variation for Musa and Dioscorea spp. under in vitro and field conditions. Results showed 99% and 91% of the total bands were polymorphic under in vitro and field conditions respectively for Musa and 100% polymorphism for Dioscorea under in vitro and field conditions. Methylation investigation via MSAP using 12 primer pair combinations showed 25% and 46% polymorphic methylated-sensitive loci, 100% and 78% of non-methylated loci of the total bands generated under in vitro and field conditions respectively. Unmethylated (HPA+/MSP+) levels were highest in TIS (0.0842) as compared to CI (0.0227) and SS (0.0161) while full methylation or absence of target (HPA-/MSP-) was lowest in TIS (0.5890) and highest in SS (0.7138). For Dioscorea , 52% and 53% methylated sensitive loci and 100% non-methylated loci were polymorphic under in vitro and field conditions respectively. Although in vitro plant tissue culture techniques led to methylation at some loci of both species, there were no observable changes in the phenotype of both crops under field conditions. This also confirmed that not all methylation events lead to phenotypic changes.

A novel temporary immersion bioreactor system for large scale multiplication of banana (Rasthali AAB—Silk)

“…The occurrence of genetic variation in micro-propagated banana plants mainly due to the use of a higher concentration of growth regulators and an increased number of subcultures as a response to the stress imposed on the plant during the culture period. It has been demonstrated in the form of DNA methylations, chromosome rearrangements and point mutations 16 , 27 , 58 , 59 . To access the genetic uniformity, DNA-based molecular markers of ISSR are widely used 60 .…”

“…The high production cost prevents small- or medium-scale laboratories with limited resources from accessing the potential benefits of plant tissue culture technology 15 . Recent studies have been proposed to utilize automated temporary immersion systems (TIS), which is less expensive and provides an optimal environment for in vitro plant cell, tissue and organ cultures with good productivity 16 – 18 . Over the years, several temporary immersion systems have been developed and successfully applied for in vitro propagation of banana 19 – 25 .…”

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

“…With the development of knowledge on tissue cultures, it has been proven that the conditions used in vitro may lead to changes in regenerated plants. Apart from morphological [ 60 ] also genetic [ 61 ] and epigenetic [ 39 , 42 , 62 ] alterations between regenerants and donor plants are observed and quantified by the metAFLP approach [ 42 ]. The metAFLP approach showed that it is sufficiently informative for analysis of even closely related species as well as tissue culture-derived regenerants [ 63 , 64 ].…”

Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

“…Some studies on in vitro culture related to conservation procedures (slow growth and cryopreservation) do not report how many DNA samples from each experimental condition were used to perform the MSAP analysis. In some cases, a single sample was used per experimental condition, it being sometimes a pool of several samples [ 111 , 121 , 122 , 123 ]. Or the fragments obtained from the different analyzed samples were counted together without considering the variability between the samples within each experimental condition.…”

“…When a statistical analysis was carried out, most of the published works used the multivariate techniques proposed in the studies of population epigenetics [ 80 , 98 , 123 ]. These methods are suitable in the context of population epigenetics, where a large number of individuals within the population are measured and the interest is to quantify diversity based on differences of DNA methylation markers between populations.…”