Estimation of Heterografting Associated DNA Methylation Changes in Tree Crops by MSAP Analysis.

The genetic incompatibility of the seedlings which are used as rootstocks (stock-scion interactions) and the mechanical stress induced by grafting are two major factors responsible for the high intraclonal variations observed in tree crops which are propagated through bud grafting. Since stress-induced DNA methylation changes associated with heterografting is a major contributor of such variations in grafted tree crops, a proper assessment of this epigenetic phenomenon is inevitable to devise strategies for the development of more uniform planting materials with minimal intraclonal variations in the future. In order to evaluate and establish the effects of heterografting on the epigenome of plants, availability of ideal plant materials and a standard procedure for testing is very essential. Development of genetically uniform own-rooted seedlings through induction of cleavage polyembryony by a novel technique of half ovulo embryo culture is the first step. Grafting of buds from these genetically and epigenetically uniform plants to genetically divergent rootstock and identification of DNA methylation polymorphism among them forms the second part of the methodology for detecting epigenetic changes associated with grafting in tree crops. Methylation-sensitive amplification polymorphism technique (MSAP), a modified version of AFLP using a pair of methylation-sensitive and insensitive isoschizomers (such as HpaII and MspI), is an ideal methodology to assess DNA methylation polymorphisms on a genomic scale in such plants. Comparative analysis of two sets of restriction digestion products (EcoRI/HpaII and EcoRI/MspI) allows the identification of DNA methylation polymorphisms induced by grafting and will aid in the detection of differentially methylated regions (DMRs) among grafted plants. This chapter describes a detailed protocol for inducing multiple embryos of single zygotic origin and regeneration of seedlings in rubber tree (Hevea brasiliensis), grafting of buds from these genetically uniform own-rooted seedlings to divergent rootstocks, identification of epigenetic changes induced by grafting or stock-scion interactions through MSAP analysis, and locating the differentially methylated genomic region. The methodology described here could be applied to any tree species commercially propagated through grafting for detecting epigenetic changes putatively associated with intraclonal variability.

Heterografting induced DNA methylation polymorphisms in Hevea brasiliensis.

MAIN CONCLUSION: Heterografting induced intraclonal epigenetic variations were detected among rubber plants. Interaction between genetically divergent root stock and scion tissues might have triggered these epigenetic changes which may eventually lead to intraclonal variability in rubber. DNA methylation in response to stress may be associated with the alteration in gene transcription leading to morphological changes in plants. Rubber tree is commercially propagated by bud grafting where the scion of a high yielding variety is grafted on to a genetically divergent root stock. Still, significant levels of intraclonal variations exist among them. Epigenetic changes associated with heterografting may be partly responsible for this conundrum. In the present study, an attempt was made to identify the impact of divergent root stock on the epigenome of scion in grafted rubber plants. Heterografts were developed by grafting eye buds from a single polyembryony derived seedling on to genetically divergent root stocks of unknown parentage. The plants were uniformly maintained and their DNA was subjected to MSAP analysis. Polymorphic DNA methylation bands corresponding to CG as well as the plant-specific CHG types of methylation were observed. Cloning of selected polymorphic regions and bisulfite sequencing confirmed the presence of methylation in the promoter and coding region of important genes including an LRR receptor kinase gene. Since divergent root stock is the major factor differentiating the grafted plants, the changes in DNA methylation patterns might have been triggered by the interaction between the two genetically different tissues of stock and scion. The study assumes importance in Hevea, because accumulation and maintenance of epigenetic changes in functional genes and promoters during subsequent cycles of vegetative propagation may contribute towards intraclonal variability eventually leading to altered phenotypes.

Genetic and epigenetic uniformity of polyembryony derived multiple seedlings of Hevea brasiliensis.

Hevea brasiliensis Muell. Arg (Para rubber tree) is a tropical tree species of Amazonian origin widely cultivated in several parts of the world for natural rubber, a highly priced commodity inevitable for the world rubber industry. Large, tree to tree variation in growth and latex yield among individual plants of high yielding Hevea clones is a common phenomenon observed in mature rubber plantations. The genetic heterogeneity of the seedlings which are used as rootstocks for propagation through budgrafting is considered as a major factor responsible for this variation. In order to minimize this variation, attempts were made to develop highly uniform rootstock material via an in vitro technique by inducing zygotic polyembryony in Hevea. Immature open pollinated fruits of a high yielding clone RRII 105 were cultured by half ovulo embryo culture technique. Multiple embryos were induced from the 8-10-week-old zygote with a novel combination of gibberellic acid (GA3), kinetin, and zeatin. Plantlets were successfully generated from the multiple embryos and raised in the field post hardening. Screening using genetic and epigenetic molecular markers revealed that the multiple seedlings developed are highly uniform and are of single zygotic origin. Development of plants having genetic and epigenetic uniformity suggests that this technique is ideal for raising uniform rootstock material in Hevea which may significantly reduce intraclonal variations. Moreover, these plants could serve as ideal material for physiological and molecular investigations towards the understanding of stock-scion interaction process in rubber.