Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
(N/A)
Non Technical Summary
Among the many types of vegetative propagation (cuttings, grafting, budding, layering, stooling, micropropagation, etc.), stem cutting is the most used method to fix and maintain desired genotypes in many woody species. This is due to features such as ease of operation and a low skill requirement. Some species, like willows and poplars, can be easily propagated without hormone induction via cutting, while others are notoriously recalcitrant, including Castanea dentata, the American chestnut (AC). This recalcitrance is due to the diminished ability of the species to form adventitious roots. Why this is so is largely unknown. An efficient rooted cutting system will allow rapid and clonal propagation of the American chestnut, a heritage species once dominating the eastern deciduous forests of the USA. This will accelerate the breeding process and facilitate germplasm conservation and restoration. The project aims to provide insights into the determinants leading to the recalcitrance to adventitious root formation, so that innovative and efficient technologies can be developed for root induction. The project employs an integrated approach that combines histology, physiology, transcriptomics, and metabolomics, and compares American chestnut with easy-to-root woody species.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
1) Identify location and cell type that form AC root primordia through a histological study.2) Reveal metabolites contributing to AC cuttings' recalcitrance to AR induction by comparison with easy-to-root woody species.3) Reveal gene modulation and endogenous phytohormones dynamics during AR induction in AC cuttings.4) Evaluate potential effects ofBacillus subtilison AR formation in AC cuttings.
Project Methods
Current-year semi-lignified shoots will be provided by the American Chestnut Foundation (see support letter). There are AC seedlings in our greenhouse. We will follow the protocol we have established. After testing different hormone treatments, humidity levels, and types of potting mix, we are now able to induce AR in AC stem cuttings. Briefly, shoots are cut into segments that are 10-15 cm in length, containing two leaves (cut to ~5 cm in length). After the basal parts of the cuttings are soaked in 0.1g/L NAA for an hour, cuttings are inserted into pots that have wet moss at the bottom and on the top with wet perlites in the middle. The pots with cuttings are kept in a temperature-controlled (22-23 °C) mist room with high humidity (mist is 14 minutes off and 6 seconds on). It takes ~1.5 months for AC AR to start emerging. Thus, maintaining a high-humidity environment is critical.1. Histology of AC AR formation:Since it takes ~1.5 months for AC AR to emerge, initially we will collect stem segments 0, 10, 20, 30, 40, and 50 days after hormone treatment. Extra collection dates will be added if necessary. Specimens are fixed in a formaldehyde/ethanol/acetic acid solution overnight at room temperature. Samples will be dehydrated in an ethanol series, infiltrated with xylene, and embedded in paraffin. Then, 15 μm-thick transverse sections will be cut with a rotatory microtome and stained with toluidine blue. Untreated cuttings will be used as a reference.2. Comparative metabolomics:Current-year semi-lignified shoots (AC, poplar, and willow) will be collected, and the basal part of each stem (1 cm in length) will be excised and quickly frozen in liquid N2. The samples will be stored at -80 °C before being ground into fine powder. Non-target metabolomics will be conducted in Clemson University's Multi-user Analytical Lab & Metabolomic Core. A total of 100 mg of each ground sample will be extracted with 1 ml of methanol. Primary metabolites (sugar, sugar alcohol, organic acids, phenolic acids, and amino acids) are analyzed using gas chromatography coupled to quadrupole time-of-flight mass spectrometer (GC-QToF). Compound identification is based on accurate mass, fragmentation pattern, and retention index. Secondary compounds are analyzed using liquid chromatograph coupled to an ultra-high resolution mass spectrometer. Putative IDs of compounds are generated through the matching of accurate mass (<2 ppm error) and fragmentation pattern with online,in silico, and in-house mass spectral libraries. As needed, absolute quantification is performed using authentic standards or isotope-labeled internal standards. Eight biological replicates will be included, and water content will be taken into account.3: Comparative transcriptomics and endogenous phytohormone dynamics of AC cuttings with and without auxin treatment:Samples of AC cuttings (basal part) with and without NAA treatment will be used for hormone analysis and RNA sequencing. Guided by the histological study results, we will incorporate four time points so that the period up to formation of the root primordia will be included. Six biological replicates will be included. Hormones will be analyzed in the Danforth Plant Science Center. RNA-seq data analysis and construction of gene co-expression networks and modules are to be conducted by the Clemson University Genomics and Bioinformatics Facility (CUGBF). Comparisons are to be performed between rooting and non-rooting samples, as well as among four timepoints. At least 40 million paired-end reads (2x 150bp) per sample will be obtained. This will give >= 15-fold genome coverage forC. dentata(800 Mb). High-quality reads are mapped to available reference genome sequences ofC. dentata(Phytozome13). Differential expression, hierarchical clustering, gene ontology (GO) and pathway enrichment analyses, and construction of gene co-expression networks and modules will be performed using the in-house pipelines in CUGBF. To validate the RNAseq results, a subset of 10 DEGs will be randomly chosen for quantitative reverse transcription-PCR (qRT-PCR). Primer design, setup of qRT-PCR reactions, and relative expression analysis will be performed. An intron-spanning primer pair will be used to detect genomic DNA contamination when possible. Four independent qPCR experiments per candidate gene for each cDNA sample will be included. Several house-keeping genes such as ACT, TUA, UBQ, and 18S, will be evaluated for their stability, and the one with a CT value that is closest to those of target DE genes will be chosen as the internal controls for normalization. The 2-ΔΔCTmethod will be used for the calculation of relative expression level of target genes.4: Evaluation of effects of B. subtilis on AR formation:We will prepare three major sets of AC cuttings, 1) NAA treatment only; 2)B. subtilisonly; and 3) NAA+B. subtilis. For treatments withB. subtilis, we will test the three strains (QST 713 and two local isolates) we have individually.Bacilluscultures are grown at room temperature overnight for an optical density of 0.6 at 600 nm wavelength in a volume of 200 ml before being poured into pots with cuttings weekly for a month. After two months, rooting rate, as well as root length and weight, will be recorded. After six months, length of new stem growth will be measured. There will be >= 10 cuttings per treatment. Cuttings are randomly arranged inside the mist room once a week. The data will be processed using SPSS software for statistical analyses.