Use of RNAi to evaluate the role of isoflavones in resistance of red clover to Sclerotinia trifoliorum and Sclerotium rolfsii

Red clover (Trifolium pratense L.) is one of the important forage crops in most temperate agricultural regions. In addition to the importance of red clover in nitrogen fixation and animal feeding, it has also drawn attention because of high levels of the secondary metabolite isoflavones which possess antifungal activity (Saviranta et al., 2008). The main disadvantage of red clover is its insufficient persistence (Hejduk, 2011). Therefore, persistence (individual plant longevity) is the main goal of most of red clover breeding programs (Taylor, 2008).Because fungal diseases are one contributing factor to the lack of persistence of red clover, improving fungal disease resistance should help to improve persistence. Using isoflavones as the plants’ own defense arsenals can be an environmentally friendly strategy to strengthen plant resistance to fungal pathogens. In this study, we decided to evaluate the possibly significant role of isoflavones in a red clover defense system against the fungal pathogens Sclerotinia trifoliorum and Sclerotium rolfsii using biosynthesis engineering of the corresponding gene. The first step toward achieving this goal is to set up effective regeneration and transformation systems, which are the prerequisite any biosynthesis engineering. In order to establish an efficient regeneration system in red clover, highly regenerative genotypes were used to evaluate the suitability of different media sequences to maximize the regeneration capacity of the plant. The media sequence KBC-SPL-SPL significantly increased the efficiency of somatic embryogenesis of red clover. To develop an efficient gene transfer system, some parameters were investigated, including evaluation of a suitable concentration of the selection agent, examination of the effect of pre-culturing of explants on the efficiency of transformation and comparison of different Agrobacterium strains for the efficiency of gene transformation. Based on the selection agent test, 75 mg/l of kanamycin sulfate was an appropriate concentration for transformant selection. The effect of pre-culturing explants was increased slightly by the frequency of gene transferring. The highly infective Agrobacterium strain C58C1 can increase the transformation efficiency for red clover to 83%. PCR and histochemical GUS assay of the transformed plants confirmed successful integration of the T-DNA into the red clover genome. In addition to an efficient gene transformation system, a tool is needed to enable careful monitoring of changes of the manipulated gene conducted by biosynthesis engineering. Quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) is the most robust tool available to detect any changes in expression of studied genes (Vandesompele et al., 2002). However, before qRT-PCR can be used in this way, one must determine the appropriate reference genes for normalization of gene expression. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All of these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. But this assumption is likely to be violated in practice. We therefore suggested a simple and novel stability index based on the analysis of variance model, which is free from the assumption made by the algorithms. We identified several reference genes with a high level of stability using our own model-based method. Then an IFS-RNAi hairpin construct was made using the gateway cloning system. Subsequently, gene transformation was successfully performed with an Agrobacterium harboring the hairpin transgene. The putative transformant lines were achieved using the regeneration protocol. Transgenic lines with a functional construct were screened using RT-qPCR. The five chosen transgenic lines which showed IFS down-regulation, plus the wild type plants, were transferred to the greenhouse for propagation. Then the collected leaves of IFS-RNAi transgenic red clover and wild type, which served as control, were exposed to the two soilborne fungal diseases S. trifoliorum and S. rolfsii in pathogenicity assays using a detached leaf method. The result obtained from orthogonal contrasts demonstrated that the difference between wild type line versus transgenic lines (isoflavone-null lines) were found to be significant for S. trifoliorum and highly significant for S. rolfsii. The present study confirms the significant role of isoflavones in disease resistance. The use of isoflavones is suggested in resistance breeding programs to improve red clover persistence

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L.) cultivars using AFLP

Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation

A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.)

Determination of appropriate reference genes is crucial to normalization of gene expression data and prevention of biased results in qRT-PCR studies. This study is the first attempt to systematically compare potential reference genes to detect the most constitutively expressed reference genes for accurate normalization in red clover tissues including leaves, stems and roots. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. However, this assumption is likely to be violated in practice. The authors therefore suggest a simple and novel stability index based on the analysis of variance model which is free from the assumption made by the algorithms. We assessed the expression stability of eight candidate reference genes including actin (ACT), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor-1alpha (EF-1 alpha), translation initiation factor (EIF-4a), ubiquitin-conjugating enzyme E2 (UBC2), polyubiquitin (UBQ10), sand family protein (SAND) and yellow-leaf-specific protein 8 (YLS8). Our results indicated that UBC2 and UBQ10 ranked as the two most stably expressed genes in leaf tissue. UBC2 and YLS8 were defined as optimal control genes for stem tissue. EIF-4a and UBC2 were found to be the most stable reference gene for root tissue. GAPDH and SAND showed relatively low stability in expression study of red clover. When all tested tissues were considered, we observed that YLS8 and UBC2 showed remarkable stability in their expression level across tissues

Planta DOI 10.1007/s00425-012-1682-2 ORIGINAL ARTICLE

A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.

Improvement of plant regeneration and Agrobacterium-mediated genetic transformation efficiency in red clover (Trifolium pratense L.)

Red clover (Trifolium pretense L.) is one of the important forage crops in most temperate agricultural regions. Red clover germplasm has limited genetic variation for disease resistance. Application of an efficient gene transformation protocol to introduce genes against diseases is considered the only approach for developing new resistant cultivars. In this study, in order to establish an efficient regeneration system in red clover, highly regenerative genotypes were utilized for evaluation of different media sequences for their suitability to utilize potentiality of regeneration capacity. Media sequence KBC-SPL-SPL significantly increased the efficiency of somatic embryo genesis of red clover. There was no relationship between regeneration ability and fresh callus weight. Also, no interaction was observed between genotypes and media sequences for fresh callus weight, number of embryos and number of developed shoots. To develop an efficient gene transfer system, some parameters were investigated, including evaluation of suitable concentration of selection agent, examination of the effect of pre-culturing of explants on efficiency of transformation and comparison of different Agrobacterium strains for efficiency of gene transformation. The effect of pre-culturing explants was increased slightly by the frequency of gene transferring. Highly infective Agrobacterium strain C58C1 can increase the transformation efficiency for red clover to 83%. PCR and histochemical GUS assay of transformed plants confirmed successful integration of the T-DNA into the red clover genome. Our established system will be a platform for genetic manipulation of red clover to broaden its genetic variation and for germplasm innovation