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Beltsville, MD, United States

Ozel C.A.,Gazi University | Kamo K.,Floral and Nursery Plants Research Unit
Acta Horticulturae

Conditions were optimized for transient transformation of Lilium longiflorum 'Nellie White' using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for/7-glucuronidase, GUS expression. Transformed bulb scales showed transient GUS expression when they had been precultured 11 days on Murashige and Skoog's (MS) medium supplemented with 2 mg/L dicamba. The outer, larger-sized bulb scales were not infected nearly as well as the inner, smaller bulb scales. Maximum GUS expression occurred when bulb scales had been obtained from plants that had been grown in the dark for at least 2 months rather than in the light indicating the importance of growing plants in the dark for Agrobacterium- mediated transient transformation of bulb scales. Basal meristems taken from plants grown 4 months in the dark showed 3 times as much GUS expression as basal meristems from plants grown in a 12 h light photoperiod. The frequency of transient GUS expression achieved in this study indicated that it should be possible to achieve stable transformation of 'Nellie White' which is the cultivar that dominates the US market of Easter lilies. Experiments for stable transformation are in progress. Source

Seo E.-Y.,Chungnam National University | Kim H.-S.,Chungnam National University | Kim J.-K.,Chungnam National University | Gotoh T.,Kyushu University | And 2 more authors.
Journal of the Faculty of Agriculture, Kyushu University

Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS vector to contain the Gateway system for high throughput cloning (Ko et al., J. Fac.Agr., Kyushu Univ., 60(1), 139-149 (2015)), and utilized this system to express a library of N. benthamiana cDNA. Random c.300 bp N. benthamiana cDNA fragments were generated by ultrasonication and inserted into the TRV VIGS vector by Gateway cloning. N. benthamiana were agroinfiltrated with randomly selected TRV cDNA constructs in Agrobacterium tumefaciens GV 2260. Distinct visible phenotypes were identified in three sets of the inoculated N. benthamiana plants. The three distinguished phenotypes showed leaf malformation and necrosis. The three expressed gene inserts were homologous to EST fragments identified as CK290013.1, CK296346.1, and AM8112161.1, and presumably these genes are related to TRV pathogenesis in N. benthamiana. Identification of the selected genes by VIGS will aid further analysis to determine the relationship between VIGS phenotype and gene function. Source

Han J.-Y.,Chungnam National University | Kim J.-K.,Chungnam National University | Cheong J.-S.,Chungnam National University | Seo E.-Y.,Chungnam National University | And 8 more authors.
Journal of the Faculty of Agriculture, Kyushu University

Due to the absence of knowledge of the distribution of Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) in apples in Korea, we carried out a survey for these viruses in Gyeongsang and Chungcheong provinces in 2014. A total of 65 samples were collected and tested by RT-PCR using ASGV and ACLSV specific primers. ASGV was detected in 22 samples, and ACLSV in three samples; two of the samples showed double infection of ASGV and ACLSV. Phylogenetic analysis suggests that Korean ASGV and ACLSV were introduced from other countries. Prevalence of ASGV and ACLSV indicates that virus prevention and control may be poorly managed in orchards. Since fruit trees remain in the orchard for many years and it is not possible to eliminate virus from infected trees, healthy scions and virus-resistant rootstocks must be used for virus control. Because it is difficult to visually distinguish ASGV-infected and ACLSV-infected apple trees from healthy trees, thorough surveys by molecular biology methods must be performed routinely. Source

Merewitz E.B.,Rutgers University | Belanger F.C.,Rutgers University | Warnke S.E.,Floral and Nursery Plants Research Unit | Huang B.,Rutgers University
Crop Science

Identification of quantitative trait loci (QTL) for drought tolerance will allow for detection of important genomic regions associated with specifi c drought tolerance traits. This study aimed to identify QTL for drought tolerance traits in a colonial bentgrass (Agrostis capillaris L.) × creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. The traits included relative water content (RWC), turf quality (TQ), electrolyte leakage (EL), chlorophyll content (CHL), normalized difference vegetation index (NDVI), and canopy temperature depression (CTD). A colonial × creeping bentgrass hybrid backcross population segregating for drought tolerance was exposed to drought stress by withholding irrigation in three different locations or environmental conditions (two greenhouse studies in two different years, 2009 and 2010, and a growth chamber study in 2010). Phenotypic trait data were collected in these studies. Significant variation in the parents and the progenies occurred for phenotypic traits and the broad-sense heritability values indicated that the traits were polygenic in nature. A total of 32 potential QTL of varying effects (31 major and 1 minor) were detected on seven chromosomes. Significant overlap of QTL was found, particularly for CHL and CTD on 1A1, CTD, CHL, EL, and NDVI on linkage group 2A1, CTD and TQ on 2A2, TQ and RWC on 5A1 and 5A2, and TQ and CHL on 5A2. The QTL with the strongest effect for each trait (highest likelihood of the odds [LOD] and percent of phenotypic variance explained by the QTL) were CTD (4.19, 18.8%), CHL (4.23, 18.9%), EL (3.25, 14.9%), and NDVI (4.12, 22.4%) on group 2A1, RWC (3.27, 15.7%) on group 5A1, and TQ (4.38, 19.5%) on group 5A2. The analysis of potential genes in the QTL regions for these physiological traits indicate that the QTL may be linked to metabolic factors involved in N metabolism and energy metabolism such as photosynthesis and respiration. The QTL regions identified here could contain important genetic factors conferring drought tolerance in bentgrass species. © Crop Science Society of America. Source

Amaradasa B.S.,University of Nebraska - Lincoln | Horvath B.J.,University of Tennessee at Knoxville | Lakshman D.K.,Floral and Nursery Plants Research Unit | Warnke S.E.,Floral and Nursery Plants Research Unit

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNAITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n 5 18), AG 2-2IIIB (n 5 30) and AG 5 (n 5 1) clustered separately. Waitea circinata var. zeae (n 5 9) and var. circinata (n 5 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n 5 16) clustered into two groups. RhizoctoRhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infraspecies level. © 2013 by The Mycological Society of America. Source

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