The metabolic pathway and function of ethylene during the senescence of many fruits and flowers have been extensively studied, the molecular basis of ethylene-insensitive flower senescence remains unknown. The ephemeral flowers of daylily (Hemerocallis) were used as a model system for the examination of ethylene-insensitive senescence. Senescence-associated cDNA clones were isolated from a cDNA library constructed from mRNA expressed in senescing tepals of daylily flowers. Up-regulated cDNA clones were identified by differentially screening the cDNA library. Sequence analysis of one of the clones, designated as SEN12, indicates that it contains a MADS box domain and an associated leucine-zipper K-box region and may be a transcription factor similar to floral homeotic genes. Northern analysis indicates that SEN12 encodes for a rare message. Therefore, reverse transcriptase polymerase chain reaction (RT-PCR) assays were used to quantitate the abundance of SEN12 transcripts during floral senescence. RT-PCR assays demonstrated that SEN12 transcripts significantly increase in abundance during the earliest stages of flower senescence and continue to increase until the end of senescence. We propose that SEN12 may be involved in controlling senescence in ethylene-insensitive flowers and we are continuing to investigate this hypothesis.
Nathan E. Lange, Victoriano Valpuesta, Carolyn A. Napoli, and Michael S. Reid
Madhurababu Kunta, J.V. da Graça, and Mani Skaria
transcriptase–polymerase chain reaction (RT-PCR) technique as a quick method to screen a large number of trees for the presence of viroids in the virus-tested budwood program to improve the budwood program and help expedite introduction of new germplasm into the
Mohsen Mohseni-Moghadam, Christopher S. Cramer, Robert L. Steiner, and Rebecca Creamer
presence in the inoculum. Samples were collected in plastic bags, placed in an ice chest, and finally stored at –4 °C until testing. Samples were analyzed to confirm the presence of IYSV by the ELISA ( Copeland, 1998 ) and RT-PCR ( Cortês et al., 1998 ). In
Sandra E. Vega and Jiwan P. Palta
Previous studies in our laboratory both in pine needles and potato leaves have shown evidence of an increase in 18: 2 (linoleate) in the purified plasma membrane fraction during cold acclimation. This increase was reversible on deacclimation, thereby suggesting a link between the accumulation of 18: 2 and acquisition of freezing tolerance. These studies suggest that the activity of specific desaturases may be modulated during cold acclimation. This study was aimed at studying the possible involvement of stearoyl-ACP desaturase (delta9) in potato cold acclimation response. Our approach was to study the induction of delta9 desaturase at the transcript level by using potato delta9 desaturase gene specific primers and reverse transcriptase. For this purpose, mRNA from S. tuberosum (cold sensitive, unable to acclimate) and S. commersonii (cold tolerant, able to cold acclimate) was extracted before and after acclimation. Sequence analysis confirmed that the amplified band was delta9 desaturase. Our results show that there is an increase in delta9 desaturase gene transcripts during cold acclimation and that this increase is associated with the cold acclimation response in potato. These results together with previous reports on the increase in 18: 2 in the plasma membrane during cold acclimation give more evidence toward the involvement of stearoyl-ACP desaturase (delta9) in the potato cold response.
Sara Spiegel, Dan Thompson, Aniko Varga, and Delano James
An apple chlorotic leaf spot virus (ACLSV) isolate was detected by TAS-ELISA and RT-PCR in an ornamental dwarf flowering almond (Prunus glandulosa Thunb.). This plant, maintained at the Centre for Plant Health, Sidney, B.C., Canada, has been showing transient leaf symptoms during the spring seasons. A 390-bp fragment and a 1,350-bp product, in the RNA polymerase and the coat protein viral coding regions, respectively, were amplified by RT-PCR from the infected plant. A sequence comparison of the 390-bp fragment of this ACLSV isolate (designated as AL1292) with other published isolates, revealed a similarity of 81% to 84% at the nucleotide level and 88% to 100% at the amino acid level. In contrast to other ACLSV isolates, AL1292 has an exceptionally narrow range of experimental herbaceous and woody hosts, as determined by mechanical and graft inoculation assays. These standard bioassays may not be effective for the detection of the AL1292 isolate because of its limited host range. The results we report in this paper confirm P. glandulosa as a natural host of this virus. Currently it is not known how ACLSV is spread, other than by bud-grafting and possibly by root grafts. The use of virus-tested source plants for the preparation of planting material will minimize its spread.
P. Martínez-Gómez, M. Rubio, F. Dicenta, and T.M. Gradziel
Sharka [(plum pox virus (PPV)] mainly affects Prunus species, including apricot (Prunus armeniaca L.), peach (Prunus persica L.), plum (Prunus salicina Lindl., Prunus domestica L.), and, to a lesser degree, sweet (Prunus avium L.) and sour cherry (Prunus cerasus L.). Level of resistance to a Dideron isolate of PPV in seven California almond [P. dulcis (Miller) D.A. Webb], five processing peach cultivars, and two peach rootstocks was evaluated. In addition, almond and peach selections resulting from interspecific almond × peach hybridization and subsequent gene introgression were tested. Evaluations were conducted in controlled facilities after grafting the test genotypes onto inoculated GF305 peach rootstocks. Leaves were evaluated for PPV symptoms during three consecutive cycles of growth. ELISA-DASI and RT-PCR analysis were also employed to verify the presence or absence of PPV. Peach cultivars and rootstocks showed sharka symptoms and were ELISA-DASI or RT-PCR positive for some growth cycles, indicating their susceptibility to PPV. Almond cultivars and almond × peach hybrids did not show symptoms and were ELISA-DASI and RT-PCR negative, demonstrating resistance to PPV. Two (almond × peach) F2 selections as well as two of three backcrossed peach selections also showed a resistant behavior against the PPV-D isolate. Results demonstrate a high level of resistance in almond and indicate potential for PPV resistance transfer to commercial peach cultivars.
Beatrice Nesi, Debora Trinchello, Sara Lazzereschi, Antonio Grassotti, and Barbara Ruffoni
the sensitivity is low compared with recently introduced polymerase chain reaction techniques ( Spiegel, 2006 ). Reverse transcriptase–polymerase chain reaction (RT-PCR) was used for detection of a Tomato Aspermy Virus isolate that infected
Parminder S. Multani, Christopher S. Cramer, Robert L. Steiner, and Rebecca Creamer
. Plant samples were collected to confirm the presence of IYSV by ELISA ( Copeland, 1998 ) and reverse transcription–polymerase chain reaction (RT-PCR) ( Cortês et al., 1998 ). Samples were stored at –80 °C for 1 month before they were analyzed. Enzyme
Jingjing Kou, Zhihui Zhao, Wenjiang Wang, Chuangqi Wei, Junfeng Guan, and Christopher Ference
). Total RNA was reverse-transcribed with the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Biomedical, Dalian, China). Quantitative real-time reverse-transcription PCR (RT-PCR) was performed using SYBR ® Premix Ex Taq™ II kit (TaKaRa Biomedical
James M. Crosslin
., 2002 ). Sequence analysis suggests that this ORF is highly conserved among American and European isolates of TRV and thus has been widely used for detection of the virus by reverse transcription polymerase chain reaction (RT-PCR) in potato foliage and