A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.
Several important biological processes are performed by distinct functional domains found on replication-associated protein (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal 669 bp (5'AC1) and C-terminal 783 bp (3'AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens to make use of an interference strategy for impairing cotton leaf curl virus (CLCuV) infection in transgenic cotton. Compared with nontransformed control, we observed that transgenic cotton plants overexpressing either N-terminal (5'AC1) or C-terminal (3'AC1) sequences confer resistance to CLCuV by inhibiting replication of viral genomic and β satellite DNA components. Molecular analysis by Northern blot hybridization revealed high transgene expression in early and late growth stages associated with inhibition of CLCuV replication. Of the eight T(1) transgenic lines tested, six had delayed and minor symptoms as compared to nontransformed control lines which developed disease symptoms after 2-3 weeks of whitefly-mediated viral delivery. Virus biological assay and growth of T(2) plants proved that transgenic cotton plants overexpressing 5'- and 3'AC1 displayed high resistance level up to 72, 81%, respectively, as compared to non-transformed control plants following inoculation with viruliferous whiteflies giving significantly high cotton seed yield. Progeny analysis of these plants by polymerase chain reaction (PCR), Southern blotting and virus biological assay showed stable transgene, integration, inheritance and cotton leaf curl disease (CLCuD) resistance in two of the eight transgenic lines having single or two transgene insertions. Transgenic cotton expressing partial AC1 gene of CLCuV can be used as virus resistance source in cotton breeding programs aiming to improve virus resistance in cotton crop.
RNA silencing technology has become the tool of choice for inducing resistance against viruses in plants. A significant discovery of this technology is that double-stranded RNA (dsRNA), which is diced into small interfering RNAs (siRNAs), is a potent trigger for RNA silencing. By exploiting this phenomenon in transgenic plants, it is possible to confer high level of virus resistance by specific targeting of cognate viral RNA. In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have constructed a chimeric expression vector containing three partial gene sequences derived from the ORF2 gene of Potato virus X, Helper Component Protease gene of Potato virus Y and Coat protein gene of Potato leaf roll virus. Solanum tuberosum cv. Desiree and Kuroda were transformed with this chimeric gene cassette via Agrobacterium tumefaciens-mediated transformation and transgenic status was confirmed by PCR, Southern and double antibody sandwich ELISA detection. Due to simultaneous RNA silencing, as demonstrated by accumulation of specific siRNAs, the expression of partial triple-gene sequence cassette depicted 20% of the transgenic plants are immune against all three viruses. Thus, expression of a single transgene construct can effectively confer resistance to multiple viruses in transgenic plants.
Late spring coldness (LSC) is critical for wheat growth and development in the Huang-Huai valleys of China. However, little is known about the molecular mechanisms for young spikes responding to low temperature (LT) stress during anther connective tissue formation phase (ACFP). To elucidate the molecular mechanisms associated with low temperature, we performed a comparative transcriptome analysis of wheat cultivars Xinmai26 (XM26: cold-sensitive) and Yannong19 (YN19: cold-tolerant) using RNA-seq data. Over 4000 differently expressed genes (DEGs) were identified under low temperature conditions (T1: 4°C) and freezing conditions (T2: −4°C) compared with control (CK: 16°C). The number of DEGs associated with two cultivars at two low temperature treatments (T1: 4°C and T2: −4°C) were 834, 1,353, 231, and 1,882 in four comparison groups (Xinmai26-CK vs. Xinmai26-T1, Xinmai26-CK vs. Xinmai26-T2, Yannong19-CK vs. Yannong19-T1, and Yannong19-CK vs. Yannong19-T2), respectively. Furthermore, to validate the accuracy of RNA-seq, 16 DEGs were analyzed using quantitative real-time RT-PCR. Several transcriptome changes were observed through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analysis in plant hormone signal transduction, circadian rhythm-plant, and starch and sucrose metabolism under low temperature. In addition, 126 transcription factors (TFs), including AP2-ERF, bHLH, WRKY, MYB, HSF, and members of the bZIP family, were considered as cold-responsive. It is the first study to investigate DEGs associated with low temperature stress at the transcriptome level in two wheat cultivars with different cold resistance capacities. Most likely, the variations in transcription factors (TFs) regulation, and starch and sucrose metabolism contribute to different cold resistance capacities in the two cultivars. Further, physiological activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) enzymes, malondialdehyde (MDA), soluble sugar (SS), and sucrose contents were evaluated to investigate the negative impacts of low temperature in both cultivars. These findings provide new insight into the molecular mechanisms of plant responses to low temperature and potential candidate genes that required for improving wheat’s capacity to withstand low temperature stress.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.