Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.

The development of a formulation for siRNA delivery enables posttranscriptional gene silencing in plants with a simple spray application.

Foliar application of dsRNA to elicit an RNA interference (RNAi) response is currently under consideration as a crop protection strategy. To access the RNAi machinery of a plant, foliarly applied dsRNAs must traverse the plant cuticle, avoid nuclease degradation, and penetrate the cell wall and plasma membrane. Application methods and co-formulants have been identified by Bayer Crop Science researchers and others that can help bypass barriers to dsRNA uptake in plants leading to an RNAi response in greenhouse grown, young plants and cell cultures. However, these advances in dsRNA delivery have yet to yield systemic RNAi silencing of an endogenous gene target required for product concepts such as weed control. Systemic RNAi silencing in plants has only been observed with the GFP transgene in Nicotiana benthamiana. Because biologically meaningful whole plant RNAi has not been observed for endogenous gene products in N. benthamiana or in other plant species tested, under growing conditions including field production, the regulatory risk assessment of foliarly applied dsRNA-based products should not consider exposure scenarios that include systemic response to small RNAs in treated plants.

The Initiation of RNA interference (RNAi) by topically applied double stranded RNA (dsRNA) has potential applications for plant functional genomics, crop improvement and crop protection. The primary obstacle for the development of this technology is efficient delivery of RNAi effectors. The plant cell wall is a particularly challenging barrier to the delivery of macromolecules. Many of the transfection agents that are commonly used with animal cells produce nanocomplexes that are significantly larger than the size exclusion limit of the plant cell wall. Utilizing a class of very small nanoparticles called carbon dots, a method of delivering siRNA into the model plant Nicotiana benthamiana and tomato is described. Low-pressure spray application of these formulations with a spreading surfactant resulted in strong silencing of GFP transgenes in both species. The delivery efficacy of carbon dot formulations was also demonstrated by silencing endogenous genes that encode two sub-units of magnesium chelatase, an enzyme necessary for chlorophyll synthesis. The strong visible phenotypes observed with the carbon dot facilitated delivery were validated by measuring significant reductions in the target gene transcript and/or protein levels. Methods for the delivery of RNAi effectors into plants, such as the carbon dot formulations described here, could become valuable tools for gene silencing in plants with practical applications in plant functional genomics and agriculture.

No abstract

Abstract22 nt miRNAs or siRNAs have been shown to specifically induce production of transitive (secondary) siRNAs for targeted mRNAs. An abrasion method to deliver dsRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for 3 endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GUN4. However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs, but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also had more transitive siRNAs produced in response to 22 nt siRNAs, but the response varied from weak (CHL-I) to strong (CHL-H). 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for GFP, CHL-H and GUN4 in N. benthamiana. The special activity of 22 nt siRNAs in producing a greater local phenotype and induction of elevated levels of transitive siRNAs was also shown in A. cruentus for the CHL-H gene. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

The cell cycle in cotton (Gossypium hirsutum) fibers is poorly understood. The objective of this study was to evaluate the cell cycle status and DNA content in developing cotton fibers. The DNA content and cell cycle distribution in fiber and hypocotyl cells were determined by flow cytometry. Expression levels of minichrosomal maintenance protein (mcm), cyclin B, and a retinoblastoma-like protein (rb) genes were determined with real-time PCR in fibers and dividing and nondividing tissues. No endoreduplication occurred, nor did genome size or percentage of G1-phase nuclei differ between hypocotyls and fibers. Approximately 13 and 17% of fiber nuclei were in the S phase in 14 days after anthesis (d) fibers and 25 d fibers, respectively. The mcm and cyclin B were expressed at higher levels in fibers than in mature leaves, but expression levels in fibers were less than 15% of meristematic tissues. Rb was expressed in fibers at levels less than 50% of mature leaves or meristematic tissues. Based on an apparent increase in S-phase cells as fibers mature and the low level of expression of genes associated with cell cycle progression, we conclude that S-phase arrest occurs in developing cotton fiber.

Main conclusion 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. Abstract RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.