Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications

Chiong, Kelvin; Damaj, Mona B.; Padilla, Carmen S.; Avila, Carlos A.; Pant, Shankar R.; Mandadi, Kranthi K.; Ramos, Ninfa; Carvalho, Denise V.; Mirkov, T. Erik

doi:10.1186/s13007-017-0255-6

Cited by 17 publications

(11 citation statements)

References 51 publications

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“…www.nature.com/scientificreports www.nature.com/scientificreports/ DnA isolation and quantitative real-time pcR (qpcR). Leaf DNA was isolated according to Rezadoost and Chiong 37,38 with minor modifications as follow: leaf samples were finely chopped, and ~200 mg of leaf tissues was taken in 2 ml screw-cap microcentrifuge tubes 18 . The samples were homogenized for 45 s at 5000 rpm with the Precellys 24 homogenizer (MO BIO Laboratories, Carlsbad, CA, USA) in the presence of two steel BB air gun beads (BB refers to the bead size, 4.5 mm-diameter) (Walmart Supercenter, Bentonville, AR, USA).…”

Section: Methods Plants Materialmentioning

confidence: 99%

Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics

Sanchez

Pant

Mandadi

et al. 2020

Sci Rep

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Raman spectroscopy (RS) is an emerging analytical technique that can be used to develop and deploy precision agriculture. RS allows for confirmatory diagnostic of biotic and abiotic stresses on plants. Specifically, RS can be used for Huanglongbing (HLB) diagnostics on both orange and grapefruit trees, as well as detection and identification of various fungal and viral diseases. The questions that remain to be answered is how early can RS detect and identify the disease and whether RS is more sensitive than qPCR, the "golden standard" in pathogen diagnostics? Using RS and HLB as case study, we monitored healthy (qPCR-negative) in-field grown citrus trees and compared their spectra to the spectra collected from healthy orange and grapefruit trees grown in a greenhouse with restricted insect access and confirmed as HLB free by qPCR. Our result indicated that RS was capable of early prediction of HLB and that nearly all in-field qPCR-negative plants were infected by the disease. Using advanced multivariate statistical analysis, we also showed that qPCR-negative plants exhibited HLB-specific spectral characteristics that can be distinguished from unrelated nutrition deficit characteristics. These results demonstrate that RS is capable of much more sensitive diagnostics of HLB compared to qPCR. Plant diseases can be caused by a variety of different pathogens, such as viruses, fungi and bacteria. Huanglongbing (HLB), or citrus greening, is a devastating disease that plagues citrus trees in Asia, Africa, and more recently the Americas 1. In the United States, HLB is associated with Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium that inhabits the plant phloem in uneven and variable titers 2,3. Limited knowledge is available on CLas bacterium due to our inability to culture it in the laboratory. The bacterium is transmitted by the highly mobile Asian citrus psyllid (Diaphorina citri) enabling proliferation of HLB on large agricultural areas. The disease is also able to spread by grafting. In late stages, HLB-infected plants have asymmetric mottling on the leaves and lopsided and green fruits. HLB causes fruit drop and poor fruit quality, e.g. smaller size, less juice yield, higher acidity and lower sugar content. Early stages of the disease, however, have minimal to no symptoms and the bacteria can remain undetectable even by quantitative polymerase chain reaction (qPCR) in the trees for several years after initial infection 4. This, wherein, lies a significant challenge in regard to HLB management. Farmers are reluctant to remove trees that have normal, healthy appearances and keep producing fruits. However, if such trees have CLas bacteria, they allow for psyllids to quickly spread this devastating disease over large distances. Therefore, it becomes critical to detect HLB at the early (pre-symptomatic) stage and develop appropriate intervention strategies for HLB containment. In disease diagnostics, the most predominant methods used to confirm a pathogen are PCR and/or enzyme-linked immunosorbent as...

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Section: Methods Plants Materialmentioning

confidence: 99%

Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics

Sanchez

Pant

Mandadi

et al. 2020

Sci Rep

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“…We verified the presence of the ShGPCR1 and bar (selectable marker) genes in co-transformed sugarcane plants by PCR using the forward primer (5'-GATGCTCACCCTGTTGTTTG-3') from the Ubi promoter and the reverse primer (5'-GACAGATCGAGCTCTGACTAGG-3') from the Tnos terminator. We performed PCR on a ProFlex™ PCR System in a total reaction volume of 25 μl using 100 ng of genomic DNA (isolated from 0.5-1 g of young leaves of 3-4-month-old plants using the protocol of Chiong et al, 2017), 0.1 μM of each target-specific primer, and 1.0 U of Taq DNA polymerase and ThermoPol™ buffer (New England BioLabs, Ipswich, MA, United States). PCR conditions were as: one denaturing cycle at 95°C for 30 s, 30 cycles each at 95°C for 15 s, 52°C for 15 s, and 68°C for 1 min, and a final extension cycle at 68°C for 5 min.…”

Section: Sugarcane Transformation and Selection Of Transgenicsmentioning

confidence: 99%

A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses

Ramasamy¹,

Damaj²,

Vargas-Bautista³

et al. 2021

Front. Plant Sci.

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Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.

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“…Controls included vector-transformed lines and non-transformed plants (tissue culture-derived). Genomic DNA was isolated by grinding leaf tissues (3 g) collected from 3-to 4-month-old transgenic sugarcane plants in liquid nitrogen as previously reported (Tai and Tanksley, 1990;Chiong et al, 2017).…”

Section: Transgenic Plant Screeningmentioning

confidence: 99%

High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane

Padilla¹,

Damaj²,

Yang

et al. 2020

Front. Bioeng. Biotechnol.

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Sugarcane and energy cane (Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg −1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 (Ubi) promoter. Co-expression of recombinant GNA from stacked multiple promoters (pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg −1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg −1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg −1 tissue). Purification by mannoseagarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications.

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Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications

Cited by 17 publications

References 51 publications

Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics

Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics

A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses

High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane

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