Name: potato virus x-based microrna silencing (vbms) in potato.
Uploaded: 2020-05-11T13:30:00
Description: Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

We thank Dr. Yule Liu from Tsinghua University for providing the PVX-LIC vector. This work was supported by a start-up fund from the Texas A&#38;M AgriLife Research and the Hatch Project TEX0-1-9675 from USDA National Institute of Food and Agriculture to JS.

1. Grow Potato Plants.
<ol>
	<li>Propagate in vitro potato plants in culture tubes (25 x 150 mm) with Murashige and Skoog (MS) media plus&#160;Gamborg&#8217;s vitamin (MS basal salt mixture, Gamborg&#8217;s vitamin, 30 g/L sucrose, 3.5 g/L agar, pH = 5.7). Place the tubes in the growth room under 20&#8211;22 &#176;C, 16 h light/8 h dark photoperiod, and light intensity 120 &#181;mol/m2&#8729;s1. 
	NOTE: New shoots and roots normally develop in 1&#8211;2 weeks from plants. Propagate plants wit...

We present a PVX-based miRNA silencing system to characterize the function of endogenous miRNAs in potato by integrating the STTM design into the PVX vector. The VbMS system proved to be effective in silencing miRNA165/166 in potato, a highly conserved miRNA family across plant species.
The TM approach has been developed to interfere with the expression of miRNAs based on an artificial miRNA target mimic that is designed to create a mismatch loop at the expected cleavage site within the miRNA ...

Plant microRNAs (miRNAs) are characterized as 20&#8211;24 nucleotide-long, nuclear-encoded regulatory RNAs1 and play fundamental roles in almost every aspect of plant biological processes, including growth and development2,3, photosynthesis and metabolism4,5,6,7, hormone synthesis and signaling8,9, biotic and abiotic responses10,11,12,13, and nutrient and energy regulation14,15. The regulatory roles of plant miRNAs are well-programmed and fulfilled typically at post-transcriptional levels by either cleaving or translationally repressing the target mRNAs.
Tremendous progress has been made towards&#160;identification, transcriptional profiling, and target prediction of miRNAs in potato16,17,18,19,20,21. However, the functional characterization of miRNAs in plants, including potato, has lagged behind other organisms due to the lack of efficient and high-throughput genetic approaches. It is challenging to perform functional analysis of individual miRNA by standard loss-of function analysis, because most miRNAs belong to families with considerable genetic redundancy22. In addition, a single miRNA can control multiple target genes23 and several different miRNAs can modulate the same molecular pathway collaboratively24,25. These properties make it difficult to characterize the function of a specific miRNA or a miRNA family.
Much of the functional analysis of miRNAs has relied heavily on gain-of-function approaches that have obvious limitations. The artificial miRNA (amiRNA) method exploits the endogenous primary transcripts (pri-miRNAs) to produce miRNAs at a high level, leading to inhibition of target gene expression26,27,28,29. However, activation tagging and miRNA overexpression using a strong constitutive 35S promoter often lead to heightened expression of miRNAs that are not representative of in vivo conditions and therefore may not reflect the endogenous function of miRNAs30. An alternative approach has been developed involving expression of miRNA-resistant forms of target genes that contain uncleavable mutations in the binding and/or cleavage sites31,32,33. But this approach can also potentially cause misinterpretation of the phenotype derived from the miRNA-resistant target transgene due to transgenic artifacts. Therefore, conclusions from these gain-of-function studies should be drawn with caution34. Another major limitation of the above-described approaches is that they require transformation, which is labor-intensive and time-consuming. Furthermore, the transgene-dependent approaches are hardly applicable for transform-recalcitrant plant species. Therefore, it is essential to develop a fast and efficient loss-of-function approach to unravel the function&#160;of miRNAs.
To bypass the prerequisite of the transformation procedure, virus-based microRNA silencing (VbMS) has been established by combining the target mimic (TM) strategies with virus-derived vectors. In the VbMS system, artificially designed TM molecules are transiently expressed from a virus backbone, offering a powerful, high-throughput, and time-saving tool to dissect the function of plant endogenous miRNAs35,36. VbMS was initially developed in N. benthamiana and tomato with the tobacco rattle virus (TRV)35,36,37 and has been extended to Arabidopsis, cotton, wheat, and maize using various other virus expression systems, including potato virus X (PVX)38, cotton leaf crumple virus (ClCrV)39, cucumber mosaic virus (CMV)40,41,42, Chinese wheat mosaic virus (CWMV)43, and barley stripe mosaic virus (BSMV)44,45.
Potato (Solanum tuberosum) is the fourth most important food crop and the most widely grown noncereal crop in the world primarily because of its high nutritional value, high energy production, and relatively low input requirements46. Several features of potato make it an attractive dicotyledonous model plant. It is a vegetatively propagated polyploid crop with high outcrossing rate, heterozygosity, and genetic diversity. However, to date, there is no report characterizing the function of miRNAs in potato using VbMS. Here, we present a ligation-independent cloning (LIC)-adapted potato PVX-based VbMS approach to evaluate the function of miRNAs in potato plants38. We selected the miR165/166 family to illustrate the VbMS assay because the miR165/166 family and their target mRNAs and Class III homeodomain/Leu zipper (HD-ZIP III) transcription factors have been extensively characterized22,47,48. The HD-ZIP III genes are key regulators of meristem development and organ polarity, and suppression of miR165/166 function leads to increased expression of the HD-ZIP III genes, resulting in pleotropic developmental defects such as reduced apical dominance and aberrant patterns of leaf polarity22,35,38,41. The readily scorable developmental phenotypes correlated with silencing of miRNA165/166 enable accurate evaluation of the effectiveness of the PVX-based VbMS assay.
In this study, we demonstrate that the PVX-based VbMS system can effectively block the function of miRNAs in potato. Because the PVX-based virus-induced gene silencing (VIGS) system has been established in a number of potato varieties49,50,51,52, this PVX-based VbMS approach can be likely applied to a broad range of diploid and tetraploid potato species.

<table border="0" cellpadding="0" cellspacing="0" style="width:691px;" width="691">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">100 &#181;M dATP and 100 &#181;M dTTP</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">TQAC136</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3 M Sodium acetate, pH 4.0.</td>
			<td style="width:188px;">Teknova, Hollister, CA 95023, USA</td>
			<td style="width:120px;">#S0297</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetosyringone</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D2666-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chloroform</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0757-950ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dimethyl sulfoxide, DMSO</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D0798-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DTT</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0281-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">E. coli DB3.1</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for maintenance of PVX-LIC and pTRV2e containing the ccdB gene</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">E. coli DH5&#945;</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for the destination constructs generated by LIC cloning</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed</td>
			<td>ICL Specialty Fertilizers, Summerville, SC 29483, USA</td>
			<td style="width:120px;">G99260</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA, USA</td>
			<td>7958960001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isoamyl alcohol</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0944-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Koptec Pure Ethanol &#8211; 200 Proof</td>
			<td style="width:188px;">Decon Labs, King of Prussia, PA 19406 , USA</td>
			<td style="width:120px;">V1005M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MES</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">M0606-250G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MgCl2</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">MFCD00149781</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">M-MuLV Reverse Transcriptase</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0253L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Nano-drop spectrometer: NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">ND-ONEC-W</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Bio-Rad MyCycler PCR System</td>
			<td style="width:188px;">Bio-Rad, Hercules, California 94547, USA</td>
			<td style="width:120px;">170-9703</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Eppendorf Mastercycler pro</td>
			<td style="width:188px;">Eppendorf, Hauppauge, NY 11788, USA</td>
			<td style="width:120px;">950030010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pH meter</td>
			<td style="width:188px;">Sper Scientific, Scottsdale, AZ 85260, USA</td>
			<td style="width:120px;">Benchtop pH / mV Meter - 860031</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0883-400ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phytagel: Gellan Gum</td>
			<td>Alfa Aesar, Tewksbury, MA 01876, USA</td>
			<td style="width:120px;">J63423-A1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVX VIGS vector: PVX-LIC</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;">Zhao et al., 2016</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">4485697</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Real-time PCR reagent: KAPA SYBR&#174; FAST qPCR Master Mix (2x) Kit</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA 01887, USA</td>
			<td style="width:120px;">7959389001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Restriction enzyme: SmaI</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td>R0141S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Reverse transcription reagents: qScript cDNA SuperMix</td>
			<td>Quanta BioSciences, Gaithersburg, MD 20877 , USA</td>
			<td style="width:120px;">95107-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNA extraction Kit: E.Z.N.A. Plant RNA Kit</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">SKU: D3485-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNase Inhibitor Murine</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0314L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNAzol RT</td>
			<td style="width:188px;">Sigma-Aldrich, St. Louis, MO 63103, USA</td>
			<td style="width:120px;">R4533</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Soil: Metro-Mix 360</td>
			<td>Sun Gro Horticulture, Agawam, MA 01001-2907, USA</td>
			<td style="width:120px;">Metro-Mix 360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">T4 DNA polymerase and buffer</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0203S</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

potato virus x-based microrna silencing (vbms) in potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Figure 2 shows the PVX-STTM165/166 potato plants (Katahdin) with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins. More severe phenotypes such as trumpet-shaped leaf formation have also been observed. In contrast, no phenotypic abnormality was observed in the PVX control plants. These results show that the VbMS system was effective in suppressing endogenous miRNA function in tetraploid potato plants and the PVX-VbMS system was a robust genetic tool to deter...

Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

The traditional approach to analyzing the function of microRNAs in plants primarily relies on the generation of stable knockdown plants. This virus-based microRNA silencing system does not require tedious and time-consuming transformation. Virus-based microRNA silencing is a rapid and efficient tool for functional characterization of microRNAs in plants that can be applied to a broad range of diploid and tetraploid potato species.Demonstrating the procedure will be Jinping Zhao, a post-doctoral research associate from my laboratory. To design the target mimic module, insert a mismatch sequence into the reverse complement sequence of the microRNA of interest at the site corresponding to the 10th to 11th nucleotides of the microRNA. To amplify the short tandem target mimic fragment, set up a PCR reaction with each primer of interest, a 48 nucleotide space oligo, 10 times PCR buffer, a dNTP mixture, high fidelity PCR DNA polymerase, and double distilled water to a final volume of 50 microliters.Then amplify the microRNA by standard PCR using the parameters as indicated. To purify the short tandem target mimics, add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate to the PCR product. Mix vigorously before centrifuging the reaction at 14, 000 times G for 10 minutes.After removing the supernatant, rinse the pellet with one milliliter of 70%ethanol and allow the pellet to dry, then dissolve the pellet in 20 microliters of double distilled water. To set up a T4 DNA polymerase reaction, mix the purified short tandem target mimic PCR product with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dATP, T4 DNA polymerase, and double distilled water to a final volume of five microliters on ice. Then incubate the mixture for 15 minutes at 37 degrees Celsius, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.To prepare the PVX-based VBMS construct, digest five micrograms of PVX ligation independent cloning plasmids with 2.5 microliters of SMA1 in a total volume of 100 microliters. Add an equal volume of phenol to chloroform to isoamyl alcohol, add a 25 to 24 to one ratio to the digested PVX ligation independent cloning products with vigorous mixing and collect the product by centrifugation. Add an equal volume of chloroform to isoamyl alcohol at a 24 to one ratio to the supernatant with vortexing before subjecting the tube contents to a second centrifugation.At the end of the spin, transfer the supernatant to a new centrifuge tube and add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate with vigorous mixing. Centrifuge the tube and rinse the pellet with one milliliter of 70%ethanol and vigorous vortexing. Centrifuge the tube again and remove the supernatant to allow the pellet to air dry.Then dissolve the digested PVX ligation independent cloning plasmid pellet in 100 microliters of double distilled water. To set up the T4 DNA polymerase reaction, mix the digested PVX ligation independent cloning vector DNA with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dTTP, and T4 DNA polymerase to a final volume of five microliters on ice. Then incubate the mixture at 37 degrees Celsius for 15 minutes, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.To clone the short tandem target mimic sequence into the PVX ligation independent cloning vector, mix five microliters of the T4 DNA polymerase-treated short tandem target mimic PCR product with five microliters of the T4 DNA polymerase-treated PVX ligation independent cloning plasmid and incubate the mixture for five minutes at 70 degrees Celsius. At the end of the incubation, cool the tube contents to 22 degrees Celsius in a PCR machine at a rate of 0.1 degrees Celsius per second, leaving the tube in the thermocycler for 30 minutes once the target temperature is reached. At the end of the incubation, transform five microliters of ligation independent cloning reaction product into E.coli DH5 alpha using standard transformation protocols and grow the transformed bacteria on an LB plate containing 50 micrograms per milliliter kanamycin.After verifying the sequences of the short tandem target mimic fragments by terminator cycle sequencing, isolate the PVX VBMS plasmids from the validated clones and transform them into the appropriate Agrobacterium strains. To inoculate four-week-old in vitro potato plants with Agrobacterium containing the PVX VBMS plasmids, pick positive transformants containing PVX VBMS vectors and inoculate the transformants in 50 milliliters of liquid LB containing 50 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin for 16 hours at 28 degrees Celsius and 220 revolutions per minute. During the incubation, streak positive Agrobacterium colonies onto at least two new LB plates containing kanamycin and rifampicin and grow the cultures at 28 degrees Celsius for one day.When the optical density at 600 nanometers reaches 1.0, collect the Agrobacterium liquid culture by centrifugation and resuspend the pellet in a sufficient volume of infiltration buffer to adjust the optical density at 600 nanometers to 1.0. After a six-hour incubation at room temperature, load a one milliliter needleless syringe with the Agrobacterium suspension and use one hand to flip and hold an expanded leaf from one potato plant. Using one finger, support the leaf lamina from the adaxial side at the desired site of infiltration.And holding the syringe vertical to the leaf surface with the other hand, infiltrate 100 to 200 microliters of Agrobacterium culture into the abaxial side of the lamina. Next, use a toothpick to collect colonies from the Agrobacterium culture plates and to scratch the stem surface of the first one or two internodes of the infiltrated potato plants and the epidermis of the stem, then grow the infiltrated plants in the greenhouse for two to four weeks. When phenotypes appear, use scissors to collect samples with phenotypes from the VBMS and control plants.Isolate the total RNA from the collected tissues and use reverse transcriptase PCR to analyze the microRNA and target mRNA expression in each sample. In these images, PVX short tandem target mimic 165/166 potato plants with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins can be observed. More severe phenotypes such as trumpet-shaped leaf formation have also been observed.In contrast, no phenotypical abnormalities are observed in PVX control plants, demonstrating that the VBSM system is effective in suppressing endogenous microRNA function in tetraploid potato plants. PVX short tandem target mimic 165/166 potato plants exhibit ectopic leaf to tissue growth from the abaxial side of the leaf lamina along the lamina veins, demonstrating that the PVX VBMS system can be applied to other potato varieties, including major potato cultivars. It is essential to simultaneously infiltrate the leafs and to inoculate the stem of the same plant with the same Agrobacterium culture.Transgenic approaches to stably introducing target mimic molecules into potato plants are recommended when the silencing state of the targeted microRNAs needs to be maintained in the offspring generations.

potato-virus-x-based-microrna-silencing-vbms-in-potato

Research

JoVE Journal

Environment

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Field Measurement of Effective Leaf Area Index using Optical Device in Vegetation Canopy

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between green components of plants and the atmosphere, and many physiological processes occur there, primarily photosynthetic uptake, respiration, and transpiration. LAI is also an input parameter for many models involving carbon, water, and the energy cycle. Moreover, ground-based in situ measurements serve as the calibration method for LAI obtained from remote sensing products. Therefore, straightforward indirect optical methods are necessary for making precise and rapid LAI estimates. The methodological approach, advantages, controversies, and future perspectives of the newly developed LP 110 optical device based on the relation between radiation transmitted through the vegetation canopy and canopy gaps were discussed in the protocol. Furthermore, the instrument was compared to the world standard LAI-2200 Plant Canopy Analyzer. The LP 110 enables more rapid and more straightforward processing of data acquired in the field, and it is more affordable than the Plant Canopy Analyzer. The new instrument is characterized by its ease of use for both above- and below-canopy readings due to its greater sensor sensitivity, in-built digital inclinometer, and automatic logging of readings at the correct position. Therefore, the hand-held LP 110 device is a suitable gadget for performing LAI estimation in forestry, ecology, horticulture, and agriculture based on the representative results. Moreover, the same device also enables the user to take accurate measurements of incident photosynthetically active radiation (PAR) intensity.

Fast and precise leaf area index (LAI) estimation in terrestrial ecosystems is crucial for a wide range of ecological studies and calibrating remote sensing products. Presented here is the protocol for using the new LP 110 optical device for taking ground-based in situ LAI measurements.

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between ...