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 with fresh MS/Gamborg&#8217;s vitamin media every month.</li>
	<li>Four weeks later, transplant in vitro plants into soil and grow them in a greenhouse under 20&#8211;22 &#176;C, 16 h light/8 h dark photoperiod, and light intensity 120 &#181;mol/m2&#8729;s1. 
	NOTE: The plants with newly developed roots and leaves are suitable for transplanting. Maintain moisture for the freshly transplanted plants for the first 3&#8211;4 days.</li>
</ol>
2. Construct the VbMS vectors.
<ol>
	<li>Design and clone the short tandem TM molecules (STTM, Figure 1)22,53 for the miRNA of interest. 
	NOTE: Acquire miRNA sequences based on experimental data or from the miRbase database54,55,56,57,58,59. The miR166 sequence used in this study has been described previously60.
	<ol>
		<li>Design the TM module by inserting a mismatch sequence, normally 5&#8217;-CTA-3&#8217;, into the reverse complement sequence of miRNA at the site corresponding to the 10th&#8211;11th nucleotides of the miRNA. 
		NOTE: For example, the Stu-miR160 sequence is 5&#8217;-UGCCUGGCUCCCUGUAUGCC-3&#8217;61, where the 10th&#8211;11th nucleotides is in bold. The reverse complement sequence (in deoxynucleotides) is 5&#8217;-GGCATACAGGGAGCCAGGCA-3&#8217; and the mismatch bulge insertion site is shown in bold. The TM molecule sequence (deoxynucleotide) should be 5&#8217;-GGCATACAGG-CTA-GAGCCAGGCA-3&#8217;.</li>
		<li>Design primers for cloning the STTM fragment (Figure 1). Use DNA oligonucleotides with the 48-nt spacer sequence as template for PCR cloning. The forward primer consists of a LIC1 linker (5&#8217;-CgACgACAAgACCgT-3&#8217;), the forward sequence of the above designed for TM molecule, and the partial 5&#8217; sequence of the 48-nt spacer (5&#8217;-GTTGTTGTTGTTATGGT-3&#8217;). The reverse primer consists of a LIC2 linker (5&#8217;-gAggAgAagAgCCgT-3&#8217;), the reverse complement sequence of the TM molecule, and a partial reverse complement to the 3&#8217; sequence of the 48-nt spacer (5&#8217;-ATTCTTCTTCTTTAGACCAT-3&#8217;). 
		NOTE: The 48-nt spacer sequence is 5&#8217;-GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAAT-3&#8217;. For example, for STTM-miR160, the forward primer should be 5&#8217;-CgACgACAAgACCgT-GGCATACAGG-CTA-GAGCCAGGCA-GTTGTTGTTGTTATGGT-3&#8217;; the reverse primer should be 5&#8217;-gAggAgAagAgCCgT-TGCCTGGCTC-TAG-CCTGTATGCC-ATTCTTCTTCTTTAGACCAT-3&#8217; (Figure 1).</li>
		<li>Amplify the STTM fragment in a volume of 50 &#181;L by PCR using the synthesized universal 48-nt spacer as the template and a high fidelity DNA polymerase.
		<ol>
			<li>Set up the PCR reaction by mixing 0.5 &#181;L of each primer (40 &#181;M), 0.5 &#181;L of 48-nt spacer oligo (40 &#181;M), 5 &#181;L of 10x PCR buffer, 1 &#181;L of dNTP mixture (10 mM each), 0.1 &#181;L of high fidelity DNA polymerase (10 U/&#181;L ), and 43 &#181;L of ddH2O to a total volume of 50 &#181;L. Perform standard PCR amplification as follows: 94 &#176;C for 3 min, 32 cycles of 94 &#176;C for 45 s, 60 &#176;C for 45 s and 72 &#176;C for 60 s.</li>
		</ol>
		</li>
		<li>Purify the STTM fragment by ethanol precipitation. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) to the PCR products. Mix vigorously and centrifuge at 14,000 x g for 10 min. Remove the supernatant and rinse the pellet with 1 mL of 70% ethanol. Dry the pellet and dissolve it in 20 &#181;L of ddH2O. 
		NOTE: Use DNA electrophoresis to check the amplification of the STTM PCR products.</li>
		<li>Set up the T4 DNA polymerase reaction on ice by mixing 2.5 &#181;L of purified STTM PCR product, 0.5 &#181;L of 10x T4 DNA polymerase buffer, 0.05 &#181;L of 1 M dithiothreitol (DTT), 0.25 &#181;L of 100 mM dATP, 0.1 &#181;L of T4 DNA polymerase (3 U/&#181;L), and 1.6 &#181;L of ddH2O to a total volume of 5 &#181;L. Incubate the mixture at 37 &#176;C for 15 min, and treat the products at 75 &#176;C for 20 min to inactivate the T4 DNA polymerase.</li>
	</ol>
	</li>
	<li>Prepare the PVX-based VbMS construct.
	<ol>
		<li>Digest 5 &#956;g of PVX-LIC plasmid38 with 2.5 &#181;L of SmaI (20 U/&#181;L) in a volume of 100 &#181;L.</li>
		<li>Add an equal volume of phenol:chloroform:isopropanol (25:24:1, pH = 6.7/8.0) to the digested PVX-LIC products and mix vigorously. Centrifuge at 14,000 x g for 10 min and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform:isopropanol (24:1) and vortex vigorously. Centrifuge at 14,000 x g for 10 min. 
		NOTE: The PVX-LIC vector harbors the LIC cassette for cloning. The LIC cassette of PVX-LIC vector contains a ccdB gene and a chloramphenicol-resistant gene and needs to be maintained/propagated in the E. coli strain DB3.1 using LB medium containing kanamycin (50 &#956;g/L) and chloramphenicol (15 &#956;g/L).</li>
		<li>Transfer the supernatant to a new centrifuge tube. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) and mix vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant.</li>
		<li>Rinse the pellet with 1 mL of 70% ethanol and vortex vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant. Dry the pellet and dissolve the digested PVX-LIC plasmid with 100 &#181;L of ddH2O.</li>
		<li>Set up the T4 DNA polymerase reaction on ice by mixing 2.5 &#181;L of digested PVX-LIC vector DNA, 0.5 &#181;L of 10x T4 DNA polymerase buffer, 0.05 &#181;L of 1 M DTT, 0.25 &#181;L of 100 mM dTTP, and 0.1 &#181;L of T4 DNA polymerase (3 U/&#181;L) in a total volume of 5 &#181;L. Incubate the mixture at 37 &#176;C for 15 min and treat the products at 75&#8201;&#176;C for 20 min to inactivate the T4 DNA polymerase.</li>
	</ol>
	</li>
	<li>Clone the STTM sequence into the PVX-LIC vector using a LIC reaction. Mix the T4 DNA polymerase-treated STTM PCR products (5 &#181;L) and the T4 DNA polymerase-treated PVX-LIC plasmids (5 &#181;L). Incubate at 70 &#176;C for 5&#8201;min, cool down to 22 &#176;C at a ramp of 0.1 &#176;C/s, and keep at 22 &#176;C for 30 min using a PCR machine. 
	NOTE: Extend the incubation time to overnight at 4 &#176;C to achieve higher efficiency of LIC cloning.</li>
	<li>Transform 5 &#181;L of the LIC reaction products into the E. coli DH5&#945; and grow on an LB plate containing 50 &#956;g/mL kanamycin62,63. Pick and verify positive colonies by PCR with the cloning primers and universal primer for the PVX-LIC vector followed by&#160;sequencing.
	<ol>
		<li>Perform colony PCR with the forward primer for PVX-LIC (5&#8217;-GTGTTGGCTTGCAAACTAGAT-3&#8217;) in combination with the reverse primer for STTM cloning to identify positive clones. The size of the PCR band should be ~300 bp. 
		NOTE: Verify the sequences of STTM fragments by terminator cycle sequencing64,65.</li>
	</ol>
	</li>
	<li>Isolate the PVX-STTM plasmids from the validated clones and transform them into Agrobacterium strains GV3101, GV2260, or EHA10562,63. Verify Agrobacterium colonies by PCR. 
	NOTE: Confirm Agrobacterium colonies by PCR using the forward primer for PVX-LIC and the reverse primer for STTM cloning.</li>
</ol>
3. Perform PVX-based VbMS assay in potato plants.
<ol>
	<li>Transplant 4-week-old in vitro potato plants into soil. The transplanted plants will be subjected to VbMS assay 3&#8211;4 days later.</li>
	<li>Inoculate potato plants with Agrobacterium containing the PVX-STTM plasmids. 
	NOTE: For the VbMS assay in potato, Agrobacterium-mediated infiltration and toothpick-scratching inoculation are performed simultaneously.
	<ol>
		<li>Pick and inoculate positive transformants containing PVX-STTM vectors into 50 mL of liquid LB containing 50 &#956;g/mL kanamycin and 50 &#956;g/mL rifampicin. Grow in a 28 &#176;C incubator at 220 rpm for 16 h until OD600 = 1.0.</li>
		<li>At the same time, streak the positive Agrobacterium colonies onto at least two new LB plates containing 50 &#956;g/mL kanamycin and 50 &#956;g/mL rifampicin and grow at 28 &#176;C for 1 day. Include the PVX-LIC38,66 vector as a control and PVX-GFP67,68 to monitor virus spread.</li>
		<li>Collect the Agrobacterium liquid culture by centrifugation at 3,400 x g for 10 min at room temperature. Resuspend Agrobacterium cells with an equal volume of infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 &#181;M acetosyringone, pH = 5.6) and adjust to OD600 = 1.0. Incubate at room temperature for 6 h.</li>
		<li>Infiltrate the Agrobacterium culture into the abaxial side of fully expanded leaves with a 1 mL needleless syringe.
		<ol>
			<li>Flip and hold a leaf with one hand, then use one finger to support the leaf lamina from the adaxial side at the infiltration site. Keep the syringe vertical to the leaf surface with the other hand and infiltrate the Agrobacterium culture into the abaxial side of lamina.</li>
		</ol>
		</li>
		<li>Scrape the Agrobacterium culture from the LB plates and scratch the stem surface of the first one or two internodes of the infiltrated potato plants with a toothpick. Gently scratch the epidermis of the stem. Avoid piercing through the stem, which may cause severe damage to the plants.</li>
	</ol>
	</li>
	<li>Grow the infiltrated plants at 22 &#176;C with a 16 h light/8 h dark photoperiod and light intensity of 120 &#956;mol/m2&#8729; s1 in a greenhouse. 
	NOTE: Phenotypes caused by miRNA silencing usually appear in 2&#8211;4 weeks postinoculation (Figure 2, Figure 3). It typically takes 10&#8211;20 days for the VbMS phenotype to appear after infiltration. The VbMS phenotype depends on the properties of the specific miRNA and target genes, growth conditions, and potato varieties.</li>
</ol>
4. Perform expression analysis.
<ol>
	<li>When phenotypes appear at 2&#8211;4 weeks postinoculation, collect tissues such as shoots, leaves, flowers, or roots with phenotypes from the VbMS plants and tissues from the control plants with scissors. Isolate total RNAs from the collected tissues.</li>
	<li>Check the RNA quality by electrophoresis62,63 and quantify the RNA concentration by measuring the OD260 absorbance with a spectrophotometer.</li>
	<li>Use stem-loop real-time reverse transcription PCR (RT-PCR) to analyze the miRNA expression.
	<ol>
		<li>For specific miRNAs, design a stem-loop reverse transcription primer. Stem-loop RT primers contain a universal 5&#8217; backbone and a 3&#8217; 6-nt extension of a specific miRNA. Design a 5&#8217; universal backbone (5&#8217;- GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGAC-3&#8217;) that forms a stem-loop structure. (The upper case corresponds to an inverse-repeated sequence and the lower case to the loop region).</li>
		<li>Part of the 5&#8217; backbone sequence that forms a loop serves as the reverse primer for subsequent PCR amplification (bold-italic sequence in the backbone sequence). Add a 6-nt extension sequence that is reverse-complementary to the 3&#8217; end of the miRNA of interest to the stem-loop primer to provide specificity for reverse transcription (Supplemental Figure 1A). 
		NOTE: Design the stem-loop reverse transcription primer as described by Chen et al.69 and Erika Varkonyi-Gasic et al.70,71,72. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5&#8217;-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGCATA-3&#8217;. The stem-loop reverse transcription primer for the potato miR165/166 family is designed as 5&#8217;-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGGG(A/G)A-3&#8217;. (The bold uppercase sequences provide the specificity of reverse transcription for specific miRNAs).</li>
		<li>Set up the reverse transcription reaction by mixing 50&#8211;200 ng of total RNA, 1 &#181;L of the stem-loop reverse transcription primer (100 &#956;M), 2 &#181;L of 10x buffer, 0.2 &#181;L of RNase inhibitor (40 U/&#181;L), 0.25 &#181;L of dNTPs (10 mM each), and 1 &#181;L of reverse transcriptase (200 U/&#181;L) on ice. Add nuclease-free ddH2O to a total volume of 20 &#181;L.</li>
		<li>Perform reverse transcription using the pulsed reverse transcription procedure. Incubate the reverse transcription reaction mixture at 16 &#176;C for 30 min, perform a temperature cycle of 30 &#176;C for 30 s, 42 &#176;C for 30 s and 50 &#176;C for 1 s, for a total of 60 cycles, and inactivate the reverse transcriptase by incubation at 85 &#176;C for 5 min.</li>
		<li>For real-time PCR analysis of miRNA expression, design the forward primer based on the miRNA sequence but do not include sequences that overlap with the above designed stem-loop reverse transcription primer. Add a 3&#8211;7-nt extension to the 5&#8217; of the forward primer to optimize the length, melting temperature, and GC content (Supplemental Figure 1). 
		NOTE: The universal reverse primer is 5&#8217;-GTGCAGGGTCCGAGGT-3&#8217;. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5&#8217;-CGGCTGCCTGGCTCC-3&#8217;. The stem-loop reverse transcription primer for miR165/166 family is 5&#8217;-CGGCTCGGACCAGGCTT-3&#8217;. The bold sequences serve as 5&#8217; extensions for primer optimization. The stem-loop reverse transcription primers and the real-time PCR primers for miRNA can be designed using the miRNA Primer Design Tool73.</li>
	</ol>
	</li>
	<li>Synthesize cDNAs of the target mRNAs by standard reverse transcription PCR (RT-PCR). Incubate the reverse transcription reaction mixture at 37 &#176;C for 60 min and inactivate the reverse transcriptase by heating to 85 &#176;C for 5 min. 
	NOTE: (1) Predict target mRNAs using the psRNATarget program74 if the target mRNAs are not known. (2) The universal reverse transcription primer for mRNA is an anchored primer 5&#8217;-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3&#8217;.</li>
	<li>Set up the real-time PCR reaction for both&#160;miRNA of interest and the target mRNAs. Mix 0.5 &#181;L of template cDNA, 5 &#181;L of 2x real-time PCR buffer with SYBR green, 0.05 &#181;L of each forward and reverse primer (40 &#181;M), and 4.4 &#181;L of ddH2O in a total volume of 5 &#181;L on ice.</li>
	<li>Incubate at 95 &#176;C for 3 min, 40 cycles of 95 &#176;C for 3 s and 60 &#176;C for 30 s. Perform a subsequent melting curve analysis as follows: Incubate at 95 &#176;C for 15 s, cool down to 60 &#176;C at a ramp of 20 &#176;C/s, keep at 60 &#176;C for 60 s, heat to 95 &#176;C at a ramp of 0.2 &#176;C/s, and keep at 95 &#176;C for 15 s. Analyze the Ct-values using the &#916;&#916;Ct methods76,77 and plot the means with standard errors. 
	NOTE: (1) The real-time PCR primers for target mRNAs can be designed as described75. (2) Potato polyubiquitin 10 gene can serve as an internal control for normalization in potato. The forward primer for the potato polyubiquitin 10 gene is 5&#8217;-ATGTTGCCTTTCTTATGTGTGGTTG-3&#8217; and the reverse primer 5&#8217;-TTATTTATTCACATAAACGACAGTTCAACC-3&#8217;. (3) For real-time PCR analysis, contamination and primer-dimer formation may generate false positive results. To monitor nonspecific amplification and increase the liability of real-time PCR analysis, it is recommended to include controls without template and reverse transcriptase for real-time PCR assays.</li>
</ol>

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

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3.0K Views.  Texas A&M University System. 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.

Video: Potato Virus X-Based microRNA Silencing VbMS In Potato.

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 ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

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 ...