This work was supported by the National Natural Science Foundation of China (31772291), the Research Project for Public Interest in Zhejiang Province (2017C32027), the Key Science Project of Plant Breeding in Zhejiang (2016C02051), and the National Program for the Support of Top-notch Young Professionals (to P.X.).

1. Seed Sterilization and Germination
<ol>
	<li>For surface sterilization, soak the bottle gourd seeds in a 500-mL beaker filled with water at 58 &#176;C with occasional stirring, until the water temperature drops to 40 &#176;C.</li>
	<li>Meanwhile, put 3 kg of peat soil into a nylon bag and, to sterilize it, autoclave it at 120 &#176;C/0.5 MPa for 20 min.</li>
	<li>Keep soaking the bottle gourd seeds for 4 - 5 h more with no stirring.
	<ol>
		<li>Once the water reaches room temperature, rinse the seeds 2x - 3x with di...

<ol>
	<li>Schwarz, D., Rouphael, Y., Colla, G., Venema, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+as+a+tool+to+improve+tolerance+of+vegetables+to+abiotic+stresses:+Thermal+stress,+water+stress+and+organic+pollutants.">Grafting as a tool to improve tolerance of vegetables to abiotic stresses: Thermal stress, water stress and organic pollutants.</a> Scientia Horticulturae. 127, 162-171 (2010).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tolerance+differences+in+cucumber+seedlings+grafted+on+rootstocks+with+different+tolerance+to+low+temperature+and+weak+light+stresses.">Mechanisms of tolerance differences in cucumber seedlings grafted on rootstocks with different tolerance to low temperature and weak light stresses.</a> Turkish Journal of Botany. 39 (4), 606-614 (2015).</li><li>Li, C. H., Li, Y. S., Bai, L. Q., He, C. X., Yu, X. 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J., Rivard, C. L., Kubota, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+fruiting+vegetables+to+manage+soilborne+pathogens,+foliar+pathogens,+arthropods+and+weeds.">Grafting fruiting vegetables to manage soilborne pathogens, foliar pathogens, arthropods and weeds.</a> Scientia Horticulturae. 127 (2), 127-146 (2010).</li><li>Asins, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+rootstock-mediated+nitrogen+(N)+uptake+and+root-to-shoot+signalling+at+contrasting+N+availabilities+in+tomato.">Genetic analysis of rootstock-mediated nitrogen (N) uptake and root-to-shoot signalling at contrasting N availabilities in tomato.</a> Plant Science. 263, 94-106 (2017).</li><li>Yin, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+protective+enzymes+in+tomato+rootstocks+to+resist+root+knot+nematodes.">Role of protective enzymes in tomato rootstocks to resist root knot nematodes.</a> Acta Horticulturae. 1086 (1086), 213-218 (2015).</li><li>Gaion, L. A., Carvalho, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Distance+Signaling:+what+grafting+has+revealed?.">Long-Distance Signaling: what grafting has revealed?.</a> Journal of Plant Growth Regulation. 37 (2), 694-704 (2018).</li><li>Turnbull, C. G., Hennig, L., K&#246;hler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+as+a+research+tool.">Grafting as a research tool.</a> Plant Developmental Biology. , 11-26 (2010).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting-responsive+miRNAs+in+cucumber+and+pumpkin+seedlings+identified+by+high-throughput+sequencing+at+whole+genome+level.">Grafting-responsive miRNAs in cucumber and pumpkin seedlings identified by high-throughput sequencing at whole genome level.</a> Physiologia Plantarum. 151 (4), 406-422 (2014).</li><li>Lakhotia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+miRNAome+in+root,+stem,+leaf+and+tuber+developmental+stages+of+potato+(Solanum+tuberosum+L.)+by+high-throughput+sequencing.">Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing.</a> BMC Plant Biology. 14 (1), 6 (2014).</li><li>Jones-Rhoades, M. W., Bartel, D. P., Bartel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+their+regulatory+roles+in+plants.">MicroRNAs and their regulatory roles in plants.</a> Annual Review of Plant Biology. 57, 19-53 (2006).</li><li>Puzey, J. R., Kramer, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+conserved+Aquilegia+coerulea+microRNAs+and+their+targets.">Identification of conserved Aquilegia coerulea microRNAs and their targets.</a> Genetic. 448 (1), 46-56 (2009).</li><li>Matthewman, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR395+is+a+general+component+of+the+sulfate+assimilation+regulatory+network+in+Arabidopsis.">miR395 is a general component of the sulfate assimilation regulatory network in Arabidopsis.</a> FEBS Letters. 586 (19), 3242-3248 (2012).</li><li>Ali, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+RNA+silencing+signal+through+grafting+confers+virus+resistance+from+transgenically+silenced+tobacco+rootstocks+to+non-transgenic+tomato+and+tobacco+scions.">Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions.</a> Journal of Plant Biochemistry and Biotechnology. 25 (3), 245-252 (2016).</li><li>Li, Y. S., Li, C. H., Bai, L. Q., He, C. X., Yu, X. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+and+target+gene+responses+to+salt+stress+in+grafted+cucumber+seedlings.">MicroRNA and target gene responses to salt stress in grafted cucumber seedlings.</a> Acta Physiologiae Plantarum. 38 (2), 1-12 (2016).</li><li>Pagliarani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accumulation+of+miRNAs+differentially+modulated+by+drought+stress+is+affected+by+grafting+in+grapevine.">The accumulation of miRNAs differentially modulated by drought stress is affected by grafting in grapevine.</a> Plant Physiology. 173 (4), 2180-2195 (2017).</li><li>Liu, N., Yang, J. H., Guo, S. G., Xu, Y., Zhang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+and+comparative+analysis+of+conserved+and+novel+microRNAs+in+grafted+watermelon+by+high-throughput+sequencing.">Genome-wide identification and comparative analysis of conserved and novel microRNAs in grafted watermelon by high-throughput sequencing.</a> PLoS One. 8 (2), e57359 (2013).</li><li>Song, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2JC-350+automatic+grafting+machine+with+cut+grafting+method+for+vegetable+seedling.">Development of 2JC-350 automatic grafting machine with cut grafting method for vegetable seedling.</a> Transactions of the Chinese Society of Agricultural Engineering. 22 (12), 103-106 (2006).</li><li>Kumar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+leaf+rust+responsive+miRNAs+in+wheat+(triticum+aestivum+l.)+using+high-throughput+sequencing+and+prediction+of+their+targets+through+degradome+analysis.">Uncovering leaf rust responsive miRNAs in wheat (triticum aestivum l.) using high-throughput sequencing and prediction of their targets through degradome analysis.</a> Planta. 245 (1), 1-22 (2016).</li><li>Kohli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+wilt+and+salt+stress-responsive+microRNAs+in+chickpea+through+high-throughput+sequencing.">Identification and characterization of wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing.</a> PLoS One. 9 (10), e108851 (2014).</li><li>Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+challenges+in+next-generation+genomics.+International+Conference+on+Scientific+and+Statistical+Database+Management.">Computational challenges in next-generation genomics. International Conference on Scientific and Statistical Database Management.</a> ACM. 2, (2013).</li><li>Guo, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+draft+genome+of+watermelon+(Citrullus+lanatus)+and+resequencing+of+20+diverse+accessions.">The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions.</a> Nature Genetics. 45, 51-58 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gourdbase:+a+genome-centered+multi-omics+database+for+the+bottle+gourd+(lagenaria+siceraria),+an+economically+important+cucurbit+crop.">Gourdbase: a genome-centered multi-omics database for the bottle gourd (lagenaria siceraria), an economically important cucurbit crop.</a> Scientific Reports. 8 (1), 306 (2018).</li><li>Wang, X. F., Liu, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Curation+of+miRBase+Annotation+Using+Integrated+Small+RNA+High-Throughput+Sequencing+Data+for+C.+elegans+and+Drosophila.">Systematic Curation of miRBase Annotation Using Integrated Small RNA High-Throughput Sequencing Data for C. elegans and Drosophila.</a> Frontiers in Genetics. 2, 25 (2011).</li><li>Bo, X. C., Wang, S. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TargetFinder:+a+software+for+antisense+oligonucleotide+target+site+selection+based+on+MAST+and+secondary+structures+of+target+mRNA.">TargetFinder: a software for antisense oligonucleotide target site selection based on MAST and secondary structures of target mRNA.</a> Bioinformatics. 21 (8), 1401-1402 (2005).</li><li>. GOATOOLS: Tools for Gene Ontology Available from: <a target="_blank" href="https://doi.org/10.5281/zenodo.31628">https://doi.org/10.5281/zenodo.31628</a> (2015)</li><li>Wang, L. P., Li, G. J., Wu, X. H., Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomic+analyses+provide+novel+insights+into+the+effects+of+grafting+wound+and+hetero-grafting+per+se+on+bottle+gourd.">Comparative proteomic analyses provide novel insights into the effects of grafting wound and hetero-grafting per se on bottle gourd.</a> Scientia Horticulturae. 200 (8), 1-6 (2016).</li></ol>

The authors have nothing to disclose. The small RNA sequencing data is deposited in the GenBank under the accession number SRP136842.

In this protocol, we described in detail a highly efficient and reproducible method to make homo- and heterografts between watermelon and bottle gourd. This method, requiring no specific equipment, is very easy to operate and typically has a very high survival rate of grafting. The method can also be used to make grafts for other cucurbits, such as between watermelon, cucumber, and pumpkin.
It is worth noting that the relative size (age) of the rootstock and scion is critical to making a succe...

Grafting has long been employed as an agricultural technique to improve crop production and tolerance to biotic and abiotic stresses1,2,3. In heterografting systems, elite rootstocks can enhance water and nutrients uptake of plants, strengthen resistance to soil pathogens, and limit the negative effects of metal toxicity4,5, which may confer the grafts an enhanced growth vigor and increased tolerance to environmental stresses. In many cases, heterografting can also impact fruit qualities in horticultural plants, leading to improved fruit flavor and increased content of health-related compounds6,7. It has been found that the long-distance transfer of phytohormones, RNAs, peptides, and proteins between the rootstock and the scion is a fundamental mechanism modulating the growth and development reprogramming of scion plants8,9,10. Grafting has been widely used in studies of long-distance signaling and transport in relation to environmental adaptation11. Grafting experiments are especially powerful for unambiguous detection of transmitted molecules in receiving tissue or vascular sap, and activation or suppression of molecular targets due to signal transmission12.
Non-coding RNAs, a big class of RNA that exert important regulatory functions in cells, have been reported to play a role in facilitating plant adaptation to abiotic stress13. miRNAs are endogenous small non-coding RNAs of about 20 - 24 nt. Studies have revealed the regulatory role of miRNAs in various aspects of plant activities, such as shoot growth, lateral root formation14,15,16, nutrient uptake, sulfate metabolism and homeostasis17, and responses to biotic and abiotic stress18. Recently, the expression of miRNAs and their target genes were related to salt stress tolerance in heterografted cucumber seedlings19. In the intervariety grafts of grape, the responses of miRNA expression to drought stress were found to be genotype-dependent20.
The rapid development and decreasing cost of high-throughput sequencing technology have provided a great opportunity for the study of miRNA regulations in agronomical plants. Watermelon (Citrullus lanatus [Thunb.] Mansf.), an important cucurbit crop grown throughout the world, is susceptible to low temperatures. Bottle gourd (Lagenaria siceraria [Molina] Standl.) is a more climate-resilient cucurbit commonly used by farmers to graft with watermelon. The primary goal of the current study is to establish a standard, efficient, and convenient method for making heterografts between watermelon (Citrullus lanatus [Thunb.] Mansf.) and bottle gourd (Lagenaria siceraria [Molina] Standl). This protocol also provides a detailed experimental scheme and analytical procedures for the study of the regulation of miRNA expressions following grafting, which is useful for revealing the mechanisms underlying heterografting advantages.
The plant materials used in this study include the watermelon cultivar and the bottle gourd landrace. Watermelon cultivar is a commercial cultivar with high yield but susceptible to low temperatures. Bottle gourd landrace is a popular rootstock for grafting with watermelon, cucumber, and bottle gourd, due to its excellent tolerance of low temperatures21.

<table>
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA-free DNase I</td>
			<td>Takara</td>
			<td>D2270A</td>
			<td></td>
		</tr>
		<tr>
			<td>Truseq Small RNA sample prep Kit</td>
			<td>Illumina</td>
			<td>RS-200-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bionalyser</td>
			<td>Agilent</td>
			<td>5067</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Polymerase</td>
			<td>Thermo Fisher Scientific</td>
			<td>F530S</td>
			<td></td>
		</tr>
		<tr>
			<td>UEA sRNA workbench 2.4-plant version&#160;(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://srna-workbench.cmp.uea.ac.uk/</td>
		</tr>
		<tr>
			<td>Rfam 11.0 database&#160;(website)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://rfam.janelia.org</td>
		</tr>
		<tr>
			<td>miRBase 22.0&#160;(website)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://www.mirbase.org/</td>
		</tr>
		<tr>
			<td>MIREAP(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>https://sourceforge.net/projects/mireap/</td>
		</tr>
		<tr>
			<td>TargetFinder&#160;(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://targetfinder.org/</td>
		</tr>
	</tbody>
</table>

generating homo- and heterografts between watermelon and bottle gourd for the study of cold-responsive micrornas

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58242/58242fig2.jpg" /> 
Figure 2: Phenotypes of various grafts at room temperature and cold-stressed conditions. (a) This panel shows homo- and heterografted seedlings at room temperature as the control. (b) This panel shows homo- and heterografted seedlings after 48 h of cold treatment. <a href="https://www.jove.com/files/ftp_upload/58242/58242fig2large.jpg" target="_blank"...

Watch this Scientific Journal Video about Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs at JoVE.com

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

This method can help answer key questions in miRNA response to ability to stress in plant grafts such as how miRNA are regulated under cold stress in watermelon bottle gourd grafts. The main advantage of this technique is that it is highly efficient and reproducible method to make homo and heterografts. It requires no specific equipment, it is very easy to perform, and typically results in a very high survival rate of grafting.Through this method, can provide insight into miRNA response to cold stress in the watermelon bottle gourd graft system. It can also be applied to other modern organisms for revealing the mechanisms of local and long distance miRNA transportation. Visual demonstration of this method is critical as the grafting steps are difficult to learn.This step required advanced skills and are key to the survival of the grafted plants. To begin, soak bottle gourd seeds in a 500 milliliter beaker of 58 degrees Celsius water. Stir the seeds occasionally until the water temperature drops to 40 degrees Celsius.While the water cools, put three kilograms of peat soil into a nylon bag and autoclave it. Once the water reaches room temperature, rinse the seeds two to three times in distilled water and drain the excess water. Allow the seeds to sprout in a gauze bag at 28 degrees Celsius in the dark growth chamber.After germination, sow the seeds into plastic pots filled with the sterilized peat soil. When the bottle gourd seedlings develop two flattened cotyledons, repeat this process with watermelon seeds. Grow the bottle gourd and watermelon seedlings in a growth chamber.Add water to the seedlings once per day in the afternoon. Next, cut the hypocotyls of the watermelon seedlings two to three centimeters below the cotyledons. Cut the top of the bottle gourd seedlings at the side, immediately above the cotyledons.Then use a toothpick to make a hole in the top of the trimmed bottle gourd seedlings. Insert the trimmed watermelon seedlings into the hole to make heterografts. After this, prepare homografts using the previously described method.First, wrap the grafted seedlings in transparent polyethylene bags to maintain a relatively high humidity. Then maintain the wrapped seedlings in a growth chamber for seven days. After the seventh day, remove the bags and allow the plants to grow under the same conditions for seven to 10 days.Divide the seedlings into two groups, one for cold treatments and one for control. Return the control seedlings to the same environmental conditions. Place the cold treated seedlings into a growth chamber set to six degrees Celsius with the same light dark conditions as the control group.After 48 hours, leave the samples of the scion and rootstocks from the grafts. Freeze the samples immediately in liquid nitrogen and store them at minus 70 degrees Celsius. Transfer the frozen sample to a two milliliter microcentrifuge tube in liquid nitrogen.Add a stainless steel bead to each sample tube and homogenize the tissues to a fine powder. For each grafting combination, take equal amounts of ground sample from 10 seedlings and mix them in a 10 milliliter centrifuge tube. Add an appropriate amount of guanidium hydrochloride reagent.Next, add RNA-free DNase one to 150 units per milliliter at 37 degrees Celsius for one hour. Then determine the total RNA quantity on a microcapillary electrophoresis system. After this, thaw the library normalization reagents and adapters.Then ligate small RNase with the five prime and three prime adapters, and elute and purify them. Reverse transcribe the five prime and three prime ligated small RNase following the manufacturer's guidelines. Next, perform PCR amplification following the manufacturer's protocol.Then use the microcaillary electrophoresis system to ensure that the RNA integrity number is greater than seven. Load one microliter of an RNA library on the microcapillary electrophoresis system to ensure that the RNA integrity number is greater than seven. Finally, sequence the small RNA libraries on a high throughput sequencing instrument.Using this protocol, a 98%survival rate for grafting and the phenotypes for room temperature and cold stress conditions were obtained. sRNAs of 24 nucleotides made up the biggest class of sRNAs in all grafting combination, regardless of temperature treatment. After 48 hours of cold treatment, 30 and 268 microRNAs were up and down-regulated, respectively.In the leaves of the scion in heterografts, conversely, in the leaves of the rootstock, 31 and 12 microRNAs were up and down-regulated, respectively. In the watermelon-watermelon homografts, 64 and 83 microRNAs were up and down-regulated, respectively. This demonstrated that heterografting caused profound reprogramming of the microRNA expressions.While attempting this procedure, it's important to remember the relative size and age of the daughter stalk and the scion is critical to making a successful graft. Following this procedure, other methods like lncRNA sequencing, proteomic profiling can be performed in order to investigate the regulation of lncRNA and protein and the coding system in the grafting system. After its development, this technique paved the way for research in the field of plant abiotic tolerance to explore the mechanism of plant grafting advantage in Cucurbitaceae and beyond.Don't forget that working with guanidine hydrochloride can be extremely hazardous and appropriate precaution should always be taken with performing this procedure.

generating-homo-heterografts-between-watermelon-bottle-gourd-for

Research

JoVE Journal

Environment

7.6K visualizações.  Zhejiang Academy of Agricultural Sciences. Este método pode ajudar a responder a perguntas-chave na resposta do miRNA à capacidade de estressar em enxertos vegetais, como como o miRNA é regulado sob estresse frio em enxertos de cabaça de garrafa de melancia. A principal vantagem dessa técnica é que é um método altamente eficiente e reprodutível para fazer homo e heteroenxertos. Não requer nenhum equipamento específico, é muito fácil de executar, e normalmente resulta em uma taxa de sobrevivência muito alta de enxerto.