Questo lavoro è stato supportato dal programma di ricerca del genoma di pianta dalla National Science Foundation (NSF-PGRP-IOS-1339388).

1. la crescita delle piante
<ol><li>Seminare i semi di soia 5-10 (Williams 82) in un vaso di 13 cm in serra in condizioni di giorno lungo (16 h luce alle 1.500 µmol m-2 s-1) a 25 ° C sul mix di terreno personalizzato per la soia (1:1:1 rapporto di sabbia del suolo, la perlite e la torpedine).</li>
</ol>2. preparazione del DNA del plasmide
<ol><li>Utilizzando una pipetta sterile o uno stuzzicadenti, scegli una singola Colonia o glicerolo congelati stock di e. coli che trasportano il plasmide contenente il gene di interesse e inoculare esso in 20 mL di terreno liquido di Luria-Bertani (LB) con gli antibiotici adatti in un pallone da 50 mL.</li>
	<li>Incubare la beuta a 37 ° C durante la notte su un agitatore. Raccogliere i batteri da centrifugazione la sospensione a 12.000 x g a temperatura ambiente per 5 minuti e scartare il surnatante. Estrarre e purificare il plasmide seguendo la procedura del produttore di un kit di preparazione del plasmide.</li>
</ol>3. isolamento di protoplasti
<ol><li>Tagliare foglie unifoliate recentemente ampliate da piantine di soia 10 - giorno di vita (Figura 1). Nota: Selezione di foglie in una fase inerente allo sviluppo appropriata è la chiave per il successo per la preparazione di protoplasti di soia. Utilizzare solo foglie appena ampliati alle prime fasi dello sviluppo. Come lascia maturo, pareti cellulari diventano più difficile da digerire.</li>
	<li>Con una lama di rasoio fresca, togliere la foglia unifoliata e quindi tagliare i resti in 0,5-1 mm strisce la nervatura centrale. Nota: Due foglie unifoliate digeriti in 10 mL di soluzione di enzima darà protoplasti sufficienti per più di 10 trasformazioni.</li>
	<li>Usando un paio di pinze, trasferimento la foglia strisce immediatamente e delicatamente in 10 mL di soluzione enzimatica preparata al momento (tabella 1) in una provetta da 15 mL. Vuoto si infiltra in foglia strisce per 15 min a temperatura ambiente.</li>
	<li>Incubare le strisce di foglia nella soluzione enzimatica con agitazione delicata (40 giri) sotto luce bassa per 4-6 h a temperatura ambiente. Assicurarsi che la soluzione di enzima diventa giallo-verde come protoplasti vengono rilasciati. Verificare la soluzione di enzima/protoplasti al microscopio (X10). Nota: I protoplasti rilasciati sono sferici a forma, mentre le cellule non digerite hanno forma ovale o irregolare.</li>
	<li>Trasferire i 10 mL di soluzione di enzima/protoplasti in una provetta da 50 mL versando delicatamente e aggiungere 10 mL di soluzione di W5 (tabella 1) a temperatura ambiente per interrompere la digestione. Capovolgere delicatamente la provetta un paio di volte. Versare delicatamente la soluzione enzima/protoplasti su una maglia di nylon pulito 75 µm posizionata sopra un tubo da 50 mL per rimuovere i tessuti della foglia non digerito.</li>
	<li>Centrifugare la soluzione di flusso continuo enzima/protoplasti a 100 x g nel tubo 50 mL per 1-2 min a temperatura ambiente. Rimuovere delicatamente il supernatante utilizzando una pipetta sierologica 10ml senza disturbare il pellet di protoplasti.</li>
	<li>Risospendere i protoplasti e contando il numero di protoplasti su un emocitometro sotto il microscopio (x10), diluire ad una concentrazione di 2 x 105 mL-1 in soluzione di W5 refrigerati a 4 ° C. Mantenere i protoplasti su ghiaccio per 30 min.</li>
	<li>Centrifugare la sospensione a 100 x g per 1-2 min a temperatura ambiente e rimuovere delicatamente la soluzione W5 utilizzando una pipetta da 1 mL senza disturbare il pellet di protoplasti. Risospendere i protoplasti nella soluzione MMG (tabella 1) ad una concentrazione di 2 x 105 mL-1 a temperatura ambiente.</li>
</ol>4. trasformazione di protoplasti
<ol><li>Fai aliquote di 100 µ l dei protoplasti (2 x 104 protoplasti a 2 x 105 mL-1) in 1,5 mL bassa adesione per microcentrifuga utilizzando puntali per pipette uncut 200 µ l. Mettere un'aliquota da parte per servire come controllo negativo. Aggiungere 10 µ l di plasmide (10-20 µ g) in ciascuna dell'aliquota resto dei protoplasti.</li>
	<li>Aggiungere lentamente 110 µ l di soluzione preparata di PEG (tabella 1) sulla parete interna del tubo per microcentrifuga da 1,5 mL e quindi capovolgere delicatamente e ruotare il tubo fino a quando la soluzione diventa omogenea.</li>
	<li>Incubare la miscela di trasformazione a temperatura ambiente per 15 min.</li>
	<li>Per fermare la trasformazione, lentamente aggiungere 400 µ l di soluzione di W5 per la provetta da 1,5 mL a temperatura ambiente e capovolgere delicatamente la provetta fino a quando la soluzione diventa omogenea. Centrifugare la provetta a 100 g per 1-2 min a temperatura ambiente e scartare il surnatante con una pipetta.</li>
	<li>Aggiungere 1 mL di soluzione WI (tabella 1) al tubo e risospendere pipettando delicatamente 1 - 2 volte. Aggiungere 1 mL di siero di vitello sterile (vol/vol) 5% in ogni pozzetto di una piastra di coltura del tessuto 6 pozzetti per rivestire la superficie ed evitare i protoplasti di attaccarsi alla piastra.</li>
	<li>Dopo pochi secondi, scartare il siero di vitello utilizzando una pipetta. Trasferire i protoplasti sedimento in un pozzetto della piastra di coltura. Coprire la piastra con un coperchio.</li>
</ol>5. protoplasti incubazione e raccolta
<ol><li>Incubare i protoplasti a temperatura ambiente per 1-2 giorni al buio. Nota: Abbiamo trovato che due-giorni di incubazione produce più forte segnale di proteina fluorescente in generale rispetto ad un giorno incubazione, e che il segnale di proteina fluorescente può durare fino a 3-4 giorni.</li>
	<li>Trasferire la soluzione di protoplasti di un tubo del microcentrifuge bassa adesione di 1,5 mL. Centrifugare la provetta a 100 x g per 1-2 min a temperatura ambiente per raccogliere protoplasti. Rimuovere il surnatante con una pipetta e trasferire 10 protoplasti µ l su una lastra di vetro.</li>
	<li>Osservare il segnale fluorescente sotto fluorescenza o microscopia confocale. Utilizzare non trasformate protoplasti come controllo negativo (non dovrebbe essere osservato nessun segnale).</li>
</ol>

<ol>
	<li>Melchers, G., Labib, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+hybridisation+of+plants+by+fusion+of+protoplasts.">Somatic hybridisation of plants by fusion of protoplasts.</a> Mol. Gen. Genet. 135 (4), 277-294 (1974).</li><li>Gresshoff, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+white+clover:+callus,+suspension,+protoplast+culture,+and+plant+regeneration.">In vitro culture of white clover: callus, suspension, protoplast culture, and plant regeneration.</a> Bot. Gaz. 141 (2), 157-164 (1980).</li><li>L&#246;rz, H., Baker, B., Schell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+cereal+cells+mediated+by+protoplast+transformation.">Gene transfer to cereal cells mediated by protoplast transformation.</a> Mol. Gen. Genet. 199 (2), 178-182 (1985).</li><li>Hayashimoto, A., Li, Z., Murai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polyethylene+glycol-mediated+protoplast+transformation+system+for+production+of+fertile+transgenic+rice+plants.">A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants.</a> Plant Physiol. 93 (3), 857-863 (1990).</li><li>Koop, H. -. U., 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=Integration+of+foreign+sequences+into+the+tobacco+plastome+via+polyethylene+glycol-mediated+protoplast+transformation.">Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.</a> Planta. 199 (2), 193-201 (1996).</li><li>Hrazdina, G., Wagner, G. J., Siegelman, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+enzymes+of+anthocyanin+biosynthesis+in+protoplasts.">Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts.</a> Phytochemistry. 17 (1), 53-56 (1978).</li><li>Lin, W., Wittenbach, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+proteases+in+wheat+and+corn+mesophyll+protoplasts.">Subcellular localization of proteases in wheat and corn mesophyll protoplasts.</a> Plant Physiol. 67 (5), 969-972 (1981).</li><li>V&#246;geli-Lange, R., Wagner, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+cadmium+and+cadmium-binding+peptides+in+tobacco+leaves.">Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves.</a> Plant Physiol. 92 (4), 1086-1093 (1990).</li><li>Chen, S., 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=A+highly+efficient+transient+protoplast+system+for+analyzing+defence+gene+expression+and+protein-protein+interactions+in+rice.">A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.</a> Mol. Plant Pathol. 7 (5), 417-427 (2006).</li><li>Wu, F. -. H., 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=Tape-Arabidopsis+Sandwich-a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method.</a> Plant methods. 5 (1), 16 (2009).</li><li>Christensen, A. H., Sharrock, R. A., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+polyubiquitin+genes:+structure,+thermal+perturbation+of+expression+and+transcript+splicing,+and+promoter+activity+following+transfer+to+protoplasts+by+electroporation.">Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.</a> Plant Mol. Biol. 18 (4), 675-689 (1992).</li><li>Marcotte, W. R., Bayley, C. C., Quatrano, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+wheat+promoter+by+abscisic+acid+in+rice+protoplasts.">Regulation of a wheat promoter by abscisic acid in rice protoplasts.</a> Nature. 335 (6189), 454-457 (1988).</li><li>Dron, M., Clouse, S. D., Dixon, R. A., Lawton, M. A., Lamb, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+fungal+elicitor+regulation+of+a+plant+defense+gene+promoter+in+electroporated+protoplasts.">Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts.</a> P. Natl. A. Sci. USA. 85 (18), 6738-6742 (1988).</li><li>Cocking, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+plant+protoplasts+and+vacuoles.">A method for the isolation of plant protoplasts and vacuoles.</a> Nature. 187 (4741), 962-963 (1960).</li><li>Zhang, H., 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=Transgenic+rice+plants+produced+by+electroporation-mediated+plasmid+uptake+into+protoplasts.">Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts.</a> Plant Cell Rep. 7 (6), 379-384 (1988).</li><li>Fromm, M., Taylor, L. P., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+genes+transferred+into+monocot+and+dicot+plant+cells+by+electroporation.">Expression of genes transferred into monocot and dicot plant cells by electroporation.</a> P. Natl. A. Sci. USA. 82 (17), 5824-5828 (1985).</li><li>Crossway, 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=Integration+of+foreign+DNA+following+microinjection+of+tobacco+mesophyll+protoplasts.">Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts.</a> Mol. Gen. Genet. 202 (2), 179-185 (1986).</li><li>Holm, P. B., Olsen, O., Schnorf, M., Brinch-Pedersen, H., Knudsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+barley+by+microinjection+into+isolated+zygote+protoplasts.">Transformation of barley by microinjection into isolated zygote protoplasts.</a> Transgenic Res. 9 (1), 21-32 (2000).</li><li>Schapire, A. L., Lois, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+and+rapid+method+for+the+isolation+and+transfection+of+Arabidopsis+leaf+mesophyll+protoplasts+for+large-scale+applications.">A simplified and rapid method for the isolation and transfection of Arabidopsis leaf mesophyll protoplasts for large-scale applications.</a> Plant Signal Transduction: Methods and Protocols. 1363, 79-88 (2016).</li><li>Niedz, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+somatic+embryos+from+sweet+orange+(C.+sinensis)+protoplasts+using+semi-permeable+membranes.">Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes.</a> Plant Cell Tiss. Org. Cult. 84 (3), 353-357 (2006).</li><li>Sheng, X., 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=Protoplast+isolation+and+plant+regeneration+of+different+doubled+haploid+lines+of+cauliflower+(Brassica+oleracea+var.+botrytis).">Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis).</a> Plant Cell Tiss. Org. 107 (3), 513-520 (2011).</li><li>Grun, P., Chu, L. -. J. <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+plants+from+protoplasts+of+Solanum+(Solanaceae).">Development of plants from protoplasts of Solanum (Solanaceae).</a> Am. J. Bot. 65 (5), 538-543 (1978).</li><li>Davey, M. R., Anthony, P., Power, J. B., Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+protoplasts:+status+and+biotechnological+perspectives.">Plant protoplasts: status and biotechnological perspectives.</a> Biotechnol. Adv. 23 (2), 131-171 (2005).</li><li>Pitzschke, A., Persak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poinsettia+protoplasts-a+simple,+robust+and+efficient+system+for+transient+gene+expression+studies.">Poinsettia protoplasts-a simple, robust and efficient system for transient gene expression studies.</a> Plant methods. 8 (1), 14 (2012).</li><li>Yoo, S. -. D., Cho, Y. -. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat. Protoc. 2 (7), 1565 (2007).</li><li>Sedivy, E. J., Wu, F., Hanzawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soybean+domestication:+the+origin,+genetic+architecture+and+molecular+bases.">Soybean domestication: the origin, genetic architecture and molecular bases.</a> New Phytol. 214 (2), 539-553 (2017).</li><li>Yang, Y., Li, R., Qi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+plant+promoters+and+transcription+factors+by+agroinfiltration+of+tobacco+leaves.">In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves.</a> Plant J. 22 (6), 543-551 (2000).</li><li>Marion, 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=Systematic+analysis+of+protein+subcellular+localization+and+interaction+using+high-throughput+transient+transformation+of+Arabidopsis+seedlings.">Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings.</a> Plant J. 56 (1), 169-179 (2008).</li><li>Wu, H. -. 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=AGROBEST:+an+efficient+Agrobacterium-mediated+transient+expression+method+for+versatile+gene+function+analyses+in+Arabidopsis+seedlings.">AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings.</a> Plant methods. 10 (1), 19 (2014).</li><li>Govindarajulu, M., Elmore, J. M., Fester, T., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+constitutive+viral+promoters+in+transgenic+soybean+roots+and+nodules.">Evaluation of constitutive viral promoters in transgenic soybean roots and nodules.</a> Mol Plant Pathol. 21 (8), 1027-1035 (2008).</li><li>Nagamatsu, 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=Functional+analysis+of+soybean+genes+involved+in+flavonoid+biosynthesis+by+virus-induced+gene+silencing.">Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.</a> Plant Biotechnol. J. 5 (6), 778-790 (2007).</li><li>Juvale, P. S., 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=Temporal+and+spatial+Bean+pod+mottle+virus-induced+gene+silencing+in+soybean.">Temporal and spatial Bean pod mottle virus-induced gene silencing in soybean.</a> Mol. Plant Pathol. 13 (9), 1140-1148 (2012).</li><li>Zhang, C., Bradshaw, J. D., Whitham, S. A., Hill, 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=The+development+of+an+efficient+multipurpose+bean+pod+mottle+virus+viral+vector+set+for+foreign+gene+expression+and+RNA+silencing.">The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.</a> Plant Physiol. 153 (1), 52-65 (2010).</li><li>Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mesophyll+protoplasts+from+mature+leaves+of+soybeans.">Isolation of mesophyll protoplasts from mature leaves of soybeans.</a> Plant Physiol. 73 (4), 1067-1069 (1983).</li><li>Yi, 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=A+single-repeat+MYB+transcription+factor,+GmMYB176,+regulates+CHS8+gene+expression+and+affects+isoflavonoid+biosynthesis+in+soybean.">A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean.</a> Plant J. 62 (6), 1019-1034 (2010).</li><li>Faria, J. 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=The+NAC+domain-containing+protein,+GmNAC6,+is+a+downstream+component+of+the+ER+stress-and+osmotic+stress-induced+NRP-mediated+cell-death+signaling+pathway.">The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway.</a> BMC Plant Biol. 11 (1), 129 (2011).</li><li>Kidokoro, S., 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=Soybean+DREB1/CBF-type+transcription+factors+function+in+heat+and+drought+as+well+as+cold+stress-responsive+gene+expression.">Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.</a> Plant J. 81 (3), 505-518 (2015).</li><li>Sun, X., 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=Targeted+mutagenesis+in+soybean+using+the+CRISPR-Cas9+system.">Targeted mutagenesis in soybean using the CRISPR-Cas9 system.</a> Sci Rep-UK. 5, 10342 (2015).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY&#8482;+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY&#8482; vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Xia, Z., 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=Positional+cloning+and+characterization+reveal+the+molecular+basis+for+soybean+maturity+locus+E1+that+regulates+photoperiodic+flowering.">Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.</a> P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Questo protocollo per l'isolamento di protoplasti di soia e l'applicazione di studi di espressione transitoria è stato accuratamente testato e funziona molto bene nel nostro laboratorio. Le procedure sono semplici e facili e richiedono apparecchiature ordinarie e minimo costo. Il nostro protocollo produce grandi quantità di protoplasti di uniforme, di alta qualità rispetto ai metodi precedentemente segnalati34,35,36,</su...

Protoplasti sono cellule vegetali che hanno rimosse le pareti cellulari. Come mantengono la maggior parte delle caratteristiche e attività di cellule vegetali, protoplasti sono un sistema di buon modello di osservare e valutare diversi eventi cellulari e sono strumenti preziosi per studiare ibridazione somatica1 e impianto di rigenerazione2. Protoplasti sono stati anche ampiamente utilizzati per impianto trasformazione3,4,5, poiché le pareti cellulari altrimenti bloccherebbe il passaggio del DNA nella cellula. Protoplasti possiedono alcune delle risposte fisiologiche e processi cellulari di piante intatte, offrendo quindi un valore fondamentale nella ricerca di base per lo studio di proteine sottocellulari localizzazione6,7,8, interazioni proteina-proteina9,10e promotore attività11,12,13 in cellule vive.
L'isolamento di protoplasti di pianta in primo luogo è stato segnalato nel 196014 e i protocolli per l'isolamento e la trasformazione dei protoplasti sono stati sviluppati e ottimizzati. Una procedura standard di isolamento del protoplasto comporta il taglio delle foglie e la digestione enzimatica delle pareti cellulari, seguita dalla separazione dei protoplasti rilasciati da detriti del tessuto non digerito. Strategie di trasformazione include elettroporazione15,16, microinjection17,18e base di polietilenglicole (PEG)4,5,19 metodi. Una vasta gamma di specie sono stati segnalati successo per l'isolamento di protoplasti, compresi agrumi20, Brassica21, Solanaceae22 e altre piante ornamentali famiglie23,24. Mentre i tipi di tessuti diversi sono utilizzati in varie specie, un sistema di espressione transiente in protoplasti di Arabidopsis mesofillo (TEAMP) isolato dalle foglie della pianta modello Arabidopsis thaliana è stato affermata25 e ampiamente adottato per diverse applicazioni.
Soia (Glycine max (L.) Merr.) è una delle proteine più importanti e olio colture26. A differenza di Arabidopsis e riso, ottenimento di piante di soia transgenica è noto per essere piuttosto difficile e basso rendimento. Agrobacterium tumefaciens-mediata infiltrazione è stato popolarmente utilizzato per studi di espressione transiente in cellule epidermiche in tabacco27 e piantine in Arabidopsis28,29, considerando che Agrobacterium rhizogenes è stato utilizzato per la trasformazione delle radici pelosi in soia30. Approcci di silenziamento genica indotta da virus sono stati utilizzati per downregulation di destinazione geni31,32 e transitori espressione33 in maniera sistematica. Protoplasti forniscono una preziosa e versatile alternativa a questi approcci. Protoplasti possono essere ottenuti da materiali aboveground di soia e consentono l'espressione del transgene rapido e sincronizzato. Tuttavia, poiché l'isolamento iniziale successo dei protoplasti di soia nel 198334, ci sono stati rapporti limitati sull'applicazione dei protoplasti in soia35,36,37, 38, principalmente a causa della relativamente basse rese dei protoplasti di soia.
Qui, descriviamo un protocollo semplice ed efficiente per l'isolamento di protoplasti di soia e la sua applicazione per studi di espressione genica transitoria. Utilizzando foglie giovani unifoliate da piantine di soia, siamo stati in grado di ottenere grandi quantità di protoplasti vitali entro poche ore. Inoltre, abbiamo ottimizzato un metodo di trasformazione di PEG-calcio-mediata che è semplice e a basso costo per trasportare DNA in protoplasti di soia con alta efficienza.

<table><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>BP1427-500</td><td></td></tr></tbody></table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Soia (Glycine max (L.) Merr.) è una specie di coltura importante ed è diventato un modello di legumi per gli studi delle vie genetiche e biochimiche. Pertanto, è importante stabilire un sistema di espressione genica transitoria efficiente in soia. Qui, segnaliamo un semplice protocollo per la preparazione dei protoplasti di soia e la sua applicazione per l'analisi funzionale transitorie. Abbiamo trovato che le foglie giovani unifoliate da piantine di soia ha provocato grandi quantità di protoplasti di alta qualità. Grazie all'ottimizzazione di un metodo di trasformazione PEG-calcio-mediata, abbiamo raggiunto l'efficienza di trasformazione alta utilizzando protoplasti unifoliate soia. Questo sistema fornisce un modello efficiente e versatile per l'esame dei complessi meccanismi di regolamentazione e di segnalazione in cellule di soia dal vivo e può aiutare per meglio comprendere diversi processi cellulari, inerente allo sviluppo e fisiologici dei legumi.

Diversi organi di soia 10 - giorno-vecchio sono stati testati per la preparazione del protoplasto (Figura 1) e i rendimenti sono stati osservati al microscopio (Figura 2). Pareti cellulari da ipocotile ed epicotyl erano appena digeriti, e alcune cellule siamo stati attaccati alla vicenda (Figura 2B, 2C). Nel cotiledone (Figura 2D) e radice (<strong class...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Abbiamo sviluppato un protocollo semplice ed efficiente per la preparazione di grandi quantità di protoplasti di soia studiare i complessi meccanismi di regolamentazione e di segnalazione in cellule vive.

Un metodo semplice per l'isolamento di protoplasti di soia e l'applicazione di analisi di espressione genica transitoria

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.9K Views.  California State University, Northridge. Abbiamo sviluppato un protocollo semplice ed efficiente per la preparazione di grandi quantità di protoplasti di soia studiare i complessi meccanismi di regolamentazione e di segnalazione in cellule vive.

Video: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Una piattaforma robotica per high-throughput Protoplast Isolamento e Trasformazione

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Ricerca

Genetica

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Transient espressione genica in tabacco con all&#39;Assemblea Gibson e Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Ambiente

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolamento di protoplasti da tessuti di 14-giorni di età piantine di Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Biologia

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotica e Image Analysis dinamica per gli Studi di espressione genica nei tessuti vegetali

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificazione di sequenze di localizzazione Plasmodesmal in proteine In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Immunologia e infezioni

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Produzione ad alta efficienza di radici pelose di soia transgenica

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Autore in primo piano: Trasformazione della radice pelosa della soia per l'analisi della funzione genica  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Trasformazione della radice pelosa di soia per l'analisi della funzione genica

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

Un metodo semplice per l'isolamento di protoplasti di soia e l'applicazione di analisi di espressione genica transitoria

Riepilogo

Esplora Altri Video

Una piattaforma robotica per high-throughput Protoplast Isolamento e Trasformazione

Transient espressione genica in tabacco con all'Assemblea Gibson e Gene Gun

Isolamento di protoplasti da tessuti di 14-giorni di età piantine di Arabidopsis thaliana

Robotica e Image Analysis dinamica per gli Studi di espressione genica nei tessuti vegetali

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in Arabidopsis thaliana

Identificazione di sequenze di localizzazione Plasmodesmal in proteine In Planta

Isolamento efficiente e rapida di embrioni nelle prime fasi di

Trasformazione della radice pelosa di soia per l'analisi della funzione genica

Trasformazione della radice pelosa di soia per l'analisi della funzione genica

Trasformazione della radice pelosa di soia per l'analisi della funzione genica