Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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This work was supported by the Plant Genome Research Program from the National Science Foundation (NSF-PGRP-IOS-1339388).

1. Growth of the plants
<ol>
	<li>Sow 5 - 10 soybean seeds (Williams 82) in a 13 cm pot in the greenhouse under long-day conditions (16 h light at 1,500 &#181;mol m-2 s-1) at 25 &#176;C on the custom soil mix for Soybean (the 1:1:1 ratio of soil, perlite and torpedo sand).</li>
</ol>
2. Preparation of Plasmid DNA
<ol>
	<li>Using a sterile pipette tip or toothpick, pick a single colony or frozen glycerol stock of E. coli carrying the plasmid containing the gene of interest and inoculate it in 20 mL Luria-Bertani (LB) liquid medium with appropriate antibiotics in a 50 mL flask.</li>
	<li>Incubate the flask at 37 &#176;C overnight on a shaker. Collect the bacteria by centrifuging the suspension at 12,000 x g at room temperature for 5 min and discarding the supernatant. Extract and purify the plasmid following the manufacturer&#39;s procedure of a plasmid prep kit.</li>
</ol>
3. Protoplast isolation
<ol>
	<li>Cut newly expanded unifoliate leaves from 10-day old soybean seedlings (Figure 1). 
	NOTE: Selection of leaves at an appropriate developmental stage is the key to the success for soybean protoplast preparation. Only use just expanded leaves at early developmental stages. As leaves mature, cell walls become harder to digest.</li>
	<li>With a fresh razor blade, remove the midrib from the unifoliate leaf and then cut the remains into 0.5-1 mm strips. 
	NOTE: Two unifoliate leaves digested in 10 mL enzyme solution will give sufficient protoplasts for more than 10 transformations.</li>
	<li>Using a pair of forceps, transfer the leaf strips immediately and gently into 10 mL of freshly prepared enzyme solution (Table 1) in a 15 mL tube. Vacuum infiltrate the leaf strips for 15 min at room temperature.</li>
	<li>Incubate the leaf strips in the enzyme solution with gentle agitation (40 rpm) under low light for 4-6 h at room temperature. Ensure that the enzyme solution turns yellow-green as protoplasts are released. Check the enzyme/protoplasts solution under the microscope (X10). 
	NOTE: The released protoplasts are spherical shaped, while the undigested cells have irregular or oval shape.</li>
	<li>Transfer the 10 mL of enzyme/protoplast solution in a 50 mL tube by gently pouring and add 10 mL of W5 solution (Table 1) at room temperature to stop the digestion. Gently invert the tube a few times. Pour gently the enzyme/protoplasts solution on a clean 75 &#181;m nylon mesh placed on top of a 50 mL tube to remove the undigested leaf tissues.</li>
	<li>Centrifuge the flow-through enzyme/protoplasts solution at 100 x g in the 50 mL tube for 1-2 min at room temperature. Gently remove the supernatant using a 10 mL serological pipette without disturbing the protoplast pellet.</li>
	<li>Resuspend the protoplasts and dilute to a concentration of 2 x 105 mL-1 in chilled W5 solution at 4 &#176;C by counting protoplast number on a hemacytometer under the microscope (x10). Keep the protoplasts on ice for 30 min.</li>
	<li>Centrifuge the suspension at 100 x g for 1-2 min at room temperature and gently remove the W5 solution using a 1 mL pipette without disturbing the protoplast pellet. Resuspend the protoplasts in MMG solution (Table 1) at a concentration of 2 x 105 mL-1 at room temperature.</li>
</ol>
4. Protoplast transformation
<ol>
	<li>Make 100 &#181;L aliquots of protoplasts (2 x 104 protoplasts at 2 x 105 mL-1) in 1.5 mL low adhesion microcentrifuge tubes using uncut 200 &#181;L pipette tips. Put one aliquot aside to serve as negative control. Add 10 &#181;L of plasmid (10-20 &#181;g) into each of the rest aliquot of protoplasts.</li>
	<li>Slowly add 110 &#181;L of freshly prepared PEG solution (Table 1) on the inner wall of the 1.5 mL microcentrifuge tube, and then gently invert and rotate the tube until the solution becomes homogeneous.</li>
	<li>Incubate the transformation mixture at room temperature for 15 min.</li>
	<li>To stop the transformation, slowly add 400 &#181;L of W5 solution to the 1.5 mL tube at room temperature and gently invert the tube until the solution becomes homogeneous. Centrifuge the tube at 100 g for 1-2 min at room temperature and discard the supernatant using a pipette.</li>
	<li>Add 1 mL of WI solution (Table 1) to the tube and resuspend by gently pipetting 1-2 times. Add 1 mL of 5% (vol/vol) sterile calf serum in each well of a 6-well tissue culture plate to coat the surface and prevent the protoplasts from sticking to the plate.</li>
	<li>After a few seconds, discard the calf serum using a pipette. Transfer the resuspended protoplasts into a well of the culture plate. Cover the plate with a lid.</li>
</ol>
5. Protoplast incubation and harvesting
<ol>
	<li>Incubate the protoplasts at room temperature for 1-2 days in the dark. 
	NOTE: We found that two-days incubation yields stronger fluorescent protein signal in general compared to one-day incubation, and that the fluorescent protein signal may last for up to 3-4 days.</li>
	<li>Transfer the protoplast solution to a 1.5 mL low adhesion microcentrifuge tube. Centrifuge the tube at 100 x g for 1-2 min at room temperature to harvest protoplasts. Remove the supernatant using a pipette and transfer 10 &#181;L protoplasts onto a glass slide.</li>
	<li>Observe fluorescent signal under fluorescence or confocal microscopy. Use non-transformed protoplasts as negative control (No signal should be observed).</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>

The authors declare that they have no competing financial interests.

This protocol for the isolation of soybean protoplasts and the application to transient expression studies has been thoroughly tested and works very well in our laboratory. The procedures are simple and easy and require ordinary equipment and minimum cost. Our protocol yields large quantities of uniform, high quality protoplasts compared to previously reported methods34,35,36,37,38. However, since there are many factors that affect protoplast yields and transformation rates, it is strongly recommended for researchers to optimize the conditions according to their experimental conditions, desirable results and materials used. While this system is extremely useful for examination of immediate regulatory and biochemical events in plant cells, it is not suitable for observation of long term cellular processes and events that occur at tissue or organismal levels.
We found that selection of vigorously growing plant materials at an ideal developmental stage was the most crucial factor in the soybean protoplast preparation. It determines not only yields of protoplasts, but quality that affects the subsequent DNA transformation. It is important to always grow soybean plants in a constant environment away from any stress, such as drought, flooding, extreme temperatures or pests. The highest protoplast yield and transformation efficiency can be achieved using just expanded unifoliate leaves from young seedlings. For beginners, it is suggested to use unifoliate leaves at different developmental stages and make a comparison to obtain the best results.
Protoplast/DNA ratio is an important factor for optimal transformation efficiency. It is usually best to start with the ratio of 2 x 104 protoplasts/10-20 &#181;g DNA, but optimization of the ratio for individual constructs is recommended25. The use of high quality DNA obtained by a DNA purification kit is strongly recommended.
For the choice of vectors for transient expression in protoplasts, high-copy number and small-sized vectors are generally preferred. Although binary vectors for Agrobacterium tumefaciens-mediated transformation of plants can be used for protoplast transformation, the large size of these vectors may result in less optimal transformation efficiency. For GFP-fusion protein expression, we usually use vectors that do not possess a selection marker for plants and are thus relatively small (6-7kb), such as p2GWF7 and p2FGW739 (https://gateway.psb.ugent.be).
Multiple vectors can be used to transform protoplasts simultaneously. We had success in expressing BiFC vectors for testing protein-protein interactions, as well as multiple fluorescent proteins for organelle and subcellular labeling in soybean protoplasts. Furthermore, simplicity and large yields of our method offers an ideal system for examination of regulatory and biochemical events, gene-targeting vector design, isolation of transiently expressed proteins and protein complex, and purification of specific organelle such as nuclei, enabling further applications to emerging genomic and proteomic approaches.

Protoplasts are plant cells that have cell walls removed. As they maintain most of features and activities of plant cells, protoplasts are a good model system to observe and evaluate diverse cellular events, and are valuable tools to study somatic hybridisation1 and plant regeneration2. Protoplasts have been also widely utilized for plant transformation3,4,5, since cell walls would otherwise block the passage of DNA into the cell. Protoplasts possess some of the physiological responses and cellular processes of intact plants, hence offering fundamental value in basic research to study subcellular protein localization6,7,8, protein-protein interactions9,10, and promoter activity11,12,13 in live cells.
The isolation of plant protoplasts was first reported in 196014 and the protocols for both isolation and transformation of protoplasts have been developed and optimized. A standard procedure of protoplast isolation involves the cutting of leaves and enzymatic digestion of cell walls, followed by separation of released protoplasts from non-digested tissue debris. Transformation strategies includes electroporation15,16, microinjection17,18, and polyethylene glycol-based (PEG)4,5,19 methods. A wide range of species have been reported successful for protoplast isolation, including Citrus20, Brassica21, Solanaceae22 and other ornamental plant families23,24. While diverse tissue types are used in various species, a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) isolated from leaves of the model plant Arabidopsis thaliana has been well established25 and widely adopted to diverse applications.
Soybean (Glycine max (L.) Merr.) is one of the most important protein and oil crops26. Unlike Arabidopsis and rice, obtaining transgenic soybean plants is known to be rather difficult and low efficiency. Agrobacterium tumefaciens-mediated infiltration has been popularly used for transient gene expression studies in the epidermal cells in tobacco27 and seedlings in Arabidopsis28,29, whereas Agrobacterium rhizogenes has been used for transformation of hairy roots in soybean30. Virus-induced gene silencing approaches have been utilized for downregulation of target genes31,32 and transient expression33 in a systemic manner. Protoplasts provide a valuable and versatile alternative to these approaches. Protoplasts can be obtained from soybean&#39;s aboveground materials and allow quick and synchronized transgene expression. However, since the initial successful isolation of soybean protoplasts in the 198334, there have been limited reports on the application of protoplasts in soybean35,36,37,38, primarily due to relatively low yields of soybean protoplasts.
Here, we describe a simple and efficient protocol for isolation of soybean protoplasts and its application for transient gene expression studies. Using young unifoliate leaves from soybean seedlings, we were able to obtain large quantities of vital protoplasts within a few hours. In addition, we have optimized a PEG-calcium-mediated transformation method that is simple and low cost to deliver DNA into soybean protoplasts with high efficiency.

<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

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

Different organs of 10-day old soybeans were tested for protoplast preparation (Figure 1) and yields were observed under the microscope (Figure 2). Cell walls from hypocotyl and epicotyl were hardly digested, and some cells stayed attached to each other (Figure 2B, 2C). In cotyledon (Figure 2D) and root (Figure 2A), cell walls were removed only in a small portion of the cells. In contrast, a large number of protoplasts were observed when unifoliate was used (Figure 2E-G). Unifoliate leaves at different developmental stages were further examined (Figure 2H). While both the unexpanded and just expanded unifoliate leaves resulted in high yields of protoplasts, the size of protoplasts from the just expanded unifoliate were more uniform (Figure 2F) than the unexpanded unifoliate (Figure 2E). For the fully expanded unifoliate, the cell walls were still intact in most of the cells (Figure 2G). We tested cell wall-digestion enzymes from three different manufacturers and obtained comparable results as described above. Based on these observations, we concluded that selection of plant materials was a crucial factor and that just expanded unifoliate leaves from young soybean seedlings were the best material for protoplast preparation.
A range of different amounts of plasmid DNA (0.1 &#181;g, 1 &#181;g, 5 &#181;g and 20 &#181;g) was tested for optimal transformation efficiency of soybean unifoliate protoplasts (Figure 3A-D). 20 &#181;g plasmid DNA showed the highest transformation efficiency with more than 50% transformation rate (Figure 3D), while 0.1 &#181;g showed the lowest (less than 1%) (Figure 3A). We obtained comparable transformation efficiency using different DNA purification kits from three manufacturers. This result suggests that larger amounts of plasmid DNA would greatly help to increase the transformation efficiency.
Figure 4 is confocal images of soybean protoplasts transformed with construct p2GWF7-E1, which expresses GFP fused to the legume-specific gene E1 (Glyma.06G207800), driven by the CaMV 35S promoter in the vector p2GWF739. E1-GFP fusion protein shows nuclear localization in soybean protoplasts, which is consistent with a previous study using the Arabidopsis protoplast system40. Given that E1 is a legume-specific gene, our result using the soybean protoplast system provides a conclusive insight in the subcellular localization of E1 protein.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57258/57258fig1.jpg" /> 
Figure 1. Illustration showing the organs of a soybean seedling that are tested for protoplast preparation in this study, including root, hypocotyl, cotyledon, epicotyl and unifoliate. After a pair of unifoliate leaves, soybean seedlings develop trifoliate leaves. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57258/57258fig2.jpg" /> 
Figure 2. Protoplast cells prepared from different organs and developmental stages of soybean seedlings. (A-D) Cells prepared from different organs of 10-day old soybean seedlings: root (A), hypocotyl (B), epicotyl (C), cotyledon (D). (E-G) Protoplast cells prepared from unifoliate leaves at different developmental stages: unexpanded (E), just expanded (F), and fully expanded (G) unifoliate, corresponding to the soybean seedlings in (H) on the left, middle and right, respectively. The scale bar is 25 &#181;m (A-G) or 25 mm (H). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57258/57258fig3.jpg" /> 
Figure 3. Confocal images showing transformation efficiency of soybean unifoliate protoplasts with different amounts of plasmid DNA. Images of GFP (represented in green) and bright field (grey) are merged. Protoplasts were transformed with different amounts of the plasmid p2GWF7-E1: 0.1 &#181;g (A), 1 &#181;g (B), 5 &#181;g (C) and 20 &#181;g (D). Fluorescence signal of GFP was monitored 24 hours after transformation under microscopy. The black scale bar is 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57258/57258fig4.jpg" /> 
Figure 4. Confocal images showing the subcellular localization of E1-GFP in the nucleus of soybean unifoliate protoplasts. The plasmid p2GWF7-E1 was used for transformation. Images of GFP (represented in green) and bright field (grey) are merged. Fluorescence signal of GFP was monitored 24 hours after transformation. The black scale bar is 5 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Enzyme solution (freshly prepared)</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>20mM</td>
		</tr>
		<tr>
			<td>Cellulase CELF</td>
			<td>2% (w/v)</td>
		</tr>
		<tr>
			<td>Pectolyase Y-23</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>0.75 M</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.2 mM</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td colspan="2">W5 solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>154 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>125 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td colspan="2">MMg solution</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>400 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>15 mM</td>
		</tr>
		<tr>
			<td colspan="2">PEG solution (freshly prepared)</td>
		</tr>
		<tr>
			<td>PEG4000</td>
			<td>20% (w/v)</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td colspan="2">WI solution</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>0.5 M</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>20 mM</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions used for soybean protoplast isolation and transformation.

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

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

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

Nanyang Technological University

Research

JoVE Journal

Biology

24.8K Views.  California State University, Northridge. We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

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.

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 ...

Genetics

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.

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

Environment

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

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.

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

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.

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

High-Efficiency Production of Transgenic Soybean Hairy Roots

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.

Author Spotlight: Soybean Hairy Root Transformation for the Analysis of Gene Function  

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

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

Bioengineering

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

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

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Chapters in this video

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

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

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

Soybean Hairy Root Transformation for the Analysis of Gene Function

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Isolation and Transcriptome Analysis of Plant Cell Types

Scalable Transfection of Maize Mesophyll Protoplasts