Abstract
The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.
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References
Appenroth KJ, Borisjuk N, Lam E (2013) Telling duckweed apart: genoty** technologies for the lemnaceae. Chin J Appl Environ Biol 19:1–10
Batta AK, Xu G, Honda A, Miyazaki T, Salen G (2006) Stigmasterol reduces plasma cholesterol levels and inhibits hepatic synthesis and intestinal absorption in the rat. Metabolism 55:292–299
Bertran K, Bertran K, Thomas C, Guo X, Bublot M, Pritchard N, Regan JT, Cox KM, Gasdaska JR, Dickey LF, Kapczynski DR, Swayne DE (2015) Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens. Vaccine 33:3456–3462
Chang WC, Chiu PL (1976) Induction of callus from fronds of duckweed (Lemna gibba L.). Bot Bull Acad Sin 17:106–109
Chhabra G, Chaudhary D, Sainger M, Jaiwal PK (2011) Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants. Physiol Mol Biol Plants 17:129–136
Christou P (1990) Morphological description of transgenic soybean chimeras created by the delivery, integration and expression of foreign DNA using electric discharge particle acceleration. Ann Bot-London 66:379–386
Christou P, Ford TL (1995) Recovery of chimeric rice plants from dry seed using electric discharge particle acceleration. Ann Bot-London 75:449–454
Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597
Dekeyser R, Claes B, Marichal M, Van MM, Caplan A (1989) Evaluation of selectable markers for rice transformation. Plant Physiol 90:217
Desai PN, Shrivastava N, Padh H (2010) Production of heterologous protein in plants: strategies for optimal expression. Biotechnol Adv 28:427–435
Dominguez A, Cervera M, Perez RM, Romero J, Fagoaga C, Cubero J, Lopez MM, Juarez JA, Navarro L (2004) Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breeding 14:171–183
Gabay O, Sanchez C, Salvat C, Chevy F, Breton M, Nourissat G, Wolf C, Jacques C, Berenbaum F (2010) Stigmasterol: a phytosterol with potential anti-osteoarthritic properties. Osteoarthr Cartil 18:106–116
Ge X, Zhang N, Phillips GC, Xu J (2012) Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol. Bioresour Technol 124:485–488
Ghosh T, Maity TK, Singh J (2011) Evaluation of antitumor activity of stigmasterol, a constituent isolated from Bacopa monnieri Linn aerial parts against Ehrlich Ascites Carcinoma in mice. Orient Pharm Exp Med 11:41–49
Hamada H, Linghu Q, Nagira Y, Miki R, Taoka N, Imai R (2017) An in planta biolistic method for stable wheat transformation. Sci Rep 7:11443
He Y, Ning T, **e T, Qiu Q, Zhang L, Sun Y, Jiang D, Fu K, Yin F, Zhang W, Shen L, Wang H, Li J, Lin Q, Sun Y, Li H, Zhu Y, Yang D (2011) Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci USA 108:19078–19083
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Stn Circ 347:1–32
Huang M, Fu LL, Sun XP, Di R, Zhang JM (2016) Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata. Acta Physiol Plant 38:122
Iatrou EI, Stasinakis AS, Aloupi M (2015) Cultivating duckweed Lemna minor in urine and treated domestic wastewater for simultaneous biomass production and removal of nutrients and antimicrobials. Ecol Eng 84:632–639
Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056–1063
Jamaluddin F, Mohamed S, Lajis MN (1994) Hypoglycaemic effect of Parkia speciosa seeds due to the synergistic action of β-sitosterol and stigmasterol. Food Chem 49:339–345
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Khanna D, Sethi G, Ahn KS, Pandey MK, Kunnumakkara AB, Sung B, Aggarwal A, Aggarwal BB (2007) Natural products as a gold mine for arthritis treatment. Curr Opin Pharmacol 7:344–351
Khvatkov P, Chernobrovkina M, Okuneva A, Shvedova A, Chaban I, Dolgov S (2015) Callus induction and regeneration in Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss Org 120:263–273
Ko SM, Sun HJ, Oh MJ, Song IJ, Kim MJ, Sin HS, Goh CH, Kim YW, Lim PO, Lee HY, Kim SW (2011) Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor. Hortic Environ Biote 52:511–515
Komari T, Kubo T (1999) Methods of genetic transformation: Agrobacterium tumefaciens. Molecular improvement of cereal crops. Springer, Dordrecht 5:43–82
Kovganko NV, Survilo VL (2000) New synthesis of ecdysteroids based on stigmasterol. Chem Nat Compd 36:377–380
Lam E, Appenroth KJ, Michael T, Mori K, Fakhoorian T (2014) Duckweed in bloom: the 2nd international conference on duckweed research and applications heralds the return of a plant model for plant biology. Plant Mol Biol 84:737–742
Les DH, Crawford DJ, Landolt E, Gabel JD, Kimball RT (2002) Phylogeny and systematics of Lemnaceae, the duckweed family. Syst Bot 27:221–240
Liu GY, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681
Mathews H, Wagoner W, Kellogg J, Bestwick R (1995) Genetic transformation of strawberry: stable integration of a gene to control biosynthesis of ethylene. In Vitro Cell Dev-Pl 31:36–43
Moon HK, Stomp AM (1997) Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (duckweed). In Vitro Cell Dev-Pl 33:20–25
Moreira AR (2007) The evolution of protein expression and cell culture. Biopharm Int 20:56–68
Oron G (1994) Duckweed culture for wastewater renovation and biomass production. Agr Water Manage 26:27–40
Ozias-Akins P, Rogers SG, Fraley RT, Horsch RB, Vasil IK (1988) Evaluation of selectable markers for obtaining stable transformants in the Gramineae. Plant Physiol 86:602–606
Panda S, Jafri M, Kar A, Meheta BK (2009) Thyroid inhibitory, antiperoxidative and hypoglycemic effects of stigmasterol isolated from Butea monosperma. Fitoterapia 80:123–126
Pandith H, Zhang X, Thongpraditchote S, Wongkrajang Y, Gritsanapan W, Baek SJ (2013) Effect of Siam weed extract and its bioactive component scutellarein tetramethyl ether on anti-inflammatory activity through NF-kappaB pathway. J Ethnopharmacol 147:434–441
Popov SV, Golovchenko VV, Ovodova RG, Smirnov VV, Khramova DS, Popova GY, Ovodov YS (2006) Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L. Vaccine 24:5413–5419
R Höfgen LW (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877
Rakosy TE, Aurori CM, Dijkstra C, Thieme R, Aurori A, Davey MR (2007) The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes. Plant Cell Rep 26:661–671
Raksha BR, Siva R, Vino S, Babu S (2016) Spatio-varietal differences in stigmasterol biosynthesis in tomato and overexpression of a sterol desaturase gene for enhanced stigmasterol production. In Vitro Cell Dev-Pl 52:571–579
Saveleva NV, Burlakovskiy MS, Yemelyanov VV, Lutova LA (2016) Transgenic plants as bioreactors to produce substances for medical and veterinary uses. Russ J Genet Appl Res 6:712–724
Schmülling T, Schell J (1993) Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras. Plant Mol Biol 21:705–708
Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811
Stefaniak B, Woźny A, Budna I (2002) Callus induction and plant regeneration in Lemna Minor L. Biol Plantarum 45:469–472
Stomp AM (2005) The duckweeds: a valuable plant for biomanufacturing. Biotechnol Annu Rev 11:69
Sundararaman P, Djerassi C (1977) A convenient synthesis of progesterone from stigmasterol. J Org Chem 42:3633–3634
Van Hoeck A, Horemans N, Monsieurs P, Cao HX, Vandenhove H, Blust R (2015) The first draft genome of the aquatic model plant Lemna minor opens the route for future stress physiology research and biotechnological applications. Biotechnol Biofuels 8:188
Vunsh R, Li J, Hanania U, Edelman M, Flaishman M, Perl A, Wisniewski JP, Freyssinet G (2007) High expression of transgene protein in Spirodela. Plant Cell Rep 26:1511–1519
Wang W, Haberer G, Gundlach H et al (2014) The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat Commun 5:3311
Whitaker BD, Gapper NE (2008) Ripening-specific stigmasterol increase in tomato fruit is associated with increased sterol C-22 desaturase (CYP710A11) gene expression. J Agr Food Chem 56:3828–3835
Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3:119–128
Wilmink A, Dons JJM (1993) Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Mol Biol Rep 11:165–185
Zhao H, Appenroth K, Landesman L, Salmean AA, Lam E (2012) Duckweed rising at Chengdu: summary of the 1st international conference on duckweed application and research. Plant Mol Biol 78:627–632
Acknowledgements
This study was supported by the National Key Technology R&D Program of China (2015BAD15B01), the National Natural Science for General Foundation of China (31770395), Key deployment projects of Chinese Academy of Sciences (ZDRW-ZS-2017-2-1), Science and Technology Service Network Initiative of Chinese Academy of Sciences (KFJ-STS-ZDTP-008); Science & Technology Program of Sichuan Province (2017NZ0018 and 2017HH0077), and the Key and Open Fund of Key Laboratory of Environmental and Applied Microbiology, Chinese Academy of Sciences (KLEAMCAS201501, KLCAS-2014-05 and KLCAS-2016-02).
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HZ, XRM, GLY and YF conceived the research. GLY and XRM performed experiments. GLY and XRM analyzed the data and wrote the manuscript. YLX, LT, QL, YL, FL, YLJ, APD, and KZH contributed partial experiments.
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Yang, GL., Fang, Y., Xu, YL. et al. Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor. Plant Mol Biol 98, 319–331 (2018). https://doi.org/10.1007/s11103-018-0778-x
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DOI: https://doi.org/10.1007/s11103-018-0778-x