Rapid and efficient production of transgenic bermudagrass and creeping bentgrass bypassing the callus formation phase
Zeng-Yu Wang A B and Yaxin Ge AA Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA.
B Corresponding author. Email: zywang@noble.org
Functional Plant Biology 32(9) 769-776 https://doi.org/10.1071/FP05083
Submitted: 7 April 2005 Accepted: 27 May 2005 Published: 26 August 2005
Abstract
Callus culture has been an inevitable step in genetic transformation of monocotyledonous (monocot) species. The induction and maintenance of embryogenic calluses is time-consuming, laborious and also requires experience. A straightforward and callus-free transformation procedure was developed and demonstrated for two monocot species, bermudagrass (Cynodon spp.) and creeping bentgrass (Agrostis stolonifera). Stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA or pTOK233 binary vectors. Green shoots were directly produced from infected stolon nodes 4–5 weeks after hygromycin selection. Without callus formation and with minimum tissue culture, this procedure allowed us to obtain well-rooted transgenic plantlets in only 7 weeks and greenhouse-grown plants in only 9 weeks. The established plants were screened by PCR; the transgenic nature of the plants was demonstrated by Southern hybridisation analysis. Expression of the transgenes was confirmed by northern hybridisation analysis and GUS staining. Based on the number of transgenic plants obtained and the number of stolon nodes inoculated, transformation frequencies of 4.8%–6.1% and 6.3%–11.3% were achieved for bermudagrass and creeping bentgrass, respectively. The rapid and efficient production of transgenic plants without callus induction is a significant improvement for genetic transformation of monocot species.
Keywords: Agrobacterium, Agrostis, bermudagrass, creeping bentgrass, Cynodon, forage and turf grass, genetic transformation, transgenic plant.
Acknowledgments
We thank Kathy Spohn for editing the manuscript. The research was supported by the Samuel Roberts Noble Foundation.
Aldemita RR, Hodges TK
(1996)
Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199, 612–617.
| Crossref | GoogleScholarGoogle Scholar |
Chen L,
Auh C,
Dowling P,
Bell J,
Chen F,
Hopkins A,
Dixon RA, Wang Z-Y
(2003) Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnology Journal 1, 437–449.
| Crossref | GoogleScholarGoogle Scholar |
Chen L,
Auh C,
Dowling P,
Bell J,
Lehmann D, Wang Z-Y
(2004) Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea). Functional Plant Biology 31, 235–245.
| Crossref | GoogleScholarGoogle Scholar |
Cheng M,
Hu TC,
Layton J,
Liu CN, Fry JE
(2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cellular & Developmental Biology. Plant 39, 595–604.
| Crossref | GoogleScholarGoogle Scholar |
Cheng M,
Lowe BA,
Spencer TM,
Ye XD, Armstrong CL
(2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cellular & Developmental Biology. Plant 40, 31–45.
| Crossref | GoogleScholarGoogle Scholar |
Chilton MD,
Currier TC,
Farrand SK,
Bendich AJ,
Gordon MP, Nester EW
(1974)
Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proceedings of the National Academy of Sciences USA 71, 3672–3676.
Cho MJ,
Choi HW, Lemaux PG
(2001) Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds. Plant Cell Reports 20, 318–324.
| Crossref | GoogleScholarGoogle Scholar |
Choi HW,
Lemaux PG, Cho M-J
(2000) Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Science 40, 524–533.
Dai S,
Zheng P,
Marmey P,
Zhang S,
Tian W,
Chen S,
Beachy RN, Fauquet C
(2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Molecular Breeding 7, 25–33.
| Crossref | GoogleScholarGoogle Scholar |
Dai WD,
Bonos S,
Guo Z,
Meyer WA,
Day PR, Belanger FC
(2003) Expression of pokeweed antiviral proteins in creeping bentgrass. Plant Cell Reports 21, 497–502.
| PubMed |
Frame BR,
Shou H,
Chikwamba RK,
Zhang Z, Xiang C , et al.
(2002)
Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiology 129, 13–22.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Goldman JJ,
Hanna WW,
Fleming GH, Ozias-Akins P
(2004) Ploidy variation among herbicide-resistant bermudagrass plants of cv. TifEagle transformed with the bar gene. Plant Cell Reports 22, 553–560.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Ha SB,
Wu FS, Thorne TK
(1992) Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. Plant Cell Reports 11, 601–604.
| Crossref | GoogleScholarGoogle Scholar |
Hanna WW, Elsner JE
(1999) Registration of ‘TifEagle’ bermudagrass. Crop Science 39, 1258.
Hartman CL,
Lee L,
Day PR, Tumer NE
(1994) Herbicide resistant turfgrass (Agrostis palustris Huds.) by biolistic transformation. Bio/Technology 12, 919–923.
| Crossref | GoogleScholarGoogle Scholar |
Hiei Y,
Ohta S,
Komari T, Kumashiro T
(1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal 6, 271–282.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Hu T,
Metz S,
Chay C,
Zhou HP, Biest N , et al.
(2003)
Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports 21, 1010–1019.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Huber M,
Hahn R, Hess D
(2002) High transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. ‘Combi’) by microbombardment of immature embryos followed by GFP screening combined with PPT selection. Molecular Breeding 10, 19–30.
| Crossref | GoogleScholarGoogle Scholar |
Janakiraman V,
Steinau M,
McCoy SB, Trick HN
(2002) Recent advances in wheat transformation. In Vitro Cellular & Developmental Biology. Plant 38, 404–414.
| Crossref | GoogleScholarGoogle Scholar |
Jauhar, PP (1993).
Ke J,
Khan R,
Johnson T,
Somers DA, Das A
(2001) High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens.
Plant Cell Reports 20, 150–156.
| Crossref | GoogleScholarGoogle Scholar |
Li L, Qu R
(2004) Development of highly regenerable callus lines and biolistic transformation of turf-type common bermudagrass [Cynodon dactylon (L.) Pers.]. Plant Cell Reports 22, 403–407.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Lichtenstein, C ,
and
Draper, J (1985). Genetic engineering of plants. In ‘DNA cloning’. pp. 67–119. (IRL Press: Oxford)
Luo H,
Hu Q,
Nelson K,
Longo C,
Kausch AP,
Chandlee JM,
Wipff JK, Fricker CR
(2004)
Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration. Plant Cell Reports 22, 645–652.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Murashige T, Skoog F
(1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497.
Popelka JC, Altpeter F
(2003)
Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.). Molecular Breeding 11, 203–211.
| Crossref | GoogleScholarGoogle Scholar |
Potrykus I
(1991) Gene transfer to plants: assessment of published approaches and results. Annual Review of Plant Physiology and Plant Molecular Biology 42, 205–225.
| Crossref | GoogleScholarGoogle Scholar |
Sahrawat AK,
Becker D,
Lütticke S, Lörz H
(2003) Genetic improvement of wheat via alien gene transfer, an assessment. Plant Science 165, 1147–1168.
| Crossref | GoogleScholarGoogle Scholar |
Sallaud C,
Meynard D,
Boxtel J,
Gay C, Bès M , et al.
(2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theoretical and Applied Genetics 106, 1396–1408.
| PubMed |
Sambrook, J ,
Fritsch, EF ,
and
Maniatis, T (1989).
Spangenberg G,
Wang Z-Y,
Wu XL,
Nagel J,
Iglesias VA, Potrykus I
(1995) Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. Journal of Plant Physiology 145, 693–701.
Spangenberg, G ,
Wang, Z-Y ,
and
Potrykus, I (1998).
Tingay S,
McElroy D,
Kalla R,
Fieg S,
Wang M,
Thornton S,
Brettell R, Wang MB
(1997)
Agrobacterium tumefaciens-mediated barley transformation. The Plant Journal 11, 1369–1376.
| Crossref | GoogleScholarGoogle Scholar |
Vasil IK
(1994) Molecular improvement of cereals. Plant Molecular Biology 25, 925–937.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vasil V,
Castillo AM,
Fromm ME, Vasil IK
(1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10, 667–674.
| Crossref | GoogleScholarGoogle Scholar |
Wan YC, Lemaux PG
(1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiology 104, 37–48.
| PubMed |
Wang Z-Y, Ge YX
(2005)
Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea Schreb.). Journal of Plant Physiology 162, 103–113.
| Crossref |
PubMed |
Wang Z-Y,
Hopkins A, Mian R
(2001) Forage and turf grass biotechnology. Critical Reviews in Plant Sciences 20, 573–619.
| Crossref | GoogleScholarGoogle Scholar |
Wang Z-Y,
Lehmann D,
Bell J, Hopkins A
(2002) Development of an efficient plant regeneration system for Russian wildrye (Psathyrostachys juncea). Plant Cell Reports 20, 797–801.
| Crossref | GoogleScholarGoogle Scholar |
Wang Z-Y,
Bell J,
Ge YX, Lehmann D
(2003a) Inheritance of transgenes in transgenic tall fescue (Festuca arundinacea Schreb.). In Vitro Cellular & Developmental Biology. Plant 39, 277–282.
| Crossref | GoogleScholarGoogle Scholar |
Wang Z-Y,
Bell J, Hopkins A
(2003b) Establishment of a plant regeneration system for wheatgrasses (Thinopyrum, Agropyron and Pascopyrum). Plant Cell, Tissue and Organ Culture 73, 265–273.
| Crossref | GoogleScholarGoogle Scholar |
Wang Z-Y,
Bell J, Lehmann D
(2004) Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells. Plant Cell Reports 22, 903–909.
| PubMed |
Warnke, S (2002). Creeping bentgrass (Agrostis stolonifera L.). In ‘Turfgrass biology, genetics and breeding’. pp. 175–185. (John Wiley & Sons, Inc.: Hoboken, NJ)
Xiao L, Ha SB
(1997) Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment. Plant Cell Reports 16, 874–878.
| Crossref | GoogleScholarGoogle Scholar |
Yu TT,
Skinner DZ,
Liang GH,
Trick HN,
Huang B, Muthukrishnan S
(2000)
Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133, 229–233.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zhang G,
Lu S,
Chen TA,
Funk CR, Meyer WA
(2003) Transformation of triploid bermudagrass (Cynodon dactylon × C. transvaalensis cv. TifEagle) by means of biolistic bombardment. Plant Cell Reports 21, 860–864.
| PubMed |
Zhao ZY,
Cai TS,
Tagliani L,
Miller M, Wang N , et al.
(2000)
Agrobacterium-mediated sorghum transformation. Plant Molecular Biology 44, 789–798.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zhong H,
Bolyard MG,
Srinivasan C, Sticklen MB
(1994) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Reports 13, 1–6.
