Agrobacterium-mediated transformation and generation of male sterile lines of Australian canola
Yan Zhang A , Mohan B. Singh A , Ines Swoboda A and Prem L. Bhalla A BA Plant Molecular Biology and Biotechnology Laboratory, ARC Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, Vic. 3010, Australia.
B Corresponding author; Email: premlb@unimelb.edu.au
Australian Journal of Agricultural Research 56(4) 353-361 https://doi.org/10.1071/AR04175
Submitted: 4 August 2004 Accepted: 4 February 2005 Published: 26 April 2005
Abstract
An efficient protocol for Agrobacterium-mediated transformation of Australian commercial canola cultivars using seedling explants is described. Seedling explants provide flexibility and reduction in labour and maintenance costs of explant sources. Five commercial genotypes of canola were successfully transformed using the developed protocol. A transformation efficiency of 67% was obtained for genotypes Oscar and RK7 from cotyledon explants, which was higher than the rate for the most commonly used cultivar Westar (33%). Comparison of different seedling explants showed that although transgenic plants could be regenerated from all explant types (cotyledons, hypocotyls, and roots) used, the number of plants regenerated per explant type varied among the cultivars. Cotyledons produced the maximum number of transgenic shoots (RK7, RI25, Oscar, and Westar cultivars), whereas root explants produced the lowest numbers of shoots. Therefore, cotyledons and hypocotyls can be considered as ideal explants for the Agrobacterium-mediated transformation of these Australian canola cultivars. Integration and expression of the introduced transgene were analysed by DNA gel blot, leaf disc test, and GUS expression assays. Analysis of progeny showed that the transgene was stably inherited. The possibility of producing male sterile lines using an antisense approach was also explored. For this, Bcp1, a gene shown to be vital for viable pollen development, was targetted. Pollen ablation and lack of seed set were observed in the transgenic plants. Histochemical tests showed an intact tapetum layer and well developed pollen in control plants, whereas degraded tapetum and ablated pollen were noted in the transgenic plants. These results indicate that it is possible to generate stable transgenic male-sterile lines of canola using this strategy.
Additional keywords: plant transformation, male-sterility.
Acknowledgments
Our sincere thanks to Dr Phil Salisbury and Mr Wayne Burton, VIDA Horsham, for providing seeds of canola genotypes. We are also grateful to Dr Johan Memelink, Leiden, for kindly providing us pBIN plus and ARC for the financial support.
Bancroft, JD ,
and
Stevens, A (1990).
Bhalla PL, Smith N
(1998) Agrobacterium tumefaciens- mediated transformation of cauliflower, Brassica oleracea var. botrytis. Molecular Breeding 4, 531–541.
| Crossref | GoogleScholarGoogle Scholar |
Bhalla PL,
Swoboda I, Singh MB
(1999) Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen. Proceedings of the National Academy of Sciences of the United States of America 96, 11676–11680.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Blume B, Grierson D
(1997) Expression of ACC oxidase promoter-GUS fusion in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli. The Plant Journal 12, 731–746.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Boulter ME,
Croy E,
Simpson P,
Shield R,
Croy RRD, Shirsat AH
(1990) Transformation of Brassica napus (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes-a comparison. Plant Science 70, 91–99.
| Crossref | GoogleScholarGoogle Scholar |
Chen JL, Beverdorf WD
(1994) A combined use of microprojectile bombardment and DNA inhibition enhances transformation frequency of canola (Brassica napus L.). Theoretical and Applied Genetics 88, 187–192.
Chevre AM,
Eber F,
Baranger A,
Hureau G,
Barret P,
Picault H, Renard M
(1998) Characterisation of backcross generations obtained under field conditions from oilseed rape–wild radish F1 interspecific hybrids: an assessment of transgene dispersal. Theoretical and Applied Genetics 97, 90–98.
| Crossref | GoogleScholarGoogle Scholar |
Christey MC, Sinclair BK
(1992) Regeneration of transgenic kale (Brassica oleracea var. acephala), rape (B. napus) and turnip (B. campestris var. rapifera) plants via Agrobacterium rhizogenes mediated transformation. Plant Science 87, 161–169.
| Crossref | GoogleScholarGoogle Scholar |
Czako M,
Wilson J,
Yu Y, Marton L
(1993) Sustained root culture for generation and vegetative propagation of transgenic Arabidopsis thaliana. Plant Cell Reports 12, 603–606.
Damgaard O,
Hollund Jensen L, Rasmussen OS
(1997) Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars. Transgenic Research 6, 279–288.
| Crossref | GoogleScholarGoogle Scholar |
Doyle JJ, Doyle JI
(1990) Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
Engelen FA,
van Molthoff JW,
Conner AJ,
Nap J-P,
Pereira A, Stiekema WJ
(1995) pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Research 4, 288–290.
| PubMed |
Evans DE,
Taylor PE,
Singh MB, Knox RB
(1992) The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napes L. pollen. Planta 186, 343–354.
| Crossref | GoogleScholarGoogle Scholar |
Fagard M, Vaucheret H
(2000) (Trans) Gene silencing in plants: how many mechanisms? Annual Review of Plant Physiology and Plant Molecular Biology 51, 167–194.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Hamilton AJ,
Lycett GW, Grierson D
(1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284–287.
| Crossref | GoogleScholarGoogle Scholar |
Heslop-Harrison J, Heslop-Harrison Y
(1970) Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technology 45, 115–120.
| PubMed |
Kazan K,
Curtis MD,
Goulter KC, Manners JM
(1997) Agrobacterium tumefaciens-mediated transformation of double haploid canola (Brassica napus) lines. Australian Journal of Plant Physiology 24, 97–102.
Kwon YW, Kim DS
(2001) Herbicide-resistant genetically-modified crop: its risks with an emphasis on gene flow. Weed Biology and Management 1, 42–52.
| Crossref | GoogleScholarGoogle Scholar |
Mailer RJ,
Wratten N, Vonarx M
(1997) Genetic diversity among Australian canola cultivars determined by randomly amplified polymorphic DNA. Australian Journal of Experimental Agriculture 37, 793–800.
| Crossref | GoogleScholarGoogle Scholar |
Mariani C,
De Beuckeleer M,
Truettner J,
Leemans J, Goldberg RB
(1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347, 737–741.
| Crossref | GoogleScholarGoogle Scholar |
Mariani C,
Gossele V,
De Beuckeleer M,
De Block M,
Goldberg RB,
De Greef W, Leemans J
(1992) A chimeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357, 384–387.
| Crossref | GoogleScholarGoogle Scholar |
Metz TD,
Dixit R, Earle ED
(1995) Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata). Plant Cell Reports 15, 287–292.
| Crossref | GoogleScholarGoogle Scholar |
Murashige T, Skoog F
(1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497.
Perez-Prat E, van Lookeren Campagne MM
(2002) Hybrid seed production and the challenge of propagating male-sterile plants. Trends in Plant Science 7, 199–203.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Poulsen GB
(1996) Genetic transformation of Brassica. Plant Breeding 115, 209–225.
Rieger MA,
Preston C, Powles SB
(1999) Risks of gene flow from transgenic herbicide-resistant canola (Brassica napus) to weedy relatives in southern Australian cropping systems. Australian Journal of Agricultural Research 50, 115–128.
Sambrook, J ,
Fritsch, EF ,
and
Maniatis, T (1989).
Stewart CN,
Adang MJ,
All JN,
Raymer PL,
Ramachandran S, Parrott WA
(1996) Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis crylAc gene. Plant Physiology 112, 115–120.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Theerakulpisut P,
Xu H,
Singh MB,
Pettitt JM, Knox RB
(1991) Isolation and developmental expression of Bcp1, an anther-specific cDNA clone in Brassica campestris. Plant Cell 3, 1073–1084.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Thomzik JE
(1995) Agrobacterium-mediated transformation of stem disks from oilseed rape (Brassica napus L.). In ‘Methods in molecular biology Vol 44: protocols’. (Eds KMA Gartland, MR Davey)
(Humana Press Inc.: Totwa, NJ)
Xu H,
Davies SP,
Kwan BYH,
O’Brien AP,
Singh MB, Knox RB
(1993) Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco. Molecular and General Genetics 239, 58–65.
| PubMed |
Xu H,
Knox RB,
Taylor PE, Singh MB
(1995) Bcp1, a gene required for male fertility in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 92, 2106–2110.
| PubMed |
Zabaleta E,
Mouras A,
Hernould M,
Suharsono S, Araya A
(1996) Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA. Proceedings of the National Academy of Sciences of the United States of America 93, 11259–11263.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
