Plant Cell, Tissue and Organ Culture57: 153–156, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Research note

Wheat regenerated from scutellum callus as a source of material fortransformation

Alison Harvey, Lisa Moisan, Suzanne Lindup & David Lonsdale∗Department of Molecular Genetics, John Innes Centre, Colney, Norwich NR2 7UH, UK (∗requests for offprints;Fax: 603-505725; E-mail: lonsdale@bbsrc.ac.uk)

Received 27 August 1998; accepted in revised form 25 August 1999

Key words:biolistics, luciferase, tissue culture

Abstract

Reports of wheat transformation efficiencies vary from less than 1% to more than 5% for individual experiments.Rarely are negative experiments reported though we estimate that between one in two and one in three of allexperiments fail to produce transformed plants. Consequently if transformation efficiencies were calculated fromthe total number of scutellum bombarded rather than from only those experiments which produced transformedplants there would be a significant fall in reported efficiencies. The use of scutellum-derived material from plantsregenerated from scutellum callus and grown in a controlled environment room significantly reduced the number ofexperiments failing to produce plants. Though there is a small but significant increase in transformation efficienciesfor individual experiments, the recovery of plants, as a direct consequence of the reduction in the number offailed experiments, increases nearly 350%, from 4.8 plants/1000 scutella from seed gown plants bombarded to 17plants/1000 embryos bombarded from tissue culture regenerated plants. There appears to be no additional gains tobe made from plants which are cycled through tissue culture more than one time.

Abbreviations: CER – controlled environment room; MS – Murashige and Skoog (1962) medium; PPT –phosphinothricin; 2,4D – 2,4 dichlorophenoxyacetic acid

Because of the importance of wheat as one of theworlds major food crops, numerous laboratories havedeveloped the capability of wheat transformation us-ing biolistics (Weeks et al., 1993; Becker et al., 1994;Nehra et al., 1994; Altpeter et al., 1996), electropor-ation (He et al., 1994) and more recentlyAgrobac-terium tumefasciens(Cheng et al., 1997), with scutel-lum being the target tissue of choice. However, despitea large number of laboratories working on wheat trans-formation since it was first reported in 1992 by Vasilet al., reported recoveries of transformed plants (trans-formation efficiency) have remained at around 1%of the number of scutellum bombarded. Variation ofthe tissue culture media, culture protocols, selectionsystems, (herbicide or antibiotic) have all failed toproduce a reproducible or sustainable improvement intransformation efficiency. In addition, those experi-

ments which fail to produce transgenic plants are in-frequently reported compared to those which succeed,so realistic transformation efficiencies are significantlylower. In the absence of the negative results, it isdifficult to draw any real conclusions on how goodthe transformation technology really is or to comparereasons as to why experiments failed. Plant material isgenerally grown in greenhouses where heat and lightare controlled or in CERs under precise regimes oflight, dark and humidity as the quality of the plant ma-terial is considered to be crucial if transformed plantsare going to be recovered.

We have been developing wheat transformation us-ing thebargene as the selectable marker and the fireflyluciferase reporter gene (Lonsdale et al., 1998a) tomonitor the transformation process. In this report wedescribe a comparison of transformation experiments,

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carried out over a period of one year, using scutellummaterial isolated from plants grown from seed to thosefrom plants which have been regenerated from scutel-lar callus. The results clearly demonstrate that callusderived from tissue culture regenerated plants gives asmall and sustainable improvement in the recovery oftransformed plants from individual experiments and,more importantly, a significant reduction in the num-ber of experiments which fail to produce transformedplants.

Triticum aestivumcv ‘Bobwhite’ seed was ob-tained from the USDA national small grains collec-tion, Aberdeen, Idaho, USA.T. aestivumcv. ‘ChineseSpring’ seed was obtained from Dr John Snape, Cer-eals Research Department, John Innes Centre, Nor-wich, UK. Seeds were germinated on damp filter paperand sown in peat and sand (1:1) potting compostin 6 cm−2 pots. Growth was initiated at approxim-ately 12 ◦C with a 12-h photoperiod for 4 weeks,after which plants were potted into one litre pots andtransferred to a CER with 18◦C day/12◦C night tem-perature, 16-h photoperiod and 70% relative humidity.Alternatively plants were obtained from regeneratingscutellum tissue which had not been subject to particlebombardment. Scutella were isolated 14 to 18 dayspost-anthesis and cultured on MS medium containing2 mg l−1 2,4-D for 5 to 10 days prior to bombardment.Details of media and regeneration protocols have beenpreviously described (Lonsdale et al., 1998b). Scutel-lum derived from plants grown from seed were termed‘cycle-0’. These plants grown in a CER provided thescutellum for the next experiments, cycle-1, and fromthese the cycle-2 material was developed. Althoughlimited numbers of plants were passaged in this waytheir phenotype always appeared normal and there wasnever any evidence of sterility. Scutella were bom-barded with plasmids containing the firefly luciferasereporter gene (Luc+) andbar expression cassettes(see Lonsdale et al., 1998a and 1998b for details).Transformed callus was selected on MS medium con-taining increasing concentrations of PPT (from 1 to3 mg l−1) and leaf material from regenerated plantswas tested for luciferase activity by the method ofOw et al. (1986). Expression of thebar gene wastested by leaf painting: A solution of PPT, 150 mgl−1, 0.1% TWEEN, was applied to a 1-cm sectionof leaf from each tiller, 3 times at 2 day intervals.Lack of necrosis, compared to controls, after 7 dayswas taken as evidence for the expression of thebartransgene. Presence of thebarand Luc+ gene was con-firmed by PCR: DNA from leaf material was isolated

using the CTAB procedure (Murray and Thompson,1980). Multiplex PCR was performed, 94◦4′ [94◦1′,59◦1′, 72◦1′]×x28, using 3 primers in a single 50µlreaction containing 20 nM of each of the gene spe-cific primers (Luc+: 5′-CGCAGGTCTTCCCGACG;bar: 5′-CGGTCTGCACCATCGTCAAC) and 40 nMof the nopaline synthase terminator primer (5′-CGCAAGACCGGCAACAGG), 320µM dNTPs and2% DMSO. The products all differing by 100 bp wereseparated by agarose gel electrophoresis, Southernblotted and probed with internal gene specific end-labelled oligonucleotides to confirm identity of theamplified band(s).

One of the important aspects of this data is thatboth sets of plants, those grown from seed (cycle-0)and those regenerated from scutellum tissue (cycle-1 and 2) were maintained in the same CER overthe same period of time and prepared for use on thedates shown (Table 1). Because of this interlacing ofthe experiments any external factors which would ef-fect the transformation competence of the scutellumat any period of time should have effected both setsof material equally and therefore the experiments asa set are directly comparable. The experiments 122(cycle-2), 123 (cycle-2) and 124 (cycle-0), failed toproduce any transformants indicating that some factoreffected both sets of material, though it was unclearwhy these experiments failed, particularly the materialfrom cycle-2 material (Table 1).

For reasons which can be ascertained between 1in 2 to 1 in 3, of experiments using scutella de-rived from seed grown plants fail to produce trans-formed plants (Table 1), despite the fact that plants aregrown in CERs. In comparison, scutella derived fromplants which had been regenerated from scutellar cal-lus provided scutellum material which behaved muchmore reproducibly. Nearly all experiments producedtransformed plants in contrast to material which hadbeen isolated from seed grown plants. In relation to1000 embryos bombarded: from seed grown mater-ial, 4.8 transformed plants were recovered comparedto 17 plants from scutellum that has been recycledthrough tissue culture. This represents about a 350%increase in the recovery of transformed plants. It is notknown why this increase in transformation compet-ence should be associated with scutella derived fromtissue culture cycled plants; though it has been previ-ously reported that in orchardgrass leaf-base explantsare much more embryogenic when derived from tissueculture grown material (Conger et al., 1983).

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Table 1. Recovery of non-clonal transgenic plants from callus.

Exp. Cycle Date of No. scutella No. EfficiencyNo. No. experiment bombarded transformed %

plants

81 ×1 11/4/96 25 3 1284 ×1 11/11/96 190 3 2.1

112 ×2 4/29/97 147 4 2.7113 ×2 5/2/97 160 1 0.6115 ×2 5/8/97 120 5 4.2119 ×2 5/16/97 50 2 6120 ×2 5/20/97 120 2 1.7122 ×2 5/28/97 120 0123 ×2 6/2/97 90 0126 ×1 7/1/97 165 1 0.6127 ×1 7/7/97 96 1 1

Totals/Ave 1283 22 1.70%

89 ×0 11/19/96 90 092 ×0 11/28/96 40 093 ×0 12/4/96 80 094 ×0 12/5/96 200 2 195 ×0 12/12/96 100 097 ×0 12/16/96 130 1 0.899 ×0 12/20/96 260 0

111 ×0 4/28/97 160 1 0.6116 ×0 5/9/97 80 0121 ×0 5/22/97 130 3 2.3124 ×0 6/3/97 110 0131 ×0 8/1/97 70 0

Totals/Ave 1450 7 0.48%

Tissue culture regenerated material or propagatedmaterial is known to generate somaclonal variation.However, in the experiments we have conducted lis-ted in Table 1, none of the plants exhibited sterilityand none exhibited phenotypic abnormalities. Thereis no clear reason why plants regenerated from tissueculture are a significantly better source of scutellummaterial for transformation experiments but we havefound that this effect is sustainable up to the presentday and is equally applicable whether the materialis derived from Bobwhite or Chinese Spring. Thisobservation suggests that the response is cultivar in-dependent. This has clear implications for many of themore recalcitrant wheat varieties.

Acknowledgements

AH and LM. were supported by MAFF research grantNo. CE0127. DML and SL acknowledge the supportand facilities provided by the BBSRC.

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