Research and Development s8

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title Agrobacterium mediated transformation of oats

MAFF project code CEO161

Contractor organisation and location Institute of Grassland and Environmental Research Plas Gogerddan Aberystwyth SY23 3EB

Total MAFF project costs £ £83,601

Project start date 01/05/98 Project end date 28/04/00

Executive summary (maximum 2 sides A4)

Improved tissue culture systems for oats amenable to T-DNA transfer and gene expression from Agrobacterium tumefaciens have been developed, parameters for T-DNA transfer have been tested and the effects of different virG genes and virG gene combinations on the efficiency of T-DNA transfer to oat cells from Agrobacterium stains AGL1 and LBA4404 determined. In general strain LBA4404 was found to be ineffective while AGL1 was effective, with high transformation frequencies under optimised conditions. Problems with eliminating Agrobacterium from explants were partially overcome with the use of alternative antibiotics, but, unlike transformation via micro-projectile bombardment, no herbicide resistant or gus expressing plants were regenerated following Agrobacterium transformation.

01 Assessment of the potential of different oat tissues and genotypes, and effects of vir gene inducers and tissue wounding for T-DNA transfer from A. tumefaciens.

 T-DNA transfer was obtained in all tissues and genotype tested. Low salt medium, absence of acetosyringone vir gene inducer, during inoculation and co-cultivation, and long co-cultivation times were found to be the most important factors for efficient T-DNA transfer in oat. Agarose and treatments such as wounding, vacuum and plasmolysis were found to increase transformation efficiency. Inoculation times lower than 6h and addition of glucose in the co-cultivation medium did not significantly affect T-DNA transfer, and high Agrobacterium inoculum densities and a lower co-cultivation temperature had a negative effect on transformation efficiency  Based on a study of GUS expression in non embryogenic tissue (derived from mature embryos and embryo axis) and in embryogenic callus, 4 to 9 weeks after transformation it appeared that :- i) Long co-cultivations (6 to 10 days) were necessary for efficient T-DNA transfer. ii) T-DNA transfer was more efficient to cells of the embryo axis than the whole embryo .

CSG 15 (Rev. 12/99) 1 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code iii) Transformation efficiency was higher when co-cultivation was carried out in the presence of 2,4D. iv) Reduction of MS salts to 1/10 of normal strength increased transformation efficiency. v) Reducing the co-cultivation temperature to 22oC decreased transformation efficiency. vi) Pre and post treatments on high osmotic medium (HOM) increased transformation efficiency.

02 – Improvement of tissue culture systems Oat tissue culture was improved by application of two published methods.  A rapid callus formation and regeneration system [1] was successfully applied to variety Millennium (winter husked), recalcitrant to traditional tissue culture. Formation of embryogenic callus from leaf bases occurred in 3 weeks, with plant regeneration in a further 3-4 weeks.  A multiple shoot system [2] was tested on three varieties Melys- spring oat variety, Millennium- winter oat and Bullion- spring naked oat. Up to 81% of isolated meristems multiplied and regenerated. Apical meristems of 7 day-old seedlings were isolated and cultured in the dark at 25oC. Growing leaves were regularly removed and after one week of culture meristems started to multiply and by 5 weeks multiple shoot meristems were apparent.  A rapid system for production of embryogenic callus and plant regeneration in rice [3] was also tested in oats on variety Melys. There was no improvement in embryogenesis using this system as compared with the traditional oat tissue culture system. This method was not subsequently used.

03 – Effect of different virG regions on transformation efficiency Agrobacterium strains carrying different virG genes or vir region on binary vector pSoup were compared for their effect on transformation efficiency.  VirGN54D was the most efficient of the three virG genes tested, followed by virG542, then by virGwt.  The Komari fragment, which contains virG542 as well as virB and virC from pTiBo542, was highly efficient compared to the strain lacking additional vir genes.  The relative position of the virG genes to the T-DNA had an importance in the efficiency of T-DNA transfer which was virG gene-dependent. Indeed, while virGwt and virGN54D led to more efficient T-DNA transfer when they were on pSoup, in trans to the T-DNA, virG542, either on its own or within the Komari fragment, was more efficient when on pGreen, in cis to the T-DNA.  Increasing the copy number of virGwt and virGN54D did not lead to an increase in transformation efficiency. A slight increase in efficiency was observed when two copies of virG542 were present in the Agrobacterium strain.

04 – Comparison of Agrobacterium strain/disarmed Ti plasmid combinations. All AGL1 Agrobacterium strains were tested , and all led to GUS expressing foci 4-6 weeks after co-cultivation in all tissue tested. Embryogenic calli were inoculated with these strains, transferred to selection on PPT, and to regeneration medium. However, no transformed plant regenerated. Following general observations made by other groups from this program at MAFF progress meeting, that LBA4404 strains were poorly efficient at wheat and barley transformation, it was commonly decided that these strains would not be tested further. This decision affects this objective in which only the AGL1 strains delivered by IPSR were fully tested. However strain LBA4404/pTOK233 was tested on embryo, embryogenic callus and multiple shoots and produced a few spots of GUS expression. Transient expression was observed in the scutellum of inoculated embryo, a non regenerable tissue in oat. Thus T-DNA transfer occurred at reasonably high frequency with all AGL1 strains tested, but at very low frequency with LBA4404 harbouring the new vectors or with LBA4404/pTOK233.

References: [1] Gless, C., Lorz, H. & JahneGartner, A. (1998). Establishment of a highly efficient regeneration system from leaf base segments of oat (Avena sativa L.). Plant Cell Reports 17, 441-445. [2] Zhang, S., Zhong, H. & Sticklen, M. B. (1996). Production of multiple shoots from shoot apical meristems of oat ( Avena sativa L). Journal of Plant Physiology 148, 667-671. [3] Toki, S. (1997). Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Molecular Biology Reporter 15, 16-21.

CSG 15 (1/00) 2 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Scientific report (maximum 20 sides A4)

01/01 – Assessment of the potential of different oat tissues and genotypes for T-DNA transfer from A. tumefaciens.

Four oat explants, (mature embryos, mature embryo axis, primary embryogenic callus and leaf base sections), were tested for their potential for transformation by Agrobacterium. The explants were inoculated with pre- induced strain AGL1/pSoup/pAL156 and co-cultivated for 6d. Transformation efficiencies for embryogenic callus and leaf base were very similar, 1.9 and 1.62 GUS foci/explant, respectively (Fig2A). Because of the much smaller size of leaf bases than calli, one leaf base offers a smaller surface than one piece of callus for the attachment of the bacteria, therefore results from these two tissues, expressed as number of GUS foci/explant, can not be directly compared. However if results were expressed relative to the surface of tissue exposed, transformation efficiency would be much higher for leaf bases than for embryogenic calli. Some transformed embryos, which had grown from a leaf base, were observed (Fig 1C). Due to the destructive nature of the GUS assay, and in order to increase the chances of recovering transformed plants, when embryo and embryo axes were transformed, transformation efficiency was determined 4 weeks after co-cultivation on the non-embryogenic tissues that had developed, while the embryogenic tissues were transferred to selection medium. Unlike embryogenic tissue, which is a solid, organised tissue, non- embryogenic tissue is very soft, disorganised, and disintegrates easily in liquid. After a few hours of incubation in X-gluc solution with shaking, the blue foci observed from non-embryogenic tissues were in fact individual GUS expressing cells rather than cell clusters which have derived from a single transformed cell as observed in embryogenic tissue. Fig 1A-B illustrates the difference of expression observed in these two tissues. The number of GUS expressing foci per explant is therefore expected to be higher for non-embryogenic tissue from embryo and embryo axis than for embryogenic tissue from callus. Transformation efficiencies for embryo and embryo axis however, are directly comparable, since the GUS assay was performed on similar tissue. In this experiment, the number of GUS foci/explant was slightly higher for embryo axis than for whole embryo (23.9 and 21 GUS foci/explant, respectively) (Fig 2A). In another experiment the transformation efficiency of embryo axes was almost four-fold higher than the transformation efficiency of embryos (Fig 2B). All tissues were transferred to selection and regeneration media, and while shoots were produced, no plants regenerated from any of the four types of tissue were obtained. Multiple shoot cultures were also transformed, but transformation efficiencies were very much lower than with other explants. The potential of cv. Melys and Bullion for Agrobacterium transformation was tested on embryo axes and leaf base sections inoculated with pre-induced Agrobacterium. Embryo axes of Melys appeared to be 50% more amenable to Agrobacterium transformation than Bullion (Fig 3). Calli derived from these tissues were transferred directly to regeneration medium under selection. However no transformed plants regenerated from these cultures.

01/02 – Effect of virG inducer and co-cultivation on transformation efficiency.

Effect of acetosyringone (AS): Embryos of Bullion were inoculated with Agrobacterium either in the absence of AS or with 200 or 400 M AS. The number of GUS foci/explant was 10-fold higher when AS was omitted from the media than with 200 M AS, even though Agrobacterium had not been pre-induced. Increasing the AS concentration to 400 M resulted in a 7-fold decrease in the number of GUS foci/explant (Fig 4A),. The development of calli was highly affected by AS at 400 M, and to a lesser extent by AS at 200 M. The negative effect of AS on transformation efficiency was confirmed in a second experiment, in which 200 M AS was included in the inoculation medium, and when tested in the co-cultivartion medium on embryo axies of var melys. Again, transformation was considerably more efficient in the absence than in presence of AS during co- cultivation (Fig 4B). The effect of the omission of AS was further investigated (see Protocol for details). Agrobacterium was induced with 200 M AS prior to centrifugation. Embryo axes were inoculated in presence or in absence of AS and both samples were co-cultivated in the presence or in the absence of AS. In this experiment, omission of AS from the co-cultivation medium alone had no effect on transformation efficiency

CSG 15 (1/00) 3 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code (Fig 4C). Omission of AS from both inoculation and co-cultivation media, however, induced a 2.3-fold increase in efficiency compared to when AS was present in both media.

Effect of Agrobacterium density: The effect of the densities of the Agrobacterium inoculum between 0.1 and 14.0 (OD600) were tested with embryos of Bullion and embryo axis of Melys with two Agrobacterium strains. Transformation efficiency was very low but decreased in embryo axes as the inoculum density increased. Highest efficiencies were obtained with densities lower than 1.0 (Fig 5).

Effect of inoculation time: Embryo axes of Melys were incubated with Agrobacterium for periods of time varying between 15 min and 15 h before they were transferred to co-cultivation medium. A 15 min inoculation produced an average of 2.2 GUS foci/explants (Fig 6). Inoculation times between 2 and 6 h slightly increased transformation efficiency. A significant increase in transformation efficiency was obtained with a 15h inoculation, reaching 4.9 GUS foci/explant. Although it was not the case in this experiment, it was feared that incubation of explants in liquid Agrobacterium inoculum for 15 h would cause problems of bacterial overgrowth during co-cultivation and would have a negative effect on the health of the tissue. Therefore a 2-3 h inoculation time was chosen for other experiments.

Effect of co-cultivation time: When embryos were co-cultivated for 2, 3, 6 or 9d, little expression was observed with 2 or 3 d co-cultivation. Some GUS foci could be seen with a 6 d co-cultivation for the highest inoculum density. Only with 9 d of co-cultivation could GUS foci be seen in all three samples (Fig 7A). Very little GUS expression was observed with a 1 d co-cultivation of embryo axis of Bullion. GUS expressing foci were detected with co-cultivations of 6 to 10 d, with a maximum of 5.8 foci/explant obtained with a 6 d co- cultivation (Fig 7B). Leaf bases of Melys also showed GUS expression only with at least 6 d of co-cultivation, with maximum expression obtained with 8 and 10 d (Fig 7C). It was clear from these results that long co- cultivations of 6 to 10 d were necessary for T-DNA transfer into oat cells.

Effect of co-cultivation temperature: The effect of two temperatures, 22 and 25oC, was tested during a 7 d co- cultivation of embryo axis of Melys. Transformation was almost four-fold more efficient at 25oC than at 22oC (Fig 8).. This result indicates that lowering the co-cultivation temperature by 3oC did not favour T-DNA transfer. Other temperatures were not tested but 25oC may not be optimal.

Effect of the composition of co-cultivation medium on transformation efficiency. Effect of low salt concentration : The salt content of MS medium in the inoculation and co-cultivation media was decreased tenfold and its effect was tested on transformation efficiency of embryogenic callus and embryo axis. Twice as many GUS foci were detected on embryogenic calli which had been inoculated and co-cultivated on low salt strength media 1 ( /10 MS2) as were detected on embryogenic calli inoculated and co-cultivated on full salt strength media (MS2). 1 1 The average number of GUS foci/explant on /10 MS2 was 47.6 (Fig 9A). On embryo axes, the effect of /10 MS2 on the transformation efficiency was dramatic, giving a 22-fold increase in GUS foci on explants (Fig 9A). 1 However /10 MS2 had an adverse effect on subsequent embryogenesis from embryo axis callus, as embryo axes 1 transformed on /10 MS2 produced less and smaller embryogenic calli than embryo axes transformed on MS2. Following this observation, experiments were carried out to test the effect on transformation efficiency of 1 shortening the time embryo axes were left on /10 MS2 during co-cultivation. 1 During an 8d long co-cultivation with Agrobacterium, embryo axes were either cultured on /10 MS2 for 8d , or 1 1 cultured for 4 d on /10 MS2 followed by 4 d on MS2, or cultured for 4 d on MS2 followed by 4 d on /10 MS2. 1 The 8 d co-cultivation on /10 MS2 gave a similar transformation efficiency (Fig 9B) to the 9 d co-cultivation of 1 Fig 9A. Reducing the time the embryo axes were cultured on /10 MS2 to four days had opposite effects 1 depending on when /10 MS2 was applied during the co-cultivation period. When embryos were first co- 1 cultivated on /10 MS2 for four days, then on MS2 for the last four days, efficiency of transformation was 1 increased compared to the 8 d-long /10 MS2 co-cultivation, whereas the transformation efficiency was lowered 1 if the /10 MS2 was applied during the second half of the co-cultivation. Although the reduction of co-cultivation 1 on /10 MS2 to the first four days increased transformation efficiency, the calli that developed from the embryo

CSG 15 (1/00) 4 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code axes were still less embryogenic than the calli that developed from embryo axes co-cultivated on MS2 (data not 1 shown). Therefore, the effect of further reducing co-cultivation on /10 MS2 was investigated. 1 Following inoculation in /10 MS2, embryo axes were co-cultivated either on MS2 for 7 d, or 1, 2 or 3 d on 1 /10 MS2 followed by 6 , 5 or 4 d on MS2, respectively, for a total co-cultivation of 7 d. Results showed that 1 transformation efficiency started to improve with a 2 d co-cultivation on /10 MS2 compared to co-cultivation on MS2 (Fig 9C). Transformation efficiency was dramatically improved when the embryo axes were left on 1 /10 MS2 for 3 days and was 6.6-fold higher than when the explants were co-cultivated on MS2. In addition, the embryo axes of this sample produced embryogenic calli which resembled those of the 7d-MS2 sample in both 1 number and quality. Therefore a 3d co-cultivation on /10 MS2 followed by a 4d co-cultivation on MS2 was optimal for efficient T-DNA transfer without loss of embryogenic potential of the embryo axis.

Effect of glucose : Glucose (10g/l) was added in either the inoculation medium alone or in both the inoculation and co-cultivation media during transformation of embryogenic calli of Melys. A slight increase in transformation efficiency was noted when glucose was present in both media (Fig 10), however this increase was not dramatic and Agrobacterium started to grow on the calli after 5 d of co-cultivation. Therefore, glucose was not included in further experiments.

Effect of the solidifying agent: The effect agar or SeaPlaque agarose in low salt co-cultivation medium on 1 transformation efficiency of embryo axes was investigated. Gelrite could not be used to solidify /10 MS2, as the salt concentration was too low to allow formation of the gel matrix. Detection of GUS foci revealed that transformation efficiency was 24% higher when the co-cultivation medium was solidified with agarose than when it was solidified with agar (Fig 11).

Effect of auxin addition during inoculation and co-cultivation. Embryogenic calli of Melys were 1 inoculated with AGL1/pAL155/pAL156 and co-cultivated on /10MS either in the presence or absence of 2 mg/l 2,4-D. GUS assay revealed that addition of 2,4-D increased transformation efficiency by more than 20% (Fig 11b)

01/03 – Effect of tissue wounding on transformation efficiency.

Embryo axes of Melys were wounded by bombardment with gold particles prior to inoculation with Agrobacterium. Although wounding the tissue increased transformation efficiency 3.5-fold, less than 1 GUS expressing focus was detected per explant (Fig 12). Other treatments aiming at improving attachment of Agrobacterium on the tissue were tested, such as vacuum infiltration and tissue plasmolysis. Vacuum was applied for 2½ h during inoculation of embryo axes. Transformation efficiency was almost 4-fold higher for embryo axes that had been vacuum infiltrated than for untreated embryo axes (Fig 12). However, as for the wounded explants, the number of GUS foci/explant was 1 very low. Embryos were also plasmolysed on /10 MS2 medium supplemented with 0.2 M mannitol and 0.2 M 1 1 1 sorbitol ( /10 HOM), for 4 h prior to inoculation with Agrobacterium in /10 HOM. Explants were left on /10 HOM 1 overnight after inoculation and transferred to /10 MS2 for co-cultivation. The effect of the plasmolysis treatment 1 was assessed against a sample that was inoculated and co-cultivated in /10 MS2. Four weeks after co- cultivation, the number of GUS expressing cells was 1.3-fold higher for plasmolysed embryos than for non plasmolysed embryos (Fig 13).

01/05 – Molecular analysis of plants regenerated from 01/01-01/03. Unlike transformation via micro-projectile bombardment, plant regeneration from oat calli was not possible following Agrobacterium transformation. Problems were encountered with the normal antibiotics used to eliminate Agrobacterium growth from the calli and the alternative antibiotic used may have been detrimental either to somatic embryogenesis, germination of the somatic embryos or plant development following embryo germination. As no mature plants could be regenerated from this material it was not possible to complete this milestone. (see also Regeneration of transformed plants - Objective 04)

CSG 15 (1/00) 5 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code 02 – Improvement of tissue culture systems. 02/01 –Development of a rapid regeneration system for oat callus.

A protocol for rapid callus induction/plant regeneration described for rice by Toki, 1997 [1], based on improvements in the culture medium and high temperature growth, was tested on oat (cv. Melys, spring oat). No improvement of tissue growth was observed when using Toki’s medium instead of the medium usually used for oat. Moreover, controlling water condensation in Petri dishes cultured at 30oC was problematic. Another system developed by Gless, 1998 [2],. which claimed callus induction from leaf bases of oat seedlings and production of plants within 8-10 weeks was also tested. This system was successful for cv. Melys, as well as cv. Millenium (winter oat), which has been recalcitrant to traditional tissue culture, however, other cultivars such as Bullion (spring naked oat), Bulwark (winter oat) and Lexicon (winter naked oat), responded poorly to this culture system.

02/02 – Development of a multiple shoot system from oat meristems.

The multiple shoot system of Zhang 1996 [3] was tested on the three oat varieties Melys, Millennium and Bullion. Up to 81% of isolated meristems multiplied and regenerated. Apical meristems of 7 day-old seedlings were isolated and cultured in the dark at 25oC. Growing leaves were regularly removed and after one week of culture meristems started to multiply and by 5 weeks multiple shoot meristems were apparent. Melys was the cv. that gave the best response to this culture system.

03 – Effect of different virG regions on transformation efficiency.

Effect of the type of virG gene: Agrobacterium strains carrying different virG genes or vir region on the binary vector pSoup were compared for their effect on transformation efficiency. Embryo axes of Melys were inoculated with pre-induced AGL1/pAL166/pAL156, AGL1/pAL155/pAL156, AGL1/pAL165/pAL156 and AGL1/pAL154/pAL156 which contain one copy of virGwt, virG542, virGN54D and the komari fragment, respectively. Strain AGL1/pSoup/pAL156, which does not carry an extra virG gene, was used as a control. The histochemical GUS assay revealed that a copy of virGwt or virG542 on pSoup did not significantly improve transformation efficiency compared to the control lacking an additional virG gene (Fig 14). VirGN54D, on the other hand, improved transformation efficiency by 71%. The strain containing the Komari fragment was only half as efficient as the strain with no additional virG, which is probably due to instability of pAL154 as observed during construction of the strain (Alison Harvey, JIC, personal communication). In other experiments however, virG542 appeared to be 2.2-fold more efficient than virGwt (Fig 15A) when the genes were on pSoup, and the Komari fragment generated up to 8.2-fold more GUS expressing foci than when no additional virG gene was present (Fig 15D). Strain LBA4404/pTOK233, which carries the Komari fragment on its binary vector, was not efficient at transforming oat, [generating only 0.3 GUS foci/explant (Fig15 D)], despite the successful use of this vector for grass transformation (Sue Dalton, IGER, personal communication). Unexpectedly, AGL1/pSoup/pAL156 (no additional virG) produced very high GUS expression (230 GUS foci/explant), up to 3.6-fold higher than strains containing virG542 (Fig15 C).

Effect of the position and copy number of virG genes: The effect of the position of an additional virG gene with respect to the T-DNA and the effect of its copy number were investigated for virGwt, virG542 and virGN54D and the Komari fragment. Embryo axis of Melys were inoculated with pre-induced strains in the absence of AS in the case of virGwt, virGN54D and the Komari fragment and in the presence of AS in the case of virG542. Transformation efficiency was 4.9-fold higher when virGwt was on pSoup, in trans to the T-DNA (strain AGL1/pAL166/pAL156), than when it was on pGreen, in cis to the T-DNA (strain AGL1/pSoup/pAL170) (Fig 15A). Similarly, virGN54D was 4-fold more efficient when situated on pSoup (strain AGL1/pAL165/pAL156) than when situated on pGreen (strain AGL1/pSoup/pAL169) (Fig 15B). VirG542 and the Komari fragment, on the other hand, were 1.2- and 1.5-fold more efficient when on pGreen (strains AGL1/pSoup/pAL157 and AGL1/pSoup/pAL186, respectively) than when on pSoup (strain AGL1/pAL155/pAL156 and AGL1/pAL154/pAL156, respectively) (Fig 15C&D).

CSG 15 (1/00) 6 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code The strains that carried two copies of virGwt or virGN54D, one on each plasmid (AGL1/pAL166/pAL170 and AGL1/pAL165/pAL169, respectively), gave a transformation efficiency that was intermediate between the efficiency of the strains that carried one copy of the gene on pSoup and the efficiency of the strains that carried one copy of the gene on pGreen (Fig 15A-B). The presence of two copies of virG542 in strain AGL1/pAL155/pAL157 induced a 28.1% and 9.3% increase in transformation efficiency compared to the two strains that had only one copy of the gene, on pSoup or on pGreen, respectively (Fig 15C).

Effect of combining virG542 and virGN54D: Strains which contained one copy of virG542 and one copy of virGN54D, each on either pSoup or pGreen, were tested and compared to strains that carried two copies of either gene. Strains AGL1/pAL155/pAL157 (two copies of virG542) and AGL1/pAL165/pAL169 (two copies of virGN54D) produced similar numbers of GUS foci/explants. Transformation efficiency was reduced by more than 40% when strains containing one copy of each virG gene were used (AGL1/pAL155/pAL169 and AGL1/pAL165/pAL157), regardless of the position of the genes (Fig 16).

04 – Comparison of Agrobacterium strain/disarmed Ti plasmid combinations.

All AGL1 Agrobacterium strains were tested , and all led to GUS expressing foci 4-6 weeks after co-cultivation in all tissue tested. Agrobacterium strains LBA4404/pAL166/pAL156 and LBA4404/pAL155/pAL157 were tested with embryo and leaf bases and produced very few spots of GUS expression (Fig 5,S1 and Fig 7). Strains AGL1/pSoup/pAL156 and AGL1/pAL155/pAL156 were compared to strains LBA4404/pSoup/pAL156 and LBA4404/pAL155/pAL156 respectively in order to test the effect of the type of disarmed Ti plasmid (agropine type pTiBo542 in AGL1 and octopine type pAch5 in LBA4404). Embryogenic calli were inoculated with these strains, transferred to selection on PPT, and to regeneration medium. However, no transformed plant regenerated. Following general observations made by other groups from this program at MAFF progress meeting, that LBA4404 strains were poorly efficient at wheat and barley transformation, it was commonly decided that these strains would not be tested further. Strain LBA4404/pTOK233 was also tested on embryo, embryogenic callus and multiple shoots and produced a few spots of GUS expression. Transient expression was observed in the scutellum of inoculated embryo, a non regenerable tissue in oat.

Regeneration of transformed plants

Tissues from all experiments, representing transformation of all type of tissue from all genotypes with all AGL1 strains plus the 4 LBA4404 strains tested, were transferred to selection medium. Some calli survived 10 weeks of selection and were transferred to regeneration medium. Calli turned green and started to produce shoots. However, no plants regenerated on 3 mg/l PPT. The histochemical GUS assay performed on green calli from two experiments revealed no GUS expression in these calli. After 5-7 weeks on PPT-containing regeneration medium, green calli from various experiments were transferred to PPT-free regeneration medium in order to produce plants which would supply sufficient material for DNA extractions. A few calli were transferred back to callus medium (MS2) to grow more tissue. DNA was extracted from leaves of three regenerated plants and two calli, and Southern blots were performed in order to verify if these tissues were transformed. No T-DNA was detected. Calli from embryo axes which had been transformed with LBA4404/pTOK233 were first selected on 50 mg/l paromomycin. As after 6 weeks of selection all calli looked as healthy as non-transformed calli grown in absence of paromomycin, the concentration of paromomycin was doubled. However numerous shoots regenerated when the calli were transferred to regeneration medium. None of the shoots tested for GUS expression appeared positive.

Conclusions and discussion

CSG 15 (1/00) 7 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Oat tissue culture was improved by application of two published methods. Callus induction and plant regeneration were obtained from leaf bases of Millenium, a high yield winter oat which was recalcitrant to tissue culture with other systems. T-DNA transfer was obtained in all tissues and genotype tested. The highest transformation efficiencies were 230 GUS foci/explant for embryo axis, 47.6 GUS foci/explant for embryogenic callus and 1.6 GUS foci/explant for leaf base. Low salt medium, absence of AS during inoculation and co-cultivation, and long co-cultivation times were found to be the most important factors for efficient T-DNA transfer in oat. Agarose and treatments such as wounding, vacuum and plasmolysis were found to increase transformation efficiency. Inoculation times shorter than 6h and addition of glucose in the co-cultivation medium did not significantly affect T-DNA transfer, and high Agrobacterium inoculum densities and a lower co-cultivation temperature had a negative effect on transformation efficiency. VirGN54D was the most efficient of the three virG genes tested, followed by virG542, then by virGwt. The Komari fragment, which contains virG542 as well as virB and virC from pTiBo542, was highly efficient compared to the strain lacking additional vir genes. No experiment was done to compare the Komari fragment and virG542. The relative position of the virG genes to the T-DNA had an importance in the efficiency of T-DNA transfer which was virG gene-dependent. Indeed, while virGwt and virGN54D led to more efficient T-DNA transfer when they were on pSoup, in trans to the T-DNA, virG542, either on its own or within the Komari fragment, was more efficient when on pGreen, in cis to the T-DNA. Increasing the copy number of virGwt and virGN54D did not lead to an increase in transformation efficiency. A slight increase in efficiency was observed when two copies of virG542 were present in the Agrobacterium strain. T-DNA transfer occurred at reasonably high frequency with all AGL1 strains tested, but at very low frequency with LBA4404/pTOK233.

Four unexpected aspects of Agrobacterium transformation of oat arose during this project: 1) No transient expression of GUS was observed immediately after co-cultivation. 2) GUS was expressed 4 weeks after co-cultivation, 3) The GUS expression detected in callus during selection was limited to spots, not large cell clusters, 4) Calli which resisted selection did not express GUS, did not regenerate plants under selection, and did not contain T-DNA. The same observations were made for wheat and barley transformation within the MAFF program (personal communications), suggesting that the problem is species independent, and may come from the Agrobacterium strains used. It is not likely that the tissue culture system was the cause of the non- regeneration of transformed plants. A possible cause for the non-regeneration of transformed plants is that the bar gene was not expressed in the transformed cells, leading to their death during selection. Non- expression of complete bar gene sequences could be due to silencing (due to the common ubiquitin promoter used to drive bar and gus gene expression in these vectors), as we have previously shown that a ubi-bar- ubi- gus construct (in the form of pAHC25) can result in low levels of co-expression and loss of bar gene function, when transformed via microprojectile bombardment in oats (MAFF contract CTO114). Lack of bar gene expression may also be due to malfunction of the construct itself. Being situated next to the left border of the T-DNA, the bar gene might be more prone to truncation than the gus gene, either during T-strand synthesis or during integration of the T-DNA into the genome.

Three major problems were encountered during the first year of the project: (1) Agrobacterium tended to grow and cover the explants during co-cultivation. The effect of the composition of the co-cultivation medium on Agrobacterium growth was studied, and a medium which slowed down Agrobacterium growth without affecting oat tissue growth was identified. (2) The growth of Agrobacterium (both LBA4404 and AGL1 stains), could not be controlled with the antibiotic cefotaxime (250 mg/l) after co-cultivation, despite being putatively sensitive to this antibiotic. Increasing the concentration of cefotaxime to 500 mg/l in MS2.TA slowed down the growth of the bacteria, but also affected the growth of the tissue, which turned yellow first, and then brown. An experiment, where the effect of cefotaxime or Timentin, on the growth of Agrobacterium was tested, showed that cefotaxime slowed down Agrobacterium growth by 66% compared with a

CSG 15 (1/00) 8 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code control lacking antibiotic, while Timentin inhibited the growth of the bacteria by 90%. Timentin was therefore used instead of cefotaxime in all subsequent experiments, although problems of removal of Agrobacterium contamination persisted whenever Timentin was removed from the cultures. (3) Very few transient expression foci were detected in tissues after co-cultivation. However GUS expressing foci were detected 4-6 weeks after transformation in calli, embryo- and embryo axis- derived calli. Therefore the effects of various parameters on transformation efficiency (objectives 01/01-01/03, 03/01 and 04/01) were determined according to the number of GUS expressing foci detected 4 to 6 weeks after transformation (stable transformation) rather than immediately after co- cultivation (transient expression). Objectives 01/04, 03/02 and 04/02 originally set are therefore combined with objectives 01/01-01/03, 03/01 and 04/01, respectively.

Plasmids used Structure pAL169 pGreen + bar + gus + vir GN54D pAL170 pGreen + bar + gus + virGWT pAL165 pSoup + vir GN54D pAL166 pSoup + vir GWT pAL156 pGreen + bar + gus pAL157 pGreen + bar + gus + vir G542 pAL155 pSoup + vir G542 pAL154 pSoup + Komari fragment pAL186 pGreen + Komari fragment pTOK233 35S-hpt +35S-gus + nosNPTII +vir C,B G542

Agrobacterium stains: LBA4404 and AGL1 (Ti+vir 542) Vector combinations Experiment numbers refered to in Figures. pSoup + pAL169 AT 53 pSoup + pAL170 AT 52 pSoup + pAL156 AT 45,53,54,55 pSoup + pAL157 AT 45 pSoup + pAL186 AT55 pAL165 + pAL156 AT 53 pAL165 + pAL169 AT 53 pAL165 + pAL157 AT 53 pAL166 + pAL156 AT 52,53 and AT 26,27 (LBA) pAL166 + pAL170 AT52 pAL155 + pAL157 AT45,53 and AT 27 (LBA) pAL155 + pAL169 AT 53 pAL155 + pAL156 AT 29, 34, 36, 37, 38 ,40, 41, 42, 43, 44, 45, 46, 51, 52, 53, pAL154 + pAL156 AT53.55

CSG 15 (1/00) 9 Protocol and summary of experimental variables tested for Agrobacterium transformation of oats.

Overnight Agrobacterium growth Vary strain and vir G regions on pSoup and pGreen vectors

+ AS

growth P r e

centrifugation centrifugation - i n d u c

t Vary explant i

Resuspend in inoculation Resuspend in inoculation o medium with AS n (embryo, embryo axis, callus, medium (no AS) leaf base, multiple shoots) and oat variety growth

g wounding Explant Induced g plasmolysis Agrobacterium g vacuum

g acetosyringone (AS) Inoculation vary inoculation time vary inoculum density vary media composition Rinse & blot dry

g 24D g AS Test different Plate on agar vary co-cultivation time antibiotics to (co-cultivation) vary temperature kill At vary media composition vary solidification agent

Transfer to callus induction media + Timentin antibiotic

Transfer to selection media (+ PPT+Timentin)

Transfer resistant calli to plant regeneration media (+PPT + Timentin)

Plant regeneration of recalcitrant shoots in absence of PPT and Timentin Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code Figure 1 - GUS expression in oat tissues transformed with Agrobacterium

A - GUS expressing cells in embryogenic calli 7 weeks after co- cultivation with AGL1/pAL155/pAL156 (experiment AT36).

C B - GUS expressing cells in embryo axis- derived non embryogenic tissue 4 weeks after co-cultivation with AGL1/pAL155/pAL156 (experiment AT37).

C - GUS expressing, transformed (t) and non transformed (n) leaf base- t derived embryos 4 weeks after co- cultivation with AGL1/pSoup/pAL156 (experiment AT54).

11 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 2 - Potential of oat tissues for Agrobacterium transformation.

A B

t 40 n . . a 20 l p x 30 t e n / i a l c p 15 o x f e / 20 i c S o f U 10 S G

U 10 b G

b 5 N N 0 0 embryo embryo callus leaf base embryo embryo axis axis

Tissues of genotype Melys were transformed with AGL1/pSoup/pAL156 (A - experiment AT54) or AGL1/pAL155/pAL156 (B- experiment AT43) according to the protocols described in Table 1.

Figure 3- Potential of oat genotypes for Agrobacterium transformation.

t n a l 6 p x .

e 5 / i c

o 4 f

S 3 U G

2 b

N 1

0 Melys Bullion

Embryo axes of each genotype were transformed with AGL1/pSoup/pAL156 (experiment AT55).

12 A B Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 4 – Effect of acetosyringone on transformation efficiency.

A B C

8 6

t 7 n a t

l 5 n

p 6 a x l e p / 5 4 i x c e / o i

f 4 c 3 o S f

U 3 S

G 2

2 U b G

N 1 b 1 N 0 0 0 200 400 0 200 AS in inoculation and co- AS in co-cultivation cultivation media (M) medium (M)

60 t n a

l 50 p x e

/ 40 i c o f

30 S U

G 20

N 10

0 0 200 AS in co-cultivation medium (M)

Embryo axes of Bullion (A) and embryos of Melys (B and C) were inoculated with AGL1/pAL155/pAL156. A - the effect of the absence or presence of AS in two concentrations was tested

13 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code during both inoculation and co-cultivation (experiment AT34). B - 200 M AS was added in inoculation medium and the effect of the absence or presence of AS was tested during co-cultivation (experiment AT41). C - the effect of the absence or presence of AS was tested during inoculation and/or co-cultivation (experiment AT44). Red bars: 0 M AS, yellow bars: 200 M AS in inoculation medium.

14 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 5 – Effect of the density of the Agrobacterium inoculum on transformation efficiency.

2 .

n 1.6 a l p x e

/ 1.2 i c o f

S 0.8 U G

b 0.4 S2 N S1 0 0.1 0.6 1 1.7 2 4 7 14

Inoculum density (OD600)

Embryos of Bullion (S1) and embryo axes of Melys (S2) were inoculated with LBA4404/pAL166/pAL156 (experiment AT26) and AGL1/pAL155/pAL156 (experiment AT40), respectively.

Figure 6 - Effect of inoculation time on transformation efficiency.

5 .

t 4 n a l p x

e 3 / i c o f

S 2 U G

N 1

0 0.25 2 4 6 15 Inoculation time (h)

Embryo axes of Melys were inoculated with AGL1/pAL155/pAL156 (experiment AT38).

15 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 7 - Effect of co-cultivation time on transformation efficiency.

A - transformation efficiency of embryos of Bullion inoculated with LBA4404/pAL166/pAL156 at OD 0.1 (green series), 0.6 (white series) or 1.7 (blue series) and co-cultivated for 2 to 9d (experiment AT27). B - transformation efficiency of embryo axis of Bullion inoculated with strain LBA4404/pAL155/pAL157 and co-cultivated for 1 to 10 d (experiment AT27). C - transformation efficiency of leaf bases of Melys inoculated with LBA4404/pAL155/pAL157 and co-cultivated for 2 to 10 d (experiment AT27)

B C

6 0.25 .

t n .

t a l

n 5

p 0.2 a l x p e / x 4 i e c

/ 0.15 i o f c

3 S f

U 0.1 S G U 2 b G

N 0.05 b 1 N 0 0 2 6 8 10 1 6 8 10 Co-cultivation time (d) Co-cultivation time (d)

16 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 8 - Effect of co-cultivation temperature on transformation efficiency.

t n a

l 1.2 p . x

e 1 / i c 0.8 o f

S 0.6 U

G 0.4

N 0.2 0 22 25

Temperature (oC)

Embryo axes of Melys were inoculated with AGL1/pAL155/pAL156 and co-cultivated for 7 d at either 22oC or 25oC (experiment AT41).

17 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 9 – Effect of low salt concentration on transformation efficiency.

50 60 .

t .

n t a 40 l

n 50 a p l x p e / x 40 i 30 e c / i o c f

o 30 f S

20 U S G U 20

G b

10 N b 10 N 0 MS2 1/10MS2 0 a b c

180

t n

. 150 a l p

x 120 e / i c

o 90 f

U 60 G

b 30 N 0 0 1 2 3

1 CNumber of days on /10MS2

A - Embryogenic calli (yellow series, experiments AT36) and embryo axis (red series, experiment AT37) of Melys were inoculated and co-cultivated with AGL1/pAL155/pAL156 either on full strength MS2 medium or

1 on low strength MS2 medium ( /10 MS2). B - Embryo axes of Melys were inoculated with AGL1/pAL155/pAL156 in full strength MS2 medium and

1 1 either (a), co-cultivated on /10 MS2 for 8 d, (b), co-cultivated on /10 MS2 for 4 d and on MS2 for 4 d, or (c), co-

1 cultivated on MS2 for 4 d and on /10 MS2 for 4 d.

1 C - Embryo axes of Melys were inoculated with AGL1/pAL155/pAL156 in /10 MS2 and co-cultivated on 1 /10 MS2 for 0-3 days and on MS2 for 7-4 days, respectively.

18 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 10 – Effect of glucose on transformation efficiency.

n . a l

p 10 x e / i

c 8 o f

S 6 U G

b 4 N 2 0 a b c

Embryogenic calli of Melys were inoculated with AGL1/pAL155/pAL156 (experiment AT29). a, no glucose added; b, 10 g/l glucose added in inoculation medium; c, 10 g/l glucose added in inoculation and co-cultivation media

Figure 11 – Effect of the solidifying agent on transformation efficiency.

t 60 n a . l

p 50 x e /

i 40 c o f

30 S

U 20 G

N 10 0 agar agarose

1 Embryo axes of Melys were inoculated with AGL1/pAL155/pAL156 and co-cultivated on /10MS2 solidified with either agar or SeaPlaque agarose (experiment AT44b).

19 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 11B – Effect of presence or absence of auxin on transformation efficiency.

50 . t n a

l 40 p x e /

i 30 c o f

S 20 U G

b 10 N 0 0 2 Concentration of 2,4-D (mg/l)

1 Embryogenic calli of Melys were inoculated with AGL1/pAL155/pAL156 and co-cultivated on /10MS either without 2,4-D or with 2mg/l 2,4-D (experiment AT36b).

Figure 12 – Effect of wounding the tissue and vacuum application on transformation efficiency.

t 1 n a l .

p 0.8 x e / i

c 0.6 o f

U 0.4 G

b 0.2 N

0 a b c

Embryo axis of Melys were inoculated with AGL1/pAL155/pAL156 (experiment AT42). a, non- wounded explants, no vacuum applied; b, wounded explants, no vacuum applied; c, non-wounded explants, vacuum applied.

Figure 13 – Effect of plasmolysis on transformation efficiency.

20 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

t n . a l

p 20 x e / i

c 15 o f

S 10 U G

b 5 N

0 a b

Embryos of Melys were inoculated with AGL1/pAL155/pAL156 (experiment AT46). a, inoculation and

1 1 co-cultivation were done in /10 MS2. b, explants were plasmolysed on /10 HOM for 4 h prior to inoculation

1 1 1 in /10 HOM. Plasmolysed explants were co-cultivated on /10 HOM overnight, then on /10 MS2.

Figure 14 – Effect of the type of virG gene on transformation efficiency.

160 140 . 120

t n a l 100 p x e / i 80 c o f

S 60 U G

b 40 N 20 0 a b c d e

Embryo axes of Melys were transformed with strains (a) AGL1/pSoup/pAL156, (b) AGL1/pAL166/pAL156, (c) AGL1/pAL155/pAL156, (d) AGL1/pAL165/pAL156 or (e) AGL1/pAL154/pAL156. Strains carried either no additional virG gene, or one copy of virGWT, virG542, virGN54D or the komari fragment on pSoup, respectively (experiment AT53).

21 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 15 - Effect of the position and copy number of virG genes on transformation efficiency. The position and copy number of. A B

200 160 .

t 160 n

n 120 a a l l p p x x 120 e e / / i i c

c 80 o o f f

80 S S U U G G 40

b b 40 N N 0 D 0 C a b c d a b c d

240 50 .

. t

t n 200 n

a 40 l a l p p x x

e 160 / e i

/ 30 i c c o f 120 o

S 20 U

80 U G

b 10 N

40 N 0 0 a b c d a b c d

A - virGWT (experiment AT52), B - virGN54D (experiment AT53), C - virG542 (experiment AT45) D the position of the komari fragment (experiment AT55) were tested on embryo axes of Melys.

A - a: AGL1/pAL155/pAL156 (virG542 on pSoup), b: AGL1/pAL166/pAL156 (virGWT on pSoup), c: AGL1/pSoup/pAL170 (virGWT on pGreen), d: AGL1/pAL166/pAL170 (virGWT on both pSoup and pGreen). B - a: AGL1/pAL155/pAL156 (virG542 on pSoup), b: AGL1/pAL165/pAL156 (virGN54D on pSoup), c: AGL1/pSoup/pAL169 (virGN54D on pGreen), d: AGL1/pAL165/pAL169 (virGN54D on both pSoup and pGreen). C- a: AGL1/pSoup/pAL156 (no additional virG), b: AGL1/pAL155/pAL156 (virG542 on pSoup), c: AGL1/pSoup/pAL157 (virG542 on pGreen), d: AGL1/pAL155/pAL157 (virG542 on both pSoup and pGreen). D - a: AGL1/pSoup/pAL156 (no additional virG), b: AGL1/pAL154/pAL156 (komari fragment on pSoup), c: AGL1/pSoup/pAL186 (komari fragment on pGreen), d: LBA4404/pTOK233.

22 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Figure 16 - Effect of combining virG542 and virGN54D.

140

n 120 . a l p

x 100 e / i

c 80 o f

S 60 U G 40 b N 20 0 a b c d

Strains containing two copies of virG542 (a, AGL1/pAL155/pAL157), two copies of virGN54D (b, AGL1/pAL165/pAL169) or one copy of each gene (c, AGL1/pAL155/pAL169 containing virG542 on pSoup and virGN54D on pGreen and d, AGL1/pAL165/pAL157 containing virGN54D on pSoup and virG542 on pGreen) were used to transform embryo axes of Melys (experiment AT53).

23 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

Table 1 - Experimental procedures of Agrobacterium transformation experiments.

1 1 HOM, osmotic medium, /10HOM, HOM with /10 of MS salts; MS2, callus induction MS5, callus induction medium 1 1 1 1 containing 5 mg/l 2,4-D; /10MS2, callus induction medium with /10 of MS salts; /10MS0, callus induction medium with /10 of MS salts and no 2,4-D; a, 200 M AS; b, 10 g/l glucose.

Pre-centrifugation Post-centrifugation Tissue Exp Ab inducing Ab inducing Co- Co- Parameter Inoculatio Inoculation erim Additi cultivation cultivation tested Inducing n medium Addition Inducing time ent on of medium time time of ASa time ASa AT2 Ab density Embry HOM + AS o/n + - HOM + - 10 min 6 co-cult. o and MS5 + 2-9 d AT2 Co-cult.time Emb. AS - MS5 + - 10 min MS5 + AS 1-10 d 7 time axis AT2 MS2 ± MS2 + AS ± 1 Glucose Callus - + 1 /2 h 2 h 10 d 9 glucoseb glucoseb

AT3 Embry 1 AS - MS2 +/- 1 /2 h 2 h MS2±AS 9 d 4 o

AT3 MS2 or MS2 + AS Low salt Callus - + 3 h 2 h 7 d 1 or 6a /10MS2 1 1 /10MS0MS2 + AT3 /10MS0 or 2,4-D Callus - + 3 h 2 h 7 d 1 AS or 6b /10MS2 1 AT3 Emb. MS2 or MS2/10MS2 + AS + Low salt - + 3 h 2 h 9 d 1 or 7 axis /10MS2 1 AT3 Inoculatio Emb. /10MS2 + - MS2 + - 15 min-15h MS2 + AS 9 d 8 n time axis AT4 Emb. Ab density - MS2 + 4 h 2 h MS2 + AS 8 d 0 axis AT4 Co-cult. Emb. 1 - MS2 + 3 h 2 /2 h MS2 + AS 7 d 1 tem- axis AT4 perature,Wound, Emb. 1 - MS2 + 3 h 2 /2 h MS2 + AS 9 d 2 vacuum axis AT4 Emb. MS2 + AS 1 Low salt - /10MS2 + 4 h 2 h 8 d 3 axis, or 1 AT4 Solidifying embryoEmb. /10MS2 + 1 1 1 1 + 2 /2 h /10MS0 - 2 /2 h /10MS2 7 d 4a agent axis AT4 Emb. 1 1 1 1 AS + 2 /2 h /10MS0 ± - 2 /2 h /10MS2 ± AS 7 d 4b axis 1 AT4 Emb. /10MS2 + 4 + 1 1 virG - /10MS0 + 2 h 2 /2 h 5 axis AS and MS2 4 d + AS

24 Project Agrobacterium mediated transformation of oats MAFF CEO161 title project code

1 1 /10HOM + AT4 Plasmolys Embry /10HOM or o/n + 6 d - + 2 h 2 h 1 AS and/or 6 is o /10MS2 or 7 d 1 1 /10MS2 + AT5 Emb. /10MS2 and 1 1 Low salt + 4 /2 h /10MS2 + - 2 h 7 d 1 axis MS2

1 AT5 Emb. /10MS2 and 3 + 1 1 1 virG + 2 /2 h /10MS2 - 2 /2 h 2 axis MS2 4 d

1 AT5 Emb. /10MS2 and 3 + 1 virG + 3 h /10MS2 - 3 h 3 axis MS2 4 d

1 AT5 /10MS2 and 3 + 1 1 Tissues Various + 3 h /10MS2 - 3 /2 h 4 MS2 3 d

1 AT5 Genotype Emb. /10MS2 and 3 + 1 1 + 2 h /10MS2 - 2 /2 h 5 s axis MS2 4 d

Published papers and presentations during project. Morris P. 1998. Opportunities for plant breeding using oat transformation. Presentation to Annual Research Strategy meeting. Svalof-Weibull. Sweden.

Perret S. 1999. Agrobacterium mediated transformation of oats. MAFF Crop Molecular Genetics Workshop 26-28 May . PBI Cambridge.

Morris P. 1999. Development of a reliable transformation system for husked and naked oats. MAFF Crop Molecular Genetics Workshop 26-28 May . PBI Cambridge.

Perret S. 1999. Genetic Transformation of oats. Seminar Department of Plant Genetics, University of Minnesota St Pauls, Minnesota USA.

Perret S and Morris P. 1999. Expression patterns of three constitutive and a seed specific promoter in transgenic oats. 16th International Long Ashton Symposium: Biotechnology of Cereals. 13-15 September IACR. Long Ashton. UK.

Leggett JM., Perret S, Harper. J, and Morris P. 1999. Chromosomal location of co-transformed transgenes in the hexaploid cultivated oat Avena sativa using FISH. Heredity 84:46-63

Perret S. 1999 Agrobacterium-mediated transformation of oats. Presentation at MAFF Crop Mol Genetics Meeting: Agrobacterium-mediated transformation of cereals July 1999. London.

Perret S. 2000. Agrobacterium-mediated transformation of oats. Presentation at MAFF Crop Mol Genetics Meeting: Agrobacterium-mediated transformation of cereals February 2000. London

Kuai B, Wan S.M., Perret S, Dalton S.J. Bettany A.J.E and Morris P. (2000) Production of fertile transgenic oat (Avena sativa L.) plants and stability of bar gene expression in T1 and T2 inbred progeny. Plant Cell Tissue and Organ Culture (in press).

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