gehou om 09h30 op 19 Julie 2002 in die PNS-Raadsaal, Rivonia
Die Voorsitter, mnr GJH Scholtemeijer heet almal teenwoordig welkom by die vergadering.
Mnr GJH Scholtemeijer PNS (Voorsitter) Dr M Griessel PNS Dr J de Kock PNS Prof D Berger Universiteit van Pretoria Dr T Watson WNNR Dr K de Ronde LNR – Roodeplaat Me B Campbell WNNR Mnr D Oelofse LNR – Roodeplaat Mnr R Laurie LNR – Roodeplaat Mnr G Keun Sekretaris
Vasstelling van die sakelys
Die volgende sake ter bespreking word op die sakelys geplaas:
- Probleme met transformasie.
- Patent – mnr Oelofse.
- Notule – vergadering rakende projek van dr Koch.
- Projekaansoek – Prof Berger.
Sake ter bespreking
Probleme met transformasie
Dr De Ronde suggested that the experiments be discussed in the following sequence:
- Glasshouse experiment.
- PGIP plants.
- Testing of PGIP plants.
Mnr Laurie berig soos volg:
- Gedurende die vergadering van 10 Oktober 2001 is genoem dat daar 3 000 nageslag sade is wat geplant gaan word. Dit sou met ignite gespuit word om te bepaal of daar enige weerstandbiedende T1 plante is;
- Daar was twee opsies, te wete om al die plante te PCR om seker te maak dat niks verlore gaan nie of om vir die BAR-geen te selekteer, aangesien dit saam met die PGIP-geen oorgedra kan wees. Aangesien die PCR opsie geweldig baie werk is, is die tweede opsie gevolg;
- Die sade wat gesaai is het nie almal opgekom nie. Die vermoede is dat daar transformasie skade was;
- 'n Bespuiting met 0,1% ignite is gedoen; en
- Ten spyte daarvan dat die plante gelyk het of hulle weerstandbiedend was, het die plante begin vrek. Daar was slegs 65 plante oor. Die kontrole plante is almal dood. Van die 65 plante het ook begin vrek en slegs 4 plante leef nog. Op die stadium is die plantmateriaal oorgedra na me Campbell.
Ms Campbell reported as follows:
- It was mentioned at the previous meeting that 704 seeds were germinated;
- Similar experiments to those of Mr Laurie were performed, although a different approach was adopted with the germination of the seed on a medium;
- Of the 704 seeds approximately 100 survived the media grow;
- The seeds were germinated for approximately 4 weeks on the medium before being transferred to the glasshouse;
- The plants were transferred from tissue culture into small seedling trays and were placed in a plant growth room and not directly into the glasshouse;
- All the remaining plants died. It looked like the plants had some sort of infection;
- During the experiment ± 20 plants were taken and DNA isolated from these plants. Together with the plants of dr De Ronde and mr Laurie, PCR screens were performed in order to determine if the plants were transgenic or not. A NAD-gene PCR was done. The plants were then screened for the PGIP transgene, BAR transgene and the GUS transgene. The plants were PGIP, BAR and GUS negative; thus
- None of the 704 seeds screened were transformed.
To a question of dr Griessel why there is no transformation, dr De Ronde replied that they don't know. She further mentioned the following:
- Similar work to the experiments done by ms Campbell was done with the same results;
- There are a lot of questions that are unanswered and tests must be done to get some answers;
- After the results became known a series of transformation experiments were done. These experiments/tests were not done in the initial process due to the fact that the idea was to get a anthracnose resistant plant as soon as possible;
- DNA extractions were performed on the T2 plants of the GUS and BAR transformants of the previous season that were planted in the greenhouse. PCR's had already been done on 23 plants and the results differ from each other in all instances; and
- It's not known if some of the T2 plants in the greenhouse are positive with the BAR and GUS-gene.
Dr Watson mentioned that although further experimentation can be done an alternative strategy must be followed to get to the final result. He mentioned that if the transformation is regarded as unsuccessful the rest of the work had been successful in so far as the fact that the promoter works. An alternative is to consider a trade off with the Australians who have had success with the transformation technology.
Prof Berger mentioned that according to him the first problem is that it is quite difficult to screen the plants and secondly that not enough energy was put into confirmation of the first GUS and BAR transformants.
The Chairperson mentioned that despite all the problems it seems that the vital missing link is a reliable test for transformation. Prof Berger responded by saying that a reliable PCR protocol needed to be developed for the BAR-gene. He stated that the test for GUS and PGIP was reliable.
To a question from the Chairperson what the alternative solutions were, dr Griessel mentioned that if it is suspected that the vacuum infiltration technique is not working, then there are only two options:
- To make use of the tissue culture technique ourselves or by the the Australians; or
- That there were problems with the vacuum infiltration and the experiment needs to be repeated.
Die Voorsitter versoek dr De Ronde om aan te dui of die fout by die tegniek lê, waarop dr De Ronde meld dat sy nie weet waar die fout is nie, maar graag die geleentheid wil hê om 'n paar toetse te doen. Vir die herhaling word ten minste 'n seisoen (6 maande) benodig.
Die Voorsitter verneem met watter werk voortgegaan kan word indien dr De Ronde 6 maande gegun word om toetse te doen, waarop mnr Oelofse meld dat hy kan voortgaan met die "promoter sequence". Die Voorsitter meld dat daar dus voortgegaan kan word met die registrasie van die patent, waarop mnr Oelofse meld dat om die werk waarmee hy tans besig is te voltooi, hy 'n verdere 6-8 maande benodig.
Die Voorsitter meld dat die tweede alternatief die "tissue culture technique" is en daar met die Australiërs kontak gemaak moet word.
Dr Griessel versoek dat daar seker gemaak word dat die werk wat die Australiërs gedoen het nie net op angustifolius was nie maar ook op albus en verneem of die totale pakket beskikbaar gestel word wanneer met die Australiërs onderhandel word. Mnr Oelofse meld dat die Asutraliërs reeds beskik oor die peroksidasegeen en volgens sy toesighouers kan die promoter nie beskikbaar gestel word nie. Dr Watson mentioned that only the PGIP construct be offered and if that does not work then something extra should be added.
Die Voorsitter meld dat daar nie te veel detail bespreek word nie, aangesien daar slegs na alternatiewe gekyk moet word. Dr De Kock meld dat 'n derde alternatief is om die projek te staak, waarop die Voorsitter noem dat die projek gestaak kan word nadat dr De Ronde 'n herhaling gedoen het en daar geen positiewe resultate is nie.
Dr Watson mentioned that they did obtain something of value in the process but are unable to put the whole process together. He further mentioned that the only problem is to put the gene and construct together with the plant.
Die Voorsitter verneem van prof Berger of hy die persone in Australië waarna verwys word reeds ontmoet het, waarop prof Berger bevestig dat hy sodanige kontak het en dat die aangeleentheid per e-pos/skrywes hanteer kan word.
Dr Watson informed the meeting that other groups also had success with the transformation technology. According to him that is the CSIRO in Australia.
Dr Griessel verneem op watter wyse die kundigheid soos die promoter en peroksidase waaroor reeds beskik word oorgedra gaan word na die plant. Prof Berger replied by explaining that the promoter can go into any plant and therefore it can be sold to any company working on sunflower, tobacco, potato, etc. Prof Berger further explained to the meeting that if the Australians agree to do the work the present constructs won't be given to them and that the best option is to wait until Mr Oelofse has connected the promoter to the peroxidase gene in the combined PGIP construct. Mr Oelofse mentioned that he is in no position to make decisions regarding the promoter.
The Chairperson mentioned that the ARC and PRF are joint owners of the promoter. He proceeded to ask the meeting what their level of confidence is that the process will constitute an anthracnose resistant lupin seed to which dr Watson replied approximately 80% and Prof Berger about 50%. The Chairperson mentioned that nothing should be done until such time the patent has been registered. He asked what would be lost if a deal was negotiated with the Australians and they were unsuccessful to which Prof Berger replied that the only thing lost will be the investment made up until now.
Prof Berger summarised the work to be done as follows:
- There are two (2) phases:
- First will be to check the T2 plants for the GUS and the BAR genes. The GUS activity should be checked (± 6 weeks). If there is ± 10 plants that are positive it tells that dr De Ronde can get the method to work. If all are negative other alternatives must be looked at. The process will not take longer than three (3) months;
- It is very important for dr De Ronde to proceed with the vacuum infiltration; and
- In the meantime the negotiations with the Australians must proceed.
Prof Berger referred to his pending negotiations with the Australians and asked whether he should enquire about marker resistance breeding specifically to albus. The meeting recommended as such.
- Dat dr De Ronde voortgaan met die herhaling van die werk wat deur hulle gedoen word.
- Dat prof Berger versoek word om met kundiges in Australië kontak te maak rakende die aangeleentheid.
Patent - Mnr Oelofse
Mnr Oelofse gave feedback regarding the promoter. The procedure and cost for the registration of the patent was also discussed.
Notule - Projek van Dr Koch
Dr De Kock verneem of dit nodig is dat dr Koch voortgaan met haar projek. Hy meld verder dat aangesien daar geen plante afkomstig is vanaf dr De Ronde en me Campell nie, daar miskien gewag moet word met die ander werk ten opsigte van die 16 cultivars deur dr Koch.
- Dat die projek van dr Koch voorlopig gestaak word.
Projekaansoek - Prof Berger
The Chairperson informed the meeting regarding the aim of the project submitted by Prof Berger. He requested Prof Berger to comment on the project taking into consideration the discussion took place under item 4.1.
Prof Berger commented as follows:
- That the submitted project is very small and builds on the current project. However, the project is not dependant on the current project but it adds value to the current project;
- The project is a more detailed project in respect of the polygalacturonase of the fungus;
- There are two benefits:
- One will be able to test other PGIP's; and
- The idea is to produce a lot of the pg protein. That is a tool that one can use to test chemicals to see if there are any chemicals which can knockout the pg.
- The proposed project is for a period of two (2) years to get hold of the gene and make the protein.
To a question of the Chairperson with regard to the fact that the current project may be stopped after a period of three months, Prof Berger replied that his proposed project will not replace the current project and has no value to the PRF.
The Chairperson explained that the project proposal submitted is a provisional application and final applications will only be heard at the end of September 2002.
Datum van volgende vergadering
Die volgende vergadering van die Antraknose Werkgroep is geskeduleer vir 7 Oktober 2002.
Aangesien daar geen verdere sake ter bespreking is nie, verdaag die Voorsitter die vergadering om 11h35.