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Plant transformation using pollen vacuum infiltrated with Agrobacterium tumefaciens

Tjokrokusumo, Donowati (1998) Plant transformation using pollen vacuum infiltrated with Agrobacterium tumefaciens. Masters by Research thesis, Murdoch University.

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Bechtold et al. (1993) obtained transformed seedlings from Arabidopsis thaliana inflorescences vacuum infiltrated with Agrobacterium tumefaciens. In this thesis vacuum infiltration of pollen with Agrobacterium tumefaciens and other pollen treatments were studied to evaluate whether these methods could achieve transformation of the dicotyledon Petunia hybrida, var. Peach Ice and the monocotyledon Zea mays, var. Super Sweet.

Pollen germination in vitro from both petunia and com was optimised. The highest germination of petunia pollen (62 %) was achieved in medium containing 20 % sucrose, 100 mg!'1 boric acid and 300 mgl’1 CaCl2. 2H20. In com, the maximum germination observed was 73 % in medium containing 10 % sucrose, 100 rngf1 boric acid and 300 mgl'1 CaCl2. 2H20.

The Agrobacterium tumefaciens used in this research carried the plasmid pCGP1258 containing the GUS gene and the bar gene both controlled by the 35S promoter.

The effect of vacuum on petunia pollen suspended in germination medium showed that germination was not significantly depressed by exposure of 20 minutes vacuum (80 kPa) and that this treatment induced an increase in pollen tube length. In com, vacuum treatment of pollen reduced germination when applied for more than 1 minute. Pollen treated for 1 minute developed longer pollen tubes than control pollen. Agrobacterium tumefaciens cells placed under vacuum for up to 30 minutes were unaffected.

Pollen of petunia which was vacuum-infiltrated with Agrobacterium for 20 minutes was used to pollinate the stigmas. Similarly com pollen vacuum treated with Agrobacterium for 1 minute was used to pollinate the silks. At the same time pollination was also carried out using untreated pollen and applying a drop of Agrobacteria solution to the stigma at the time of pollination. The resultant T, seedlings were screened in vitro on medium containing 3 mg Y of the herbicide Basta for petunia, while seedlings of com were screened by applying 20 mg 1-1 of Basta R to the leaves of 15 day old plants.

In petunia 9 % of 1806 seedlings tested were resistant to BastaR, while in com 32 % of 372 plants tested were resistant to BastaR. BastaR resistance was seen in seedlings resulting from both the vacuum and the drop treatments.

Putatively transformed T] plants of petunia were grown further in the glasshouse and 10 % of plants showed GUS expression in tissues of the leaf, pistil and young anthers. Amongst putatively transformed T, com plants 20 % showed GUS expression in the silks.

PCR analyses showed that for the T1 putatively transformed petunia plants, 66 % were positive for the presence of the GUS gene although only 10 % expressed GUS. For com plants 44 % of putatively transformed T1 plants showed the presence of the GUS gene through PCR, compared with 20 % from GUS expression. No carryover bacterial contamination of the transformed plants could be detected.

Southern hybridisation analyses showed that iscoRI-digested DNA of putatively transformed petunia plants tested with the bar gene probe hybridised twice to genomic DNA fragments of approximately 6.5 kb and 4.3 kb.

Some T2 plants were produced by crossing two putatively transformed T1 plants. Amongst the T2 progeny; 5 % were resistant to Basta , 5 % of these expressed GUS, and PCR analysis showed 61 % of those resistant to Basta were positive for the presence of the GUS gene. Two different T2 plants and the putatively transformed ^ plants showed almost identical banding patterns when a bar gene probe was used in a Southern hybridisation, indicating inheritance of the transgene.

In summary it was shown that vacuum infiltration of pollen with Agrobacterium is unnecessary to achieve transformation of petunia and com. Transformation can be achieved if there are bacteria present while the germinating pollen is growing on the stigma and style. It seems likely that this was also the mechanism through which vacuum infiltrated inflorescences of Arabidopsis produced the transformed progeny reported by Bechtold et al. (1993).

Item Type: Thesis (Masters by Research)
Murdoch Affiliation(s): Division of Science
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): McComb, Jen and Heinrich, Tatjana
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