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Molecular approaches to plant virus resistance in lupins

Li, Hua (2000) Molecular approaches to plant virus resistance in lupins. PhD thesis, Murdoch University.

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Narrow-leafed lupin (L. angustifolius) is the main legume crop grown in Western Australia. Yellow lupin (L. luteus) is a new crop for Western Australia which can tolerate more marginal acid soils than narrow-leafed lupin. Because lupins as crop plants are relatively undeveloped, there is potential to introduce additional traits into lupin species by conventional breeding or by gene transfer technology. Resistance to bean yellow mosaic virus (BYMV) and tolerance to herbicides are of commercial and environmental value.

The aim of this work was to optimise conditions for tissue culture and regeneration of transgenic lupin plants, specifically to introduce the NIa gene derived from BYMV for BYMV resistance, and the bar gene for tolerance to the herbicide glufosinate. Two existing L. angustifolius regeneration and transformation methods were assessed, and improved methods were developed to regenerate and transform L. angustifolius and L. luteus.

Experiments were carried out to determine optimal conditions for culture and gene transfer to examine the most effective A. tumefaciens strain, the period of precultivation, the period of cocultivation, the best method to wound the meristem, the optimum time of incubation before transfer to selection medium and the concentration of glufosinate in the selection medium.

Results from the above experiments were combined into a standard transformation procedure. The embryonic axes from mature seeds were excised and punctured 10-15 times with a fine needle. The explants were co-cultivated with A. tumefaciens strain AgL0, or LBA4404 for 4 days on IL medium without selection. Explants were then transferred to IL medium with antibiotic for shoot regeneration. After four days a drop of glufosinate (2000 mg/l) was applied on the apical region of the explant. Two to three weeks later, shoots that developed were excised individually from the shoot apices and placed on IL medium with 20 mg/L glufosinate and Timentin™.

After subculture every 10-14 days for 4-6 months, induction of roots in both lupin species was achieved by incubation in medium containing either NAA (2mg/l) or IBA (3 mg/l) (rooting medium). Thirty to fifty percent of L. angustifolius explants and 10 % of L. luteus explants developed roots in vitro. Those explants that did not develop roots were grafted onto 10-14-day old L. angustifolius Merrit or Myallie seedlings in vitro. This technique was successful in about 98% of cases. T0 plants were matured in liquid medium with a hydroponics system or in sand in the glasshouse.

A total of 13,460 L. angustifolius explants from 7 genotypes were cocultivated with A. tumefaciens with 9 different gene constructs. Twenty-four independent L. angustifolius primary transformants of 5 genotypes, transformed with one of 4 constructs were obtained. A total of 35,034 L. luteus explants from 9 genotypes were cocultivated with A. tumefaciens with 14 different constructs. Forty-eight independent L. luteus primary transformants of 4 genotypes, transformed with one of 6 constructs were obtained.

Southern analysis of 14 T. angustifolius primary transformants showed that they contained 1-4 copies of a transgene. Three L. luteus transformants contained 3-4 copies of the transgene.

All 24 L. angustifolius events and 26 of the 34 L. luteus events tested expressed PAT and acetylated glufosinate in PAT assays. Plants that expressed PAT usually tolerated leaf application of 100 mg/1 glufosinate. In 8 L. luteus T0 events, the bar gene was inactivated. Two L. luteus transformants were highly tolerant to glufosinate and showed no tissue damage after application of 1000 mg/1 glufosinate.

A PCR product from a T1 L. luteus plant, of part of the virus-resistance transgene (Nla) was sequenced. It was 3 nucleotides shorter than expected, 417 vs 420, because of a deletion. A 1,939 bp PCR product consisting of the regulatory elements 35S promoter, CMV 5’UTR and CaMV 3’UTR, and the full-length Nla gene, was similarly isolated from a T3-generation L. angustifolius plant. There were 4 nucleotide substitutions in the Nla gene, but no changes to the regulatory elements.

Plant lines derived from 17 different L. angustifolius transformation events were challenged by sap inoculation of BYMV-Mi in the glasshouse, 5 events were challenged by aphid inoculation of BYMV-Mi in a screenhouse. Manual inoculation was more effective than aphid inoculation as determined by the infection rate of non-transgenic controls. In glasshouse trials, nine transgenic events showed no resistance to systemic infection. The rates of systemic virus infection in plant lines from 6 primary transformants ranged from 60-94%. After aphid inoculation in screenhouse trials, 77-100% of non-transgenic controls became infected, and 50-100% of transgenic plants became infected. Conclusive results were therefore not obtained from aphid inoculation experiments.

Twenty six lines from 5 L. luteus transformation events were challenged by manual sap inoculation of BYMV-Mi in a glasshouse, 6 lines from 4 transformation events were challenged by aphid inoculation in a screenhouse, and 8 lines from 5 transformation events by aphid inoculation in a small field trial. In the glasshouse trials, 90-100% the nontransgenic control plants became infected after 5 virus inoculations. The rates of systemic virus infection in plant lines from 20 lines ranged from 18-86%. After aphid inoculation of the virus in the screenhouse trials, 28-57% of non-transgenic controls were infected, and 0- 70% of transgenic events were infected. In the line without systemic infection, only 28% of controls became infected. In a small field trial, 37-69% of control plants and 36-64% of the transgenic plants became infected. As with aphid inoculation experiments with L. angustifolius, conclusive results were not obtained from aphid inoculation of L luteus.

In conclusion, an improved system for in vitro culture, selection and regeneration of transgenic plants of L. angustifolius has been developed and applied to generate a series of transgenic plants with different synthetic virus resistance constructs. A similar L. luteus transformation procedure has been developed, with production for the first time of transgenic plants of L. luteus. Some individual plants from both species were highly resistant to BYMV. However, other plants within the same lines were susceptible. Transgenic plants tolerant to high levels to glufosinate, and of potential commercial use, were obtained for both species. Inheritance of transgenes was followed up to the T5 generation.

Item Type: Thesis (PhD)
Murdoch Affiliation: Division of Science and Engineering
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): Jones, Michael
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