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Investigation of alternative approaches to narrow-leafed lupin (Lupinus angustifolius) genetic transformation

Ratanasanobon, Kanokwan (2014) Investigation of alternative approaches to narrow-leafed lupin (Lupinus angustifolius) genetic transformation. PhD thesis, Murdoch University.

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Narrow-leafed lupin (Lupinus angustifolius) is one of the top six crops that contribute value to the Australian economy. Gene transfer technology has been studied as a strategy to improve lupin varieties against diseases to improve yield, production and seed quality. However, the established method used for transformation of lupin is based on Agrobacterium and embryonic axes as explants is a method of low efficiency. The aim of this project was to investigate the alternative genetic transformation methods for genetic manipulation of narrow-leafed lupin (L. angustifolius) to improve the transformation efficiency. Two potential genetic transformation methods were investigated: particle bombardment (direct gene transfer), and in planta transformation (Agrobacterium-based transformation). In addition, lupin-Agrobacterium interactions were studied to provide information of the factors limiting transformation, and whether the involvement of an additional virG using construct carrying virGN54D (constitutive virG mutant carrying Asn-54 to Asp amino acid substitution) improved lupin transformation efficiency.

In this project, a genetic transformation protocol using particle bombardment for narrow-leafed lupin was accomplished. The following conditions were identified as being optimal for transformation via particle bombardment using a helium inflow particle gun with lupin embryonic axes as target explants:

a) Embryonic axes used as explants were pre-treated in MS media supplemented with 5 mg/L BAP and 0.5 mg/L NAA, 3% sucrose and 0.7% agar for 3 days in the dark at 25oC.
b) Pre-treated explants were placed onto MS media with 0.3 M mannitol as osmoticum 4 h prior to bombardment.
c) Bombardment was carried out by:
• A precipitation protocol using plasmid DNA prepared at 2 ng DNA per μg tungsten particles.
•Bombardment was carried out twice at 400 psi with a 7 cm target distance with 10 μL coated particles.
d) Bombarded explants were kept on osmoticum media (MS media with 0.3 M mannitol) for 4 h then transferred to pre-/post-treatment media for post-treatment for another 3 days and kept in the dark at 25oC.
e) After post-treatment, explants were transferred to selection media (MS medium with 1 mg/L BAP and 0.1 mg/L NAA, 3% sucrose, 0.7% agar and 10 mg/L PPT) for 8 weeks with subculture every two weeks. Surviving shoots were transferred to rooting media and analysed for presence of the transgene by PCR.

A transformation efficiency of 0.4% for T0 production was achieved as confirmed by amplification of a gus gene by PCR. However those transformed explants did not form roots.

In planta transformation of seedlings and flowers of narrow-leafed lupin was investigated. For seedling transformation, factors essential for delivering A. tumefaciens to the target tissues (L2 layer of apical meristem of seedings) and to enhance the ability of A. tumefaciens cells to transform plant cells were studied and optimised. Sonication and vacuum infiltration facilitated penetration of A. tumefaciens cells to the target tissue, sonicating seedlings 15 min before 10 min infiltration with A. tumefaciens cells gave the best overall balance of both gene transfer determined by GUS staining and seedling survival rate. The Agrobacterium induction condition and infiltration medium was developed after testing and optimising of media and Agrobacterium growth. Modified LB medium with glucose 30 g/L was the best medium that gave the highest percentage of shoots showing GUS expression, at 35±5% which was significantly higher than the control infiltration media used for Medicago and A. thaliana in planta transformation at the 0.05 level Tukey HSD. The combined optimised conditions were further tested. Some shoots, picked at random, were positive for GUS expression, including the whole apical area and parts of leaves of new shoots, indicating gene transfer and stable transformation although chimeric. However, transformants were not obtained. Further investigations suggested that there may not have been enough viable A. tumefaciens on seedling shoots for successful transformation. Survival of A. tumefaciens cells on the plant tissues was about 103 times less than routinely used for transformation of lupins in the established in vitro lupin transformation method.

In in planta transformation of lupin flowers, experiments were designed to deliver Agrobacterium cells to lupin ovules as target tissues. Factors reported to contribute to success in this type of transformation, such as using surfactant, infiltration period and times under vacuum and composition of infiltration medium were tested and optimised for lupins. Thirty plants with floral inflorescences having flowers ranging from the dome to open stages were infiltrated twice for 3 min each with MS liquid medium containing 10 mM glucose, 0.01% Silwet L-77 and A. tumefaciens cells in early exponential stage at concentration of OD 1.87. Pod set was 10.82 %. No transformant was obtained. The same infiltration media and conditions were used with Arabidopsis thaliana cv Columbia as control plants and transformants obtained at 0.255%. Histology studies of lupin flower structure by SEM and wax-embedded sectioning revealed that there did not appear to be a physical channel for A. tumefaciens cells to gain access to the ovule via the stigma or style before anthesis. Furthermore, Agrobacterium cells could not gain access to the ovule through the immature carpel of young flowers as the developing carpel closed while the ovule developed inside.

Interactions between Agrobacterium and lupin were studied to determine which stages limit transfer of genes from Agrobacterium to lupins, and which might be modified to achieve and/or improve transformation efficiency by A. tumefaciens in a genotype-independent fashion. The stages studied were: the attachment of Agrobacterium to the lupin explants, T-DNA transport across the cell wall and through the cell membrane. In addition, the effect of an extra virG was examined to find out if it would increase T-DNA transport. The interaction studies were done by comparing reactions to gene transfer in lupin cultivars Merrit and Quillinock which have significant difference in transformation efficiency (6.5% for cv Merrit and ~1% for cv Quillinock). The results of the attachment of Agrobacterium cells experiment showed no significance statistically in the number of bacterial cells attached to the explants (half embryonic axes) of six cultivars of narrow-leafed lupin (cvs Merrit, Quilinock, Belara, Illyarrie, Yorrel and Danja), indicative that the attachment stage was not the factor limiting gene transfer. T-DNA transport through the cell wall and cell membrane was evaluated through gus expression in experiment using cell suspensions (cells with cell walls) and protoplast (cells without cell walls) with T-DNA transfer determined by the relative intensities of the RT-PCR amplicons. The results showed, unexpectedly, that cv Merrit had less T-DNA transferred by A. tumefaciens than cv Quillinock in cell suspensions but not in protoplasts. The results were supported with MUG assays of transient expression of the gus reporter gene in cell suspensions of both cultivars. This indicated that the differences in cell wall composition between these two cultivars played an important role in gene transfer, but the factors limiting transformation success in Quillinock were downstream from T-DNA transfer to the cytoplasm of the host cell, possibly involving in T-DNA integration and/or expression, or selection and recovery of whole plants. The effect of an extra virG was examined with lupin, virGN54D increased transient expression of gus only in cv Quillinock cells, not in cv Merrit cells. Constructs carrying virGN54D may, therefore, be of some use as a component of a transformation protocol for cv Quillinock, and possibly other recalcitrant lupin cultivars.

This work has confirmed the relative difficulty of transforming narrow-leafed lupins, and it is concluded that despite investigating a series of alternative approaches, the method based on ‘stab inoculation’ of apical meristems, followed by selection of chimeric tissues to generate transgenic inflorescences, appears to be the most reliable approach. However, it is strongly genotype dependant, and improvements in efficiency and reduced genotype dependence are still desirable.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Wylie, Steve and Jones, Michael
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