TY - JOUR
T1 - An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes
AU - Nguyen, An Hoai
AU - Hodgson, Leon M.
AU - Erskine, William
AU - Barker, Susan J.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75 % and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.
AB - For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75 % and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.
KW - Agrobacterium tumefaciens
KW - Delayed selection methodology
KW - Green fluorescent protein
KW - Lupinus angustifolius legume transformation
KW - Mericlinal and periclinal chimera
KW - Narrow leafed lupin
KW - Regeneration
KW - Shoot axillary bud transformation
UR - http://www.scopus.com/inward/record.url?scp=84988603532&partnerID=8YFLogxK
U2 - 10.1007/s11240-016-1087-1
DO - 10.1007/s11240-016-1087-1
M3 - Article
AN - SCOPUS:84988603532
SN - 0167-6857
VL - 127
SP - 623
EP - 635
JO - Plant Cell, Tissue and Organ Culture
JF - Plant Cell, Tissue and Organ Culture
IS - 3
ER -