Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: COMPARISON OF THREE METHODS FOR SUCCESSFUL MICROSPORE CULTURE
OF WHEAT
COMPARISON OF THREE METHODS FOR SUCCESSFUL MICROSPORE CULTURE
OF WHEAT
M. Patel, N.L. Darvey and D.R. Marshall, Department of Crop
Sciences, The University of Sydney, N.S.W. 2006, Australia.
Microspores of four wheat cultivars or advanced breeding
lines (Grebe, Bindawarra, Wt2 and QT 4336) were successfully
cultured to give rise to embryoids by using one of three culture
techniques. The main factors relevant to the successful
application of this technology were: (1a) pre-culture of anthers
in mannitol plus macroelements for 7 days, isolation of
microspores via a magnetic stirrer in modified MC17 medium with
PAA, and resuspension in the medium after filtration OR (1b)
mechanical isolation via blending of cold treated spikelets,
filtration and resuspension in modified MC17 medium after
gradient centrifugation at 2500 rpm and (2) culture of
microspores at a density of 5xl0^5 microspores per ml as dense
droplets surrounded by approximately 20 wheat ovaries. The yield
of embryoids per dish varied according to the treatment and
cultivars involved. Mechanical isolation via the blender with
gradient centrifugation gave a mean of 107, 2, 41 and 84
embryoids per dish for the above mentioned cultivars
respectively. The anther pre-culture technique with magnetic
stirring gave approximately half of the above cultivar responses,
whereas the 3rd technique involving pre-culture of anthers,
shedding of microspores in mannitol and simple centrifugation in
the medium also yielded a moderate number of embryoids.
Microspores commenced division 5-6 days after isolation via
blending, and after 7-9 days from pre-cultured anthers. A longer
cold pre-treatment (up to 28 days) prior to blending reduced the
time for the first division to 3-4 days. Approximately 50% of
cultured embryoids yielded plants on the regeneration medium;
however the majority (86%) were albinos. Improvements in the
green to albino ratio via modifications to the media or culture
technology will be required before this technique is routinely
used for transformation technology of single cell microspores or
microspore cell clusters.
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