Plant Genome I Conference
Town & Country Conference Center, San Diego, CA, November, 1992.
PG-I: 44pg1
WHEAT SOMATIC EMBRYOS DEVELOP FROM AGROBACTERIUM-TREATED
EMBRYOGENIC CELLS IN SIMULATED OVULES
John G. Carman and Elizabeth E. Hood, Plants, Soils and
Biometeorology Dept and Biology Dept, respectively, Utah State
Univ., Logan, UT 84322-4820.
Wheat somatic embryos often are inviable. We are testing
the hypothesis: viable somatic embryos form from transformed
embryogenic cells when such cells are exposed to dynamic
simulations in vitro of the physical, nutritional and hormonal
conditions in ovules. We have 1) characterized nutrients,
hormones, dissolved O2, pH and osmolality in ovular fluids, 2)
formulated media that simulate these fluids, 3) evaluated zygotic
and somatic embryo development in simulated ovules, and 4)
produced somatic embryos, in simulated ovules, from embryogenic
cells treated with Agrobacterium tumefaciens. Caryopses were
ruptured, centrifuged and the apoplastic supernatant collected
and analyzed. Levels of nutrients in mmol kg -1, compared with
Murashige and Skoog medium, were (in situ/MS): N, 105/60; P,
16/1.8; K, 50/20; Ca, 6/3; Mg, 8/1.3; S, 11/1.5. About 85 % of
total N was from free amino acids. Water soluble carbohydrates
varied during development but were on average (in mmol kg -1):
mao-inositol, 15; glucose, 100; fructose, 200; sucrose, 10; and
trisaccharides, 95. Dissolved O2 at the embryo surface from 3 to
18 d post-anthesis (DPA) varied diurnally and generally ranged
from 2.6 to 4.8 mmol kg -1 (approx. 35 to 65% saturation). The
pH of ovule sap increased from 6.3 at 0 to 6 DPA to 6.9 at 18
DPA. The osmolality of the cavity sap decreased from about 800
mmol kg -1 at 0 to 6 DPA to 400 mmol kg -1 at 12 DPA and then
increased to nearly 500 mmol kg -1 by 18 DPA. In ovule levels of
cytokinins, ABA and IAA were elevated at different times during
early stages of embryogenesis (0 to 6 DPA). Thereafter total
cytokinin levels declined while total ABA and IAA increased. At
12 DPA and thereafter cytokinin levels were very low. Zygotic
and somatic embryo development improved in media that simulated
ovular conditions. Results will be discussed in terms of
potential improvements in transformation techniques. Research
supported by USDA, NRI-CGP, award no. 91-37300-6457.
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