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HORT 515
Haploids In Vitro
Day 0 - emasculation
Day 2 - pollination with H. bulbosum pollen
Day 3 (to 5) - 40% of the embryonic cells are haploid, endosperm
abortion occurs, GA3 treatment enhances retention of florets
Day 11 - 94% of the embryonic cells are haploid
Day 14 (to 16) - embryos are dissected and cultured in the dark at
18 to 22 C, embryos develop in vitro
Day 22 (to 28) - embryos are transferred to light for seedling
development
Day 50 - plants
Cross (VV x BB)
Progeny: V VV VB VBB
n= (7) (14) (14) (21)
1517 0 26 0
Barley Monoploid Production
X
H. vulgare H. bulbosum
(n=7) (n=7)
Hybrid Zygote
History:
Tulecke (1953) - haploid callus (but no plants) derived Ginkgo biloba
Microspore Tetrad
First Mitosis
Similar Nuclei
Vegetative
Generative
Germination
Haploid Proembryo
Haploid Embryo
Factors affecting the development of haploid plants in
vitro
Anther stage - most responsive cells for haploid embryo formation are
those between the tetrad stage of microsporogenesis to just past the
first pollen mitosis, example
Actively growing plants and the first set of flowers are most
responsive
Anther stage - most responsive cells for haploid embryo formation are
those between the tetrad stage of microsporogenesis to just past the
first pollen mitosis, example
Actively growing plants and the first set of flowers are most
responsive
3 to 5°C
Vegetative Microspore
Similar nuclei
Generative
3 to 5°C
Embryo
Cold Pretreatment of Anthers Enhances the Embryogenic
Response
Cold treatment imposed prior to the first pollen mitosis increases the
frequency of symmetric divisions of the microspore leading to embryo
formation, control – room temperature.
Tobacco
Producing Embryos
100 3°C
w/identical nuclei
80 5°C for 72 h
% Anthers
5°C
% Pollen
60 C 10
5 Control
40
C
20 0
0 3 7 12
0 Days in Culture
Tobacco Datura
Culture medium