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11. Wearing sterile latex
gloves, a mask and a watchmaker's 2x headband enlarger, excise the
haploid embryos from the disinfected GPCs under a laminar flow hood.
(Fine scalpels fashioned from discarded dentist's needles are best
for this purpose; their surgical steel enables repeated flaming, and
cooling in cold sterile water. Lift each green caryopsis from the
McCartney bottle by inserting the sterilized scalpel into its
attachment end. Hold the brush end between the thumb and forefinger
and cut along the lateral grooves at the attachment end, fold back
the dorsal pericarp and green integuments, and lift out the haploid
embryo).
12. Five excised embryos are placed in each 100 ml tissue culture jar
containing 20 ml of autoclaved IRM (the barley anther culture medium
of Olsen (1987) modified by Daniel (1990) and us. This modified
MS-medium contains 165 mg
l-1
instead of 1650 mg l-1
NH4NO3, 20000 mg l-1
sucrose, 100 mg l-1
myoinositol, 0.4 mg
l-1
thiamine HCl, 0.5 mg l-1
BA, 1 mg l-1
IBA and 2300 mg l-1
Gelrite; the pH is adjusted to 5.8 with KOH).
13. Seal the transparent lids of the tissue culture jars containing
the rescued embryos with clear plastic wrap. Place the jars in the
dark for 10 days at 6-8C and then at 25C until the regenerating
shoots are 1 cm long. Transfer them to a 25C growth cabinet with cool
white fluorescent tubes on a 10 h light/14 h dark cycle. The
regenerating plantlets reach the three leaf stage after four to six
weeks.
14. Transplant the plantlets in micro-irrigated, sand-filled 2-liter
pots (6-8 per pot) in a 16C growthroom on a 10 h light/14 h dark
cycle, and water once with a dilute seaweed extract (2 ml/l Kelpak)
to stimulate growth. (Plantlets transplanted in a greenhouse should
be covered with a 50% shade screen for a week).
15. Lift the plants from the pots as soon as they have developed two
to three sprouts (usually after a month), and trim their washed roots
to 10 em. Treat the plants in an aerated (use an aquarium pump)
aqueous solution of 0.05% colchicine (BDH), without DMSO, for 24
hours at 22C. Rinse the plants in running water for an hour and
transfer them to aerated water in a refrigerator at 4C for three
days. (The colchicine solution can be used again; filter and store in
a refrigerator).
16. The shoots of the
treated plants are cut back to 15 cm and the plants repotted (2-3 per
pot) in the 16C growthroom. Water once with the Kelpak solution.
17. After 4-6 weeks the + or - 95% plants that survived the
colchicine treatment will have formed new shoots. Transfer the potted
spring wheats to a greenhouse on a 18C day/12C night cycle, but
vernalize the winter wheats first for at least six weeks at 4C.
18. Nearly all the colchicine treated plants will produce a few
spikes with fertile doubled haploid (DH) sectors. Cover the fertile
spikes to prevent cross pollination. These spikes will give rise to
homozygous DH lines from which new cultivars can be selected.
References
Bajaj YPS (ed) (1990) Biotechnology in agriculture and
forestry 12. Haploids in crop improvement I. Springer Verlag,
Berlin.
Barclay IR(1975)
High frequency of haploid production in wheat (Triticum aestivum)
by chromosome elimination. Nature 256: 410-411.
Daniel G (1990)
Einfluss
verschiedener Faktoren auf die Pflanzenregeneration in der
Antherenkultur bei@Sommerund Wintergerste. Bayer Landw Jahrbuch
67: 609-617.
Henry Y and De Buyser J (1990) Wheat
anther culture. In: Bajaj YPS (ed) Biotechnology in agriculture and
forestry 13, Wheat.
Springer-Verlag, Berlin, 285-352.
Inagaki MN and Tahir M
(1990) Comparison of
haploid production frequencies in wheat varieties crossed with
Hordeum
bulbosum L. and maize. Jpn J Breed 40:209-216.
Jensen CJ (1977)
Monoploid
production by chromosome elimination. In: Reinert J and Bajaj YPS
(eds) Applied and fundamental aspects of plant cell, tissue and organ
culture. Springer Verlag, Berlin, 299-340.
Kisana NS, Nkongolo KK, Quick JS and Johnson DL (1993) Production of
doubled haploids by anther culture and wheat x maize method in a
wheat breeding programme. Plant Breed 110: 96-102.
Laurie DA and Bennett MD (1988) The
production of haploid wheat plants from wheat x maize crosses. Theor
Appl. Genet 76: 393-397.
Laurie DA and Reymondie S (1991) High
frequencies of fertilization and haploid seedling production in
crosses between commercial hexaploid wheat varieties and maize. Plant
Breed 106:
182-189.
Matzk F and Mahn A (1994) Improved
techniques for haploid production in wheat using chromosome
elimination. Plant Breed 113: 125-129.
Mujeeb-Kazi A, Riera-Lizarazu 0 and William MDHM (1995)
Production of polyhaploid wheat plants using maize and
Tripsacum. CIMMYT
Research Report 2:
47-65.
Olsen, FL (1987)
Induction of
microspore embryogenesis in cultured anthers of Hordeum vulgare.
The effects of ammonium nitrate, glutamine and asparagine as
nitrogen sources. Carlsberg Res Commun
53: 393-404.
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