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Wheat Information
Service
Number 85: 49-51 (1997)
Research information
A
reliable protocol for doubled haploid accelerated wheat breeding
R de V.
Pienaar, M. Horn and A.J.G. Lesch
SENSAKO, Stellenbosch Branch, P.O. Box 3079, 7602 Matieland,
South Africa.
Doubling the chromosome number of haploids produces instant
homozygotes called doubled haploids (DHs). These give rise to pure
breeding lines that advance breeding programmes aimed at homozygosity
by four to six generations. Furthermore, the recovery of a specific
recombinant from x independently segregating loci is
2x times more efficient with the DH method than
with conventional genetic methods (Jensen 1977). The production of
DHs from heterozygous material therefore became the preferred method
for breeding autogamous crops (Bajaj 1990).
Wheat haploids can be produced by means of anther culture (Henry and
De Buyser 1990) or by chromosome elimination following wide crosses
(Barclay 1975, Laurie and Bennett 1988, Inagaki and Tahir 1990, Matzk
and Mahn 1994). The 'wheat x maize'method is usually more reliable
than anther culture (Kisana et al. 1993, MuJeeb-Kazi et al. 1995).
Unfortunately most South African wheats responded poorly to the
protocols generally employed.
The following protocol was developed over the past five years after
evaluating 250 growth regulator solutions as wheat haploidizers
(WHs), and 50 embryo rescue media (ERM) for their ability to
regenerate plants from the induced haploid embryos. The method of
Laurie and Reymondie (1991) served as control. This protocol resulted
in up to 25 haploid embryos and 15 haploid plants per spike.
Protocol for DH production by the wheat x maize method
1. Plant the heterozygous wheat kernels in moistened, sand-filled
2-liter pots (2 kernels/pot) in a greenhouse with a 12-14 hour
photoperiod on a 18C day/12C night cycle.
2. Plant the maize
parent likewise in a greenhouse with a 16 hour photoperiod on a 28C
day/18C night cycle. (We use a maize line developed from Seneca 60.
Two pots are planted twice weekly. Anthesis occurs six weeks later.
Four plants produce sufficient pollen per day for 20 wheat
spikes).
3. After germination
micro-irrigate (40 ml) the pots three to five times a day (depending
on plant size and greenhouse temperature) with the nutrient solution
of Maree (pers. commun.) to obtain luxuriant plants. This nutrient
solution is made up by stirring 13 liters (l) of each of two stock
solutions into a tank containing 1000 l water. Stock solution A
consists of 540 g KN03, 1746 g
KH2P04, 1331 g K2S04,
3375 g MgSO4,150 g Microplex (a commercial micro-element
complex), and 1.5 l liquid NH4NO3 (19%) in 100
l water, whereas stock solution B consists of 5775 g CaN03
in 100 l water). Aphids are controlled with 2g
l-1
Gaucho 70 WS (Bayer), thrips with 2g l-1
Mesurol (Bayer) plus
3 g sucrose, red spider with 0.35 ml l-1
Agrimec (Merck), and powdery mildew with 1g
l-1
BAS- 49015 (BASF) or 1 ml l-1
Afugan (AgrEvo) plus 1 ml l-1
Sporkill (Hygrotech) alternated with 2 g
l-1
Diathane M.45 (Rohm
& Haas). All sprays are deleterious to embryo development and
should be discontinued at least one week before pollination. Cover
the wheat plants with a 50% shade screen during summer.
4. As soon as the primary spikes of healthy, vigorous plants have
emerged from the boot, emasculate the two outer florets of their 15
best spikelets (enter from the side with the forceps). Remove the
upper and the two basal spikelets as well as all the other florets.
Cover the emasculated spikes with glassine or plastic bags and record
the date of emasculation on the bags.
5. Remove the upper halves of the florets at the stage anthesis would
normally have commenced, i.e. when the florets of the central
spikelets begin to open (usually 3-5 days after emasculation).
6. Collect fresh maize pollen by tapping the tassels over a smooth
sheet. Transfer the pollen and some dehisced anthers (to prevent
pollen clogging) immediately to a small receptacle with a lid.
Pollinate the wheat pistils liberally using a fine brush (0 Rolfes
279 Oron VC 1372, England, being the best). Replace the glassine or
plastic bags with brown paper bags and record the date of pollination
on the bags.
7. After 24-30 hours fill all the floral cups with the WH growth
regulator solution by means of an insulin syringe to induce embryo
formation (wear protective clothing). Cover the spikes again with the
brown paper bags and record the treatment date. (The WHs which induce
the most regenerable embryos are:
WH10 = 30 mg
l-1
picloram + 4 mg
l-1
BA
WH12 = 20 mg
l-1
picloram + 25 mg
l-1
2,4,5-T + 6 mg
l-1
BA
WH13 = 50 mg l-1
2,4-D + 100 mg
l-1
GA3.
The WHs containing
dicamba, e.g. WM6 (30 mg l-1
dicamba + 10 mg
l-1
BA), induce
high frequencies of embryos, but their regeneration is very
poor).
8. Harvest the treated spikes 16-20 days after pollination if the
greenhouse temperature is > 22C, but at 21-25 days if it is <
18C. (The spikes may be kept at 6C for 24 hours in a plastic
bag).
9. Remove the green parthenocarpic caryopses (GPCs) from the florets
by bending them backwards with a forceps. This prevents damage to the
attachment site; damage causes microbial contamination of the
embryos. Discard the white and necrotic caryopses as they contain no
embryos.
10. Surface sterilize
the GPCs in sterile McCartney bottles by rinsing in 70% ethanol for 1
minute and then in 1% sodium hypochlorite for 10 minutes. Rinse four
times in sterile distilled water, each lasting 3 minutes. (Changing
solutions is facilitated by placing sterilized gauze over the mouths
of the bottles).
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