(1) Soaked seeds were treated according to the method of Gayen et al (1994). The seeds were soaked in water 48 hours at 18 C. A small portion of the plumule tip was cut off before treatment. Treatment with 0.06% colchicine concentration was carried out at 18 C. After treatment the seeds were washed in running water, and then kept in the dark at 18 C for two days. Then they were planted in the field nursery.
(2) 2-3 day seedlings were treated with colchicine according to the method proposed by Deimling et al. (1997). The seeds were germinated in a thermostat at 26 C up to the stage when their coleoptile had grown to the length of about 1 cm. The top of the coleoptile was cut, and long roots were shortened to 2-3 cm. The seedlings were placed in a vessel with colchicine. The treatment was carried out in the dark with 0.06% colchicine at 18 C during 12 hours. After treatment the seedlings were washed with running water and kept in the dark for two days. Several modifications of this method were tried. First, the effect of 0.02% colchicine concentration was examined. Second, the two temperatures of post treatment, 18 C and 26 C were tried. The seedlings were kept in the dark for two days at these two temperatures after treatment. Then the seedlings were planted in the field.
(3) Seedlings were treated according to the method of Zabirova et al. (1996) at the stage of 3-4 leaves. The 0.125% colchicine solution was injected approximately 3-5 mm above the apex. After the injection the seedlings were kept in the dark for two days at 18 C and then planted in the field.
During the experiment the effect of colchicine was considered to be successful in each case if several anthers with fertile pollen appeared on a tassel of a haploid plant. Fertility of pollen was evaluated visually. The Table presents the results obtained. The methods developed by Deimling et al. (1997) and Zabirova et al. (1996) were rather efficient under our conditions. Using the method of Deimling et al. (1997) allowed us to obtain 28.8% and 31.8% of haploids with fertile pollen. Our attempt to modernize the method gave no positive results. Reducing the colchicine concentration from 0.06% to 0.02% resulted in the fact that not a single haploid treated had anthers.
It was difficult to judge on degree of importance of temperature on treatment of the seedlings with colchicine. Unfortunately after planting the seedlings in the field they were attacked by rooks and the number of haploid plants under study was decreased significantly. The small number of plants in the variant with the temperature of the post-treatment of 26 C did not allow determining its difference from the treatment at the temperature of 18 C. Haploids with fertile pollen were obtained with approximately similar frequency in both cases.
Use of injection of colchicine according to the method of Zabirova et al. (1996) resulted in 27.5% haploid plants with fertile pollen. This frequency was quite comparable with the frequency resulting from using the method of Deimling et al. (1997).
In the control, 211 haploid plants were used. Not a single one of these plants had anthers with fertile pollen. We assume that it was the result of rather unfavorable environmental conditions which prevailed in summer 1999 in our region. Strong drought was combined with prolonged high air temperature in the period before tasseling, during tasseling and during flowering. Usually, when environmental conditions are optimal, we obtain, without using any doubling factors, 2-5% haploid plants which have a certain quantity of anthers with fertile pollen. Strong drought and hot conditions led to the fact that in the control all the haploid plants were sterile.
Thus it can be noted that the methods developed by Deimling et al. (1997) and Zabirova et al. (1996) allowed us to obtain fertile pollen in haploid plants even in unfavorable drought and hot conditions. These methods can be successfully used for obtaining doubled haploid lines in a mass quantity.
CLARIFYING NOTE. In the previous article, Chalyk S.T. 1999. Creating new haploid-inducing lines of maize, MNL 73, p. 53-54, it was shown that in our experiments the highest proportion of haploids occurred at the top of an ear. The lowest frequency of haploids was obtained at the bottom of an ear. During the analysis of these data the author referred to the fact that in the article of S. Chase (1969) a similar conclusion was made. However this was a mistake. In his article S. Chase made a directly contrary conclusion. In this connection the author apologizes to Dr. S. Chase and readers for the mistake made.
Table. Frequency of haploid plants obtained
with fertile pollen.
|Material||Conc. of colchicine, %||Temperature, C||Total haploids||Haploids with fertile pollen|
|Before treatment||After treatment||Number||%|
|Seeds soaked for 48 hours||0.06||18||18||21||0||0|
|2-3 day seedlings||0.02||26||18||27||0||0|
|2-3 day seedlings||0.02||26||26||19||0||0|
|2-3 day seedlings||0.06||26||18||52||15||28.8|
|2-3 day seedlings||0.06||26||26||22||7||31.8|
|Seedlings with 3-4 leaves||0.125||26||18||40||11||27.5|
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