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Pollen tube behaviour and effect of wheat genotypes on embryo induction in wheat x maize crosses

Mukta Sirohi, Navneeta Kaushik and V.K. Khanna

Department of Genetics and Plant Breeding,G.B. Pant University of Agriculture & Technology, Pantnagar, Uttarachal-263 145, India.


Six wheat genotypes were crossed with a composite maize variety “Kanchan” and frequencies of haploid embryo formation were compared. All the wheat genotypes produced seeds and embryos following post- pollination treatments with 2,4 –D and GA3. Seed set percentage among six wheat genotypes varied from 77.5 to 93.36 % while the frequency of embryo formation ranged from 2.2 to 9.53 % across all genotypes. Analysis of variance clearly revealed the effect of wheat genotypes on the percentage of embryo formation. Reasons for the genotypic effect of wheat need to be investigated to increase the efficiency of the wheat x maize haploid production system. Using normal microscopy and aniline blue as a dye for staining, pollen tube behaviour was studied. Correlation studies were done between various pre- fertilization and post- fertilization factors. Number of embryos obtained showed a highly significant positive correlation with average pollen tube length and a highly significant negative correlation with abnormal pollen tubes.

Media Summary

Responses of the wheat genotypes significantly differed for the embryo formation frequency and pollen rube behaviour in wide crosses between wheat and maize.

Key Words

Wheat x maize hybridization, wheat polyhaploids, embryo rescue, Pollen germination, widehybrids


Wide crosses followed by elimination of the genome of one parent have been an alternative method for the induction of haploid zygotic embryos and subsequent plants. Bulbosum technique, originally developed for barley haploid production was extended to wheat by Barclay (1975) who reported that high frequency of haploids could be recovered from wheat Hordeum bulbosum crosses after the elimination of H. bulbosum chromosomes early in the embryo development but the effect of crossability genes Kr1 and Kr2 in wheat has restricted the use of the bulbosum technique for the poly-haploid production in wheat (Snape et al., 1979). The production of haploid plants from inter-generic crosses between wheat x maize was reported by Laurie and Bennett (1988). Since then, it has emerged as the system of choice for the poly-haploid production in wheat. Fertilization by maize has been found to be relatively insensitive to the action of dominant alleles at Kr loci. Thus, haploids can be recovered across different genotypes. For the success of hybridization the interaction between pollen and pistil and various pre-fertilization factors affect crossability. Therefore, the aim of this work was to study the influence of wheat genotypes on the haploid embryo formation in wheat x maize crosses and to find out the correlation between various pre- fertilization factors with embryo formation frequency.

Materials and methods

Experimental Material:

The experimental material used in the present investigation included six wheat genotypes viz. UP 2003, PBW 373, PBW 343, PBW 175, PBW 65 and Synthetic 56, out of which first five are Triticum aestivum while the sixth one has Triticum turgidum and Triticum tauschii as it’s parents. A composite maize variety “Kanchan” was used as the pollen donor. Emasculation of wheat spikes was carried out using cut glume method. At the time of anthesis, when the stigmas had become feathery, they were pollinated with the freshly collected maize pollen. The pollinated spikes were sprayed with 75 ppm 2, 4–D one and two days after pollination and 300 ppm GA3 on the third day. The spikes were left on the plants in the field for sixteen days.

Embryo Rescue:

Sixteen days after pollination, seeds were collected and seed set was recorded for all the genotypes. The embryos were dissected out from the sterilized seeds and were cultured on the half strength MS media (Murashige and Skoog, 1962) and maintained at 4C in dark. After 5-6 days cultures were transferred to 25C and 16/8 hours dark/ light cycle. When the regenerated embryos grew to become plantlets 5- 10 cm in height, they were potted in the soil.

Statistical Analysis:

To test the influence of wheat genotypes on the embryo formation √X+ 0.5 transformation was used in completely randomized design with unequal number of replications.

Pre-fertilization studies:

Different pre-fertilization factors like pollen fall, pollen germination, pollen tube growth and abnormal pollen tubes were studied with nomal microscopy using 1 % aniline blue as a dye for staining.

Results and discussion

As a result of crossing wheat with maize pollen coupled with post- pollination treatments with 2,4-D and GA3, seed like structures were formed. These seeds generally lack endosperm and if it is present, it is abnormal. The haploid embryos obtained from such crossed seeds were invariably smaller than the selfed seeds of wheat. The embryos formed in wheat x maize crosses have poor viability, if left to develop on the plants, probably because of absence or early abortion of the endosperm (Zenkteler and Nitzsche, 1984; Laurie and Bennett, 1987 and Sun et al., 1992). This shows the necessity of embryo rescue for the production of haploid plants. All the wheat genotypes were crossable with maize. However, the frequency of seed set and haploid embryo formation varied considerably across different wheat genotypes. The wheat genotypes produced seeds with frequencies ranging from 77.50 to 93.36%.

According to the analysis of variance results, there were highly significant differences among the six investigated wheat genotypes for percentage of embryo formation (Table 1). In the present investigation the grand average of embryo formation over all six genotypes and replications was 6.53% (Table 2), which is lower than some of the other reports (Sun et al., 1992; Matzk and Mahn, 1994). The reason for this may be attributed to the fact that this study was conducted in open field conditions and late in the season unlike other wheat x maize studies which are generally conducted in green houses with controlled temperature, humidity and light requirements. Cambell et al., (1998) established the significant influence of temperature and light intensity on fertilization and embryo development in wheat x maize crosses.

These results clearly show that response of different wheat genotypes differed for haploid embryo formation in wheat x maize crosses. Several other workers have also reported the genotypic influence of wheat parents on the percentage of embryo formation. (Suenaga et al., 1991; Bitsch et al., 1998). Haploid nature of the plantlets obtained was determined by chromosome counting of root cell preparations. The superiority of wheat x maize system has already been established over bulbosum technique due to insensitivity of maize to Kr complement of wheat (Laurie and Bennett, 1987) and over anther culture because no recalcitrant genotypes have been reported in this system. (Islam and Shepherd, 1994). The wheat genotypes, which did not respond to anther culture, proved to be good parental material in wheat x maize system (Kisana et al., 1993). Though wheat x maize system is less genotype specific then anther culture, still there is some mechanism in this system too, which makes some wheat genotypes respond better than others in terms of embryo formation frequency. This mechanism needs to be investigated so that we may be able to select for more suitable wheat parents for efficient production of haploid wheat plants.

Pre-fertilization Studies:

Mean pollen germination showed no direct correlation with embryo formation frequency. The genotype with longest mean pollen tube length showed highest embryo formation frequency while the one with the least showed lowest embryo formation frequency. Abnormal behaviour of pollen tube was studied in terms of coiling, twisting of pollen tubes, swelling of pollen tube tips and bursting of pollen tube. A higher percentage of abnormalities were recorded in crosses as compared to selfings. Number of embryos obtained showed a direct negative correlation with the abnormal pollen tubes.

Table 1 Analysis of variance for percentage embryo formation

Sources of variation

Degrees of freedom

Sum of squares

Mean sum of squares













**Significant at 0.01 level of probability

Coefficient of variation: 8.14%

Table 2. Responses of six wheat genotypes for seed set and embryo formation


No. of florets pollinated

Seeds Obtained

Induced embryos

UP 2003


486 (87.25)

24 (4.93)

PBW 373


317 (77.50)

7 (2.20)

PBW 343


562 (90.06)

46 (8.18)

PBW 175


619 (93.36)

59 (9.53)

PBW 65


351 (88.63)

22 (6.26)

Synthetic 56


280 (84.33)

13 (4.64)









Values in parenthesis are expressed in percentage.


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