Materials and methods
Plant materials: Five F1 hybrids of hexaploid wheat were used as female parents in crosses with maize. These F1 wheats referred to as W1-W5 were produced from crossing of different parents (Table 1). Maize genotype Kanchan, which is a composite cultivar, was used as a pollen parent in the wheat x maize crosses. Planting conditions, wheat x maize crossing, embryo rescue and regeneration of plantlets: Wheat genotypes (W1-W5) were planted under field conditions of Pantnagar University at two planting dates in November. Maize plants were planted in pots at two planting dates in glasshouse under natural temperature and light conditions. Eight more plantings of maize were done under outdoor conditions at weekly intervals during October to December to ensure availability of pollen. Wheat spikes of W1-W5 were emasculated manually using cut glume method two days before anthesis and pollinated with fresh pollen of maize cultivar two days after emasculation. Viability of collected pollen was checked by KI treatment, which led to black appearance in viable pollen grains and light color for inviable pollen grains. The pollinated spikes were treated with 75 ppm solution of 2,4-D for two days and 300 ppm gibberellic acid solution on the 3rd day after pollination, each day with one spraying on each side of spike. Embryos were excised 14-16 days after pollination and cultured on half strength basal MS medium (Murashige and Skoog 1962) supplemented with 0.5 mg/l nicotinic acid, 0.1 mg/l thiamine HCl, 0.5 mg/l pyridoxine HCl, 2 mg/l glycine and 30 g/l sucrose in aseptic conditions. The cultured embryos were kept at 4C for 5-6 days in darkness and then transferred to incubation room with approximately 25C with 16/ 8 hours light/darkness. When the plantlets of 5-15 cm were obtained they were potted in an off-season nursery in glasshouse to obtain haploid plants.
Verification of haploidy: The squashed root tips of the regenerated plantlets were prepared according to Love and Love (1975) by staining with 2% carmine and mitotic metaphase chromosome complement was counted and photographed.
Pollen tube growth study: Pollen tube growth was studied using spikes of W1 and W5 crosses which were fixed 30 minutes after pollination according to D'Souza (1972). For this, wheat pistils which were fixed in 1:2 lactic acid: ethyl alcohol solution, were first treated with 1N HCl for 10 minutes and then stained for 1- 2.5 minutes with a solution of 1% cotton blue containing lactic acid, phenol, glycerol and distilled water with 1:1:1:1 ratio. The samples were destained with a solution of 40% acetic acid, orthophosphoric acid and distilled water in 1:1:1 ratio for 15-20 minutes. Stigma of these pistils was cut and one drop of lactic acid was added on slide for microscopy. The length of pollen tube was measured in W1 and W5 crosses with an ocular micrometer.
Statistical methods : Since five wheat parents (W1-W5) were crossed by maize in different number of replications the completely randomized design with unequal number of replications was used to analyze variance for difference among genotypes according to Gomez and Gomez (1984). The analyzed traits included seed set (percentage of seed containing florets) and percentage of embryo formation (number of embryo containing seeds/total number of obtained seeds). In case of two other characteristics, ie, percentage of embryo germination (number of germinated embryos/number of cultured embryos) and plantlet recovery rate (number of pInatlets obtained /number of cultured embryos), three wheat parents, W1, W3and W5, were included in the analysis. Data were transformed to are sin (x)l/2 and (x+0.5)1/2 before analysis of variance accordingly for each character. To compare means of pollen tube length between W1 and W5, Student's-t distribution was used according to Steel and Torie (1960).