Haploids are of great value as genetic and breeding tools in crop improvement. The major benefit of haploids is the compression of time required to generate homozygous genotypes. So, this technique provides breeders a very fast way of producing pure breeding lines. The production of wheat haploids from the intergeneric hybridization of wheat (Triticum aestivum L.) with maize (Zea mays L.) has been exploited to rapidly achieve homozygosit in wheat breeding programs.
To be efficient as a breeding tool, doubled haploids need to be produced in high frequency across all genotypes. In addition, production needs to be cost efficient, and the products need to be competitive with those produced by conventional breeding methods. In order to increase the efficiency of haploid production using the wheat x maize system, present studies were conducted in the Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan during 1999-2000 to 2002-2003 to increase the output with respect to seed set and embryo formation.
Hand emasculation of wheat spikes is a highly labour intensive and time consuming process. Studies of different emasculation methods (Chapter 4), indicated that the laborious hand emasculation method can be replaced by a non-emasculation or hot water emasculation method (43 °C for 4 minutes).
Tiller culture was also evaluated as an alternative technology aimed at reducing the space requirements for the pollinated wheat heads, thereby enabling increased turnover (a reduced cycle time) for greenhouse materials. It was found that wheat spikes can be successfully cultured in a liquid medium just prior to anthesis, and up to embryo rescue. The grain growth ad embryo development in the detached wheat spikes in liquid culture closely resembled that observed in intact plants (Chapter 4). The technique of detached tiller culture was proved successful in wheat x maize crossing. This technique has many advantages over intact plant method like, time and space saving, delaying of pollination by storing tillers under cooler conditions, easy hot water emasculation of detached tillers and easy application of growth hormones. A liquid medium containing 40 g L-1 sucrose, 100 mg L-1,2,4,-D and 8 mlL-1 sulphurous acid was identified as the best medium for culture of wheat x maize crossed tillers.
To improve seed set, several hormones were used to treat pollinated florets. Treatment with a high concentration of 2,4-D (100 mg L-1) was effective; however, when combinations of 2,4-D + GA3 were applied, the seed set approached 100%. Similarly, an addition of 50 mg L-1 ABA to standard doze of 2,4-D (100 mg L-1) almost doubled the rate of haploid embryo formation.
Experiment conducted in the field to study the effect of pollen parents (Chapter 5) indicated that pollen sources significantly affected embryo formation. Crosses among selected pollen sources, ‘FSH-399’,’Composite-20 ‘Akbar’ and ‘73Q3’, identified pollen parents which gave levels of embryo formation as high as 53.0% when crossed on to wheat. The results of crosses between the best pollen sources and various female wheat parents of different ploidy levels (hexaploid and tetraploid) (Chapter ) indicated that hexaploid wheat perform better than tetraploid in crosses with maize. However, successful production of embryos at a relatively high frequency could be achieved in tetraploid wheats with ‘FSH-399’. The highest frequency of embryo formation (59.0%) was produced from a cross between the hexaploid wheat F1 ‘Cross-5 and ‘FSH-399’.
In Chapter 5 (Experiment-3), different pollen sources were examined to evaluate the efficiency of embryo formation. The results showed that crosses between the wheat F1 ‘Cross-3’ and the maize cultivar ‘Sadaf’ yielded on average 59.6% embryos/pollinated florets, and that ‘Sadaf as superior to other pollen sources. There were marked differences in embryo formation (p<0.01) among the synthetic maize cultivars (Chapter 4). The synthetic maize cultivar ‘Neelum produced the highest embryo formation (52.0%).The frequency of regenerated plants per pollinated floret was higher in the wheat x synthetic maize cross than for the wheat x hybrid or inbred line maize crosses. Hence, it was recommended that synthetic (open pollinated) maize cultivars should be preferred for use in crosses with maize
A series of hormones and hormone combinations were evaluated for their impact on embryo formation (Chapter 7. It was found that plant hormones affected embryo development in vivo following crosses between wheat and maize. The combination of GA3 (75 ppm) + 2,4-D (100 ppm) considerably improved embryo foration. ABA treatment likewise had a highly significant positive effect on embryo formation with 50 ppm of ABA being superior among all plant hormones and hormone combinations. Fifty ppm of ABA increased embryo formation to nearly double that produced in the control.
The development of improved culture media for the small for the small undeveloped embryos was also considered an important factor for effective plant regeneration (Chapter 8). The gelling agent Gel-gro, as well as the presence of copper and KNO3 were found to have a marked influence on embryo regeneration. All genotypes which performed poorly on basal media (MS, LS, B5 and half strength of MS) performed better on modified media. Eighty-five haploid plants from 100 cultured embryos were recovered using modified B5 medium, whereas the control (half strength MS) only produced 58.4% embryo regeneration.
There was also a significant effect of the plant development stage on fertility at the time of colchicines treatment. Plants at the 5-7 tillers stage showed improved plant fertility and reduced plant mortality than those treated at the 2-3 tillers stage.
One of the most important factors in chromosome doubling is the seeds obtained per treated plant, since this in turn determines how quickly these lines can be used in breeding programs. The addition of colchicines to media resulted not only in higher plant fertility than did conventional methods, but furthermore led to a huge increase in the number of doubled haploid seeds.
Cryogenic storage of maize pollen was considered as an alternative to growing maize plants year round in the green house (Cahpter 5). The viability of stored maize pollen indicated that, with refinements, pollen stored in liquid nitrogen has excellent potential for use in this system as compared to other storage methods (e.g. freeze or refrigerator). The results indicated that the fertilizing ability of maize pollen could be maintained for the duration of the experiment (180 days), provided that the pollen was dried to a 11-13% moisture content prior to storage.