Doubled Haploid in Maize Breeding: Analysis of Prospects and Constraints in Indian Context

In its simplest terms doubled haploid (DH) is that individual which is having two exactly identical sets of chromosomes with each and every gene locus identical to each other. Doubled haploid should not be confused with diploid in which two sets of chromosomes are also there but it is not necessary that those two sets are exactly identical at every gene locus. DH have been produced in more than 300 species of plants, with remarkable to mention in some vegetalble crops like Brassica spp., cereal crops like rice, maize, wheat, barley etc. Generally DH is produced by two methods - (i) in vivo method and (ii) in vitro method.

In in vivo method DH plants are produced by haploid plant by the method of CHROMOSOME ELIMINATION. e.g. In case of barley haploid plants are obtained by crosses of Hordeum vulgare x Hordeum bulbosum (The Bulbosum Technique). In this cross the H. vulgare which is the cultivated species of barley, is crossed as female with H. bulbosum, which is a wild species of barley. After the successfull crossing the chromosomes of H. bulbosum got eliminated selectively during the embryo development and the haploid embryo will be obtained with only chromosomes of cultivated species which can be cultured on appropriated media and later treated with colchicine to get DH barley plants.

DH can also be obtained from the in vitro method. In this method there are two ways through which DH could be recovered. To recover DH first a haploid tissue has to be generated on appropriate media. Based on the selection of culture explant, two methods under  in vitro technique are: (a) Androgenesis and (b) Gynogenesis. In case of androgenesis explant used for culture is either anthers or microspores whereas in case of gynogenesis the explant will be ovary or ovule. The method of androgenesis is more preferable as evidenced by its success frequency as compared to gynogenesis, in several crops. The haploid explant (anthers/ovary) will be grown on a suitable media and a haploid callus or embryoid will be generated on the culture media. These hapoloid tissues are treated with colchicine and regenerated into a plant with doubled chromosome number. Sometimes there will be no need to treat the tissue with colchicine as there are several species in which the tendency of spontaneous doubling of chromosomes is very high, even upto 70%.

In case of maize both in vitro and in vivo  technique is used for DH production. DH production in maize is going to prove a very relevant area of research in near future, especially in South East Asia and Africa where maize is one of the most important crop. DH research in maize could be justified by the fact that maize is a cross pollinated crop and development of inbred line in maize and their maintenance required a lot of attention, labour, resources, time etc. If a suitable and resource efficient technology could be developed for DH production in maize, it will bring a boom in single cross hybrid (SCH) breeding of maize, which require development and testing of lots of inbred lines in maize every year. With the production of lots of inbreds throuh DH technolgy in a relatively less time will provide new combination of genes with in a very short duration to the breeders to test in SCHs.

The constraint is that the DH research in maize has not been taken seriously by Govt. agencies and research centres as evidenced by the absence of any publication on DH production in maize in India. As discussed earlier that there are two tecniques which could be employed for the production of DH in maize, but the major constraints are there associated with both the techniques. In case of in vitro techniques there are protocols availabel for anather cultue as well as ovary culture but no remarkable success has been achieved except a few small success. Especially in India there are no publication regarding this technique. The most important contraint in this technique is the availability of responsive genotypes. As reported by the several authors the anther culture or microspore culture response in maize is highly genotype dependent, but unfortunately there are no sure shot markers available for the selection of this trait and even no studies have been taken to sort out some responsive genotypes except a few unpublished work done at some centres in India. In this case the only reliable system for the screening of the responsive genotypes is the tissue culture media itself. Thus one has to screen a responsive genotype as well as a suitable media composition for the selection of genotypes. This will make the work so tedious that no one want to take this system for the production of DH. Besides all the above constraints one of the major constrraint is the unpredictably low (as low as 1%) response frequency. All these constraints make this system less favourable among the maize researchers as the system of choice for maize breeders.

The another method, which is gaining impetus these days, is the in vivo  method. In this method the normal maize lines were crossed with some specific haploid inducer stocks to get the haploid seeds. These stocks were developed by the pioneer work of Chase in late 1940s, Coe in late 1950s and Kermicle in late 1960s. In this method the haploid seeds were set on the maternal plant which is pollinated with these inducer lines. The seeds could be easily identified with the help of phenotypic marker associated with the haploid seed which is the appearance of purple colour on the crown region of the maize kernel. This is the most easy method of DH development in maize as it does not require a very sophesticated tissue culture laboratory or lots of resourses which is required in in vitro method. In this method a haploid seed could be obtained at field just by simple crossing with inducer stock. The haploid seeds will be sown in next season and the seedlings will be treated with colchicine to get DH. The frequency of DH obtained from this method is quite encouraging which is as high as about 15% .  Being an easy and resource efficient technique it could be easily accepted by traditional breeders also who can't go for a anther culture method. But there are some constraints also associated with this method. The most important is the availability of the inducer lines. Unfortunately there are no inducer lines available in the public sector maize breeding institutes or research centres. Thus the story ends prior to its begining.

But, no, its not the end of the story. The good news is that the International Maize and Wheat Improvement Centre, Mexico(CIMMYT) will shortly going to introduce this technology in developing countries. They are working for the improvement of these inducer lines so that they can't loose their genetic integrity in tropical or sub-tropical conditions as the origin of these lines are of temperate and reportedly they are not doing well in hot humid climate. Till now these lines are used by some of the private sector seed companies like Syngenta, Monsanto etc. for the production of DH with the only aim to earn the monitory profits. With the introduction of these inducers in public sector, it will revolutionize the maize breeding research in India and other developing counties of South East Asia and Africa.

It can be realized with the above discussion that how much this technology could add in our future breeding programmes especially in maize. In a country like India where maize is the third  most important crop the DH technology could bring the revolution in the production of this crop by providing good inbred in a very short time and hence add to food security of this nation also.