The significance of haploids ~ in genetics and plant breeding has been realized for a long time. However, their exploitation remained restricted because of the extremely low frequency with which they occur in nature (usually 0.001-0.01%).

Spontaneous production of haploids usually occurs through the process of parthenogenesis (embryo development from an unfertilized egg). Rarely, however, they reproduce the characters of the male parent alone, suggesting their origin through 'ovule androgenesis '2 (embryo development inside the ovule by the activity of the male nucleus alone).

Until 1964 the artificial production of haploids was attempted through: (a) distant hybridization, (b) delayed pollination, (c) application of irradiated pollen, (d) hormone treatments, and (e) temperature shocks. None of these methods, however, proved dependable. Therefore, the development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.

The technique of haploid production through anther culture ('anther androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.

During the last decade considerable success has been achieved with the induction of androgenesis in isolated pollen culture and androgenic haploids have been used to breed new cultivars of crop plants

Anther cultureThe experimental plants for anther and pollen culture should ideally be grown under controlled conditions of temperature, light and humidity and anthers should be taken from young plants.

Generally, with increasing age of the donor plants the androgenic response declines and abnormalities appear.

In plants grown under controlled environmental conditions it may be possible to draw a rough correlation between the stage of pollen development and certain visible morphological features of the bud, such as the length of the corolla tube, emergence of corolla from the calyx, and the like. These external markers can be used for selecting buds of approximately the required stage.

However, the correlation is never absolute and, therefore, it is always necessary to crush one of the anthersfrom each bud to assess the exact stage of pollen development. The selected buds are surface sterilized with a suitable disinfectant

Anthers along with their filaments are excised under aseptic conditions and placed on a sterilized petriplate. One of the anthers is crushed in acetocarmine to test the stage of pollen development and if it is found to be of the correct stage the anthers of the remaining stamens are gently detached from their filaments, without injuring the anthers, and placed horizontally on the medium (anther culture).

When dealing with plants having minute flowers, such as Asparagus, Brassica and Trifolium, it may be necessary to use a stereoscopic microscope for dissecting the anthers. Alternatively, only the perianth may be removed and the rest of the bud, with the stamens intact, inoculated. In some cases complete inflorescences have been cultured to obtain androgenic haploids. However, this simplified approach can be tried with only such genotypes where androgenesis occurs on simple nutrient media containing mineral salts, vitamins and sugars, because on such a medium the chances of the sporophytic tissue proliferating are very remote.

However, where growth hormones are essential to induce androgenic development of pollen grains the sporophytic tissues should be removed as far as possible. The gap between bud collection and anther/pollen culture should not exceed 2h. The anther cultures are generally maintained in alternating periods of light (12-18 h; 5000-10 000 lx m 2) at 28~ and darkness (12-6 h) at 22~ .

However, optimal storage conditions need to be determined for individual systems. For example, the anther cultures of Brassica species are very sensitive to light and, therefore, should be maintained in the dark throughout.

In responsive anthers, the wall tissues gradually turn brown and, depending on the species, after 3-8 weeks they burst open due to the pressure exerted by the growing pollen callus or pollen embryos .

The embryos may germinate on the original medium or require transfer to another medium to form plantlets. After they have attained a height of about 3-5 cm, the individual plantlets or shoots are excised and transferredto a medium which would support good development of the root system. The rooted plants are transferred to sterilized potting-mix in small pots or seed trays.

The plants arising from different pollen grains in an anther would be genetically different. To achieve rapid clonal multiplication of the desired genotypes, for breeding or other experimental purposes, micropropagationthrough shoot multiplication may be tried

Isolated pollen cultureThere are many problems associated with raising haploids through anther culture. The pollen grains within an anther lobe being genetically heterogeneous, the plants arising from an anther would constitute a heterogeneous population. Mixing of calli of different pollen origin within an anther lobe enhances the chances of regenerating chimeric plants. Furthermore, if the proliferation of anther wall cells occurs concomitant with the callusing of pollen the tissue finally derived would not be purely ofgametophytic origin.

In the cultures of anthers showing asynchronous pollen development the older grains may suppress the androgenic response of younger grains by releasing toxic substances as observed in Brassica napus (Kott et al., 1988). Isolated pollen culture cannot only circumvent these problems but also offers many other advantages:

(a) it is a haploid, single cell system, (b) a homogeneous population of pollen grains at the developmental stage most suitable for androgenesis can be obtained by gradient centrifugation (c) isolated microspores,can be genetically modified by exposing them to mutagenic treatments or insertion of foreign genes before culture and the new genotypes selected at an early stage, and (d) according to Aslam et al. (1990) and

Siebel and Pauls (1989), in rapid cycling B.napus, pollen culture is 60 times more efficient than anther culture in terms of embryo production.

The technique of pollen culture has been considerably improved and androgenic plants through isolated pollen culture have been raised for many crop plants, The initial success with isolated pollen culture was based on the use of some kind of a nurse tissue or its extract. Sharp et al. (1972) raised haploid tissue clones from isolated pollen grains of tomato by plating them on small filter paper pieces placed over cultured anthers of the same species.

The observations suggest that anther wall provides a suitable environment necessary to trigger androgenesis. Based partly on the analysis of non-embryogenic anthers (taken directly from flower buds) and embryogenic anthers (cultured for about 7 days), Nitsch (1974) highlighted the importance of glutamine, /-serine and myo-inositol as constituents of media for ab initio pollen culture of Datura and tobacco.

To improve the efficiency of isolated pollen culture for the production of haploids, Wenzel et al. (1975) introduced the technique of density gradient centrifugation which allows the separation of embryogenic grainsfrom a mixture of embryogenic and non-embryogenic grains obtained after crushing the anthers. The anthers of barley were collected at the proper stage of development and gently macerated to obtain a suspension of pollen grains. After removing the debris by repeated filtration and centrifugation the suspension was layered on 30% sucrose solution and centrifuged at 1200 g for 5 min. The androgenic, vacuolated pollen grains formed a band at the top of the sucrose solution. Rashid and Reinert (1980) slightly modified the technique and used 55% Percoll and 4% sucrose solution, instead of 30% sucrose, for the separation of starch-free, embryogenic grains of tobacco. Percoll gradient centrifugation was found very useful to collect highly embryogenic grains of maize (Gaillard et al., 1991). The grains collected at the interface of 40/50% Percoll showed maximum androgenic response.

Isolated pollen culture is not only more efficient but also more convenient than anther culture. The tedious process of dissection of individual anthers is avoided. Instead, the entire buds within a suitable size range are crushed and the embryogenic grains are then separated by gradient centrifugation

GYNOGENESISDevelopment of plants from unfertilized cells of the female gametophyte (embryo sac), in floret, ovary or ovule culture is becoming an attractive alternative to anther culture for haploid production.

The gynogenic plants may arise through direct embryogenesis, or the gametic cells may form a callus followed by plant regeneration on another medium. Although the available data on in vitro gynogenesis is not enough to make generalisations the key factors affecting it areExplantYoung flowers, ovaries or ovules have been used as the explant to produce gynogenic haploids. Generally, ovules attached to the placenta respond better than isolated ovules. The stage of the female gametophyte at culture is crucial. Mostly, the explants cultured at nearly mature embryo sac stage gave best resultsPre-treatmentA beneficial role of cold treatment on gynogenesis has been reported. Pretreating the capitula of sunflower at 4~ for 24-48 h before culture significantly increased the induction frequency, cold treatment of panicles of rice 7~ for I day before ovary culture. In Cucumis melo pollination of pistils with irradiated pollen was essentialto obtain ovules capable of forming gynogenetic haploids.

Culture mediumThe culture media used for the production of gynogenetic haploids vary considerably. The most widely used basal medium in these studies happens to be N6. However, N6 and MS were equally good for the production of maternal haploids of maize

Generally, sucrose is used at higher levels: 3-10% for barley, 8-14% for wheat, 5-12% for maize and 3-6% for rice and gerbera. For the graminaceous species the induction medium is generally supplemented with 2,4-D (2 mg 1-1) or MCPA (0.125-0.5 mg 1-1).

The best combination of growth regulators for parthenogenetic haploid production in ovary/ovule culture of Beta vulgaris was found to be 0.3 mg 1-1 BAP, 0.1 mg 1-1 NAA and 0.05 mg 1-1 2,4-D.

In sunflower, on the other hand, a hormone-free medium gave higher induction frequency than with MCPA. Mostly solid media have been used to induce gynogenesis. However, in rice at the induction stage liquid medium proved superior to solid medium

On the other hand, for maize solid medium was more productive than liquid medium. The cultures are initially stored in the dark.

In vitro gynogenesis is a multistage process. The special requirements of each stage ought to be studied carefully. For example, rice ovary culture comprises at least two stages. The first stage of induction is characterized by float culture on liquid medium supplemented with exogenous auxin and cultured in dark while the second stage of regeneration requires transfer of the callus to solid medium, reduction in auxin concentration and keeping the cultures in light . Rarely, a gametophytic cell may directly form an embryo capable of germination on the original medium.

Practical importanceThe production of haploids via gynogenesis is more tedious, less efficient and so far restricted to a very few species compared to androgenesis. However, it may prove

valuable as an alternative to anther culture under special situations: (1) In plants, such as sugar beet, onion, and melon, where anther culture has not been successful, gynogenesis has yielded haploids. (2) In male sterile plants the value of haploid production via gynogenesis is obvious. (3) Albinism is a limiting factor in anther culture of some cereals. In these crops ovary/ovule culture may provide relatively higher proportion of green haploids.

DIPLOIDIZATION TO RAISE HOMOZYGOUS DIPLOIDSHaploids may grow normally up to the flowering stage but in the absence of homologous chromosomes meiosis is abnormal and, consequently, viable gametes are not formed. To obtain fertile, homozygous diploids for analysing the progenies and the breeding behaviour of the pollen plants the chromosome complement of the haploids must be duplicated.

Spontaneous duplication of chromosomes in pollen-derived plants has been observed but its frequency is fairly low. In wheat and barley 20- 50% and 25-70% of the pollen plants respectively, exhibited spontaneous diploidization.

However, generally, the rate of spontaneous duplication of chromosomes may be very low. This can be significantly enhanced by using artificial means. For Nicotiana tabacum a 0.4% solution of colchicine is recommended to diploidize the pollen plants. In practice, the young pollen-derived plants are immersed in a filter-sterilized solution of colchicine for about 96 h and then transferred to a culture medium to allow their further growth.

Alternatively, the treatment is given in the form of a lanolin paste. It is applied to the axils of the upper leaves and the main axis is decapitated to stimulate the axillary buds to grow into diploid and fertile branches.

chromosome doubling in B. napus is induced by injecting 2% solution of colchicine into the secondary buds or by immersing the roots into 0.1-0.2% solution of colchicines..Besides bringing about chromosome duplication, colchicine treatment may also result in chromosome and gene instabilities. Therefore, the frequent occurrence of spontaneous duplication of chromosomes in differentiated plant cells (cortex, pith) and callus cells in long term cultures has also been exploited to raise homozygous, fertile diploids from haploid plants. In this method pieces of vegetative parts such as stem, root or petiole segments are cultured in a suitable medium to induce callusing. The initial callus may have some diploid cells but their frequency would increase in repeated subcultures. Such calli are transferred to the plant regeneration medium.

APPLICATIONSHomozygous lines of the cross pollinating species and hybrids are highly desirable to increase the efficiency of selection and production of homozygous plants.

The conventional method to produce homozygous plants is lengthy and laborious, requiring 7-8 recurrent cycles of inbreeding. Moreover, this approach is impractical for self-incompatible and male sterile plants and tree species. On the other hand, homozygous plants can be obtained in a single generation by diploidization of the haploids.

Haploids are also extremely useful for detecting recessive mutants which may not express themselves in the heterozygous diploid background and, therefore, can be easily lost.

Of the three in vitro methods of haploid production described in this chapter, anther/pollen culture is the simplest and hitherto most efficient.

Shortening of breeding cycleThe most important application of androgenic haploids is in the production of stable, homozygous dihaploids (DH) in a single generation equivalent to the F~ generation of pedigree breeding and, thus, considerably shortening the breeding cycle.

Normally, in a hybridization programme evaluation of lines is possible only after 4-5 years of backcrossing (Fs-F6 generations) and it takes another 4-5 years to release a new variety. By anther culture of F1 hybrids the various genotypes of gametes can be fixed and evaluated in the first generation. Anther culture can itself generate new recombinations and fix them simultaneously.

Nakamura et al. (1974) raised three promising doubled haploid lines through anther culture of the hybrids between MC-1610 and Coker-139. The new lines exhibited higher resistance to bacterial wilt, black shank and black root rot without losing the agronomic and chemical traits of the MC-1610.

Haploid breeding, involving anther culture, has been most successfully used in China to produce several agronomically superior varieties of wheat, rice, maize and pepper, many of which are under large scale cultivation.

Gametoclonal variationsBesides yielding haploids, in vitro androgenesis provides a unique opportunity to screen the gametophytic variation, caused by recombination and segregation during meiosis.MutagenesisDetection and isolation of recessive mutants in the haploid state and rapid obtainment of the mutated gene in a homozygous diploid state is a special merit of haploidy in higher plants. Application of mutagenic treatment at the microspore stage, which is a single celled structure, has the added advantage of obtaining solid mutants.

Genetic transformationAgrobacterium is a superior vehicle for transformation of dicots but its use with monocots is very limited. Therefore, alternative methods, such as microinjection, electroporation etc., are being tried. Another problem with somatic protoplasts/cell transformation in most monocots and some dicots is the poor regenerability of plants after DNA insertion. In such cases immature pollen embryos, which exhibit high regenerative capacity may offer excellent recipient cells. The pollen embryos being haploid the proof of a successful transformation is facilitated because it is expressed homozygously.

LIMITATIONSThe practical applications of androgenic haploids are still limited as the desired success with anther and pollen culture has been largely restricted to only a few families, such as Brassicaceae, Poaceae and Solanaceae.

Low y i e l d sGenerally only 5-8% of the total pollen grains in a responding anther undergo androgenic development and a very small proportion of these androgenic grains develop into full sporophytes. This turns out to be a serious limitation in obtaining full range of gametic segregants of interest to plant breeders.

Conversion of pollen embryos into plantsOne of the major problems in anther/pollen culture of Brassica species is the structural, physiological and biochemical abnormalities of pollen embryos so that 70-80% of the embryos are incapable of normal germination.

Albinism in cerealsA serious problem associated with the application of anther/pollen culture to cereal improvement is the occurrence of albinos with high frequencies.

Genetic changesInstability of genetic material during androgenesis could be an hindrance in the use of haploids. In tobacco the androgenic plants exhibited growth depression, reduced agronomic performance and abnormalities such as reduced leaf and flower size and tumerization of leaf cells, which could be correlated to amplification of DNA