Haploidy: Classification and Origin

In this article we will discuss about:- 1. Classification of Haploids 2. Origin of Haploids 3. Meiosis.

Classification of Haploids:

According to their cytological features, haploids have been classified into different types (Fig. 17.1), a brief description of which is given below.

I. Euhaploid:

The chromosome number of such a haploid is an exact multiple of one of the basic numbers of the group. Euhaploids are of two types:

(i) Mono-haploid or monoploid:

It arises from a diploid species (2x) and possesses the basic chromosome number of the species; it is designated as “x”.

(ii) Polyhaploid:

Haploid individuals arising from a polyploid species are called polyhaploids; they may be di-haploids, tri-haploids and so on. Based on their auto- or allo-ploid condition, polyhaploids have been divided into two groups:

(a) Auto-polyhaploid : Autopolyploid species give rise to auto-polyhaploids. For example,

Autotetraploid (Ax = AAAA)———-> Auto-di-haploid (2n = AA)

Auto-hexaploid (6x = AAAAAA)———–> Auto-ri-haploid (3x = AAA)

(b) Allopolyhaploid : Such individuals arise from allopolyploid species. For example,

Allotetraploid {Ax = AABB)———-> Allodihaploid (2x = AB)

Allohexaploid (6x = AABBDD)———–> Allotrihaploid (3x = ABD)

II. Aneuhaploid:

The chromosome number of such a haploid is not an exact multiple of the basic number of the group.

Aneuhaploids have been classified into five types:

(i) Disomic haploids (n + 1):

Such haploids have one chromosome in disomic condition, i.e., there is one extra chromosome belonging to the genome involved.

(ii) Addition haploids (n + few alien):

The extra chromosome(s) in the haploid is an alien chromosome.

(iii) Nullisomic haploids (n-1):

In such haploids, one chromosomes is missing from the haploid complement.

(iv) Substitution haploids (n-1 + 1 alien):

One or more chromosomes of the haploid complement are substituted by alien chromosomes.

(v) Misdivision haploids:

Such haploids contain telocentric or iso-chromosomes resulting from misdivision of centromere(s).

Origin of Haploids:

Haploids arise spontaneously, and can be experimentally induced. In nature, they arise through the development of egg cells without fertilization (parthenogenesis) or through pseudogamy. There are many animals where haploidy is common and is involved in sex determination.

For example, in insects of the order Hymenoptera (honey bees, wasps etc.), unfertilized eggs (haploid) develop into males, while fertilized eggs (diploids) develop into females. Spontaneous and induced haploidy has been reported in several animals such as Drosophila frog, mouse, chicken and newt.

Several plant species, such as, flax, cotton, rape, coconut, tomato, pearl millet, wheat, barley, are known to produce haploids spontaneously. In einkorn wheat (Triticum monococcum) Smith reported the occurrence of haploid plants at a rate of 1/1000.

The rate increased up to 20/1000 in interspecific pollination and up to 200/1000 when combined with delayed pollination. Some genotypes show very high frequency of haploid production.

The first report of induced haploidy in plant was that by Blakeslee et al. in 1922, through cold treatment of young buds of Datura stramonium. Later, haploids were induced in several other plant species through different techniques.

In general, there are three main approaches for the production of haploids:

(1) Parthenogenesis and apogamy,

(2) Somatic reduction and chromosome elimination, and (

3) Anther, pollen and ovule culture.

Parthenogenesis and Apogamy:

The term parthenogenesis is used for the development of embryo from ovum/egg cell without fertilization, whereas the term apogamy is used for the development of an embryo from a vegetative cell of the embryo sac without fertilization.

The haploids arising from the maternal cells in the embryo sac are called “gynogenetic haploids” whereas, those arising from the male (sperm) nucleus in the embryo sac are called “androgenetic haploids”.

The various methods used to produce parthenogenetic haploids are as follows:

(i) Temperature treatments

(ii) Irradiation

(iii) Chemical treatments

(iv) Delayed pollination

(v) Wide hybridization

(vi) Alien cytoplasm

(vii) Inducing genes

(viii) Haploid initiator genes

(ix) Semi gamy

(x) Selection of twin seedlings.

(i) Temperature treatments:

Parthenogenetic haploids may be produced by high or low temperature treatments. In Datura stamonium, Blakeslee and coworkers obtained haploids using cold treatment of young buds. Randolph obtained haploids in corn (Zea mays) through heat treatment just after pollination. Pollination with heat-treated pollen has also been employed for producing haploids.

(ii) Irradiation:

Haploids may be produced through irradiation of the embryo sac or of the pollen. It is presumed that the irradiated pollen grains loose their ability to fertilize but they stimulate the egg to develop into embryo parthenogenetically.

(iii) Chemical treatments:

Certain chemical agents have been used for induction of haploidy in plants. The chemical toluidine blue, which inactivates the sperm nuclei, could induce haploidy in some plant species such as, Vinca tomato, maize and Populus. Nitrous oxide was found to stimulate embryogenesis in the cells of embryo sac resulting in haploid production in Capsicum annum.

(iv) Delayed pollination:

Haploids may be produced using delayed pollination. In Triticum monococcum, high frequency (200 per 1000 plants) of haploids were obtained through this method by Smith in 1946.

(v) Wide hybridization:

Interspecific and inter-generic hybridizations may lead to the production of haploids in various plant species. In potato, di-haploids are produced by crossing tetraploid Solatium tuberosum (♀) with diploid Solatium phureja (♂); in this cross the male gamete forms a restitution nucleus which fertilizes the fused polar nuclei, while the egg cell is stimulated to develop pertheno-genetically.

Solatium phureja (2x) has also been used as pollinator for haploid production in other tuber bearing Solanum species. The rate of haploid production is influenced by the genotypes of both the female and male parents.

(vi) Alien cytoplasm:

This method of haploid production was reported by Kihara and Tsunewaki in wheat. Haploid are produced when the nuclei of strain Salmon of bread wheat (T. aestivum) are placed into the cytoplasm of certain species of Aegilops, e.g., Ae. caudata. Along with haploids, diploid seedlings were also observed (n/2n twins). The 2n seedlings were produced from fertilization of the synergid cells by the male gamete, while the egg cell was stimulated to develop parthenogenetically into haploid embryo.

(vii) Inducing genes:

Certain genes are known to induce haploidy in plants. The gene ‘indeterminate gametiphyte’ (ig) induces hapioids of both female (gynogenetic) and male (androgenetic) origin in maize (Zea mays). There is an irregular development of the mega gametophyte in plants homozygous for this gene (ig ig)’, their embryo sacs contain 1-5 egg cells and 1-7 polar nuclei. This gene leads to the production of 3 per cent haploids among which the gynogenetic and androgenetic haploids occur in the ratio of about 2:1. Combination of the ig gene with certain marker genes, such as, r^g (colourless scutellum and green plants) and R”^j (purple pigmented kernel crown, scutellum, plumule and seedlings) enables the easy identification of androgenetic and gynogenetic haploids.

For example, in the cross ig ig r^g r^g♀ x Ig Ig R^nj R^nj♂, the haploids showing colourless scutellum and green seedlings will be gynogenetic, whereas in the cross ig ig R^nj R^nj♀ x Ig Ig r⁸r^g ♂, the haploids with colourless scutellum and green seedlings will be androgenetic.

(viii) Haploid initiator genes:

A gene for haploid induction was reported by Hagberg in barley; this gene is called “haploid initiator gene” (hap). When plants homozygous for the hap gene are used in crosses as females or are selfed, they produce 15-40 per cent haploid progeny. However, when the hap plants are used as male parent, no haploid progeny are produced.

The heterozygous F₁ plants (Hap hap) obtained from the cross hap hap ♀ X Hap Hap ♀ produce 3-6 per cent haploid progeny. In tiie F₂ generation, the genotypes of diploid plants are Hap Hap, Hap hap and hap hap in the ratio 1 : 2 : 1; plants with Hap Hap genotype produce no haploid progeny, those having Hap hap genotype produce 3-6 per cent haploids, while hap hap plants produce 15-40 per cent haploids (Fig. 17.2). In contrast, 50% of the haploid F₂ plants will possess hap allele, while the rest 50% will have the Hap allele; the haploids possessing Hap allele are of value in breeding programmes.

(ix) Semi gamy:

In semi gamy, sperm nucleus enters the egg cell but nuclear fusion does not occur. Thus semi gamy falls between the two extremes, the syngamy (fusion of male and female gametes) and pseudogamy (development of embryo after pollination without involvement of male gamete). In semi gamy, the egg and sperm nuclei may begin to divide synchronously and the resulting embryo may produce plants that are chimeric in that they have distinct sectors containing either the sperm or the egg nucleus.

Occasionally, haploids of either maternal or paternal genotype may be produced. Although, semi-gametic production of haploids has been reported in several plant genera, it is mainly of interest in cotton (Gossypium) where extensive studies have been made. Semi gamy is genetically controlled, and in cotton it is caused by a dominant gene. Haploids have been produced in Gossypium hirsutum, G. barbadense, G. tomentosum and G. klatzschianum through the process of semi gamy.

(x) Selection of twin seedling:

Twin seedlings arise due to polyembrayony and may be x -x, 2x-x or 2x-2x twins. Twin seedlings are produced when a synergid cell is stimulated to divide in addition to the egg. Thus one embryo develops from the egg and the other from the synergid resulting in the production of twin embryos.

Lacadena in 1974 reported the occurrence of twin seedlings in 42 plant species. The occurrence of twin seedlings is also genetically influenced. In Capsicum frutescens, the frequency of twin seedlings is controlled by the genotype of the female parent. Frequencies of haploid-diploid (x-2x) twins vary in different species; in Lilium, this frequency is 1%, while in pepper, it is as high as 30%.

Somatic Reduction and Chromosome Elimination:

(a) Somatic reduction:

Somatic reduction is the phenomenon which results in the reduction of somatic chromosome complements and involves the segregation of whole genome. Its mechanism may involve the abnormalities of spindle such as multipolar spindle formations. It was first described in insects and later it was found to occur spontaneously in plants and plant tissues.

Somatic reduction may give rise to haploid cells during the para-sexual cycle of certain fungi. It can be induced by certain chemicals in different plants e.g., by chloramphenicol in barley root tips, by parafluorophenylalanine (pFPA) in grape seedlings, fungi and yeast.

(b) Chromosome elimination:

In certain plants, interspecific or inter-generic hybridization leads to the gradual elimination of chromosomes of one of the parental genomes resulting in the production of haploids. The mechanism of elimination may involve failure of chromosome congression at metaphase leading to chromosome lagging at anaphase and formation of micronuclei. This process may also lead to the production of androgenetic haploids.

In 1970, Kasha and Kao obtained haploid barley plants when they crossed Hordeum vulgare with H. bulbosum, since there was a gradual elimination of the chromosomes of H. bulbosum from the young hybrid embryos few days after fertilization (Fig. 17.3). The chromosomes of H. bulbosum are eliminated irrespective of its use as female or male parent; about 95% of the progeny are haploid.

However, in some of the progeny plants, all the bulbosum chromosomes are not eliminated and aneuploidy occurs. Subrahmanyam and Kasha in 1973 observed that there was gradual elimination of H. bulbosum chromosomes from the developing embryo. Subsequently, Subrahmanyam reported haploid production through chromosome elimination in various interspecific crosses in Hordeum.

Different species differ in the relative strength of chromosome elimination. Chromosome elimination is controlled by genetic factors present in both the species involved in a cross. The factors influencing the elimination of H. bulbosum chromosomes are located on chromosomes 2 and 3 of H. vulgare, the extent of elimination being also influenced by the genotype of H. bulbosum.

Inter-generic crosses involving Hordeum bulbosum can also produce haploids through selective chromosomes elimination. By crossing with H. bulbosum, haploids have been produced in Tridcum aestivum and Aegilops. In 1975, Barclay obtained haploids of T. aestivum var. Chinese Spring by crossing wheat with either 2x or 4x H. bulbosum.

Anther, Pollen and Ovule Culture:

Haploids have been produced by using anther, pollen and ovule cultures.

(a) Anther and pollen culture:

Haploids of several plant species are produced in very high frequencies by culturing anther or microspores on suitable culture media. In 1964, Guha and Maheshwari for the first time reported the production of Datura innoxia haploids through anther culture.

They noted the appearance of numerous embryoids inside the cultured anthers; subsequently these embryoids developed into haploid plantlets. Haploid production through anther culture has been successfully extended to a large number of plant genera and species, e.g., barley, rice, Brassica and tobacco. The pollen present within anthers may either directly develop into embryoids or it may form a callus from which plantlets may differentiate.

Haploids can also be produced through the culture of isolated pollen (microspore), e.g., in Datura innoxia, Nicotiana tabacum, Petunia, Oryza sativa, barley, tomato, maize etc. The factors affecting haploid production through anther culture are: physiology of the donor parent the stage of development, temperature and culture medium etc.

Anthers of most plant species are cultured at the bi-nucleate stage of the microspore, but in some cases, the uninucleate stage is the most appropriate, while in some other species the best results are obtained at the tri-nucleate stage. In nature, mitotic division of the microspore nucleus produces two nuclei: one relatively larger vegetative nucleus and other smaller generative nucleus. The generative nucleus divides once more to yield two sperm nuclei.

When anthers/microspores are cultured, the development of pollen embryos or calli may follow one of the following four pathways:

(i) In this pathway, there is an equal division of the microspore which yields two similar cells that are not differentiated into vegetative and generative cells. Both the cells divide and contribute to embryo/callus development, e.g., in Datura innoxia.

(ii) Unequal division of the microspore leads to the formation of distinct vegetative and generative cells. The callus/embryo arises from the repeated division of the vegetative cell, while the generative cell usually degenerates, for example, in Hordeum vulgare and Nicotiana tabacum.

(iii) In this pathway also vegetative and generative cells are formed by unequal division of the microspore. However, the embryo/callus develops from the generative cell only, e.g., in Hyoscyamus niger.

(iv) Alternatively, both the vegetative and generative cells, produced by an unequal division of microspore contribute to the formation of callus/embryo.

(b) Ovule culture:

Haploids have been obtained by culturing unfertilized ovaries of a number of plants, such as, barley, wheat, tobacco and rice. In rice, the pro-embryos develop from the egg cell while in barley, they originate from the egg cell as well as other cells of the embryo sac.

Meiosis in Haploids:

Monoploid or Mono-haploid (x):

In a monoploid, all the chromosomes present in a nucleus are non-homologous. Therefore, only univalents are expected at MI. But chromosome pairing has been observed in monoploids of some plants, e.g., barley, maize, rice and tomato. Synaptinemal complexes have also been observed at pachytene.

Non-homologous chromosome pairing (of small chromosomal segments) has been assigned to small duplication and genetic redundancy. In barley monoploids (2n = x = 7), Sadasivaiah and Kasha in 1971 reported the occurrence of rod bivalents with a frequency of 0.03 to 0.05 per PMC. In 1983, Kasha and Seguin-Swartz made the following three main observations regarding chromosome pairing and chiasma formation in barley monoploids.

(i) Chiasma formation is mainly dependent on duplication of chromosomes or segments of chromosome rather than on the large content of highly repetitive DNA sequences (over 70%) in the barley genome.

(ii) Chiasma frequency is highly influenced by the duplicated regions and their location on the chromosomes.

(iii) The very low frequency of association, e.g., 0.03-0.05 bivalent per PMC, could be due to random breakage and crossing over.

In some species, all the chromosomes present at MI and AI may be included in a single restitution nucleus following the telophase I; this nucleus undergoes a normal second meiotic division to produce two normal haploid spores. Therefore, after pollination by a diploid plant the monoploid may produce diploid progeny.

Chromosome pairing and crossing over in the duplicated region of non-homologous chromosomes of the monoploid will lead to the diploid progeny carrying a heterozygous translocation. Using this system, several interchanges between the chromosomes 6 and 7 and between 2 and 6 in Zea mays have been obtained; it has been concluded that the repeated interchanges were produced due to crossing overs in the duplicated segments.

In mono-haploids, the distribution of chromosomes at AI may be random. In some cases, all the chromosomes divide at AI forming a dyad each cell of which possesses n chromosomes as was observed in Datura mono-haploids (2n = x = 12) by Belling and Blakeslee in 1927.

In the species where haploidy is a regular feature, e.g., in Hymenoptera, meiosis in the haploids differs from one species to the other. The first meiotic division is completely absent and the second division (meiotic mitosis) leads to the formation of haploid sperm cells.

In male honey bee, all the chromosomes are included in one cell and cytoplasmic bud (polar body first) is pinched off. In the second division, chromosomes divide mitotically and two daughter cells of unequal size are produced. The sperm is formed from the larger cells.

Polyhaploids:

In auto-polyhaploids, two or more homologous chromosomes are present for the entire genome. Therefore, Homologous chromosome pairing is a common feature. In auto-di-haploids (2n = 2x = 24) of potato and those (2n = 2x = 16) of alfalfa, mostly bivalents with very few univalents are observed. However, fertility and vigour of these auto-polyhaploids are often lower than those of their polyploid counterparts, possibly due to inbreeding.

In case of allopolyploids, there should be no chromosome pairing since the chromosomes are not homologous. But a low frequency of bivalent formation occurs due to homoeologous pairing. In wheat (T. aestivum), 21-chromosome allotrihaploids (ABD); In = 3x = 21) showed 0.02^III and 1.69^II per cell.

The frequency of pairing increased in the nullisomic 5B-allotrihaploids (having 20 chromosomes) with the average frequency of 2.0 to 3.5^II percent; this is expected since the gene ph present in 5B is known to suppress homoeologous chromosomes pairing. In polyhaploids from auto-allopolyploids, homologous chromosomes undergo pairing, while the homoeologous chromosomes generally remain unpaired e.g., in Solatium demissum polyhaploids (2n = 3x = 36).

Secondary association:

Secondary association of univalents is also observed in haploids; the association may be side-to side, end-to-end or end-to-side. The reasons for secondary association may be duplication, homoeology and the presence of highly repetitive DNA sequences.

Phenotypic Effect of Haploidy:

Mono-haploid plants are weaker, shorter and less vigorous than the respective diploids. They are highly sterile. Their leaves, flowers and other parts are smaller as compared to those of diploids. Stomatal size is smaller but the number of stomata per unit area is higher than that in the diploids. However, in certain plant species, such as, pepper, haploids may be comparable in size and vigour to diploids.

Possible Uses of Haploids:

Haploids may be utilized for various investigations of both fundamental and applied importance as briefly described below:

(1) Haploids are used to study the chromosome behaviour during meiosis. Study of chromosome pairing in mono-haploids indicates the presence of duplications in the chromosomes.

(2) Study of chromosome pairing in haploids indicates the origin of different species of a plant. For example, in Brassica, chromosome pairing in haploids indicated that the basic chromosome number in the genus is x = 6, and different species originated through dysploidy.

(3) Information on the ancestry of species can be obtained through the study of homoeologous chromosome pairing in the haploids of different allopolyploid species.

(4) One of the most important uses of mono-haploids and polyhaploids is the production of homozygous lines in the shortest possible time. This is achieved by extracting haploids from heterozygous plants, followed by chromosomes doubling of such haploids; the resulting plants/lines are called doubled haploids or homodiploids. Chromosome doubling may occur naturally or may be induced using colchicine or some other suitable treatment. Doubled haploids may be used directly as cultivars. Cultivars derived from haploid systems have been produced in various crops such as wheat, rice, rapeseed, barley and tobacco.

(5) In cross-pollinated species, haploidy is an effective method for selecting viable combinations of genes which are then used as inbreeds after chromosome doubling.

(6) There is no segregation of genes in the homodiploids and therefore, it permits selection for quantitative characters; thus selection efficiency increases.

(7) In cases of self-incompatibility, inbred lines are readily produced by doubling the chromosome number of haploids.

(8) Haploid tissues can be maintained in vitro in undifferentiated condition and they provide a source of suspension of haploid cells. Like micro-organisms, these haploid cells of higher plants can also be used to carry out new genetic researches such as mutational studies at physiological levels and biochemical analyses.

Monoploids can be used efficiently in mutational studies because they possess only a single set of genes. Therefore, both the dominant and recessive mutations are expressed in the M₁ generation itself. Desirable mutants can be selected from among haploid cells cultured in vitro or from haploid plants and fertile homodiploids with all desirable mutations can be obtained through chromosome doubling.

(9) New genotypes can be incorporated into alien cytoplasm through androgenetic haploidy (androgenetic haploids may be produced by semi gamy and disruption of egg nucleus by irradiation). This method enables the transfer of a new genotype into the cytoplasm possessing factors for male sterility.

(10) Transfer of genes from wild diploid species to cultivated species can be done through di-haploids of polyploid species.

(11) Haploidy can be used in specific breeding schemes for dioecious plants, such as, Asparagus officinalis. Asparagus has the XX-XY system of sex determination, the male (XY) being more valuable commercially as they produce spears with a lower fibre content. An inbred population produced through sib-mating between pistillate (XX) and staminate (XY) plants consists of 50% males and 50% female plants. Androgenetic haploids, derived especially through anther culture, are used to produce homozygous female (XX) and super-male (YY) lines; crossing of such female and super-male plants yields an all male population, which is commercially superior to the conventional “50% male + 50% female” populations.

(12) Monoploids obtained from di-haploids through parthenogenesis of androgenesis can be used to select and evaluate various genomes to be put together through protoplast fusion in asexually propagated crop like potato.

(13) Di-haploids may be used in selection or crossing at diploid level before chromosome doubling (autotetraploid) as suggested by Chase in 1963 in the “analytical breeding” scheme for potato (Solatium tuberosum).

(14) Through chromosome doubling in haploids, homozygous lines can be produced for various climatic regions in one laboratory.

(15) Haploids may be used to produce translocation stocks and aneuploid stocks which are of cytogenetic importance and can be used in improvement of crop plants.