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In this article we will discuss about:- 1. Origin and Occurrence of Autotetraploids 2. Chromosome Pairing and Chiasma Formation in Autotetraploids 3. Fertility 4. Genetics.
Origin and Occurrence of Autotetraploids:
Autotetraploids originate both spontaneously and can be experimentally induced; the various means of their origin are briefly described below:
A. Spontaneous origin:
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(1) In diploid (2x) plants, sometimes a tetraploid (4x) cell may arise due to a failure in nuclear division during mitosis; this cell may divide to form a tetraploid sector. Such cases have been reported in Datura and other plant species.
(2) Spontaneous chromosomes doubling in the zygote just after fertilization produces tetraploid embryos.
(3) Unreduced gametes may be produced due to abnormal meiosis; union between such male and female gametes will produce 4x plants. In maize, tetraploids were obtained in ♀ 2x x 4x ♂ crosses.
(4) Tetraploids have been produced in maize by crossing asynaptic (as) or elongate (el) plants with 4x plants. Both asynaptic and elongate plants produce a high frequency of unreduced gametes which, after fertilization by pollen from 4x plants, produce tetraploid progeny.
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(5) Crosses of mixoploid and tetraploid plants may produce tetraploids in the progeny. For example, 3.6% tetraploid progeny were obtained from such crosses in barley. The mixoploid barley plants produce both haploid (x) and diploid (2x) pollen.
(6) Adventitious shoots arising from the callus tissue developed following decapitation of shoots may show a relatively high frequency of polyploidy, e.g., in Solanaceous plants.
B. Artificial production of tetraploids:
Experimental induction of auto-tetraploidy exploits both physical and chemical agents which usually operate by disturbing the spindle function.
I. Physical means:
(a) Temperature shocks:
Extreme temperature changes can produce polyploid cells. Blakeslee and Belling in 1924 found 4x branches in Datura as a result of cold treatment. High temperatures are reported to induce chromosomes doubling in a number of plant species like barely, rye, wheat and sweet clover.
(b) Centrifugation:
Centrifugation causes changes in chromosome number in both plants and animals. Kostoff in 1935 obtained change in chromosome number in Nicotiana induced by centrifugation of the seedlings. In silk worm (Bombyx mori), Kawaguchi in 1936 obtained triploids, tetraploids and hexaploids by centrifuging the eggs at the time of fertilization and at the first division of zygote.
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II. Chemical means:
Several chemicals are known to produce tetraploid cells through c-mitosis, the most effective of them being colchicine. Colchicine is an alkaloid extracted from the seeds and bulbs of the wild meadow saffron or autumn-flowering crocus (Colchicum autumnale L.) belonging to the family Liliaceae.
This chemical constitutes 0.2-0.8% of seed dry weight or 0.1-0.5% or corm dry weight. The chemical formula of colchicine is C22H25O6N. It has a tricyclic chemical structure with two fused 7-membered rings (Fig. 17.5). cold water (but not in hot water).
The effects of colchicine on mitosis were first studied on mice at Dustin’s laboratory in Brussels. In 1937, Nebel and Blakeslee working independently reported the use of colchicine in inducing the polyploidy. Colchicine inhibits spindle formation and prevents anaphase chromosome movement due to which all the chromosomes of a dividing cell are included in a single restitution nucleus; this leads to chromosomes doubling.
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Late metaphase chromosomes of colchicine-treated cell appear longitudinally divided and look like pairs of parallel rods since their chromatids, although separated, are unable to move towards the poles due to a lack of spindle, organisation.
Levan in 1938 termed this appearance as Colchicine mitosis or C-mitosis. Colchicine induces polyploidy in plants as well as in several animals like rabbit, swine etc. Calchicine has no effect on Colchicum upto the concentration of 1%, while in most plant species 0.1-0.5% colchicine is effective in inducing polyploidy.
Methods of use of colchicine in plants:
Colchicine is used either as an aqueous solution or as a paste prepared by mixing with lanolin or sugar. Wetting agents may be added to increase the effectiveness of colchicine. Aqueous solutions are most effective when they are freshly prepared. General methods of treatment are described below :
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(i) Seed treatment:
Actively germinating seeds are treated with colchicine using the concentration ranging from 0.001 to 1.6% but 0.2% is the most common concentration. Seeds are soaked in the colchicine solution for a period of 1 to 10 days.
(ii) Seedling treatment:
Germinated seeds are treated with colchicine solution for 3 to 24 hours. To avoid damage to the roots, the root ends of seedlings are placed on a strip of absorbent cotton that is wet with water; the seedlings along with the cotton are then rolled into a bundle. The shoots of seedlings are immersed in a suitable colchicine solution kept in a vial; care is taken to keep the roots out of contact with the colchicine solution.
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(iii) Treatment of growing shoots and buds of herbaceous plants:
Colchicine solution is brushed or dropped on the exposed shoot tips once or twice daily for few days. Cotton plugs moisted with colchicine solution may be put on the growing buds. The cotton plugs are moistened daily with the colchicine solution. In another method, a mixture of 0.5 to 1.0% colchicine in lanolin is smeared on the shoot tip; this is repeated 2-3 times per weak.
(iv) Treatment of buds of woody or semi-woody plants:
For woody plants higher concentrations of colchicine solution are necessary. Colchicine solution of 1% is prepared either in 10% aqueous solution of glycerine or in distilled water. A small quantity of wetting agent may be added to give better penetration.
The glycerine solution is applied at one or two-day intervals, whereas the aqueous solution is applied twice a day for 3 days on rapidly growing materials. Treatment is continued for a longer period of time if the material is slow growing.
Special methods of colchicine treatment:
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Several special methods of colchicine treatments are used for cereals and grasses. These methods aim to bring the chemical in contact with the growing shoot tip.
(a) A special technique was developed by Jensen in 1975 to double the chromosome number of barley monoploids. In this technique, vigorous plants at 3-4 tiller stage are uprooted and their roots are washed. The roots are cut about 3 cm below the crown and the plants are placed in glass vials.
The colchicine solution is prepared by dissolving 5 g of colchicine in 20 ml of dimethylsulfoxide (DMSO) and the final volume of one litre is made up with water. A few drops of Tween-20 or Tween-80 are added to this solution. The colchicine solution is added to the vials having the plants so that the plant crown and a part of their leaves are covered. These plants are kept in a growth room at 20°C in light for 5 hr. The treated plants are transferred directly into a light soil.
(b) In another method developed by Bell in 1950, tillers of young cereal plants are cut off an inch or so above the growing points. Tightly fitting capillaries filled with colchicine solution are then slipped over the cut ends of the tillers and held in place by a wire that is pushed into the ground; the upper end of the wire being wound around the capillary.
(c) Gerrish developed a technique in 1956 for inducing polyploidy in maize. In this method, germinating maize kernels are suspended from a string in an Erlenmeyer flask in such a way that only their coleoptiles are submerged in the colchicine solution. The cork of the flask is connected to a vacuum pump and the air of the flask is exhausted. Consequently, the coleoptile is filled with the colchicine solution. The seedling is then transferred to a nutrient solution for a few days before being planted in the field.
(d) In 1951, Luong used a special method for colchicine treatment in rice. The upper portion of rice seedlings is split longitudinally reaching down to the growing point; a small wad of absorbent cotton soaked in colchicine solution or a 0.5 mm square of blotting paper piece is then inserted so as to be in contact with the growing point. Colchicine solution is dropped at that point as often as required.
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(e) In case of underground stems, such as, bulds, corms, runners etc., colchicine is introduced into the growing regions by a hypodermic needle.
Other chemical agents:
Besides colchicine several other chemicals, e.g., acenaphthene, naphthalene-acetic acid, sodium cacodylate, sanguinarine, 8-hydroxyquinoline and nitrous oxide, are known to induce polyploidy. Different chemicals are effective indifferent species. In Colchicum, colchicine has no effect up to the concentration of 1 per cent, but acenaphthene has been found to affect- mitotic division.
Chromosome Pairing and Chiasma Formation in Autotetraploids:
In auto-tetraploids, there are four homologues for each chromosome; therefore, multivalents are formed during meiosis. Depending on the chromosome pairing and chiasma formation, the following different configurations may occur for each chromosome: a quadrivalent (ring or chain of four chromosomes), a trivalent plus a univalent or two bivalents (Fig. 17.6).
It is assumed that there is equal chance for a chromosome to pair with each of its remaining three homologues. S1, S2, S3, S4 and L1, L2, L3, L4 represent the short and long arms of the four homologues. “Trivalent plus a univalent,” may also observed if one chromosome remains unpaired.
If short arm of the four homologues of a chromosome is represented by S1, S2, S3 and S4, while the long arm is denoted as L1, L2, L3 and L4 respectively, each chromosome arm of the four homologues could pair in following three different ways (it is assumed that the pairing is initiated only at the chromosome ends):
(i) S1 : S2, S3 : S4; (ii) S1 : S3, S2 : S4; and (iii) S1 : S4, S2 : S3 (Fig. 17.6). Similar possibilities exist for the long arm as well. If partner exchange is free, that is, the pairing of the short arm of one chromosome with one homologue in any way does not affect the probability of the pairing of its long arm with the remaining two homologues, a total of 9 combinations of short and long arm associations would occur with equal probability.
These nine associations may be described as follows:
(i) S1 : S2, S3 : S4 and L1 : L2, L3 : L4
(ii) S1 : S3, S2 : S4 and L1 : L3, L2 : L4;
(iii) S1 : S4, S2 : S3, and L1 : L4, L2 : L3 :
(iv) S1 : S2, S3 : S4 and L1 : L3, L2 : L4;
(v) S1: S2, S3 : S4 and L1 : L4, L2 L3;
(vi) S1 : S3, S2 : S4 and L1 : L2, L3 : L4,
(vii) S1 : S3.S2 : S4 and L1 : L4, L2 : L3;
(viii) S1 : S4, S2 : S3 and L1 : L2, L3 : L4, and
(ix) S1 : S4, S2 : S3 and L1: L3, L2 : L4.
Of these, first three combinations of short and long arm associations will produce “bivalent + bivalent” configuration, while the remaining six combinations will produce quadrivalents. There may be more points of pairing initiation which may lead to partner exchange, and therefore, a high frequency of quadrivalent formation.
The shapes and configurations of the quadrivalents at diakinesis and MI depend upon the number and location of chiasmata, the chromosome pairing pattern and the degree of terminalization of chiasmata (Fig. 17.7).
Fertility of Autotetraploids:
Autotetraploids show reduced fertility due to irregular separation of chromosomes at Al leading to the formation of aneuploid gametes which are usually nonfunctional. Unequal chromosome separation occurs due to (i) unequal orientation of quadrivalents resulting in a 3 : 1 segregation, and (ii) reduction of chiasma frequency as a result of partner exchange causing the formation of univalents with a high frequency.
Presence of quadrivalents has detrimental effect on fertility in several crop plants, but in rye there is a regular orientation of the quadrivalents at MI so that the fertility of tetraploid is not reduced. The frequency of quadrivalents can be reduced by selection for high fertility, e.g., in maize, Brassica campestris, barley etc. Successful selection for increased fertility has been reported in linseed auto-polyploids, bajra and some other plant species.
In some cases, normal development of aneuploid spores and zygotes may also result in increased fertility of auto-tetraploids; such a development is dependent upon genotypic and environmental factors. For instance, a tetraploid variety of rye that showed about 65% seed set in Sweden had exhibited more than 90% seed set in California; the higher seed set in California resulted from the development of aneuploid gametes and embryos. In such cases, therefore, the progeny contain a high frequency of aneuploids, which is not desirable.
Genetics of Autotetraploids:
Autotetraploids show tetrasomic inheritance. Since there are four homologues for a given chromosome, 5 different genotypes are possible for the two alleles of a single gene (Table 17.3). In contrast, there are only 3 possible genotypes in diploids, while 4 genotypes are possible in triploids.
Each genotype, particularly in polyploids is give a distinct name, e.g., quadruplex, triplex, duplex etc.; these terms are based on the number of dominant alleles present in the genotypes concerned. The zygotic combinations in tetraploids arise from fusion of any two of the possible 3 different types of gametes, viz., AA, Aa and aa; the proportion of these gametes would depend on the genotype of the plant producing them and on some other factors (see, later).
Phenotypic expression of various genotypes in an autotetraploid depends on the type of gene action. If A is completely dominant, only two phenotypic classes, dominant (A) and recessive (a) are observed. But in case of cumulative effect of the gene A, every genotype will produce a separate phenotype; this would result in five phenotypes for a single gene.
In case of incomplete dominance, the genotype AAAA (quadruplex) will show the dominant trait, while the genotype aaaa (nulliplex) will show the recessive phenotype. The other genotypes, viz., AAAa (triplex), AAaa (duplex) and Aaaa (simplex), will show different degrees of intermediate expression between the quadruplex and nulliplex phenotypes.
An example of expressions of different genotypes in case of complete and incomplete dominance has been reported by Dawson in 1962 in Primula sinensis. In this plant, green stigma (G) is completely dominant to red (g); therefore, only two phenotypes are observed: green stigma (genotypes GGGG, GGGg, GGgg and Gggg) and red stigma (genotype gggg).
However, in the case of the gene for anthocyanin formation, the allele for full colour (D) is incompletely dominant to suppression of anthocyanin formation (d), therefore, different grades of anthocyanin formation is observed in the various genotypes. In this case, genotypes DDDD produces full colour (anthocyanin), while dddd produces no colour (white); other three genotypes (DDDd, DDdd, Dddd) produce different intermediate colours.
Genetic consequence:
The types and frequencies of gametes produced in auto-tetraploids depend on the relative proportion of the following two types of segregations:
(1) Random chromosome assortment
(2) Random chromatid assortment.
Random chromosome assortment:
This type of segregation assumes that:
(a) The gene in question is so close to the centromere that crossing over cannot occur between them,
(b) Each chromosome has an equal chance of pairing with each of its remaining three homologues, and
(c) Separation at AI is regularly two-by-two. In this type of assortment, both the chromatid of a chromosome go to the same pole at AI, while they separate and go to opposite poles at All. Therefore, sister chromatids (sister alleles) never end up in the same gamete.
As depicted in the Fig. 17.8, the gametic ratio in a duplex individual showing 100% random chromosome assortment is 1 AA : 4Aa : 1 aa. Similarly, the gametic ratios for triplex and simplex individuals will be 1 AA : 1 aa and 1 Aa: 1 aa, respectively.
Different zygotic combinations and phenotypic ratios will be obtained in the selfed progeny of these individuals. For example, the zygotic combination obtained on selfing of a duplex individual will be as given in Fig. 17.9. If A is completely dominant, the phenotypic ratio becomes 35A : 1a. The expected gametic, genotypic and phenotypic ratios for different types of mattings, assuming random chromosome assortment and complete dominance are given in Table 17.4.
Random chromatid assortment:
This theory assumes that:
(a) There is regular quadrivalent formation and
(b) Crossing over always occurs between the gene and the centromere.
Therefore, each chromatid, especially its region containing the gene, has equal chance to be present in a given spore, and hence sister alleles become included in the same gamete. The occurrence of sister alleles in the same gamete is called double reduction or equational segregation at the locus. For double reduction to occur, it is necessary that (i) a single crossover (or any number of crossovers) occurs between the locus and the centromere so that sister chromatids get attached to two different centromeres, (ii) the two chromatids resulting from crossing over must pass to the same pole at Al, thus sister alleles have a chance to be included in the same gamete, and (iii) subsequently, the sister chromatids move to the same pole at All as well.
Gamete formation as per this scheme in a triplex individual is presented in the Fig. 17.10. The expected frequencies of different types of gametes under random chromatid segregation can be calculated as shown in Fig. 17.10. Thus as per random chromatid segregation, the gametic ratio in a triplex will be 15A4 : 12Aa : 1 aa. In this example, the gametes A1A2, A3A4, A5A6 and a7a8 are formed due to double reduction.
Similarly, the gametic ratios for duplex and simplax individuals will be 3AA; 8Aa : 3aa, and 1AA : 12Aa : 15aa, respectively. (Table 17.5). The different genotypes and their frequencies in the progeny of a triplex individual subjected to selfing is shown in Fig. 17.11. In case complete dominance of A, the phenotypic ratio will be 783A :1a. The expected gametic and phenotypic ratios in different types of mating, assuming random chromosome and random chromatid assortments are summarised in Tables 17.4 and 17.5, respectively.
Double reduction occurs for both the alleles A and a but the recessive (aa), gametes can easily be identified. In triplex individuals the formation of aa gamete is possible only through double reduction (Fig. 17.10). But in the case of duplex and simplex individuals, the frequencies of recessive gametes increase due to double reduction. An example of double reduction was presented by Blakeslee and associates in 1923 in the case of autotetraploid Datura stramonium.
In this plant, armed capsule (A) is dominant over inermis (a) and purple flower (P) is dominant over while flower (p). The data obtained from selfed and test-cross progeny indicated the occurrence of double reduction for both the genes, A and P (Table 17.6). In the cross of triplex for capsule shape (PPPp) with the nuliplex (pppp), a very low frequency (0.6%) of recessive plants was observed.
But in similar crosses involving the gene for flower colour (AAAa x aaaa), the frequency of recessive individuals (aaaa) was comparatively higher (2.3%). These results not only revealed the occurrence of double reduction, but also indicated that gene a is located farther away from the centromere as compared to gene p.
Parameter alpha (a):
The frequency of inclusion of two sister alleles in the same gamete, that normally would have been separated (in different gametes) is represented by “alpha” (a). The minimum value of alpha is zero (a = 0); this occurs when quadrivalents are never formed or when there is always random chromosome assortment, or when 100% alternate orientation occurs.
The alpha can be calculated for any gene by taking into account the following points:
(i) The occurrence of crossing over between the gene and the centromere; this has two extremes of no chiasma formation (= 0), and chiasma formation in all the cases (= 1).
(ii) The frequency of two sister chromatids passing to the same pole; this frequency is 50% (= 1/2) which is the maximum possible when there is 100% adjacent orientation. If we consider that alternate and adjacent orientations occur with equal frequency (50% each), then the frequency of movement of sister alleles to the same pole at MI becomes only 1/4 (= 1/2 x 1/2).
(iii) At All, the frequency of (originally) sister alleles going to the same pole is 50% (= 1/2). On the above basis, alpha can be calculated as: “frequency of occurrence of chiasma X 1/2 (MI quadrivalent orientation) x 1/2 (All segregation”). Thus the maximum possible value of alpha would be 1 x 1/2 x 1/2 = 1/4, while its average value will be, 1 x (1/2 x 1/2) x 1/2 = 1/8 or 0.125 (assuming equal frequencies of alternate and adjacent orientations).
When the homologues show differential affinity, selective pairing occurs in auto-tetraploids. In case of complete selective pairing, gametes of certain combinations only would be formed. Incomplete selective pairing would produce a variable frequency of the other types of gametes as well.
Double reduction is also affected by selective pairing. If A preferentially pairs with a, double reduction would be increased. But in case there is preferential pairing of A with A and a with a, double reduction would be decreased. During evolution, gradual changes occur in chromosomes and there is an accumulation of small changes; this causes selective pairing between chromosomes.
As a result, auto-polyploids may show a tendency towards diploidization during the long course of their evolution. Selective pairing may be observed in some long established auto-tetraploids, such as potato and alfalfa.
