Gossypium arboreum (2n = 26, A2) is a diploid species with limited production in acreage com-pared with G. hirsutum and G. barbadense. However, its unique traits such as insect and disease resistance contribute an important germplasm to cotton breeding. So polyploid manipulation for G. arboreum is an effective approach of germplasm development. This research focused on tetraploid induction of G. arboreum by colchicine. Morphology and cytology identifications for obtained mutants were also conducted. The seedling growth and development of all mutants was more stunted than controls. According to preliminary morphological characteristics, mutant rates in different treatment were statistically estimated and the highest mutant rate was 42.31% under the treatment of 0.1% colchicine for 24 hours. The chromosome number of most mutants was 2n = 4x = 52, while the chromosome number of diploid controls was 2n = 2x = 26 by cytology observation of root tip cells. By microscope observation of low leaf epidermis, there were significant differences for stoma area between tetraploids and diploids. The meiosis behavior of the induced tetraploid was much more complex than that of the diploid. At diakinesis, some univalent, trivalent and polyvalent were also observed besides bivalent and quadrivalent. There were different kinds of polyad in tetraspore period of mutants. The dissociate chromosomes existed during metaphase I and II, the unbalance separation of chromosomes existed during anaphase I and II. As a result, tetraploid mutants of G. arboreum were identified and their desirable traits would be further evaluated to incorporate into next breeding program.
Polyploidy has been an important factor in eukaryote evolution [
Cotton is an important natural fiber crop traceable in tropic aridity regions, which was developed from originated a kind of perennial xylophyta into an annual crop growing in subtropical and temperate zone today [
Methods using colchicine for polyploidy induction are common for a range of plant species [
G. arboreum came from National Wild Cotton Nursery in China . Field experiments and inside experiments were finished in Shanxi Agricultural University . The first generation M1 acted as material for meiosis examination, while M2 was suitable for mitosis examination in this study.
Totally 102 pots were chosen for colchicine treatments and 25 pots were left as controls. All seeds were delinted by H2SO4 before use and approximately 3 seeds were planted in every separate pots with sunshine potting soil. When seedlings were at the cotyledon stage, the suitable plant (subsample) in each pot was treated by modified method of 1% agar with colchicine of different concentration semi-solid (26 pots for 0.1%, 27 pots for 0.2%, 24 pots for 0.3% and 25 pots for 0.4%) for 24 hours. A single drop (2 - 4 μL) of the warm (~50˚C) semi-solid was painted between cotyledons of each seedling to cover the apical bud. The treated pots were then covered by a plastic cup under a high humidity growth chamber at 25˚C. After each treatment, plastic cup was uncovered and the residue was carefully removed with tweezer. Sprinkling water and nutritive solution (0.3 g CON2H4 and 0.5 g KH2PO 4 in 1 L distilled water) were applied once a day to help recovering the seedling growth for two weeks and then seedlings were transplanted in the field.
Morphological characteristics of four treatments were examined during the seedling growth period. Retardant growth, crimpled and dark green leaf were measured as mutant preliminary indices and mutant rates of different treatments were estimated.
The ploidy level of G. arboreum was also estimated by chromosome counting of root tips from putative mutants earlier determined by morphological characteristics. Chromosome spreads were made according to the method of [
The ploidy level of G. arboreum was estimated either by stoma measurement. Low leaf epidermis was torn from obtained tetraploids and diploid controls in the same internode (the forth) and mounted on a clean slide with a drop of distilled water respectively. Another drop of 1% I-KI solution was added to stain the stoma and covered with cover slip. Slides were examined with a OLYMPUS BX51 using a 40× objective. The density and size of stoma under the same magnified scope were measured. At least 10 scopes were chosen and mean numbers were statistically calculated.
Flower buds for meiotic studies were collected from each individual plant in the field between 9:00 and 10:30 am and fixed in freshly prepared Carnoy’s fixative solution II (ethanol: acetic acid: chloroform = 5:3:2) for 12 h, then transferred to 70% alcohol, and stored under refrigeration for use. Meiotic analysis was carried out on flower buds of a suitable size; after washing the fixed buds in distilled water, anthers were squashed on slides in carbol fuchsin solution. Photographs were taken from freshly prepared slides using an Olympus BX51 microscope with automatic camera. Meiosis was studied using a minimum of 30 PMCs. With reference to the abnormal chromosome behaviors [
The experimental design was completely randomized and data analyses were performed using DPS software.
The growth rate of mutant plant with lower strain height, smaller internode length as well as deformed and crimpled leaves were all slower than diploid control in different period (
Karyotype of diploid controls was shown in
Concentration | Number of treated plants | Number of survivals | Number of mutants | Survival rate (%) | Mutation rate (%) |
---|---|---|---|---|---|
CK | 25 | 21 | - | 84.00 | - |
0.1% | 26 | 11 | 11 | 42.31 | 42.31 |
0.2% | 27 | 4 | 4 | 14.81 | 14.81 |
0.3% | 24 | 6 | 6 | 25.00 | 25.00 |
0.4% | 25 | 3 | 1 | 12.00 | 4.00 |
Number | Relative Length/(%) (S + L = T) | Index of Relative Length | Centromere Index % | Arm Ratio | Category | ||
---|---|---|---|---|---|---|---|
Long arm | Short arm | Total length | |||||
1 | 5.89 | 5.01 | 10.90 | 1.42 | 45.95 | 1.18 | m |
2 | 6.19 | 3.53 | 9.72 | 1.26 | 36.36 | 1.75 | sm |
3 | 4.71 | 4.27 | 8.98 | 1.17 | 47.54 | 1.10 | m |
4 | 4.71 | 3.68 | 8.39 | 1.09 | 43.86 | 1.28 | m |
5 | 4.42 | 3.98 | 8.39 | 1.09 | 47.37 | 1.11 | m |
6 | 3.98 | 3.68 | 7.66 | 1.00 | 48.08 | 1.08 | m |
7 | 3.98 | 3.68 | 7.66 | 1.00 | 48.08 | 1.08 | m |
8 | 3.68 | 3.39 | 7.07 | 0.92 | 47.92 | 1.09 | m |
9 | 3.09 | 2.36 | 5.45 | 0.71 | 43.24 | 1.31 | m |
10 | 3.53 | 2.80 | 6.33 | 0.82 | 44.19 | 1.26 | m |
11* | 6.04 | 2.80 | 8.84 | 1.15 | 31.67 | 2.16 | sm (SAT) |
12* | 3.98 | 1.47 | 5.45 | 0.71 | 27.03 | 2.70 | sm (SAT) |
13* | 3.98 | 1.18 | 5.15 | 0.67 | 22.86 | 3.38 | st (SAT) |
Note: *Sat-chromosome. The length of satellites is not included in the chromosome length.
on short arms of chromosome 11, 12, 13. IRL = 4L + 8M2 + 8M 1 + 6S. L stands for that IRL of the chromosome is more than 1.26, M2 for IRL ranges from 1.01 and 1.25, M1 for IRL ranges from 0.76 to 1.00, S for IRL is less than 0.75.
Karyotype of tetraploids was shown in
Number | Relative Length/(%) (S + L = T) | Index of Relative Length | Centromere Index % | Arm Ratio | Category | ||
---|---|---|---|---|---|---|---|
Long arm | Short arm | Total length | |||||
1 | 5.39 | 5.34 | 10.73 | 1.39 | 49.79 | 1.01 | m |
2 | 5.79 | 4.01 | 9.80 | 1.27 | 40.91 | 1.44 | m |
3 | 4.63 | 4.10 | 8.73 | 1.13 | 46.94 | 1.13 | m |
4 | 4.59 | 4.05 | 8.64 | 1.12 | 46.91 | 1.13 | m |
5 | 4.01 | 3.78 | 7.79 | 1.01 | 48.57 | 1.06 | m |
6 | 3.96 | 3.43 | 7.39 | 0.96 | 46.39 | 1.16 | m |
7 | 3.96 | 3.43 | 7.39 | 0.96 | 46.39 | 1.16 | m |
8 | 3.78 | 3.56 | 7.35 | 0.07 | 48.48 | 1.06 | m |
9 | 3.12 | 3.12 | 6.23 | 0.81 | 50.00 | 1.00 | M |
10 | 3.03 | 2.58 | 5.61 | 0.73 | 46.03 | 1.17 | m |
11* | 4.01 | 4.01 | 8.01 | 1.04 | 50.00 | 1.00 | M (SAT) |
12* | 4.01 | 3.12 | 7.12 | 0.93 | 43.75 | 1.29 | m (SAT) |
13* | 2.80 | 2.40 | 5.21 | 0.68 | 46.15 | 1.17 | m (SAT) |
Note: *Sat-chromosome. The length of satellites is not included in the chromosome length.
Stoma studies can give a quick view to judge the ploidy level of G. arboreum. In this study, two epidermis from strictly comparable leaves in the same internode (the forth) showed an obvious difference in the size of stoma between the obtained tetraploids and diploid controls in
Observations were made on chromosome morphology and behavior during meiosis of PMCs for induced tetraploids. The steps of induced tetraploidy in meiosis were similar to diploid controls, but much more complex. It was noticed that clustering of chromosomes as unit into separated groups formed in leptotene. At diakinesis, the disorder synapsis of homology segment made homologous chromosomes appear as abnormal styles like o, v, x, ∞ (
Sample | Stomatal density/ 200 μm × 200 μm | Stomatal aspect ratio | Stomatal approximate area (μm2) | Chloroplast number per stomatal |
---|---|---|---|---|
Control group | 14.83 ± 1.60aA | 1.30 ± 0.18aA | 453.99 ± 44.13aA | 38.83 ± 3.19aA |
Induced group | 6.50 ± 1.38bB | 1.41 ± 0.09bB | 822.99 ± 76.56bB | 46.00 ± 1.90bB |
Rangeability | −56.17% | +8.46% | +81.28% | +18.47% |
Note: Small letters stand for significance at 0.05 level and capital letters stand for significance at 0.01 level.
cluster and laggard chromosomes were observed when the homologous chromosomes separated (
The pollen grains of controls developed uniformly and nearly spherical with the average diameter of 83.04 ± 11.16 μm (
Induction of polyploidy may create wider amplitude of genetic variation which is necessary to study various cytogenetic backgrounds relating to its improvement as an ideal and alternative pulse crop in different geographical regions. Polyploids have immense importance in maintaining these aneuploids in population. Successful crossing between tetraploids and diploids often results in triploid plant which can produce different trisomics, 3n + 1 or 3n − 1 individuals in progeny. There are evidences that doubling the chromosome number may change magnitude of sterility, pattern of sexuality, plant habit and increase winter hardiness [
From this study, Gossypium arboreum (2n = 26, A2) was manipulated polyploid induction by colchicine. Morphology, cytology and other stoma, meiosis identifications all showed that the obtained mutants were tetraploids and their desirable traits would be further evaluated to incorporate into next breeding program.
Sample | Pollen grain diameter (μm) | Proportion of abnormal pollen grain (%) |
---|---|---|
Control group | 83.04 ± 11.16aA | - |
Induced group | 116.79 ± 9.54bA | 4.28 |
Rangeability | +40.64% | - |
Note: Small letters stand for significance at 0.05 level and capital letters stand for significance at 0.01 level.
This research was supported by National Natural Science Foundation of China (No. 31171599), Scientific and technological project in Shanxi province (No. 20130311004-1) and Innovation Project for Shanxi Agricultural University (No. 2013YJ45).