Regular Article
Cytogenetical Investigation of a Translocation Heterozygote Induced by Gamma Rays in Coriandrum sativum L.
2019 Volume 84 Issue 3 Pages 211-214
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2019 Volume 84 Issue 3 Pages 211-214
Seeds of Coriandrum sativum L. (Coriander) cultivar CO-2 were irradiated with different doses (100, 200, 300, 400 and 500 Gy) of gamma rays. During the meiotic analysis of pollen mother cells (PMCs), a plant having segmental exchanges between non-homologous chromosomes was isolated in populations raised from 100 Gy irradiated seeds. This paper documented with structural heterozygosity along with cytological behavior of translocation heterozygote induced and the probable cause of its occurrence in the coriander. Morphologically, the plant was weak with short height in contrary to control and irradiated populations. In the plant cytological manifestation of chromosome configurations at diakinesis and metaphase I revealed the predominance of ring octavalents with tetravalents and bivalents. In a few PMCs, the variable number of associations in the form of multivalents along with univalents and bivalents were also screened out. The induced translocation heterozygote showed unequal segregation of chromosomes, laggards, and bridges along with normal 11 : 11 separation at anaphase I. Furthermore, due to chromosomal anomalies, pollen fertility was declined to 37.36% as an overwhelming adjacent orientation (53.16%). Translocation heterozygotes may provide a source for creating aneuploids with novel gene combinations.
Mutation breeding employing gamma irradiation is one of the important tools for the improvement and modification in a genome to break through the limitations of creating variability in a short period of time. Induced mutagenesis is initiated to explore its potential for genetic upgradation as well as to elucidate the feasibility of creating translocation and multiple interchange stock, which has tremendous value in linkage analysis (Micke et al. 1985). Plants bearing translocations are known as heterozygotes as only one of the chromosomes, of a non-homologous pair, is involved (Kumar and Singh 2003). Chromosomal breakage is a common feature both in plants and animals (Mahama et al. 1998) and these broken segments, if unable to join or restitute, might result in inversions, a kind of chromosomal aberration that leads to the formation of bridges during anaphase. The observed effects of such abnormalities include the non-transmission of gametes through pollen and increased pollen and ovule sterility in plants (Phillips 1978, Minocha et al. 1982). Translocation heterozygotes, having the potential for creating and conserving specific combinations, are generally identified by reduced reproductive ability and the presence of multivalents during meiosis (Sharma and Gohil 2011). In some dioecious plants, permanent translocation heterozygosity has been produced, at least in the male plants, through the association of the translocations with the sex determination system and a translocation multivalent is characteristic of the heterozygous sex (Barlow 1981). C. sativum is a plant of immense pharmaceutical importance. Basically, it is cultivated as an important spice crop worldwide and contains essential oils rich in linalool, α-pinene, α-terpinene, limonene, and n-cymene together with various non-linalool alcohols and esters. One of the plants from gamma ray irradiation exhibited structural heterozygosity due to translocation as confirmed by the presence of multivalents and reduced pollen fertility during the course of cytological study. In the present work, an attempt has been made to explore the cytological behavior of an induced translocation heterozygote and its consequences on the fertility of C. sativum L., as a limited knowledge is available in present crop.
Healthy and fresh seeds of Coriandrum sativum L. cultivar CO-2 were collected from Centre for Research on Seed Spices, Jagudan, Gujarat, India. Seeds were packed in polythene bags and subjected to different doses (100, 200, 300, 400 and 500 Gy) of gamma irradiation using gamma chamber with Co60 at the dose rate of 7.247 KGy h−1 at National Botanical Research Institute (NBRI), Lucknow. Control sets were maintained without any treatment. After that, the seeds were sown in replicates along with their respective controls.
Meiotic analysisFor meiotic studies, young floral buds were fixed in freshly prepared glacial acetic acid–ethanol (1 : 3) solution for 24 h and stored in 90% ethanol in a refrigerator until use. Slides were prepared using anther squash technique with 2% acetocarmine. Slides were analyzed and suitable cells were photographed under a Nikon research photomicroscope. Observations were recorded on chromosome configurations at metaphase I with the interchanged complex as a ring or chain along with multiple chromosomal associations. Pollen fertility was also estimated using 2% acetocarmine. Pollen grains with stained nuclei were recorded as fertile whereas undersized and unstained pollen grains without nuclei were considered sterile. For statistical analysis, SPSS16.0 software was used.
In the control plants, meiosis was found to be perfectly normal and showed a regular formation of eleven bivalents (n=11) at diakinesis (Fig. 1A) and metaphase I (Fig. 1B) followed by normal separation (11 : 11) at anaphase I (Fig. 1C). However, in case of gamma-irradiated plant population; one plant of the 100 Gy set was isolated as translocation heterozygote with lower seed yield as compared to control. Further translocation heterozygote was characterized by the occurrence of ring or chain configurations in PMCs at diakinesis-metaphase I that could be clearly distinguished from normal ones. An array of chromosomal abnormalities like laggards, bridges and various multiple associations along with univalents were found on cytogenetic analysis of translocation heterozygote that exhibits various ring and chain configurations (Fig. 1D–I). Table 1 gives an account of variations of chromosomal configurations and their frequencies at diakinesis-metaphase I of the translocation heterozygote.
| Configurations | No. of PMCs | Frequency (%) |
|---|---|---|
| 11 II | 22 | 11.70 |
| 8II+1VI | 19 | 10.11 |
| 7II+1VIII | 12 | 6.38 |
| 5II+1IV+1VIII | 8 | 4.25 |
| 4II+2IV+1VI | 18 | 9.57 |
| 3II+2IV+1VIII | 36 | 19.15 |
| 3II+1IV+2VI | 29 | 15.42 |
| 2II+1IV+2VI+2I | 7 | 3.72 |
| 2II+1IX | 6 | 3.19 |
| 2II+2IV+1X | 5 | 2.66 |
| 1II+5IV | 17 | 9.02 |
| 1IV+1VIII+1X | 9 | 4.79 |
PMCs with ring configuration were structurally categorized as bi rings (8-shaped) and open rings while PMCs having zig-zag pattern or side by side orientations were screened out as chains (Fig. 1D–I). During the cytological analysis of induced translocation heterozygote, a total of 188 PMCs were scored at diakinesis-metaphase I out of which 36 deciphers the occurrence of octavalent with two tetravalent and four bivalents while 29 showed two hexavalents with one tetravalent and three bivalents. The induced translocation heterozygote thus exhibited the formation of ring or chain associations along with bivalents and some univalents in most of the PMCs. Besides this, 19 PMCs showed hexavalents with eight bivalents. A clear dominance of rings (62.53%) over the chains (37.46%) was observed in which open rings and bi rings were approximately 27.32% and 35.21%, respectively (Table 2). Hence, the total adjacent orientations (53.16%) have preponderance over alternate orientations (46.84%). Besides octavalent and hexavalent along with bivalents, trivalents, pentavalents, heptavalents, nonavalents, and decavalents were also screened out. However, the frequency of decavalents (2.66%) and nonavalents (3.19%) was less.
| Dose | Rings (%) | Chains (%) | Adjacent orientation (%) | Alternate orientation (%) | Pollen fertility (Mean±S.E.) | ||
|---|---|---|---|---|---|---|---|
| Open | Bi-ring (8-shaped) | Adjacent | Zig-zag | ||||
| Control | — | — | — | — | — | — | 98.30±0.33 |
| 100 Gy | 27.32 | 35.21 | 25.83 | 11.63 | 53.16 | 46.84 | 37.36±0.17 |
In a large percentage of PMCs the anaphase I was found to be regular. However, various abnormalities were also screened out with unequal separation (10 : 12, 9 : 13 and 8 : 14) and laggards (25.17%) being the most regnant (Fig. 1J–L, Table 3). Bridges (10.20%) were also reported at anaphase I. Pollen fertility was found to be 98.30±0.33% in control which was reduced to 37.36±0.17% in translocation heterozygote.
| Segregations | No. of PMCs | Frequency (%) |
|---|---|---|
| 11 : 11 | 42 | 28.57 |
| Unequal separations* | 53 | 36.05 |
| Laggards | 37 | 25.17 |
| Bridges | 15 | 10.20 |
| Total | 147 |
*It includes variable types of unequal separations like 10 : 12, 9 : 13 and 8 : 14.
In the present study, one induced translocation heterozygote of coriander show a mutual exchange of chromosome segments between two non-homologous. Translocation heterozygotes often had associations of chromosomes in rings and chains of octavalents during microsporogenesis, along with the bivalents and univalents suggesting a double interchange complex involving four non-homologous chromosomes, the centromeres of which are oriented either alternately or adjacently. The prevalence of ring interchange complexes may be because of the greater length of interchanged parts as well as chiasmata associations in all the arms of the interchange chromosomes. Sybenga (1967) classified diagrammatically, four types of interchange patterns into two types of orientations. Open the ring and adjacently attached chromosomal arms were related to the adjacent orientation, while zig-zag pattern and bi-rings (8-shaped) was a consequence of alternate orientation. The alternate orientation of centromeres and their subsequent disjunction at anaphase yield balanced gametes; whereas adjacent orientation and subsequent disjunction results in unequal separation and yield unbalanced gametes, each disomic for a chromosomal segment of one of the involved chromosomes and nullisomic for a segment of the other chromosome (Ji et al. 1999). During metaphase I, non-co-orientation of the chromosomes of a translocation heterozygote can give rise to duplicate-deficient gametes following 2 : 2 segregation or aneuploid gametes following a 3 : 1 segregation (Hagberg 1954). Adjacent orientations seem to be more responsible for the formation of laggards where there is always a possibility of unequal and delayed separation (Kumar and Singh 2003). In translocation heterozygote, some PMCs showed open types of configurations that suggested the involvement of a sufficient length of the pairing segments, and few PMCs showed closed configurations which may be the result of short pairing segments (Muller 1976). In general, the percentage of alternate orientations in translocation multivalents has a direct influence on fertility percentage. However, chromosomal rearrangements can be found in the whole fertility range from 0 to 100% (Searle et al. 1974). A homogeneous proportion of alternate and adjacent orientation may lead to 50% fertility while an increment in adjacent orientations may perturb the fertility up to less than 50%. If adjacent chromosomes pass to the opposite poles, each microspore should possess a complete set and will be capable of further development however if it passes to the same pole, the resulting microspores should be sterile owing to deficiency of chromosome segment (Sax and Anderson 1932). Presumably, the involvement of more number of different non-homologous chromosomes in translocation resulted in increased meiotic disturbances through irregular segregation of the number of translocated chromosomes in a single plant. This led to much higher pollen sterility and a concomitant decrease in seed production. Induction of translocation heterozygotes through gamma rays was known in many plant species, e.g., Pennisetum typhoides (Pantulu 1967), Vicia faba (Sjodin 1971), Vinca rosea (Sudhakaran 1971), Oryza sativa (Reddi and Redi 1975), Plantago (Padha et al. 1998) and poppy (Kumar and Naseem 2012). We have reported translocation heterozygosity for the first time in coriander, it is of immense interest to study whether this characteristic will pass on to successive generation or remain as recessive. Translocation heterozygotes are of great interest as they provide a source for raising aneuploid offspring with some novel gene combinations. However, further studies, are needed to confirm this result and to identify all the chromosomes involved in translocations; for their efficient utilization in classical and molecular linkage studies in coriander.
The authors would like to express their gratitude and special thanks to CRSS for providing seeds of C. sativum to conduct this experiment successfully. Secondly, the authors would like to thank NBRI, for providing gamma irradiation facilities. One of the authors (A.P.) debts sincere thanks to all members of Plant Genetics Laboratory for their encouragement and support who helped in completing the experiment within the time limit.
