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. 2019 Jan 7;19:10. doi: 10.1186/s12870-018-1596-7

Meiotic abnormalities affect genetic constitution and pollen viability in dicots from Indian cold deserts

Dalvir Kaur 1,✉, V K Singhal 2
PMCID: PMC6323802  PMID: 30616590

Abstract

Background

Meiotic abnormalities lead to morphological and genetic variations which caused not only to evolution but also intraspecific reproductive barriers. During present study of detailed meiotic course in dicotyledonous plants sampled from Indian cold deserts, various meiotic abnormalities have been detected. For this, the plant materials fixed in Carnoy’s fixative and studied detailed meiotic course by standard squash method in 1% acetocarmine.

Results

Meiotic abnormalities have been presently detected in 71 species which include multiple associations in diploids (Achillea millefolium L.), multivalents and univalents in polyploids (4 species), cytomixis (40 species), chromosome stickiness (20 species), nonsynchronous disjunction of bivalents (32 species), interbivalent connections (15 species), synaptic mutants (2 species), syncyte meiocytes (2 species), abnormal spindles (7 species), and fusion of pollen grains (1 species), laggards and chromatin bridges, hypo-, hyperploid PMCs, monads, dyads, triads, tetrads with micronuclei and polyads.

Conclusions

Consequently, variable sized apparently fertile pollen grains and considerable amount of sterile pollen grains are resulted as end products which lead to different genetic constitution (aneuploids and polyploids) and curtailed sexual reproductive success in these species.

Keywords: Meiosis, Meiotic abnormalities, Pollen grains, Aneuploids, Polyploids, Sexual reproduction

Background

Meiosis, [1] a critical process has a key role in the reproduction and life cycle of flowering plants involves homologous chromosomes pair, synapsis, recombination and segregation that reduces the chromosome number by half and ensures the operation of Mendel’s law of heredity [2]. The normal and harmonious course of meiosis in pollen mother cells including regular bivalent formation and normal cytokinesis ensures 100% pollen viability [3]. Any abnormality in course of meiosis causes the formation of sterile gametes and low percentage of pollen viability [4]. An array of genes is known to involve in each and every step of meiotic process [3, 5–7]. Further alongwith genic factors, various environmental factors determine the harmony of this process [8]. To investigate the evolutionary trends occurring in the species, a detailed meiotic analysis could be a strong parameter which includes the nature of chromosomes pairing, chromosome behaviour during segregation and microsporad formation [9]. Formation of genetically different gametes due to abnormal meiotic processes could lead to effective reproductive barriers between the species [10]. During the evaluation of meiotic behavior in the dicot plants sampled from Kinnaur district of Himachal Pradesh (India) which is known for its rugged mountains, cold deserts, high altitudes, harsh climatic conditions and remote district in India, 71 species showed various meiotic abnormalities which curtailed the considerable amount of pollen fertility and led to the production of heterogeneous sized fertile pollen grains.

Results

In our present studies total 71 taxa (22 polyploids and 49 diploids) showed abnormal meiotic abnormalities (Table 1) which include chromosomal multiple associations in diploids and polyploids, chromatin stickiness, deviant pairing and disjunction of bivalents, cytomixis, amalgamated meiocytes, abnormal spindle, laggards and chromatin bridges, anomalous sporads, and united pollen grains. Inconstant sized fertile gametes and significant pollen sterility are the final outcome of all these abnormalities in this present study.

Table 1.

A list of cytologically investigated species with locality, accession number, meiotic chromosome number, meiotic abnormalities and pollen grains

Taxon Locality (altitude in metre) Accession number (PUNa) MCN (Ploidy level) Meiotic irregularities Pollen grains
C CS U M AM ND L CB AbM PS HSPG FPG
1. FAMILY: RANUNCULACEAE
Anemone rivularis Buch.-Ham. ex DC. Kalpa, 2760 50898 8 (2x) + + - - - - + + + + + -
2. Aquilegia fragrans Benth. Chittkul, 3450 53994 7 (2x) + - - - - - - - - - - -
3. Clematis grata Wall. Bhabnagar, 1900 50905 8 (2x) + + - - - - + + + + - -
4. C. graveolens Lindl. Thangi, 2700 53858 8 (2x) + + - - - + + + + + + +
5. C. orientalis L. var. acutifolia Hook. f. et Thoms. Nako, 3660 49994 16 (4x) + + - - - + + + + + + -
6. Delphinium roylei Munz Sangla, 2680 53998 8 (2x) - - - - - + - - - - - -
7. Ranunculus laetus Wall. ex Royle Sangla, 2680 50946 14 (4x) + + - - - + + + + + + -
8. R. sceleratus L. Nichar, 2150 54006 16 (4x) - + - - - + + + + + + -
9. Thalictrum cultratum Wall. Nichar, 2150 53847 21 (4x) + - - - - + + + + + + -
10. T. foetidum L. Sangla, 2680 50947 21 (6x) + - - - - - + + + + + -
11. T. minus L. Chittkul, 3450 53887 7 (2x) + - - - - - - - - - - -
12. FAMILY: BERBERIDACEAE
Berberis kunwarensis Royle Sangla, 2680 54025 14 (2x) - + - - - + - + - - - -
13. FAMILY: PAPAVERACEAE
Papaver dubium L. Sangla, 2680 50952 14 (4x) - - - - - + + + + + + -
14. FAMILY: CARYOPHYLLACEAE
Dianthus angulatus Royle ex Benth. Sangla, 2680 50486 15 (2x) + - + - + - + - + + + -
15. Myosoton aquaticum (L.) Moench Nichar, 2150 53734 14 (2x) - + - - - - - - - - - -
16. Silene edgeworthii Bocquet Sangla, 2680 50961 12 (2x) + - - - - - - - + + + -
17. Spergularia diandra (Guss.) Heldr. & Sart. Chango, 3050 53741 18 (4x) + + - - - - + - + + + -
18. FAMILY GERANIACEAE
Geranium pratense L. Chittkul, 3450 50924 28(4x) - + - + - - - - + + + -
19. FAMILY: BALSAMINACEAE
Impatiens brachycentra Kar. et Kir. Reckong Peo, 2670 50888 7 (2x) - - - - - + - - - - - -
20. FAMILY: PAPILIONACEAE
Astragalus grahamianus Royle ex Benth. Rakchham, 3115 53673 8 (2x) + - - - - - + - + + - -
21. A. graveolens Buch.-Ham. ex Benth. Sangla, 2680 50900 8 (2x) + + - - - - + + + + + -
22. Colutea nepalensis Sims Khab, 2800 53684 8 (2x) - - - - - - + + + - -
23. Indigofera heterantha Wall. ex Brandis Rakchham, 3115 50912 24 (6x) + - - - - - - - - + - -
24. Lotus corniculatus L. Chitkul, 3450 53690 6 (2x) + - - - - - + + + + - -
25. Medicago falcata L. Thangi, 2700 53695 8 (2x) + - - - - - - - + + - -
26. Melilotus alba Lamk. Thangi, 2700 53699 8 (2x) + - - - - - - - + + - -
27. Trifolium repens L. Sangla, 2680 50916 16 (4x) + - - - - - - - + + - -
28. Trigonella emodi Benth. Chitkul, 3450 53713 8 (2x) + - - - - - - - + + + -
29. T. pubescens Edgew. ex Baker Rakchham, 3115 53716 8 (2x) + - - - - + + + + + + -
30. Vicia pallida Turcz. Palingi, 1900 53708 12 (4x) + + - - - + + + + + + -
31. V. rigidula Royle Kalpa, 2760 50919 12 (4x) + + - - - + + + + + + -
32. V. sativa L. Kuppa, 2600 53709 6 (2x) - - - - - + - + - - - -
33. V. tenera Grah. Sangla, 2680 51952 7 (2x) - - - - - + + + + + - -
34. FAMILY: ROSACEAE
Fragaria nubicola (Hook.) Lindl. ex Lacaita Chittkul, 3450 53797 7 (2x) + - - - - - - - + - - -
35. Potentilla atrisanguinea Lodd. var. atrisanguinea Chittkul, 3450 53840 28 (8x) + - - - - - - - + + + -
36. FAMILY: APIACEAE
Chaerophyllum acuminatum Lindl. Chittkul, 3450 53824 11(2x) + - - - - - + + + + - -
37. C. villosum Wall. Kuppa, 2600 53826 11 (2x) - - - - - + + - + + - -
38. Eriocycla caespitosa (Edgew.) H. Wolff Sangla, 2680 53823 11 (2x) + - - - - - + - + + - -
39. Heracleum candicans Wall. ex DC. Rakchham, 3115 53828 11 (2x) + + - - - - + + + + + -
40. SUB CLASS - GAMOPETALAE
FAMILY: ASTERACEAE
Achillea millefolium L. Chittkul, 3450 53873 9 (2x) - - - - - - + + + + - -
41. Anaphalis virgata Chittkul, 3450 53876 14 (4x) - - - - - + - - - - - -
42. Aster molliusculus Wall. Pooh, 2840 53914 9 (2x) - - - - - + - - - - - -
43. Carduus nutans L. Kamru, 2650 53893 20 (4x) - - - + - - - - - - - -
44. Cosmos bipinnatus Cav. Pangi, 2790 53892 12 (2x) - - - - - + - - - - - -
45. Crepis multicaulis Ledeb. Kothi, 2670 53897 5 (2x) + - - - - + - + + + - -
46. C. sancta (L.) Babcock Kuppa, 2600 53908 5 (2x) - - - - - + - + - - - -
47. Erigeron acer L. Sangla, 2680 53915 9 (2x) - - - - - + + - + + - -
48. E. annuus (L.) Pers. Sangla, 2680 53917 27 (3x) - + + - - - + + + + + -
49. Inula cappa DC. Palingi, 1900 53937 10 (2x) - - - - - + - + + + + -
50. I. cuspidata C.B. Clarke Tapri, 1680 52513 10 (2x) - - + - + + + + + + + -
51. Lactuca orientalis (Boiss.) Boiss. Nako, 3660 53948 9 (2x) - - - - - + + - + + - -
52. Saussurea albescens Hook. f. et. Thoms. Ropa, 3000 53969 17 (2x) - - - - - - + - - + - -
53. Senecio krascheninnicovi Schischk Nako, 3660 53961 10 (4x) + - - - - + + - + + + -
54. Taraxacum officinale F. H. Wigg. Ropa, 3000 55765 16 (4x) - - - + - - + + + + + -
55. Youngia glauca Edgew. Nichar, 2150 53896 8 (2x) - - - - - + + - - + - -
56. FAMILY: BORAGINACEAE
Cynoglossum zeylanicum (Wall.) Thunb. ex Lehm. Moorang, 2590 53835 12 (2x) + - - - - - + + + + - -
57. FAMILY: CUSCUTACEAE
Cuscuta reflexa Roxb. Ponda, 1980 54073 16 (4x) - + - - - + + + - - - -
58. FAMILY: SOLANACEAE
Datura stramonium L. Kalpa, 2760 50968 12 (2x) - - - - - - + + - + - -
59. Hyoscyamus niger L. Nako, 3660 53780 17 (2x) - - - - - - - + - - - -
60. Nicotiana tabacum L. Tapri, 1680 53784 24 (4x) + + - - - - - + + + - -
61. Physalis minima L. Ponda, 1980 53785 24 (4x) - - - - - + + - + + - -
62. FAMILY: SCROPHULARIACEAE
Leptorhabdos benthamiana Walp. Kalpa, 2760 54112 7 (2x) + - - - - + + + + + + -
63. Pedicularis bicornuta Klotzsch Sangla, 2680 50936 8 (2x) + + - - - + + + + + + -
64. FAMILY: LAMIACEAE
Calamintha clinopodium Benth. Chittkul, 3450 54125 10 (2x) + - - - - - + - - + - -
65. Mentha longifolia (L.) Huds. Pooh, 2840 54139 12 (2x) + - - - - - - + - - - -
66. Nepeta erecta (Royle ex Benth.) Benth. Kuppa, 2600 53606 9 (2x) + - - - - - - + + + - -
67. Salvia nubicola Wall. ex Sweet Sangla, 2680 50930 8 (2x) + - - - - + + + + + - -
68. Thymus linearis Benth. Kuppa, 2600 53631 13 (2x) + - - - - - + - + + + -
69. SUB CLASS - MONOCHLAMYDAE
FAMILY: PHYTOLACCACEAE
Phytolacca acinosa Roxb. Sangla, 2680 54074 36 (8x) - - - - - - + + - + - -
70. FAMILY: POLYGONACEAE Rumex hastatus D. Don Kothi, 2670 50895 9 (2x) - - - - - - - + - - - -
71. FAMILY: ELAEAGNACEAE
Hippophae rhamnoides L. Kalpa, 2760 49377 9 (2x) + + - - - + + + + + + -
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Symbol + (presence) and – (absence) of meiotic irregularities

a Code of Herbarium maintained by the Department of Botany, Punjabi University, Patiala, India as per “Index Herbariorum” by Holmgren and Holmgren (1998)

MCN Meiotic Chromosome Number; C Cytomixis; CS Chromosome Stickiness; U Univalents; M Multivalents; AM Asynaptic Mutant; ND Nonsynchronous Disjunction; L Laggard; CB Chromatin Bridge; AbM Abnormal Microspore; PS Pollen Sterility; HSPG Heterogenous Sized Pollen Grains; FPG Fused Pollen Grains

Chromosomal multiple association in diploids

Among the 49 diploid taxa, Achillea millefolium L. (n=9; 2x) have been noticed for the first time with chromosomal multiple associations which is collected from Chittkul region of Kinnaur district at the altitude of 3450m. In this species chromosomes were involved in the formation of chain, zigzag and ring type quadrivalents (Fig. 1a, b). These multivalents showed delayed segregation as comparative to normal bivalents during anaphases which become the cause of abnormal microspore formation and low pollen fertility.

Fig. 1.

Fig. 1

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Various Meiotic deformities during meiosis.  a) Achillea millefolium (2n=18); A PMC with 7II+1IV (typical ring, arrowed) at diakinesis. b) A. millefolium (2n=18); PMC with 7II+1IV (typical ring, arrowed) at M-I. c) Erigeron annuus (2n=27); A PMC with 27I at M-I. d & e) Carduus nutans (2n=40); PMCs showed multivalents (arrowed) at M-I. f) Geranium pratense (2n=56); PMC with multivalents (arrowed) at M-I. g & h) Taraxacum officinale (2n=32); PMCs showed multivalents (arrowed) at M-I. i) Two PMCs involved in chromatin transfer (arrowed) at P-I. j) Two PMCs involved in chromatin transfer (arrowed) at M-I. k) Two PMCs involved in chromatin transfer (arrowed) at A-I. l) Two PMCs involved in chromatin transfer (arrowed) at T-II. m) Group of PMCs involved in cytomixis (arrowed). n) Fusion of two monads. o) Intermicrosporal chromatin transfer among microspores of three tetrads. p) Cytoplasmic channels among microspores of a tetrad forming ring. q) Hypoploid (2n=24) and Hyperploid (2n=28) PMCs in Thymus linearis (2n=26). r) An enucleated PMC. s) A PMC at M-I showing pycnotic materials (arrowed). t) A PMC at M-I showing chromatin stickiness. u) Two PMCs at M-I showing early disjunction of bivalents (arrowed). v) A PMC at A-I showing late disjunction of chromosomes (arrowed). w) A PMC at A-I showing chromatin bridges (arrowed). x) Dianthus angulatus (2n=30); A PMC with 30I at M-I. y) Inula cuspidata (2n=20); A PMC with 20I at M-I

Chromosomal multiple association in polyploids

Four polyploids named Erigeron annuus (L.) Pers. (2n=27; 3x) (Fig. 1c), Carduus nutans L. (2n=40; 4x) (Fig. 1d, e), Geranium pratense L. (2n=56; 4x) (Fig. 1f), and Taraxacum officinale F. H. Wigg. (2n=32; 4x) (Fig. 1g, h) detected to be imbalanced polyploids having irregular pattern of pairing of chromosomes. The analysis of variable frequency of multivalents and univalents provide the knowledge about the nature of polyploidy. In Erigeron annuus, the presence of high frequency of unpaired chromosomes specified the allopolyploid nature of studied taxa. Presence of some multivalents in Carduus nutans (4x), Geranium pratense (4x) and Taraxacum officinale (4x) indicated towards the segmental allopolyploid nature in which 34.83%, 6.47% and 42-52% chromosomes were recorded to be involved in multivalent formation respectively. Resultant of which irregular chromosomes segregation during anaphases, abnormal sporad formation, unequal sized fertile pollen grains and also unstained/sterile pollen grains were observed.

Cytomixis

The phenomenon of cytomixis involving inter PMCs transfer of chromatin material had been reported to occur in 40 species (Table 2). The chromatin transfer in these species involving varying number of PMCs (2-28) takes place during all the stages of meiosis-I and II (Fig. 1i-m). Interestingly, the cytoplasmic channels showing chromatin transfer are also recorded among microspore units of sporads in Clematis orientalis var. acutifolia, Dianthus angulatus and Heracleum candicans (Fig. 1n-p). As a result of partial or complete chromatin transfer between meiocytes, hypo-, hyperploid and enucleated PMCs have been observed in majority of these species (Fig. 1q, r). In the present investigations, nucleolus also gets transferred along with the chromatin material from the donor PMC to the recipient PMC and the resultant PMC are depicted with two nucleoli as observed in Astragalus grahamianus, Salvia nubicola, Thalictrum cultratum, Trigonella pubescens, and Vicia pallida. All these taxa showed a variety of meiotic abnormalities included fragmentation and pycnosis of chromatin (Fig. 1s), interbivalent connections, irregular segregation of bivalents, aberrant microspores, and pollen sterility and heterogeneous sized fertile pollen grains in connection with this phenomenon of chromatin transfer.

Table 2.

Consolidated data on cytomixis in the presently studied species

S.No. Taxa Meiotic chromosome number (n) Ploidy level % age of PMCs with cytomixis No. of PMCs involved in cytomixis Meiotic stages Meiotic course Pollen fertility %age Pollen size
1. Anemone rivularis 8 2x 1.28 2 AI/II, TI/II Abnormal 94 Uniform
2. Aquilegia fragrans 7 2x 4.2 2-3 EPI, MI Normal 99 Uniform
3. Astragalus grahamianus 8 2x 71.27 2-group MI-TII Abnormal 92 Uniform
4. A. graveolens 8 2x 17.02-20.55 2 PI-TI Abnormal 96-97 Variable
5. Calamintha clinopodium 10 2x 7.20 2 MI, TI Abnormal 99 Uniform
6. Chaerophyllum acuminatum 11 2x 17.44 2-6 AI-TII Abnormal 96 Uniform
7. Clematis grata 8 2x 3.68 2-3 AI, TI Abnormal 88-98 Uniform
8. C. graveolens 8 2x 0.56-66.67 2-28 PI-TII Abnormal 91-98 Variable
9. C. orientalis var. acutifolia 16 4x 9.33-29.80 2-3 EPI-Tetrad Abnormal 84-94 Variable
10. Crepis multicaulis 5 2x 7.67-11.10 2-10 PI-AI Abnormal 92-96 Uniform
11. Cynoglossum zeylanicum 12 2x 1.56 2 A-I Abnormal 92 Uniform
12. Dianthus angulatus 2n=30 2x 14.65 2-4 MI-Tetrad Abnormal 58-62 Variable
13. Eriocycla caespitosa 11 2x 1.88 2-3 PI-TII Abnormal 97 Uniform
14. Fragaria nubicola 7 2x 17.45 2-8 MI-AI Normal 95 Uniform
15. Heracleum candicans 11 2x 26.53 2 EPI-Tetrad Abnormal 95-96 Variable
16. Hippophae rhamnoides 9 2x 7.95-43.90 2-4 EPI-TI Abnormal 93-99 Variable
17. Indigofera heterantha 24 6x 21.11-26.00 2-3 EPI-TI Normal 98-99 Uniform
18. Leptorhabdos benthamiana 7 2x 9.45 2 MI Abnormal 96 Variable
19. Lotus corniculatus 6 2x 18.34 2-3 PI-TII Abnormal 88 Uniform
20. Medicago falcata 8 2x 15.43-52.11 2-4 EPI-MI Abnormal 89-92 Uniform
21. Melilotus alba 8 2x 8.67 2-3 EPI-MI Abnormal 95-96 Uniform
22. Mentha longifolia 12 2x 2.67 2-5 MI Normal 99 Uniform
23. Nepeta erecta 9 2x 8.11-14.17 2-group EPI Abnormal 92-98 Uniform
24. Nicotiana tabacum 24 4x 22.80 2-3 EPI-MI Abnormal 87 Uniform
25. Pedicularis bicornuta 8 2x 11.93-31.63 2 MI-TI Abnormal 96-97 Variable
26. Potentilla atrisanguinea var. atrisanguinea 28 8x 38.56 2-5 EPI Abnormal 94-96 Variable
27. Ranunculus laetus 14 4x 22.51-24.05 2-4 EPI-MI Abnormal 89-90 Variable
28. Salvia nubicola 8 2x 26.02-30.88 2-4 EPI-MI Abnormal 96-97 Uniform
29. Senecio krascheninnicovi 10 4x 18.29 2-11 MI-TI Abnormal 96 Variable
30. Silene edgeworthii 12 2x 13.87 2-3 AI-TII Abnormal 83 Variable
31. Spergularia diandra 18 4x 2.87 2 MI, TII Abnormal 92 Uniform
32. Thalictrum cultratum 21 6x 47 2-3 EPI-MI Abnormal 91-94 Variable
33. T. foetidum 21 6x 1.70-43.20 2-6 EPI-TII Abnormal 78-98 Variable
34. T. minus 7 2x 2.04 2-3 EPI-MI Normal 98 Uniform
35. Thymus linearis 13 2x 26.45 2-3 MI-TI Abnormal 93 Variable
36. Trifolium repens 16 4x 5.34-6.02 2-4 MI, AI Normal 90-91 Uniform
37. Trigonella emodi 8 2x 15.88-17.24 2-3 EPI-AI Abnormal 93-94 Variable
38. T. pubescens 8 2x 47.05-62.88 2-group EPI-TII Abnormal 38-54 Variable
39. Vicia pallida 12 4x 1.52-2.20 2-3 AI/II, TI Abnormal 83-89 Variable
40. V. rigidula 12 4x 6.52 2-3 EPI, TI Abnormal 80-81 Variable
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Chromosome stickiness

The phenomenon of chromosome stickiness causing the chromosome aggulination or sticky appearance of chromosomes reported for the first time in maize [11] and attributed it to a mutation caused by a recessive gene called sticky (st). Chromosome stickiness was presently scrutinized more frequently in the PMCs during M-I in Anemone rivularis (n=8), Astragalus graveolens (n=8), Berberis kunwarensis (n=14), Clematis grata (n=8), C. graveolens (n=8), C. orientalis var. acutifolia (n=16), Cuscuta reflexa (n=16), Erigeron annuus (2n=27), Geranium pratense (n=28), Heracleum candicans (n=11), Hippophae rhamnoides (n=9), Lotus corniculatus (n=6), Myosoton aquaticum (n=14), Nicotiana tabacum (n=24), Pedicularis bicornuta (n=8), Ranunculus laetus (n=14), R. sceleratus (n=16), Spergularia diandra (n=18), Vicia pallida (n=12) and Vicia rigidula (n=12) (Fig. 1t). In these species, the severe chromatin stickiness enhanced the formation of pycnotic nuclei and delayed separation of bivalents at A-I/II, laggards and chromatin bridges, micronuclei, and ultimately resulted into sterile pollen grains.

Nonsynchronous disjunction

Nonsynchronous disjunction of bivalents (early and late) is either found in hybrid taxa or the species having different sized chromosomes and rate of chiasma terminalization [12, 13] or associated with other meiotic abnormalities [14]. Some of the presently studied species showed either precocious disjunction (Ranunculus laetus, n=14; Impatiens brachycentra, n=7; Chaerophyllum villosum, n=11; Aster molliusculus, n=9; Cosmos bipinnatus, n=12; Crepis multicaulis, n=5; Salvia nubicola, n=8) (Fig. 1u) or late disjunction (Clematis graveolens, n=8; C. orientalis var. acutifolia, n=16; Delphinium roylei, n=8; Papaver dubium, n=14; Trigonella pubescens, n=8; Vicia pallida, n=12; V. rigidula, n=12; V. sativa, n=6; V. tenera, n=7; Anaphalis virgata, n=14; Crepis sancta, n=5; Erigeron acer, n=9; Inula cuspidata, n=10; Lactuca orientalis, n=9; Youngia glauca, n=8; Cuscuta reflexa, n=16; Physalis minima, n=24; Leptorhabdos benthamiana, n=7; Pedicularis bicornuta, n=8; Hippophae rhamnoides, n=9) (Fig. 1v, w) of some bivalents. On the other hand, Ranunculus sceleratus, n=16; Thalictrum cultratum, n=21; Berberis kunwarensis, n=14; Inula cappa, n=10; and Senecio krascheninnicovi, n=10 showed both early and late disjunction of 1-2 bivalents.

Early disjunction of bivalents normally does not affect the normal distribution of chromosomes at A-I. While the late disjuction of bivalents in Ranunculus sceleratus, Papaver dubium, Vicia rigidula, V. sativa, Erigeron acer, Inula cappa, I. cuspidata, Lactuca orientalis, Youngia glauca, Cuscuta reflexa and Physalis minima causes lagging of chromosomes, chromatin bridges and consequently reduced pollen fertility [4, 15, 16].

In Delphinium roylei, Vicia pallida, V. sativa, Inula cappa, I. cuspidata, Cuscuta reflexa, Lactuca orientalis and Youngia glauca, larger sized bivalents scrutinized with decelerated segregation at A-I/II. In Delphinium roylei, Vicia sativa, and Cuscuta reflexa, this abnormality does not seem to affect the pollen fertility but in Vicia pallida, Inula cappa, I. cuspidata, Lactuca orientalis and Youngia glauca, late disjunction have resulted in the presence of laggards which may originate micronuclei at telophase-I/II and leading to reduced pollen fertility.

Asynaptic mutants

The lack of chromosome pairing and inability to generate or retain chiasmata during P-I led to asynaptic and desynaptic mutant formation. In Dianthus angulatus (2n=30) (Fig. 1x) and Inula cuspidata (2n=20) (Fig. 1y), chromosomes remain unpaired as univalents which disorganized into more than standardized number of chromatin poles during segregation of chromosomes at A-I/II. A high frequency of aberrant microspores including monads, dyads, triads, tetrads with micronuclei and polyads, sterile gametes and heterogeneous sized pollen grains are the major consequences.

Syncyte PMCs

The fusion of two or more meiocytes presently scrutinized in Clematis graveolens (2n=16) (Fig. 2a) and Dianthus angulatus (2n=30) during the early stages of meiosis-I. However the occurrence of these synmeiocyte was found to be at very low frequency. These PMCs with double content of genome undergo the meiosis and led to the formation of polyploid gametes. In both cases, the male gametes are larger in size and well fertile and able to fertilize the female gamete and might play significant role in the origin of intraspecific polyploids.

Fig. 2.

Fig. 2

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Various Meiotic deformities during meiosis. a) Clematis graveolens (2n=16); a syncyte PMC (arrowed). b) Inula cuspidata (2n=20); chromosomes disorganized into unequal sized 3 groups at A-I. c) Geranium pratense (2n=56); chromosomes disorganized into unequal sized 8 groups at A-II. d) Dianthus angulatus (2n=30); chromosomes disorganized into unequal sized 8 groups at T-II. e) A group of dyads with and without micronuclei. f) A monad with two micronuclei. g & h) Triad. i) A tetrad with one micronucleus (arrowed). j) A pentard. k) A polyad. l) Fertile and Sterile pollen grains. m) Apparently fertile heterogeneous sized pollen grains. n) Fused fertile pollen grains. o) Fused sterile pollen grains

Abnormal spindle

Spindle organization were found to be abnormal during meiosis in Colutea nepalensis (2n=16), Dianthus angulatus (2n=30), Erigeron annuus (2n=27), Geranium pratense (2n=56), Inula cuspidata (2n=20), Papaver dubium (2n=28) and Taraxacum officinale (2n=32). Here, the chromosomes fail to assemble on the spindle fibres during metaphases which is the essential property for their synchronous segregation, so they finally congregate into disparate sized chromatin nuclei at anaphases and telophases (Fig. 2b-d). As a consequence, abnormal microspores (Fig. 2e-k) are resulted which lead to low pollen fertility (Fig. 2l) and pollen grains of heterogeneous sizes (Fig. 2m).

Fused Pollen grains

Fusion among 2-3 pollen grains resulting into large sized pollen grains have been observed in Clematis graveolens (n=8) (Fig. 2n, o). In this species, 2-3 pollen grains are fused by forming cytoplasmic channels.

Discussion

Analysis of meiotic chromosome associations in diploid and polyploid plants can give precise knowledge of chromosome homology and synapsis during meiosis [17]. Inter/intraspecific hybrid origin of a plant might contemplate the level of chromosome homology. Structural heterozygosity for reciprocal translocation arguably denote the configuration of typically ring, zigzags and chains of four chromosomes in Achillea millefolium (n=9; 2x). Further, persistence of multivalents is also determined by the presence of homologous gene sequences and keep possession of chiasma in multiple associated chromosomes. Occurrence of univalents and multivalents in complex and instable polyploid genome of Erigeron annuus (2n=27; 3x), Carduus nutans (2n=40; 4x), Geranium pratense (2n=56; 4x), and Taraxacum officinale (2n=32; 4x) with distorted behaviour during gamete formation led to low reproductive success. Such chromosomal associations occur in nature at a low rate, may arise spontaneously or induced by a variety of factors including chemical or irradiation treatments [18]. The inverse correlation between frequency of chromosome associations and pollen fertility also reported in Hyoscyamus muticus [19] and Brassica campestris var. toria [20].

Cytomixis, an evolutionary, panoramic, cytological phenomenon which is a possible cause of aneuploidy and polyploidy in species [7], and produce unreduced pollen grains as reported in Hordeum species [21], Dianthus angulatus [22], and Houttuynia cordata [23] Chlorophytum borivilianum [24]. Many workers have pointed of view that the reduction in pollen viability is due to cytomixis [23, 24]. Till date, there is no clear cut opinion regarding the origin and nature of the cytomixis. Some interpretation which are thought to be responsible for cytomixis include the action of chemical agents such as colchicines [25], the use of herbicides [26], physiological and environmental factors [27], stress factors and genetic control [28]. In presently studied taxa, genetic factors and pressure of stress environment conditions seems to be the originator of this phenomenon of cytomixis [28–30].

Chromosome stickiness co-occurred with cytomixis appear to be commenced the pycnosis and degeneration of chromatin material as earlier reported by other workers [22, 31–33]. Several workers have reported that chromosome stickiness may be either under genetic control [34] or due to improper folding of chromosome fibres [35], or may also have been caused by other factors such as X-rays [36] and low temperature [37]. The presence of aluminum in the soil, besides the genetic factor, may have also caused chromosome stickiness in maize [32].

It becomes necessary for all the chromosomes to get synchronously disjucted during anaphases for the genome stability in the species. Expeditious rate of chiasma terminalization and least genic homology caused early disjunction of bivalents. Delayed segregation in Delphinium roylei, Vicia pallida, V. sativa, Inula cappa, I. cuspidata, Cuscuta reflexa, Lactuca orientalis and Youngia glauca during anaphases was due to having large sized bivalents which has also been reported in Cyathocline purpurea and Blumea spp. [38]. High chiasma frequency and their slow terminalization in large sized chromosomes might be the reason for delayed segregation.

A crucial phenomenon, synapsis, during early prophase stages is essential for gene recombination and evolutionary traits in the taxa [5]. Synaptic mutant might be due to mutations in genes controlling the chromosome pairing process and inability to generate or retain chiasmata between homologous chromosomes [5]. Several researchers have been reported for its spontaneous origin [39] and influenced by many factors like temperature, humidity and chemicals [5]. Presently the individuals of Dianthus angulatus and Inula cuspidata were growing under same climatic conditions detected with and without synaptic mutation so this make seem likely that a particular another factor might be involved. A concept of interspecific origin or non homology between two genomes could be the reason for synaptic mutation among the presently investigated taxa. A high frequency of unpaired elements in the genome of these taxa led to sterile and 2n fertile gamete formation in the end of meiosis as has also been reported in higher plants [39–41].

The spindle apparatus is normally bipolar and playing a crucial role in the accurate segregation of chromosomes during mitosis and meiosis [42]. Several mutants are known to cause failure of the spindle or impair disjunction mechanisms, like dv, ms28 and ms43 mutants as reported in maize [43]. Multipolar Spindle 1 (MPS1), a plant-specific protein which is involved in spindle organization in meiocytes has been identified in Arabidopsis thaliana [44]. Improperly aligned chromosomes on distorted spindle face either unequal or total failure of segregation during anaphases which might to be taken to lead into restitution nuclei or polyploidy or aneuploidy in resultant gametes.

Also this low temperature stress conditions prevailing in the presently investigated area might lead the amalgamation of PMCs and pollen grains. These syncyte PMCs have earlier recorded in Brachiaria jubata [45], Chrysanthemum [46], and Lindelofia longifolia var. falconeri [47]. The fusion of cell wall of pollen grains has also been recorded in intergeneric hybrids [48, 49]. The origin of intraspecific polyploidy in these taxa cannot be changed in spite of the low frequency of such fused PMCs and pollen grains.

Conclusions

Production of unreduced 2n gametes (double sized pollen grains) are the major consequence of these meiotic abnormalities in these studied taxa. Further these 2n male gametes fertilize the female gamete of respective species and led to origin of polyploidy. These all presently detected in the plants growing under the natural conditions particularly freezing temperature prevailing in the area which leads to low sexual reproductive success. The adoption of vegetative mode of reproduction may be the better regeneration method in such plants.

Methods

Collection and submission of samples

Study materials were collected from Kinnaur district of Himachal Pradesh (India) during the months of April to September for five years (2007-2011).Voucher specimens of the cytologically worked out individuals were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).

Cytological Analysis and Photomicrographs

For cytological study, young floral buds of dicot plants were fixed in carnoy’s fixative (6 ethanol: 3 chloroform: 1 acetic acid) and then stored at 4°C in 70% alcohol. Standard squash method (1% acetocarmine) was applied for observing all the meiotic stages clearly. Meiocytes, sporads and pollen grains were photomicrographed from the freshly prepared slides using Leica Qwin Digital Imaging System and Nikon Eclipse 80i microscope at laboratories situated in Department of Botany, Punjabi University, Patiala.

Acknowledgements

The authors are thankful to Head, Department of Botany, Punjabi University, Patiala (India) for providing necessary laboratories, herbarium and library facilities. The author also extends thank to Patel Memorial National College, Rajpura (Punjab) India for providing internet facility.

Funding

No funding was obtained for this study.

Availability of data and materials

All data analyzed during this study are included in this article.

Abbreviations

A-I/II

Anaphase-I/II

M-I/II

Metaphase-I/II

P-I/II

Prophase-I/II

PMC

Pollen Mother Cell

T-I/II

Telophase-I/II

Authors’ contributions

DK collected the sample, performed the experiment, analyzed the data, and interpreted the results. VKS designed the experiment. DK drafted the manuscript. DK and VK reviewed and edited the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Dalvir Kaur, Email: dalvircyto@gmail.com.

V. K. Singhal, Email: vksinghal53@gmail.com

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