Cytogenetical Investigations in Colchicine-Induced Tetraploids of Brassica Fruticulosa: an Important Wild Relative of Cultivated Brassicas

Cytogenetical Investigations in Colchicine-Induced Tetraploids of Brassica fruticulosa: An Important Wild Relative of Cultivated Brassicas

Arun Kumar*, Binay Kumar Singh, Hari Singh Meena, Bhagirath Ram, Vijay Veer Singh and Dhiraj Singh

Directorate of Rapeseed-Mustard Research, Bharatpur-321 303, Rajasthan, India

Received October 4, 2014; accepted January 12, 2015

Summary Successful induction of autotetraploidy has been achieved in five plants of B. fruticulosa Cyr. subsp. fruticulosa (2n=16, FF), a wild relative of cultivated brassicas. The diploid seedlings of B. fruticulosa were treated with different concentrations of aqueous colchicine using the cotton-swab method for 8–15 h for 2 to 3 d. The highest percentage of success was recorded when the seedlings were treated with 0.2% aqueous colchicine for 12 h within 2 d. The synthesized plants showed remarkable enhancement in several morphological and floral characters making them more robust. Cytologically, among all the associations, bivalent chromosome associations were observed more frequently (7.20 to 8.76 per cell) in various plants. However, quadrivalent frequency ranging from 2.28 to 3.14 and univalent frequency ranging from 3.44 to 4.09 per cell were characteristic of the colchicine-induced tetraploids. The number of chiasmata ranged from 15 to 36 with an average number of 21.28 and 23.60 and their terminalisation coefficient ranged between 0.73 and 0.86. The anaphase I and II disjunction of bivalents/chromosomes was leading more or less regularly and equally to the formation of seeds from the synthesized plants. Significant enhancement in morphological traits as revealed in colchicine-induced plants and normal meiotic behaviour leading to a good seed set may ultimately result in providing the plant breeder with more variability. The synthesized autotetraploids will have great opportunities to be utilized in hybridization with Brassica juncea and related species for introgression of desirable traits, especially tolerance for mustard aphid.

Key words Chiasmata, Cytology, Induced polyploidy, Meiotic behaviour, Quadrivalent.

Brassica juncea L. Czern & Coss., commonly known as Indian mustard, is the predominantly cultivated Brassica species in India with yield potential of 15–30 q/ha and 38–42% oil content. This crop often encounters severe biotic and abiotic stresses emerging as a result of unprecedented change in environmental conditions at local, regional and global levels (Chopra et al. 1996). Amongst the biotic stresses, mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a perpetual annual threat leading to productivity losses up to 70%, depending upon the severity of outbreak (Bandopadhyay et al. 2013). Present methods of aphid control in mustard are primarily based on synthetic chemical insecticides. These chemicals, besides aggravating environmental pollution, can also be toxic to friendly insects. In this sense, a resistant cultivar is a more sustainable and environment-friendly option. The development of an insect-resistant cultivar requires a heritable and transferable resistance (Stoner and Shelton 1988). However, cultivated Brassica germplasm has failed so far to provide any source of resistance against mustard aphid. Fortunately, Brassica coenospecies has been bestowed with nearly 100 species of wild and weedy relatives, possessing extensive genetic variability for many defence traits. These species can be effectively utilized to

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.223 224 A. Kumar et al. Cytologia 80(2) introduce economically important traits to cultivated species as well as development of potential wide hybrids (Prakash 2001, Singh et al. 2011, Singh et al. 2012). Brassica fruticulosa Cyr., a wild relative of crop brassicas, has been reported to possess resistance against cabbage aphid, Brevicoryne brassicae (Cole 1994a, Ellis and Farrell 1995, Ellis et al. 2000) and a higher concentration of lectins was suggested to be the underlying mechanism of resistance in this species (Cole 1994b). Earlier we have reported the synthesis of an inter-specific hybrid between B. rapa (2n=20, AA) and B. fruticulosa (2n=16, FF) through sexual hybridization (Kumar et al. 2013). However, inter-specific hybridization between homoploid B. fruticulosa and heteroploid B. juncea (2n=36, AABB) could not be achieved. It is evident that when heteroploid species with different ploidy levels are crossed, hybridization between them is relatively more difficult than when species with the same ploidy level are mated. Keeping these in view, present investigations are undertaken to induce polyploidy, particularly colchi-autotetraploidy in B. fruticulosa using aqueous colchicine, and their subsequent utilization in inter-specific hybridization with B. juncea. To the best of our knowledge, this is the first report of synthesis and utilization of autotetraploid B. fruticulosa in Brassica crop improvement programmes.

Materials and methods

Seed samples of B. fruticulosa were obtained from the germplasm section of the Directorate. The seeds were directly sown in earthen pots filled with peat, perlite and sand (1 : 1 : 1, v/v/v). Cotton swab method was employed for colchicinization. Sterilized cotton swabs immersed either in 0.1 or 0.2% aqueous colchicine were placed on emerging apical tip between two cotyledonary leaves. To avoid an increase in the concentration, colchicine was added drop by drop at regular intervals on the cotton swabs with the help of sterilized syringe. Such treatment was done for 2 to 3 d for 3 to 4 h per day. The data on various morphological attributes such as plant height, primary and secondary branches, main shoot length, leaf length, siliqua length, length and breadth of petals, etc. of both diploid and colchicine-induced plants were recorded by physical measurements with centimetre scale or micrometre as the case may be. For meiotic studies flower buds of an appropriate size were collected from selected mature plant and fixed on the spot in freshly prepared carnoy’s fluid (ethanol : chloroform : acetic acid̶6 : 3 : 1), supplemented with a drop of ferric chloride solution, for a minimum of 24 h at room temperature and subsequently stored in 70% alcohol at 10°C. Anthers were squashed in 1% acetocarmine solution with ferric chloride solution as mordant. On average 25 Pollen Mother Cells (PMC) were analyzed at diakinesis/metaphase I to estimate the range of chromosome associations and recombinational frequencies by chiasma analysis. At anaphases I/II on average 15–20 cells were analysed to study the distributional pattern of chromosomes.

Results

Efficiency of colchicine treatment B. fruticulosa seedlings at cotyledonary leaves stage were treated with aqueous colchicine (0.1% and 0.2% v/v) for 2 to 3 d for 3 to 5 h per day. The highest induction percentage (15.79%) of putative polyploidy was recovered with 0.2% aqueous colchicine (counted as the number of colchicine-induced tetraploids recovered from total number of seedlings treated with aqueous colchicine) (Table 1). A total of ﬁve colchicine-induced putative polyploids were synthesized. These plants are numbered as P1, P2, P3, P4 and P5, while diploid plants are numbered as P. 2015 Cytogenetical Investigations in Colchicine-Induced Tetraploids of Brassica fruticulosa 225

Table 1. Efﬁciency of colchicine treatment in inducing polyploidy by cotton swab method in B. fruticulosa.

Colchicine Duration of No. of days of No. of seedlings No. of seedlings No. of colchicine- Percentage of concentration (%) treatment (h) treatment treated survived induced tetraploids tetraploids

0.1 12 3 30 22 1 4.54 15 3 20 8 ̶ 0.0 0.2 8 2 25 12 1 8.33 10 2 25 19 3 15.79

Table 2. Morphological features of diploid and colchicine-induced tetraploids of B. fruticulosa.

Sample P P1 P2 P3 P4 P5 Character No. 2x 4x 4x 4x 4x 4x

1. Plant height (cm) 58.5 48.5 60 49.2 51.6 36.5 2. Primary branches per plant 5 5 5 4 3 4 3. Secondary branches per plant 6 10 8 12 5 8 4. Main raceme length (cm) 32.7 25 35 25.5 29.2 28.4 5. Length of petiole (cm) 2.3 2.5 2.6 2.4 2.5 2.3 6. Length of odd leaﬂet (cm) 8.5 12.3 13.6 11.7 12.0 9.4 7. Width of odd leaﬂet (cm) 6.2 8.5 7.8 8.1 7.6 6.8 8. Corolla length (cm) 1.0 1.3 1.5 1.4 1.3 1.2 9. Corolla width (cm) 0.5 0.6 0.8 0.7 0.6 0.5 10. Siliqua length (cm) 3.0 3.4 3.5 3.2 3.4 3.8 11. Seeds per siliqua (mean) 16 4 10 7 6 7

Figs. 1–3. Comparison of morphological attributes in diploid and colchicine-induced tetraploids of B. fruticulosa, Fig. 1. Leaf, 2. Flower, 3. Siliquae.

Morphology All the five colchicine-induced plants were robust from the initial stages of development and it was maintained till maturity, although different responses to colchicine were evident in all the five plants (Figs. 1–3, Table 2). All the colchicine-induced tetraploid plants showed reduction in plant height except one (P2) when compared with diploids. Secondary branches per plant, length of petiole, length and width of leaflet, corolla length and width and siliqua length showed considerable increase in comparison with the corresponding diploids. As for primary branches per plant in tetraploids, three plants (P3, P4 and P5) showed reduction, whereas in two plants (P1 and P2) numbers of primary branches per plant were found to be similar to diploids. The number of seeds per siliqua decreased in all the synthesized colchicine-induced polyploid plants. Among the five colchicine-induced tetraploid plants, P2 showed considerable increase in all the morphological attributes accept seeds per siliqua which were comparability less and primary branches per plant which was equal to the diploid plants (Table 2). 226 A. Kumar et al. Cytologia 80(2)

Table 3. Average number and range of chromosome associations at diakinesis/metaphase I in diploid and colchicine-induced tetraploids of B. fruticulosa.

No. of cells Quadrivalents Bivalents Univalents Plant No. Ploidy analyzed Mean Range Mean Range Mean Range

P 2x 25 ̶ ̶ 7.60±0.69 6–8 0.80±1.39 0–4 P1 4x 25 2.60±0.20 1–4 8.44±0.27 7–10 3.50±0.62 0–10 P2 4x 25 2.28±0.16 1–3 8.76±0.68 7–12 3.44±0.49 0–8 P3 4x 22 3.14±0.26 1–5 7.20±0.18 6–8 4.09±0.64 0–10 P4 4x 25 2.52±0.16 1–4 7.24±0.22 6–9 3.76±0.62 0–12 P5 4x 25 2.72±0.19 1–4 8.16±0.28 6–10 3.68±0.60 0–10

Table 4. Average number and frequency of multivalents in colchicine-induced tetraploids of B. fruticulosa.

No. of cells No. of cells with multivalent frequency of Mean number of Plant No. 2n analyzed 1 2 3 4 5 multivalent per cell

P1 4x 25 3 10 6 6 ̶ 2.60 P2 4x 25 5 8 12 ̶ ̶ 2.28 P3 4x 22 2 5 6 6 3 3.14 P4 4x 25 2 8 12 3 ̶ 2.52 P5 4x 25 2 10 6 7 ̶ 2.72

Table 5. Mean number, range of chiasmata and terminalization coefﬁcient in diploid and colchicine- induced tetraploids of B. fruticulosa.

Chiasmata No. of cells Terminalization Plant No. Ploidy Total Terminalized Unterminalized analyzed coefﬁcient Mean Range Mean Range Mean Range

P 2x 25 15.04±1.46 12–16 13.12±2.14 9–16 1.92±1.76 0–5 0.87 P1 4x 25 21.28±4.10 16–30 18.20±2.74 15–22 3.08±3.67 0–13 0.86 P2 4x 25 21.80±4.71 16–33 9.49±3.41 14–30 3.36±3.30 0–12 0.85 P3 4x 22 22.18±6.17 15–40 17.77±4.79 10–34 4.41±3.34 0–11 0.80 P4 4x 25 22.76±5.35 18–36 17.08±5.21 10–30 5.68±3.07 2–12 0.75 P5 4x 25 23.60±5.69 20–36 17.32±5.55 8–30 6.28±3.33 1–14 0.73

Cytology of diploid and colchicine-induced tetraploids Meiotic data of diploid and colchicine-induced tetraploid plants has been summarized in Tables 3–6 and some representative cells are illustrated in Figs. 4–19. From the colchicine-induced polyploids, five plants were found to be tetraploid upon cytological investigations. P: In diploid B. fruticulosa, the majority of Pollen Mother Cells (PMCs) analyzed at diakinesis/metaphase I showed normal bivalent formation and few univalents were encountered. However, their number never exceeded four per cell. The average number of chromosome associations was 7.60II+0.80I. The mean number of chiasmata per cell was 15.04 out of which 13.12 were terminalized giving terminalisation coefficient of 0.87. The equal distribution of chromosomes (8 : 8) was observed in all the cells analyzed at anaphase I (Tables 3, 5, 6) P1: The average chromosome association determined for this plant was 2.60IV+8.44II+3.50I per cell, their range being 1–4, 7–10, 0–10, respectively. Out of 25 cells analyzed, the maximum number of quadrivalents observed per cell was two, besides having other chromosome associations. The chiasmata per cell ranged from 16 to 30, their mean being 21.28, out of which 18.20 were terminalized giving the terminalization coefficient of 0.86. Four out of 15 cells analyzed at 2015 Cytogenetical Investigations in Colchicine-Induced Tetraploids of Brassica fruticulosa 227

Table 6. Anaphase I distribution in diploid and colchicine-induced tetraploids of B. fruticulosa.

Plant No. 2n No. of cells analyzed Chromosome distribution No. of cells Percentage

P 16 15 8 : 8 15 100.00 P1 32 15 16 : 16 11 73.33 15 : 17 2 13.33 14 : 18 2 13.33 P2 32 15 16 : 16 14 93.33 15 : 17 1 6.67 P3 32 20 16 : 16 12 80.00 14 : 2U : 17 3 20.00 P4 32 15 16 : 16 15 100.00 P5 32 15 16 : 16 12 80.00 15 : 1U : 16 3 20.00

Figs. 4–19. Documentation of cytological examinations in diploid and colchicine-induced tetraploids of B. fruticulosa, Figs. 4–7. B. fruticulosa diploid, Diakinesis 4. 8II, 5. 7II+2I, 6. Metaphase I 8II, 7. Anaphase I 8 : 8. 8–19 B. fruticulosa tetraploids, 8, 9. Diakinesis 8. 4IV+8I, 9. 2IV+10II+4I, 10–15. Metaphase I 10. 3IV+8II+4I, 11. 3IV+9II+2I, 12. 3IV+7II+6I, 13. 1IV+9II+10I, 14. 4IV+7II+3I, 15. 2IV+11II+2I, 16–19. Anaphase I 16, 17. 16 : 16 (equal distribution), 18, 19. Anaphase I with laggards (quadrivalents marked by arrow heads and laggards with arrow). Bar: 10 μm. 228 A. Kumar et al. Cytologia 80(2) anaphase I had the unequal distribution (15 : 17, 14 : 18) of chromosomes without any lagging univalent or bivalent. The remaining cells had the equal distribution of chromosomes (16 : 16) indicating the course of meiosis was normal (Tables 3–6). P2: All the cells analyzed had the average association in the form of 2.28IV+8.76II+3.44I per cell and their average was 1–3, 7–12, 0–8, respectively. Maximum numbers of three quadrivalents per cell were observed in 12 cells, besides other having mixture of both bivalents and univalents. The mean number of terminalized chiasmata ranged from 14 to 30 giving terminalisation coefficient of 0.85. The distribution of chromosomes at anaphase I was found to be equal in 14 cells and only one cell had unequal distribution of 15 : 17 (Tables 3–6). P3: In this plant the mean number of quadrivalents and bivalents was 3.14 and 7.20, respectively, and that of univalents was 4.09 giving an average of 3.14IV+7.20II+4.09I per cell. The chiasma frequency per cell ranged 15 to 40 and mean number was 22.18. The mean number of terminalized chiasmata ranged from 10 to 34 giving terminalisation coefficient of 0.80. The maximum number of cell showed the occurrence of either three or four quadrivalents, besides other chromosome associations. The distribution of chromosomes (16 : 16) at anaphase I was found in 12 cells and three cells had unequal distribution of lagging two univalents (Tables 3–6). P4: The average association in this plant was observed as 2.52IV+7.24II+3.76I per cell which was in the range of 1–4, 6–9, 0–12, respectively. Out of 25 cells analyzed, 12 cells were observed to consist of three quadrivalents per cell in addition to other chromosome associations. The average number of chiasmata per cell was 22.76, ranging from 18 to 36. Interestingly, in this plant all the cells analyzed at anaphase I had shown equal distribution of chromosomes (Tables 3–6). P5: All the cells analyzed had the average association in the form of 2.72IV+8.16II+3.68I per cell and their range being 1–4, 6–10, 0–10, respectively. Out of 25 cells, 10 cells had two quadrivalents besides mixture of both bivalents and univalents. The chiasmata per cell ranged from 20 to 36, their mean being 23.60, in which 17.32 were terminalized giving the terminalization coefficient of 0.73. Eleven out of 15 cells analyzed at anaphase I had the equal distribution of chromosomes (16 : 16). One cell had unequal distribution (15 : 17) with or without any laggard and two cells had unequal distribution with the presence of lagging univalents (Tables 3–6).

Discussion

Induction of polyploidy using colchicine and their cyto-morphological and genetic characterization has been a subject of immense interest among geneticists and breeders for a long time (Otto and Whitton 2000, Reiseberg 2001, Ramsey and Schemske 2002, Mable 2003). In the present investigation, some morphological features of the colchicine-induced autotetraploids, viz. secondary branches/plant, length of petiole, length and width of leaflet, corolla length and width and siliqua length, etc., showed an increase in dimension while few characters, viz. primary branches per plant and seed per siliqua, followed a reverse trend. Among the tetraploid plants, P2 showed considerable increase in all the morphological attributes except seeds per siliqua and primary branches per plant which were comparable to the diploid plants. Such observations have been previously demonstrated in a large number of plant species (Srivastava and Raina 1983, Mishra 1986, Bewal et al. 2009). Application of colchicine beyond the threshold limits may also have attributed to a negative effect on morphological features of the plants (Lawton-Rauh 2003). In autotetraploidy, each chromosome theoretically represents itself four times, due to which mostly the quadrivalent associations would be expected. In earlier literature it has been stated that the frequency of quadrivalents in comparison with bivalents shows gradation from high quadrivalent associations to perfect bivalent formations (Zadoo et al. 1975, Pal and Khoshoo 1977, Madhusoodanan and Arora 1979). Sometimes quadrivalent frequency per cell shows considerable variation even at intraspecific level (Gupta and Sinha 1978). In the present investigations, meiotic 2015 Cytogenetical Investigations in Colchicine-Induced Tetraploids of Brassica fruticulosa 229 analysis of synthesized tetraploid plants of B. fruticulosa showed low quadrivalent frequency and high bivalent average per cell. The highest mean value was observed in P3 (3.14) and ranged from one to five, while in most of the PMCs they ranged between one and four. The low quadrivalent average has been attributed to several factors. It is suggested by few workers (Pal and Pandey 1982, Pandey 1993) that autotetraploids containing small chromosomes have low quadrivalent frequency than the plants with larger chromosomes. Low frequency of quadrivalents may be possibly due to structural changes (heterozygosity) in the initial material. The heterozygosity may reduce the qudrivalent frequency in colchitetraploids to considerable extent because it is well known that even small cryptic structural changes are considered to trigger the preferential pairing. The maximum mean value (23.60) of chiasma frequency was observed in P5 and the minimum (21.28) was observed in P1. These high values of terminalized chiasma frequency indicate that the chiasmata were localized rather than randomized (Kumar and Rao 2002). Due to the presence of localized chiasmata, quadrivalents were observed mostly in the form of rings, whereas chain quadrivalents were present in low frequencies. Localized chiasmata are considered to reduce the formation of multivalents in autoploids (Srivastava and Raina 1983, Bewal et al. 2009). Similar, observations were made in present study also. All the induced tetraploids had some cells at anaphase I with unequal distribution and/or lagging bivalents or univalents one plant, viz. P4 resulting in high viable seed set. It is obvious that the seed set in the present material is not dependent upon quadrivalent frequency but may be due to the chromosomal abnormalities occurring in significant numbers during anaphases I/II (Sybenga 1972, Leitch and Bennett 1997). A maximum of four colchicine-induced tetraploid plants were recovered in 0.2% and only one plant in 0.1% colchicine, and all the recovered plants produced few seeds. The highest seed set (10) was observed in P4, and the lowest (4) in P1. Seeds obtained from the tetraploid plants few were shrivelled and shrunken. Partial or total seed sterility is one of the commonest features in colchicine-induced tetraploids and there are only few reports where the seed set was equivalent or slightly lower than in their respective diploids (Singh 1993). The production of nonviable and unbalanced gametes which arise due to meiotic anomalies, viz. mis-disjunction of multivalent, lagging bivalents or univalents and other abnormalities, were found to be the main reasons for partial or total seed sterility (Srivastava and Raina 1983) whereas some authors stated contrary to these views that cytological cause might be the sole criterion for seed sterility (Srivastava and Raina 1983, Bewal et al. 2009). As already mentioned earlier, when species with different ploidy levels are crossed, hybridization between them is relatively more difficult than when species with the same ploidy level are mated. Researchers have developed induced polyploids with the ultimate goal of making crosses not possible at the original ploidy level of the species in ornamentals (Dhooghe et al. 2009). However, reports of successful implementation of this technique in Brassica crops are lacking. Colchicine-induced tetraploid plants obtained in the present investigations are ideal for further utilization in the hybridization programme with B. juncea and other related species for introgression of traits of economic importance, especially mustard aphid in crop improvement programmes of brassicas.

Acknowledgments

We sincerely acknowledge Director, Directorate of Rapeseed-Mustard Research, Sewar, Bharatpur-321 303 (Rajasthan) India, for providing ﬁnancial support and the facilities to carry out this research work. 230 A. Kumar et al. Cytologia 80(2)

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