Some Cytomorphological Evidence for Synthesis of Interspecific Hybrids

Some Cytomorphological Evidence for Synthesis of Interspeciﬁc Hybrids between Brassica juncea and Autotetraploid B. fruticulosa

Arun Kumar*, Hari Singh Meena, Bhagirath Ram, Priyamedha, Anubhuti Sharma, Sushma Yadav, Vijay Veer Singh and Pramod Kumar Rai

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

Received February 2, 2018; accepted July 24, 2018

Summary Four successful interspeciﬁc hybrid plants were obtained through sexual hybridization between Brassica juncea cv. Rohini, Laxmi, and Varuna (2n=4x=36, AABB), and an autotetraploid B. fruticulosa (2n=4x=32, FFFF) induced by colchicine using the latter as a pollen parent. Morphological and cytological

analyses were carried out to conﬁrm the hybrid nature of F1 plants. The F1 plants (2n=34) were intermediate for most of the morphological attributes. Although, the F1s showed poor pollen fertility, nevertheless, few seeds were obtained from open pollination. Meiotic analysis of F1 plants showed a predominance of univalents, a typical feature of hybrids. The occurrence of bivalents, trivalent, and quadrivalent in PMCs of the F1s indicated homeologous pairing among the three genomes. The study suggests that B. fruticulosa has partial genome homeology with B. juncea which could be exploited in crop improvement programmes, particularly breeding for biotic stress especially, tolerance/resistance to the mustard aphid.

Key words Homeologous pairing, Meiotic analysis, Pollen fertility, Wide hybridization, Autotetraploid, Bras- sica fruticulosa, Brassica juncea.

Indian mustard, B. juncea L. Czern & Coss. is the ative species with extensive genetic variability (Prakash predominantly cultivated Brassica species in the Indian et al. 2004). These species can be effectively utilized subcontinent with yield potential of 15–30 q ha-1 (Meena to introduce economically important traits to cultivated et al. 2014, 2015, 2017). It is a natural species allotetra- species by crossing (Prakash 2001, Singh et al. 2012). ploid between B. rapa (AA, 2n=20) and B. nigra (BB, A wild cultivated relative B. fruticulosa (2n=16, FF), 2n=16), (Nagaharu 1935). Amongst the biotic stresses, has been reported to possess resistance against cabbage mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a aphid, Brevicoryne brassicae (Cole 1994a, Ellis and Far- severe perpetual annual threat that leads damage to rell 1995, Ellis et al. 2000) and a higher concentration of the crop in the range of 9–96% across different agro- lectins suggesting the underlying mechanism of resis- climatic conditions of India depending on its severity of tance in this species (Cole 1994b). Though interspecific outbreak (Hasan and Singh 2011, Atri et al. 2012, Ban- hybrids between B. rapa (2n=20, AA) and B. fruticu- dopadhyay et al. 2013). Present methods of aphid control losa (2n=16, FF) through sexual hybridization have been in mustard cultivation are primarily based on synthetic reported (Kumar et al. 2013). However, interspecific chemical insecticides. However, the effectiveness of hybridization between allotetraoploid B. juncea (2n=36, chemical insecticides in protecting crops, however, has AABB) and diploid B. fruticulosa (2n=16, FF) could not masked the negative impacts associated with their use. be achieved. It is evident that when allotetraploid species Besides, chemicals aggravating environmental pollution are crossed with plants of different ploidy levels, hy- may also be toxic to friendly insects. In this sense, a bridization between them is relatively difficult (Prakash resistant cultivar is a more sustainable and environment- et al. 2009, Prakash 2010). friendly option. The development of an insect-resistant The present study was aimed to develop an interspe- cultivar requires a heritable and transferable resistance cific hybrid of B. juncea and B. fruticulosa (2n=4x=32, (Stoner and Shelton 1988). However, cultivated Brassica FFFF, autotetraploid induced by colchicine) having germplasm has failed so far to provide any source of re- useful genetic attributes of parents, to increase genetic sistance against mustard aphid. variability and to introduce genes with special refer- The genus Brassica has about 100 wild and weedy rel- ence to tolerance/resistance against mustard aphid. This investigation reports successful synthesis of four inter- * Corresponding author, e-mail: [email protected] specific hybrids between B. juncea cv. Rohini, Laxmi DOI: 10.1508/cytologia.83.421 and Varuna and the B. fruticulosa (2n=32, FFFF), and 422 A. Kumar et al. Cytologia 83(4) some evidences for its successful establishment through for stainable pollen grains on five slides. morphological and cytological analyses. Results Materials and methods

Hybridization and morphological characteristics of F1 Plant material and hybridization hybrids Seed samples of B. juncea varieties and B. fru- A total of eight popular B. juncea, cultivars viz. Pusa ticulosa were obtained from the Germplasm Section of bold, Bio-902, Varuna, Laxmi, Rohni, NRCDR-2, Maya the ICAR-Directorate of Rapeseed-Mustard Research, and Vasundhra were selected on the basis of breeder Bharatpur, India. Interspecific crosses between plants seed index as maternal parents were used in the hy- of the cultivated B. juncea (eight popular cultivars viz. bridization and B. fruticulosa (FFFF), used as a pollen Pusa bold, Bio-902, Varuna, Laxmi, Rohini, NRCDR-2, donor. Out of eight B. juncea, cultivars, used in crossing Maya and Vasundhra), and autotetraploid B. fruticulosa autotetraploid B. fruticulosa the cultivars Rohini, Laxmi (2n=4x=32, FFFF) developed by aqueous colchicine us- and Varuna showed pollinated seeds, in Rohini 19 seeds ing the cotton-swab method (Kumar et al. 2015a) ware were obtained from 120 flower buds, in Laxmi 15 seeds attempted to produce interspecific hybrids. Unopened were obtained from 128 flower buds and in Varuna 14 flower buds of B. juncea were covered with paper bags seeds were obtained from 110 flower buds. Seeds of par- in the afternoon, and pollinated with freshly collected ents and crosses were sown in earthen pots under natural pollens of B. fruticulosa (2n=4x=32, FFFF), in follow- open field condition during next winter season. Out of ing morning and covered again. The seeds collected these 19, 15 and 14 seeds, only six, nine and seven seeds from the crossed plants and parents were sown in earth- were germinated and all the plants survived till maturity. en pots under field conditions. Morphological compari- Four plants were found to be true hybrids [one: B. juncea sons were made for identifying the F1s and ascertained cv. Rohini×B. fruticulosa (FFFF), two: B. juncea cv. cytologically. Laxmi×B. fruticulosa (FFFF), one: B. juncea cv. Varuna ×B. fruticulosa (FFFF)].

Cytological analysis The F1 hybrid plants were medium in height, pro- For meiotic observations flower buds of an appropriate fusely branched and intermediate to their parents for size were collected from mature plant and fixed in fresh- most of the morphological and inflorescence attributes ly prepared Carnoy’s fluid (ethanol : chloroform : acetic (Table 1, Fig. 1). The size and shape of leaves of F1 acid, 6 : 3 : 1), supplemented with a drop of ferric chlo- plants were closer to B. juncea. The leaves were dark ride solution, for a minimum of 24 h at room temperature green in color with sparse hairy, petiolate, lobed and and subsequently stored in 70% ethanol at 10°C. For lyrately pinnatified. The dentation of leaf margin was meiotic analysis, anthers were squashed in 1% aceto- noted to be the sinuate–dentate type with obtuse tip and carmine and a total of 78 PMCs were analyzed at diaki- petals were dark yellow in color and slightly larger in nesis/metaphase I stages of meiosis in F1 hybrid plants. size. The F1 plants exhibited the enhancement in values For pollen stainability, the pollen grains were stained in of attributes like primary and secondary branches, main glycerine–acetocarmine (1 : 1) mixture and were scored raceme length and delay in days to maturity in compari-

Table 1. Comparison of morphological characters of parents and the F1 hybrids of [B. juncea cv. Rohini, Laxmi, and Varuna×B. fruticulosa (FFFF)].

B. juncea B. fruticulosa F1 hybrids Characteristics cv. Rohini cv. Laxmi cv. Varuna cv. Rohini cv. Laxmi cv. Varuna

Plant height (cm) 112 118 110 60 98 105 93 Days to 50% ﬂowering 56 58 61 50 68 70 65 Days to maturity 110 108 115 110 125 135 122 Primary branches per plant 5.6 6.2 5.4 5 11 11 8 Secondary branches per plant 7 7 8 8 17 20 15 Main raceme length (cm) 58 55 52 35 51 47 39 Siliqua length (cm) 5.4 5.5 4.2 3.6 3.5 3.9 2.8 Seeds per siliqua 15 16 13 10 2.4 2.4 2.5 Corolla length (cm) 1.3 1.4 1.3 1.5 1.5 1.7 1.4 Corolla width (cm) 0.6 0.7 0.6 0.8 0.7 0.8 0.8 Siliqua texture Smooth Smooth Undulated Constricted Smooth Smooth Smooth Seed color Dark brown Dark brown Dark brown Brown Light brown Light brown Light brown Leaf color Medium green Medium green Medium green Dark green Dark green Dark green Dark green Leaf hairiness Sparse Sparse Sparse Sparse Sparse Sparse Sparse Leaf lobes Present Present Present Present Present Present Present Petal color Yellow Yellow Yellow Yellow Dark yellow Dark yellow Dark yellow 2018 Synthesis of Interspeciﬁc Hybrids between Brassica juncea and B. fruticulosa (4x) 423

Fig. 1. Comparison of morphological attributes of parent and F1 hybrid. a. Leaf, b. Flower, c. Siliqua P1 B. juncea (cv. Laxmi), P2 B. fruticulosa (FFFF), F1 B. juncea (cv. Laxmi)×B. fruticulosa (FFFF).

Table 2. Chromosome conﬁguration at diakinesis/metaphase I in PMCs of F1 hybrids [B. juncea cv. Rohini, Laxmi, and Varuna × B. fruticulosa (FFFF)].

Chromosome associations No. of

F1 hybrids PMCs Univalents Bivalents Trivalents Quadrivalents observed No. Mean Range No. Mean Range No. Mean Range No. Mean Range

B. juncea cv. Rohini 30 384 12.80±5.34 4–26 198 6.60±2.76 2–10 18 0.72±0.77 0–3 21 0.70±0.52 0–2 ×B. fruticulosa (FFFF) B. juncea cv. Laxmi 26 358 13.76±5.63 6–26 206 7.92±1.26 4–10 22 0.84±1.16 0–3 20 0.76±1.12 0–4 ×B. fruticulosa (FFFF) B. juncea cv. Varuna 22 232 10.55±4.88 4–22 166 7.54±2.38 4–12 8 0.36±0.39 0–1 15 0.68±0.79 0–3 ×B. fruticulosa (FFFF)

Table 3. Chromosome distribution at anaphase I in PMCs and percentage pollen stainablity of F1 hybrids of [B. juncea cv. Rohini, Laxmi and Varuna × B. fruticulosa (FFFF)].

No. of cells Chromosome F hybrids 2n No. of cells Percentage 1 analyzed distribution

B. juncea cv. Rohini 34 20 17 : 17 12 60.00 ×B. fruticulosa (FFFF) 16 : 2U* : 17 8 40.00 B. juncea cv. Laxm 34 15 17 : 17 10 66.67 ×B. fruticulosa (FFFF) 15 : 6U : 16 1 6.67 16 : 2U : 17 2 13.33 14 : 18 2 13.33 B. juncea cv. Varuna 34 15 17 : 17 10 66.67 ×B. fruticulosa (FFFF) 16 : 1B** : 17 1 6.67 14 : 18 4 26.66

* U=Univalents, ** B=Bivalents son to parents. The seeds of hybrids were smaller in size most of these observations are shown in Fig. 2. Meiotic and light brown in color. The hybrid plants had smaller analysis of the F1 hybrids (2n=34) between B. juncea siliquae as compared to the parents. Few seeds were pro- (cv. Rohni, Laxmi, Varuna) and B. fruticulosa (FFFF) duced in the F1 plants due to open pollination in the sur- showed a mixture of univalents, bivalents, trivalents and roundings of B. juncea. However, no seed was achieved quadrivalents in a total of 78 PMCs analyzed at diaki- under self-pollination. The F1 plants harbored lower nesis/metaphase I. The mean number of univalents were aphid populations under ﬁeld conditions than the parents found to be more frequent and ranged from 10.55 to B. juncea (cv. Rohini, Laxmi, and Varuna). 13.76. The mean value number of bivalents per PMC of

F1 plants were ranged between 7.92 and 6.60. The occur- Meiotic characteristics and pollen stainability of F1 hy- rence of univalents was frequent, however, two bivalents brids were observed in cross B. juncea cv. Rohini×B. fru-

The meiotic details of F1 hybrids viz. chromosome ticulosa (FFFF) and maximum of twelve bivalents were associations at diakinesis/metaphase I have been sum- observed in B. juncea cv. Varuna×B. fruticulosa (FFFF). marized in Table 2. The distribution pattern of chro- Bivalents were not aligned at the equatorial plate but mosomes at anaphase I and II are detailed in Table 3, were randomly distributed. A heteromorphic associa- 424 A. Kumar et al. Cytologia 83(4)

Fig. 2. Representative illustrations of meiotic analysis of F1 hybrid plants showing chromosome associations at metaphase I and chromosome distribution at anaphase I and II. 1–3. B. juncea (cv. Rohni)×B. fruticulosa (FFFF). 1–2. Metaphase

I, 1. 8II+18I, 2. 1IV+2III+8II+8I. 3. Anaphase II (showing laggards). 4–6. B. juncea (cv. Laxmi)×B. fruticulosa (FFFF). 4. Metaphase I, 2IV+2III+8II+8I, 5. Anaphase I (showing laggards) 6. Anaphase II (showing intermixing of chromatids at one pole). 7–10. B. juncea (cv. Varuna)×B. fruticulosa (FFFF), 7–8. Metaphase I, 7. 3IV+9II+4I, 8. 8II+18I, 9. Anaphase I. 10. Anaphase II (showing laggards). (Trivalents marked by arrow heads quadrivalents marked by arrow and laggards with arrow). Scale bar=10 µm. tion of chromosome was few. Multivalents in the form of relatively more difﬁcult than when species with the same trivalents which ranged between 0–1 to 0–3 and quadri- ploidy level are mated. Researchers have developed in- valents ranging from 0–2 to 0–4 were also observed in duced polyploids with the ultimate goal of making cross-

F1 plants. es not possible at the original ploidy level in horticulture Numerous distributional abnormalities including late crops (Dhooghe et al. 2009). However, reports on the disjunction of univalents, bivalents, and laggards were successful implementation of this technique in Brassica observed at anaphase I in all three hybrids (Table 3). crops are lacking (Prakash et al. 2009, Prakash 2010). Laggards in the form of univalents (2–6) and bivalents Thus, colchicine-induced tetraploid plants obtained in B. (one bivalent), as well as few PMCs with unequal dis- fruticulosa were further, utilized in hybridization with tribution of chromosomes with 14 : 18 also encountered B. juncea. in hybrids. In anaphase II few PMCs with laggards and In the present study, four interspecific hybrid plants one cell intermixing of chromatids at one pole was also were obtained through sexual hybridization between B. observed in B. juncea cv. Laxmi×B. fruticulosa (FFFF) juncea (cv. Rohini, Laxmi, Varuna) and B. fruticulosa (Fig. 2). However, a few cells were recorded with a nor- (FFFF), induced colchi-autotetraploidy. Interspecific hy- mal distribution of bivalents at anaphase I resulting in brid plants were obtained when B. fruticulosa (FFFF) some fertile pollen grains in the hybrids. were used as a male parent, and all the hybrids grew up Interspecific hybrids are mostly pollen sterile or have to maturity. The perusal of literature available indicates very little pollen fertility. Similar results were also re- the production of interspecific hybrids in Brassica most- corded in the F1 hybrids which showed a drastic reduc- ly via embryo rescue techniques (Chandra et al. 2004, tion in pollen stainability, recording only 18.5, 16.4, and Kaneko et al. 2009). Moreover, interspecific/intergeneric 15.8%, respectively in hybrids, with different cultivars. hybrids are successfully produced only when wild species are used as female (Chandra et al. 2004, Chen et al., Discussion 2011, Tsuda et al. 2014). Contrary to this, the present investigation reports the successful hybridization be- The wild relatives of crop plants often carry many tween B. juncea and B. fruticulosa (FFFF) even when, useful agronomic traits for resistance/tolerance to vari- it is involved as a male parent. It is quite useful in main- ous biotic/abiotic stresses and interspecific hybridiza- taining the cytological background of the crop species in tion offers a potential tool for introgression of these distant hybridization programmes. The F1 hybrid plants desired traits from wild to the cultivated forms (Prakash thus obtained were observed to be intermediate in terms and Chopra 1988). The utilization of this secondary of many phenotypic features of both the parents. These gene pool is extremely important in crop improvement observations are in congruence with earlier published re- programmes (Jena and Khush 1990, Warwick et al. ports (Chen et al. 2011, Choudhary and Joshi 2012). The 2000). As mentioned earlier, when species with different occurrence of characteristics from both progenitor spe- ploidy levels are crossed, hybridization between them is cies in the hybrids indicates that the F1 plants inherited 2018 Synthesis of Interspecific Hybrids between Brassica juncea and B. fruticulosa (4x) 425 genomes of both parental species crossed. This is an ad- S. 2012. Development and characterization of Brassica juncea- vantage since it would allow better selection for specific fruticulosa introgression lines exhibiting resistance to mustard aphid (Lipaphis erysimi Kalt). BMC Genet. 13: 104. attributes in segregating progenies. Bandopadhyay, L., Basu, D. and Sikdhar, S. R. 2013. Identification Cytological analysis of F1 hybrids (2n=34) not only of genes involved in wild crucifer Rorippa indica resistance re- confirmed their hybridity but also indicated the extent sponse on mustard aphid Lipaphis erysimi challenge. PLoS ONE of genome homoeology between the parents. Mei- 8: e73632. otic analysis of F hybrids showed the predominance Chandra, A., Gupta, M. L., Banga, S. S. and Banga, S. K. 2004. Pro- 1 duction of an interspecific hybrid between Brassica fruticulosa of univalents that ranged between 4–22 to 6–24, which and B. rapa. Plant Breed. 123: 497–498. is the typical feature of wide hybrids (Kumar et al. Chen, J. P., Ge, X. H., Yao, X. C., Feng, Y. H. and Li, Z. Y. 2011. Syn- 2013, 2015b). It was observed that PMCs had chromo- thesis and characterization of interspecific trigenomic hybrids and allohexaploids between three cultivated Brassica allotetra- some association on an average of 0.70IV+0.72III+6.60II ploids and wild species Brassica fruticulosa. Afr. J. Biotechnol. +12.80I, 0.76IV+0.84III+7.92II+13.76I and 0.68IV+0.36III+ 7.54 +10.55 , respectively in F hybrids. The observa- 10: 12171–12176. II I 1 Choudhary, B. R. and Joshi, P. 2012. Crossibility of Brassica cari- tion of bivalents which ranged from 2–10 to 4–12 in nata and B. tournefortii, and cytomorphology of their F1 hybrid. various PMCs in the hybrids could be interpreted to be Cytologia 77: 453–458. due to archaic homology within the chromosomes of the Cole, R. A. 1994a. Locating a resistance mechanism to the cabbage same genome or because of intergenomic homoeology. aphid in two wild Brassicas. Entomol. Exp. Appl. 71: 23–31. However, the occurrence of higher associations in the Cole, R. A. 1994b. 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