Introduction
Doubled haploidy is a potent plant breeding technique to significantly shorten the breeding cycle needed to achieve the ‘line fixation’ stage for enhancing genetic gain needed for the development of new varieties. In-vivo haploidization through wide hybridization via chromosome elimination is an efficient approach for doubled haploids (DH) production in wheat and related species. Since the first report of the production of haploid plants in wheat (Laurie and Bennett, Reference Laurie and Bennett1988) the technology has been utilized to produce DH derived wheat lines/varieties (Ho and Jones, Reference Ho and Jones1980; DePauw et al., Reference DePauw, Knox, Mccaig, Clarke and Clarke2011; Saulescu et al., Reference Sãulescu, Ittu, Giura, Mustãţea and Ittu2012; Graf et al., Reference Graf, Beres, Laroche, Gaudet, Eudes, Pandeya, Badea and Randhawa2013; Scheeren et al., Reference Scheeren, da Rosa Caetano, Caierao, Silva, do Nascimento, Eichelberger, Miranda de and Brammer2014; Khan et al., Reference Khan, Bhardwaj, Gangwar, Prasad and Rathore2017). Haploid induction potential of maize, a well-known cereal crop in wheat (Inagaki and Tahir, Reference Inagaki and Tahir1992; Inagaki and Muzeeb-Kazi, Reference Inagaki and Mujeeb-Kazi1995; Zhang et al., Reference Zhang, Friebe, Raupp, Harrison and Gill1996; Verma et al., Reference Verma, Bains, Mangat, Nanda, Gosal and Singh1999; Singh et al., Reference Singh, Behl and Punia2005; Santra et al., Reference Santra, Wang, Scifert and Haley2017) and Imperata cylindrica, a rhizomatous grass (Chaudhary et al., Reference Chaudhary, Sethi, Singh, Partap and Sharma2005; Chaudhary, Reference Chaudhary2008a, Reference Chaudhary, Taniguchi and Zhang2008b, Reference Chaudhary2013; Patial et al., Reference Patial, Pal, Thakur, Bana and Patial2017) have been well established, however, the comparative advantage of I. cylindrica over maize is evident due to its potential to induce haploids not only in hexaploids but also in tetraploids, triticale × wheat and wheat × rye derivatives in which maize failed to produce desirable results (Chaudhary et al., Reference Chaudhary, Sethi, Singh, Partap and Sharma2005; Pratap et al., Reference Pratap, Sethi and Chaudhary2005; Chaudhary, Reference Chaudhary2008a, Reference Chaudhary, Taniguchi and Zhang2008b, Reference Chaudhary2013; Kishore et al., Reference Kishore, Chaudhary, Chahota, Kumar, Sood, Jeberson and Tayeng2011; Mahato and Chaudhary, Reference Mahato and Chaudhary2015; Kapoor et al., Reference Kapoor, Chaudhary, Relan, Manoj, Singh and Sharma2020). Apart from bread wheat which covers a major area under wheat, durum wheat is of economic importance and demands for genetic improvement of traits like improved agronomic performance, disease resistance and improved quality. Relatively low frequency of haploid induction in durum wheat has been achieved through wide hybridization which has been mainly attributed to the absence of D genome as well as low ploidy level as compared to hexaploids (Niu et al., Reference Niu, Jiang, Hammad, Oladzadabbasabsdi, Xu, Mergoum and Elias2014). However, success has been achieved in recovering a higher frequency of haploids in durum wheat utilizing I. cylindrica as pollen source (Mahato and Chaudhary, Reference Mahato and Chaudhary2015). Triticale ( × Triticosecale) an amphiploid of wheat and rye is a hardy cereal combining hardiness and nutritional efficiency of rye and high grain yield and nutritional qualities of wheat (Dennett et al., Reference Dennett, Cooper and Trethowan2013). Hexaploid triticale (2n = 42 = AABBRR) is commonly used as bridging species for the transfer of rye traits to wheat due to its cross-compatibility, genomic stability and better adaptation as compared to triticales of other ploidy levels (Ammar et al., Reference Ammar, Mergoum, Rajaram, Mergoum and Gomez-Macpherson2004; Oettler et al., Reference Oettler, Tams, Utz, Bauer and Melchinger2005; Hao et al., Reference Hao, Luo, Zhang, Yuan, Yang, Wu, Chen, Zheng, Zhang and Liu2013). Stable triticale × wheat-derived lines are desirable having a combination of characters of both the species for better resilience to biotic and abiotic stresses. With increasing area and demand for triticale globally, success achieved through doubled haploidy breeding in hexaploid and tetraploid species needs to be replicated in other economic species of the wheat gene pool for deriving desirable lines in a shorter period. It has been well known that the rate of haploid induction through the chromosome elimination approach depends on the genotype of both male and female parents (Inagaki and Tahir, Reference Inagaki and Tahir1990; Verma et al., Reference Verma, Bains, Mangat, Nanda, Gosal and Singh1999; Chaudhary et al., Reference Chaudhary, Singh and Sethi2002). Diversity in plant genetic resources provides an opportunity for breeders to select for desired traits and further utilized them for various genetic and genomic studies with wider applications. Most of the studies on DH in wheat have either utilized improved maize composites, inbred lines or hybrids as a pollen source. Due to cross-pollinated nature of both maize and I. cylindrica, considerable diversity exists as heterozygous populations in remote locations of North-Eastern and North-Western Himalayan region which have rarely been tested for haploid induction potential. Therefore the present study was undertaken with the aim to assess haploid induction efficiency of diverse genotypes of Himalayan maize and I. cylindrica in inter and intra-specific crosses of hexaploids, tetraploids and triticales.
Materials and methods
Experimental material
The material for the present investigation consisted of twenty-six lines (female) which comprised of four genotypes of hexaploid wheat, two of tetraploid, two of triticale, seven F1s, five BC1F1s and six BC1F2 genotypes generated from crossing among the parental lines. The male parents (testers/pollen parents) consisted of twenty-one maize populations collected from different locations of North-Western and North-Eastern Himalayas and two genotypes of I. cylindrica growing locally around wheat fields (Tables 1 and 2). Lines were planted in open field conditions at three different sowing dates keeping 15 day interval for better synchronization with the male parents while maize genotypes were raised under polyhouse conditions at two different sowings maintaining 15 days interval. During hybridization period, pollen from maize plants was collected freshly in petri plates from polyhouse sowing while that of I. cylindrica was collected from the wild vegetation around wheat fields. All the field activities i.e. generation of F1s, backcross populations, wide hybridization with maize and I. cylindrica was conducted at the Experimental Farm while embryo culture work was done in Molecular Cytogenetics and Tissue Culture Laboratory (MCTL) of the Department of Crop Improvement, CSK Himachal Pradesh Agricultural University (CSK HPAU), Palampur, Himachal Pradesh, India. Generation advancement and hybridization work for generating intra and inter-varietal crosses was also done during off-season at Highland Agricultural Research & Extension Centre, Kukumseri (Lahaul & Spiti), Himachal Pradesh located at 2672 m amsl. Wide hybridization work was carried out with maize and I. cylindrica following the protocol of Laurie and Bennett (Reference Laurie and Bennett1986, Reference Laurie and Bennett1988) and Chaudhary et al. (Reference Chaudhary, Sethi, Singh, Partap and Sharma2005), respectively.
Table 1. Parentage and source of female genotypes used for haploid induction
Table 2. Parentage and source of pollen parents (male) used for haploid induction
a HPMC, Himachal Pradesh maize Collection; L&S, Lahaul & Spiti.
Methodology
Two spikes of each line were crossed with each of the twenty-one maize and two I. cylindrica genotypes. In all total of 1196 spikes were hybridized with both the pollen species (52 spikes per pollen source). Two different concentrations of 2,4-D i.e. 100 ppm for bread wheat type and 250 ppm for durum wheat, triticale and triticale × wheat crosses were injected in the uppermost internode of culms for three consecutive days at 24, 48 and 72 h after pollination. Doses of 2, 4-D for hexaploids, tetraploids and triticale × wheat derivatives were standardized based on studies carried out in our lab (Chaudhary et al., Reference Chaudhary, Mahato, Kaila and Rather2015; Badiyal et al., Reference Badiyal, Chaudhary, Jamwal and Hussain2016). The pseudoseeds were screened for the presence of an embryo (Bains et al., Reference Bains, Mangat, Singh and Nanda1998) under a source of light in which embryo positive seeds were retained and carried forward for embryo culture after washing with Tween −20. The embryos were excised carefully from sterilized seeds and embryo culture protocol was performed in a laminar air flow chamber under completely sterile conditions. Embryos were then transferred to test tubes containing MS medium (Murashige and Skoog, Reference Murashige and Skoog1962) supplemented with 0.5 mg/l kinetin, 150 mg/l glutamine and 20 mg/l each of L-arginine, L-cystine and L-leucine. The pH of the medium was maintained at 5.8 and after autoclaving, the embryos were cultured in 150 × 20 mm glass tubes. Tubes of cultured embryos were kept at 4°C in dark for 24 h for cold treatment and thereafter shifted to the dark chamber and kept at 20 ± 2°C until regeneration. The regenerated plantlets were then shifted to the normal light conditions and grown at 20 ± 2°C with 10/14 h light/dark profile with 75 per cent relative humidity for proper development of plantlets. Plantlets were transferred to a rooting medium containing NAA (Napthalene acetic acid) and IBA (Indole butyric acid) at three to the four-leaf stage for rooting and then to soil potting mixture for further growth.
Observations recorded and statistical analysis
Observations were recorded on four haploid induction parameters viz., pseudoseed formation frequency (PFF) (i.e. a percentage of a number of pseudoseeds formed out of a total number of florets pollinated), EFF (embryo formation frequency (EFF) i.e. a percentage of a number of pseudoseeds carrying embryo out of a total number of pseudoseeds formed), haploid regeneration frequency (HRF) (i.e. a percentage of haploid embryos regenerated out of a total number of embryos cultured) and haploid formation frequency (HFF) (i.e. a percentage of green haploid plantlets developed out of a total number of florets pollinated). Data recorded on all the four haploid induction parameters were subjected to line × tester analysis (Kempthorne, Reference Kempthorne1957). As the data were recorded in per cent it was used for analysis after arcsine transformation (Gomez and Gomez, Reference Gomez and Gomez1984). Two- sample t test (Snedecor and Cochran, Reference Snedecor and Cochran1989) was applied to determine the significant differences among frequencies of haploid induction parameters obtained upon crossing with maize and I. cylindrica.
Results
The results showed a variable response of both lines and testers towards haploid induction. Results of the two sample't’ test showed significant differences between both the pollen species i.e. I. cylindrica and maize for EFF and HFF when pooled over all the lines. Mean PFF, EFF, HRF and HFF obtained with both the pollen species is shown in Table 3. Mean PFF obtained with I. cylindrica and maize were 54.07 and 49.24%, respectively. Almost at par values were recorded for HRF with I. cylindrica and maize (33.60 and 35.09%, respectively). However, the frequency of embryos obtained with I. cylindrica (18.39%) was significantly higher as compared to maize (4.08%). In the case of HFF also I. cylindrica outperformed maize with a frequency of 3.85% as compared to 1.16% obtained with maize. The highest frequency of pseudoseeds with I. cylindrica were recorded in TL-2908 × DH-86 × DH-86(BC1F2) while in the case of maize it was with DH-84 (79.62%) and DH-100 (79.48%). No pseudoseeds were formed in triticale parental line TL-2908 and F1s of triticale × wheat when crossed with I. cylindrica while the same were obtained in all the genotypes crossed with maize with very low frequency. Cross DH-40 × DH-65 (F1) was most responsive to embryo formation (64.71%) with I. cylindrica followed by DH-100 (41.3%), while with maize highest percentage of embryos were obtained in genotype DH-84 (11.6%), followed by DH-86 (9.97%), PDW-314 (9.70%) and A-9-30-1 (9.67%). Genotypes DH-40, triticales, F1s of triticale × wheat and BC1F1 of (TL2900 × DH86) × DH86 remained unresponsive to embryo formation with I. cylindrica while embryos were recovered in all the genotypes crossed with maize except triticale genotype TL-2908. All the embryos obtained with I. cylindrica in crosses DH-40 × A-9-30-1 × DH-40 (BC1F2), DH-40 × PDW-314 (F1) and DH-40 × A-9-30-1 (F1) were able to regenerate, while with maize highest regeneration was recorded in embryos recovered from tetraploid genotypes PDW-314 (68.57%) and A-9-30-1 (66.08%).
Table 3. Mean haploid induction frequencies in different generations obtained with twenty-one maize and two Imperata genotypes (Ic)
Values in Bold are group wise mean values.
When the performance of both the pollen species was compared across generations, I. cylindrica outperformed maize in all the haploid induction parameters (Table 3). Comparatively maximum embryo induction frequency was observed with I. cylindrica in hexaploid × hexaploid F1s ( + 27.84%) followed by hexaploid × hexaploid BC1F1 ( + 27.36%) and in hexaploid parents ( + 19.58%) as compared to maize. However, F1s of hexaploid × tetraploid and triticales showed slightly better results with maize. Comparatively higher HFF was observed with I. cylindrica in hexaploid × hexaploid BC1F1 ( + 5.76%) followed by hexaploid wheat parents ( + 5.18%). With I. cylindrica highest frequency of haploid plants were recovered in genotype DH-100 (11.07%), DH-86(9.56%), DH-40 × DH-65 × DH-40 (BC1F1) and DH-84(8.97%), while with maize maximum recovery percentage was recorded in DH-84 (3.65%) followed by A-9-30-1(3.07%).
General combining ability (GCA) of both male and female genotypes was computed to find out the best general combiners for various haploid induction parameters (Table 4 and Supplementary Table 4). Among the testers, genotypes with significantly positive GCA for pseudo seed frequency were HPMC-11, HPMC-21, HPMC-53, HPMC-57, HPMC-60, HPMC-62, HPMC-64, Ic-ye and Ic-br. The highest values of GCA for PFF were recorded with HPMC-60 (7.58) and HPMC-53 (5.32), whereas GCA values of I. cylindrica genotypes for the same were 3.53 and 2.87 for Ic-br and Ic-ye, respectively. Among lines, significantly positive GCA values for PFF were recorded for DH-40, DH-84, DH-86, DH-100, A-9-30-1, DH-40 × PDW-314, DH-40 × A-9-30-1, DH-40 × DH-100, DH-40 × DH-65, DH-40 × DH-65 × DH-40 (BC1F1), DH-40 × DH-100 × DH-40 (BC1F1) and DH-40 × DH-65 × DH-40 (BC1F2). In the case of embryo formation, GCA of both the I. cylindrica genotypes was significantly higher as compared to maize (16.48 and 12.17%, respectively for Ic-ye and Ic-br) while in the case of maize only one genotype HPMC-53 was having significantly positive GCA value (3.25) for embryo formation. Among lines DH-84, DH-86, DH-100, PDW-314, A-9-30-1, DH-40 × DH-100 (F1) and its BC1F1, DH-40 × DH-65 (F1) and its BC1F1 were having positively significant GCA for EFF. In the case of HRF maize genotypes HPMC-11, HPMC-14, HPMC-53, HPMC-57, HPMC-60, HPMC-62, HPMC-63 and HPMC-64 possessed positive GCA while among lines all the parental lines except DH-40, TL-2900 and TL-2908, DH-40 × PDW-314 (F1), DH-40 × DH-100 (F1), DH-40 × DH-65(F1) and their BC1F1s, BC1F2s of (DH-40 × DH-65) × DH-40, (TL-2900 × DH-86) × DH-86 and (TL-2908 × DH-86) × DH-86 were having positively significant GCA. In the case of HFF tester genotypes HPMC-14, HPMC-60, HPMC-64, Ic-ye and Ic-br were having positively significant GCA with the highest values for Ic-ye (6.05) and Ic-br (3.75) while among maize significant GCA values were recorded in HPMC-60 (1.58), HPMC-64 (1.39) and HPMC-14 (1.11). Among lines DH-84, DH-86, DH-100, A-9-30-1, PDW-314, DH-40 × PDW-314 (F1), DH-40 × DH-100(F1), BC1F1 of (DH-40 × DH-100) × DH-40 and (DH-40 × DH-65) × DH-40 recorded positive GCA for HFF.
Table 4. GCA effects for haploid induction parameters of maize and Imperata genotypes
*Significant at 5% level of significance. Values in Bold are group wise mean values.
Discussion
In-vivo haploid induction in wheat via chromosome elimination mediated approach has been followed globally using maize and I. cylindrica as an efficient pollen source. This system of haploid induction have been successful and surpassed several bottlenecks associated with in-vitro methods. Furthermore, the superiority of I. cylindrica over maize is evident due to its potential to induce haploids not only in hexaploid wheat but in tetraploids, triticale × wheat and wheat-rye derivatives which have been already proved in earlier studies (Chaudhary et al., Reference Chaudhary, Sethi, Singh, Partap and Sharma2005; Kishore et al., Reference Kishore, Chaudhary, Chahota, Kumar, Sood, Jeberson and Tayeng2011; Badiyal et al., Reference Badiyal, Chaudhary, Jamwal, Hussain, Mahato and Bhatt2014; Mahato and Chaudhary, Reference Mahato and Chaudhary2015; Kapoor et al., Reference Kapoor, Chaudhary, Relan, Manoj, Singh and Sharma2020) and further supported by our results in the present study where the frequency of haploids obtained with I. cylindrica are significantly higher as compared to maize. Moreover, its natural coincidence of flowering with wheat and ample pollen shed advocates its advantage over maize which has to be raised and managed under protected conditions during the winter season, especially in sub-temperate areas.
Results obtained by hybridization of diverse lines with both the pollen species indicate the superiority of I. cylindrica over maize in haploid induction. The mean frequency of embryos and green haploid plantlets obtained upon crossing with I. cylindrica were 20.34 and 3.71%, respectively as compared to frequencies of 3.90 and 1.14% obtained with maize. Although both the I. cylindrica genotypes (Ic-ye and Ic-br) were statistically at par in their haploid induction efficiencies but the value of mean frequencies obtained with genotype Ic-ye was relatively higher as compared to Ic-br, thus genotype Ic-ye can be utilized as a relatively efficient pollen parent over Ic-br. Genetic background of I. cylindrica influence the haploid induction efficiency in wheat (Rather et al., Reference Rather, Chaudhary and Kaila2014). Poor response of triticale lines to embryo formation may be attributed to failure to form pseudo seeds due to poor imbibition of post pollination treatment of growth hormone 2,4-D required to nurse the developing embryo. Backcross progenies obtained in triticale-wheat derivatives responded well to embryo formation as well as green haploid plantlets. A higher success rate in later segregating generations is mainly due to an increase in the frequency of D genome in triticale-wheat derived lines results in improving the morphology of plants similar to hexaploid lines. Poor response of pure triticales and triticale F1s is due to genomic incompatibilities of wheat (AABBDD) and rye (AABBRR) genomes. D genome has been reported to be the triggering factor in chromosome elimination and haploid induction in wheat × I. cylindrica system (Mukai et al., Reference Mukai, Okamoto, Kiryu and Yamamoto2015). In a similar study, Kaila (Reference Kaila2013) reported paternal chromosome elimination in wheat × I. cylindrica hybrids as the outcome of B and D genomes of hexaploid wheats, where the relative effect of D genome is higher than the B genome of hexaploid wheat. Potential of I. cylindrica to induce a higher frequency of haploid embryos in durum wheat have been reported by Mahato and Chaudhary (Reference Mahato and Chaudhary2015). Unlike parental durum wheat lines, crosses with durum wheat were less responsive to haploid induction which may be due to the formation of F1 pentaploid hybrids having a unique chromosomal constitution. Parental combinations used in pentaploids crosses generally influence the degree to which D genome chromosomes are retained (Padmanabhan et al., Reference Padmanabhan, Zhang, Hare Ray, Sutherland Mark and Martin2017). On the basis of potential to induce embryos, in case of I. cylindrica, genotype Ic-ye identified best with mean EFF of 30.55, 14.48 and 25.43%, respectively for hexaploids, tetraploids and triticale × wheat derivatives whereas in case of maize for hexaploids HPMC-60 identified best with EFF of 12.61%, for tetraploids HPMC-58 (12.58%) and for triticale × wheat derivatives HPMC-16 performed relatively better with 8.91% EFF. Eight maize genotypes i.e. HPMC-16, HPMC-25, HPMC-27, HPMC-53, HPMC-56, HPMC-58, HPMC-60 and HPMC-64 were early flowering thereby providing an opportunity to initiate crossing work early in the season and thus prolonging the period of wide hybridization for enhancing the haploid induction efficiency. In a similar study, Kapoor et al. (Reference Kapoor, Chaudhary, Relan, Manoj, Singh and Sharma2020) identified Himalayan maize and cogon grass genotypes with higher haploid induction efficiencies from hybridization with eight parental lines comprised of four genotypes of hexaploid wheat, two each of tetraploid wheat and triticale. Significant differences were observed in haploid induction potential among Himalayan maize genotypes in earlier studies in hexaploid wheat (Dhiman et al., Reference Dhiman, Rana and Chaudhary2012) and in durum wheat (Mahato and Chaudhary, Reference Mahato and Chaudhary2015). Significant variation for haploid induction in wheat has also been observed among maize hybrids, inbred lines and improved populations (Verma et al., Reference Verma, Bains, Mangat, Nanda, Gosal and Singh1999; Chaudhary et al., Reference Chaudhary, Singh and Sethi2002; Kour et al., Reference Kour, Bhatt, Grewal, Singh and Khanna2008). On the basis of recovery of green haploid plantlets in crossed generations, BC1F1 generation of hexaploid × hexaploid followed by BC1F2 of hexaploid × hexaploid, triticale × wheat (BC1F2) and triticale × wheat (BC1F1) responded best.
Results of the study put forth superiority of I. cylindrica over maize in haploid induction in hexaploid and tetraploid wheat, triticales and their crosses signifying its potential to induce haploids over different ploidy groups and species. The reason seems most plausible for I. cylindrica to induce a higher frequency of haploids is due to better genomic compatibility with wheat including members of its gene pool and relatively longer pollen viability due to its hardy nature. Maize populations used in the study varied in haploid induction potential. Although overall maize genotypes have relatively low haploid induction potential as compared to I. cylindrica, maize genotypes having positive GCA for haploid induction parameters can be utilized as efficient genotypes. Sub-temperate areas where I. cylindrica flourishes well in the wild shall be utilized as a pollen source for wide hybridization with wheat for recovering higher frequency of haploid embryos with preference to genotype Ic-ye, whereas maize genotypes HPMC-14, HPMC-53, HPMC-60, HPMC-64 may be utilized for haploid induction through wheat × maize system of haploid induction. Maize and I. cylindrica genotypes with high haploid induction efficiency identified in the study are valuable genetic resources and need to be conserved both in-situ and ex-situ for long term conservation after proper characterization and documentation. Maize genotypes can be shared among workers after germplasm registration. Further cryo-preservation studies on the pollen of I. cylindrica may be carried out for making its viable pollen available to labs where I. cylindrica is not available for carrying out wide hybridization with wheat.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262121000642
