Chickpea (Cicer arietinum L.), the second most important dietary legume after common bean, is a rich source of proteins, carbohydrates, micronutrients, and vitamins (Jukanti et al. 2012). It is a potential staple food crop in about 55 countries. India is the largest producer of chickpea with an annual production of 9.9 million tonnes (FAOSTAT 2019). Chickpea production worldwide is affected by biotic and abiotic stresses. Because there is limited genetic variation in the cultivated chickpea germplasm, it is necessary to utilize wild Cicer species for genetic improvement. Wild Cicer species are strongly resistant to major biotic stresses like Ascochyta blight (Stamigna et al. 2000; Collard et al. 2001; Croser et al. 2003; Rao et al. 2003; Shah et al. 2005; Pande et al. 2006), Botrytis gray mold (Stevenson and Haware 1999; Rao et al. 2003; Pande et al. 2006), and Fusarium wilt (Infantino et al. 1996; Croser et al. 2003; Rao et al. 2003), and tolerant to abiotic stresses such as drought (Croser et al. 2003; Kashiwagi et al. 2005; Toker et al. 2007), cold (Croser et al. 2003; Toker 2005; Berger et al. 2012), and combined drought and heat (Canci and Toker 2009). Wild Cicer species also have desirable nutrition-related traits such as high seed protein and mineral contents (Rao et al. 2003; Sharma et al. 2021).
Various incompatibility barriers, linkage drag, and poor viability and sterility of F1 hybrids and progenies mean that potential wild Cicer species are underutilized in chickpea breeding programs. Two annual wild Cicer species, Cicer reticulatum and Cicer echinospermum, are crossable with cultivated chickpea. However, the sterility of F1 hybrids and progenies has limited the utilization of C. echinospermum in crossing programs. Little is known about the crossability of the other six annual wild Cicer species with cultivated chickpea. To utilize those species in chickpea improvement, specialized techniques such as the application of growth hormones, ovule culture, and embryo rescue are required (Badami et al. 1997; Mallikarjuna 1999; Mallikarjuna and Jadhav 2008; Lulsdorf et al. 2005).
Few attempts have been made to generate interspecific hybrids between Cicer arietinum from the primary gene pool and wild Cicer pinnatifidum from the tertiary gene pool (Badami et al. 1997; Mallikarjuna and Jadhav 2008). Systematic crossing efforts involving diverse parental combinations are required to advance the production of viable interspecific hybrids involving tertiary gene pool species. The aim of this study, therefore, was to evaluate the crossability and geneflow between three cultivars of C. arietinum and four wild C. pinnatifidum accessions originating/collected from Turkey and Syria.
Three chickpea cultivars (ICCV 10, ICC 4958, and ICCV 96030) and four wild accessions (ICC 17126, ICC 17276, ICC 17200, and ICC 17269) belonging to the tertiary gene pool species C. pinnatifidum were used (Table 1). The seeds of all wild accessions were scarified by incising the hard seed coat. All seeds were treated with fungicides (2 g thiram + 1 g carbendazim kg-1 seed) before sowing in pots in a 2:1:1 mixture of sterilized black soil, farmyard manure, and sand. Seed sowing was staggered to synchronize the flowering of cultivated genotypes and wild accessions. At 1 month after germination, C. pinnatifidum seedlings were exposed to an 18-h light/6-h dark photoperiod with light supplied by 60-W incandescent lights to induce early flowering (Sharma and Upadhyaya 2019). Interspecific crosses were made using the three cultivars as female parents and the four wild accessions as pollen parents. ICCV 10 and ICC 4958 were each crossed with all four C. pinnatifidum accessions, and ICCV 96030 was crossed only with two accessions, ICC 17126 and ICC 17269. The flower buds of the female parents were emasculated and tagged between 3:00 p.m. and 4:00 p.m., and then pollinated with fresh pollen from wild accessions the following morning between 8:00 a.m. and 9:00 a.m. Each day for 3 consecutive days, a mixture of growth hormones (50 mg L-1 gibberellic acid + 10 mg L-1 naphthalene acetic acid + 10 mg L-1 kinetin, 1:1:1) was applied to the base of the peduncle of the pollinated buds to prevent flower drop and pod abscission. Selfed pods on the same branch were removed to encourage growth of the crossed pods. We recorded the number of pollinations and number of fully developed pods generated in each cross (Table 2).
Yellowing pods were harvested and the ovules were cultured in liquid Murashige and Skoog (MS) medium containing 3% w/v sucrose, 0.25 mg/L indole acetic acid, and 1 mg/L zeatin. After 3 weeks, the cultured ovules were subcultured into fresh ovule culture medium and cultured until the embryos emerged from the ovules. The embryos were transferred to shoot growth medium (liquid MS containing 3% w/v sucrose, 0.25 mg/L indole acetic acid, and 1 mg/L kinetin). Well-grown shoots were cultured on root-induction medium (half-strength MS basal salts, 1.5% w/v sucrose, and 0.5 mg/L indole butyric acid). We recorded the number of ovules cultured and number of plantlets generated through ovule culture (Table 2).
One fully mature pod with a healthy F1 seed was harvested from the ICC 4958 × ICC 17269 cross. The F1 seed resembled that of the cultivated parent (ICC 4958) with respect to size, color, texture, and shape. The mature F1 seed was sown in a mixture of soil, sand, and vermiculite (3:1:1). The F1 seedling had a leaf shape similar to that of the wild C. pinnatifidum parent ICC 17269, confirming true hybridity (Figure 1). Thus, the generation of a healthy and functional F1 seed in the ICC 4958 × ICC 17269 cross was not prevented by pre-fertilization barriers such as failure of pollen germination, pollen incompatibility, arrested pollen tube growth in the stigma or style, failure of the pollen tube to penetrate the ovule, or arrested growth of the pollen tube within the ovule; or by post-fertilization barriers such as embryo abortion, or shriveled or immature F1 seeds. However, this seedling became albino (lacked chlorophyll) at 4–5 days after germination (Figure 1). This albinism is attributed to defective chloroplasts with poorly developed thylakoids and few and disorganized grana (Badami et al. 1997; Clarke et al. 2011). Our attempts to multiply this albino-type plant by regeneration through callus induction and culture of different explants (leaves, stem cuttings, and nodes) on basal MS medium containing 0.5 mg/L benzylaminopurine and 0.5 mg/L naphthalene acetic acid were unsuccessful. Thus, although geneflow in the ICC 4958 × ICC 17269 cross was not hindered by the pre- or post-fertilization barriers reported elsewhere (Badami et al. 1997; Ahmad and Slinkard 2004; Mallikarjuna and Jadhav 2008; Clarke et al. 2011), it was hindered by the albinism of F1 hybrid plants. It will be possible to generate more healthy F1 pods and seeds from this cross by increasing the number of pollinations and using different combinations of plant growth hormones. However, efforts are needed to address the problem of albinism in F1 seedlings.
Unlike other crosses, interspecific crosses involving ICCV 96030 resulted in fully developed, mature pods (Table 2). However these pods lacked mature seeds. Most of the pods contained minute to small-sized colorless ovules. Thus, the pods developed normally but the ovules inside did not (Figure 2).
Pod development begins after fertilization. In this study, C. pinnatifidum pollen successfully fertilized ICCV 96030, leading to the differentiation of the ovary into the pod wall. However, ovules did not successfully differentiate into seeds due to some intrinsic reasons. This kind of hybrid embryo response has not been reported for other chickpea interspecific crosses. The incompatibility between cultivated chickpea ICC 96030 and all the C. pinnatifidum accessions used in this study is due to a post-zygotic barrier, specifically defective embryos that could not develop into functional seeds. Post-zygotic barriers hindering interspecific hybridization between C. arietinum and C. pinnatifidum have also been reported by Badami et al. (1997). Of the three cultivated chickpea cultivars, ICCV 96030 yielded the highest number of mature, fully developed pods, including a few with enlarged embryos, when pollinated with C. pinnatifidum. It will be possible to harvest mature pods with seeds from ICCV 96030 × C. pinnatifidum crosses by increasing the number of pollinations in each cross, adjusting plant growth hormone treatments to facilitate embryo/seed development, by crossing in different directions, and/or by using other C. pinnatifidum accessions, e.g., ICC 17276 and ICC 17200, as the pollen parent. In addition, immature embryos can be rescued by ovule culture.
The aborting ovules were cultured from 7–8 days after pollination. The tiny ovules did not grow upon culturing, but one larger ovule (derived from ICCV 96030 × ICC 17269) grew normally and the embryo regenerated into a seedling (Figure 2). Although the shoot was initially green, the newly formed leaves lacked chlorophyll and the albino seedling died after 2 weeks. Defective chloroplasts are the major barrier in generating interspecific hybrids between C. arietinum and C. pinnatifidum (Clarke et al. 2011). In the crosses involving ICCV 10, flower drop was the major obstacle. Most of the pollinated flower buds dropped within 1–2 days of pollination, despite the use of plant growth hormones. Although some cross combinations formed a few pods, they turned yellow within 3–4 days of pollination, and ovules from these pods did not develop further in vitro because of their small size.
Interspecific hybridization between chickpea cultivars and C. pinnatifidum produced one fully mature F1 seed (from ICC 4958 × ICC 17269). None of the other cross combinations yielded fully mature F1 seeds. Although ICCV 96030 formed the most pods, followed by ICC 4958, only one ovule from the ICCV 96030 × ICC 17269 cross regenerated into a plantlet in ovule culture. None of the three chickpea cultivars formed pods when pollinated with C. pinnatifidum ICC 17276. On the basis of the pod, ovule, and seed formation of the interspecific crosses, we concluded that the chickpea cultivars ICC 4958 and ICCV 96030 and the C. pinnatifidum accessions ICC 17269 followed by ICC 17126 and ICC 17200 exhibited good crossability.
To our knowledge, this is the first report of a fully mature F1 seed derived from an interspecific cross between cultivated chickpea and C. pinnatifidum without using embryo rescue. Our results show that the parents’ genotypes affect crossability between C. arietinum and C. pinnatifidum. The successful development of a mature healthy F1 seed from the interspecific ICC 4958 × C. pinnatifidum ICC 17269 cross confirmed the absence of pre- and post-fertilization barriers. Instead, albinism of F1 hybrids was the major obstacle hindering geneflow between C. pinnatifidum and cultivated chickpea. Embryo abortion occurred after interspecific crosses involving the chickpea cultivar ICCV 96030 and all C. pinnatifidum accessions. Using an ovule culture technique, one albino plantlet was regenerated from the ICCV 96030 × ICC 17269 cross. The interspecific crosses between chickpea cultivar ICCV 10 and C. pinnatifidum accessions were unsuccessful due to excessive flower drop and poor pod formation. These variable genotype-specific responses of pod and seed development suggest that more genotypes should be included when testing for cross-compatibility. The cultivated genotypes used here originate from central and southern agro-geographical areas of India. Including more genotypes from other parts of India may be useful for identifying those that are readily crossable with C. pinnatifidum, preferably without producing albino progeny.
Although pod and seed formation in crosses between cultivated chickpea and C. pinnatifidum can be improved using various techniques, it will be difficult to improve geneflow between these two species because of the genetic factor that confers albinism in the F1 hybrids. These results show that different parental genotype combinations have different crossabilities in inter-specific crosses, indicating that some genetic factors are important for the efficient production of interspecific hybrids involving C. pinnatifidum.