Albinism in F1 Hybrids Hinders Geneow Between Cultivated Chickpea (Cicer arietinum L.) and the Tertiary Gene Pool Species, Cicer Pinnatidum

Wild Cicer species, especially those in the tertiary gene pool, carry useful alleles for chickpea improvement. The aim of this study was to evaluate the crossability and geneow between three chickpea cultivars (as female parents) and four cross-incompatible Cicer pinnatidum accessions (as pollen parents) from the tertiary gene pool. Ten crosses were conducted. One fully developed healthy F 1 seed was harvested in vivo from the ICC 4958 × ICC 17269 cross, but the seedling developed an albino phenotype at 4–5 days after germination. Unlike other crosses, those involving the cultivar ICCV 96030 generated a large number of pods with comparatively large ovules. One albino plantlet was obtained from the ICCV 96030 × ICC 17269 cross by embryo rescue. Crosses involving ICCV 10 resulted in ower drop and poor pod set. These variable genotype-specic responses of pod, ovule, and seed development indicate that genetic factors affect the formation of interspecic hybrids. Although pod and seed formation in these interspecic crosses can be improved, geneow between these materials is hindered by a strong genetic factor conferring albinism in the F 1 hybrids.


Introduction
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.
Various incompatibility barriers, linkage drag, and poor viability and sterility of F 1 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 F 1 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 interspeci c hybrids between Cicer arietinum from the primary gene pool and wild Cicer pinnati dum 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 interspeci c hybrids involving tertiary gene pool species. The aim of this study, therefore, was to evaluate the crossability and gene ow between three cultivars of C. arietinum and four wild C. pinnati dum 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. pinnati dum were used ( Table 1). The seeds of all wild accessions were scari ed 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 owering of cultivated genotypes and wild accessions. At 1 month after germination, C. pinnati dum seedlings were exposed to an 18-h light/6-h dark photoperiod with light supplied by 60-W incandescent lights to induce early owering (Sharma and Upadhyaya 2019). Interspeci c 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. pinnati dum accessions, and ICCV 96030 was crossed only with two accessions, ICC 17126 and ICC 17269. The ower 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 ower 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 F 1 seed was harvested from the ICC 4958 × ICC 17269 cross. The F 1 seed resembled that of the cultivated parent (ICC 4958) with respect to size, color, texture, and shape. The mature F 1 seed was sown in a mixture of soil, sand, and vermiculite (3:1:1). The F 1 seedling had a leaf shape similar to that of the wild C. pinnati dum parent ICC 17269, con rming true hybridity ( Figure 1). Thus, the generation of a healthy and functional F 1 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 F 1 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 gene ow 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 F 1 hybrid plants. It will be possible to generate more healthy F 1 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 F 1 seedlings.
Unlike other crosses, interspeci c 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. pinnati dum 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 interspeci c crosses. The incompatibility between cultivated chickpea ICC 96030 and all the C. pinnati dum accessions used in this study is due to a post-zygotic barrier, speci cally defective embryos that could not develop into functional seeds. Post-zygotic barriers hindering interspeci c hybridization between C. arietinum and C. pinnati dum 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. pinnati dum. It will be possible to harvest mature pods with seeds from ICCV 96030 × C. pinnati dum 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. pinnati dum 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 interspeci c hybrids between C. arietinum and C. pinnati dum (Clarke et al. 2011). In the crosses involving ICCV 10, ower drop was the major obstacle. Most of the pollinated ower 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.
Interspeci c hybridization between chickpea cultivars and C. pinnati dum produced one fully mature F 1 seed (from ICC 4958 × ICC 17269). None of the other cross combinations yielded fully mature F 1 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. pinnati dum ICC 17276. On the basis of the pod, ovule, and seed formation of the interspeci c crosses, we concluded that the chickpea cultivars ICC 4958 and ICCV 96030 and the C. pinnati dum accessions ICC 17269 followed by ICC 17126 and ICC 17200 exhibited good crossability.
To our knowledge, this is the rst report of a fully mature F 1 seed derived from an interspeci c cross between cultivated chickpea and C. pinnati dum without using embryo rescue. Our results show that the parents' genotypes affect crossability between C. arietinum and C. pinnati dum. The successful development of a mature healthy F 1 seed from the interspeci c ICC 4958 × C. pinnati dum ICC 17269 cross con rmed the absence of pre-and post-fertilization barriers. Instead, albinism of F 1 hybrids was the major obstacle hindering gene ow between C. pinnati dum and cultivated chickpea. Embryo abortion occurred after interspeci c crosses involving the chickpea cultivar ICCV 96030 and all C. pinnati dum accessions. Using an ovule culture technique, one albino plantlet was regenerated from the ICCV 96030 × ICC 17269 cross. The interspeci c crosses between chickpea cultivar ICCV 10 and C. pinnati dum accessions were unsuccessful due to excessive ower drop and poor pod formation. These variable genotype-speci c 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 agrogeographical areas of India. Including more genotypes from other parts of India may be useful for identifying those that are readily crossable with C. pinnati dum, preferably without producing albino progeny.
Although pod and seed formation in crosses between cultivated chickpea and C. pinnati dum can be improved using various techniques, it will be di cult to improve gene ow between these two species because of the genetic factor that confers albinism in the F 1 hybrids. These results show that different parental genotype combinations have different crossabilities in inter-speci c crosses, indicating that some genetic factors are important for the e cient production of interspeci c hybrids involving C. pinnati dum. Ovule culture to rescue hybrid embryo from ICCV 96030 × ICC 17269; a. Well-developed pods containing ovules that failed to develop; b. Ovule cultured in liquid medium.