Experimental

Capsicum (chilli peppers) is a new world genus within the nightshade family Solanaceae, with approximately 30 species, of which Capsicum annuum L., C. frutescens Mill., C. baccatum L., C. chinense Jacq. and C. pubescens Ruiz and Pavon are domesticated and currently cultivated worldwide (Csilléry, Reference Csilléry2006; Moscone et al., Reference Moscone, Scaldaferro, Grabiele, Cecchini, Garcia, Jarret, Davina, Ducasse, Barboza and Ehrendorfer2007). Of the five above-domesticated species, C. annuum L. is the most economically important one with a large number of mild and pungent types of cultivar distributed in various parts of the world. Most of the species and cultivars of Capsicum so far studied through chromosomal analysis have revealed 2n = 2x = 24 chromosome number. While 2n = 2x = 26 has been reported in a few species, only one reference to natural wild polyploidy accession of C. annuum with 2n = 48 chromosome is available (Pickersgill, Reference Pickersgill and Pochard1977; Pozzobon et al., 2006). Thus, the present study reports for the first time a genetically unexplored cultivar of C. annuum with 2n = 48 somatic and n = 24 gametic chromosomes through detailed karyomorphological observation.

Mature fruits and plants of the cultivar were collected from a nursery of South Sikkim (7000 feet, 27°10′N 88°22′E) during the month of September. This cultivar is a biennial shrub, which grows up to 1 m in height. Leaves are simple, large, opposite, acuminate, entire, with wavy margins. Flowers are small, white-coloured, in axillary clusters. Fruits are medium-sized, ovoid, green, become red when ripe, and contain many compressed yellowish-white seeds [Fig 1a (i–iii)]. The herbarium of the plant was identified as a cultivar of C. annuum from the Botanical Survey of India, Howrah, West Bengal. The voucher specimen is kept in the Department of Botany, Presidency University, Kolkata.

Fig. 1

Exomorphology, somatic and gametic chromosomes of C. annuum L. cultivar with 2n = 48 chromosomes. (a) Field-grown plant with flower (i), fruit (ii) and seeds (iii). Bar = 8 cm and 0.5 cm. (b, c) Mitotic metaphase plate showing 2n = 48 chromosomes with orcein staining. Bar = 10 μM. (d) Mitotic metaphase plate showing 2n = 48 chromosomes with giemsa staining. Bar = 10 μM. (e) Diakinesis of prophase I showing 24 bivalents (n = 24). Bar = 3 μM. (f) Meiotic metaphase I showing 24 bivalents (n = 24). Bar = 3 μM. (g) Detailed karyotype. Bar = 5 μM (A colour version of this figure can be found online at http://www.journals.cambridge.org/pgr).

Somatic chromosome analysis was carried out from the root tips of seeds of the donor plant (D) and micropropagated plants (R₀). Root tips of approximately 1 cm in length were excised and pre-treated with 0.1% colchicine for 5½ h at 10–12°C, fixed in acetic methanol (1:3) and stored at − 20°C. The root tips were hydrolyzed in 1 N HCl for 15–20 min at 37°C and subsequently stained with 2% aceto-orcein for 2–3 h and squashed in 45% acetic acid to obtain well-scattered metaphase plates. Similarly for Giemsa staining, enzymatic maceration and air-drying of fixed roots were performed as described by Fukui (Reference Fukui, Fukui and Nakayama1996) with required modifications (Jha and Yamamoto, Reference Jha and Yamamoto2012). Gametic chromosome analysis was performed from flower buds after overnight fixation in Carnoy's solution at 10–12°C. Dissected anthers were smeared with 2% aceto-carmine on a clean glass slide to obtain good meiotic plates. Haploid chromosome numbers per pollen mother cell were determined by counting the number of bivalents from diakinesis and from metaphase I plates. At least 5–10 well-scattered metaphase plates per root tip were studied. Photographs were taken using Carl Ziess, Axiostar plus Photomicroscope. Detailed karyotype and meiotic chromosome analysis were carried out using camera lucida and digital calipers. Karyomorphological data in the present study are established using parameters such as total chromatin length, centromeric index and arm ratio. Stebbins' Category (Stebbins, Reference Stebbins1971) of the karyotype was determined by using the short arm length and long arm length of each chromosome.

Analysis from more than 50 root tips and at least ten metaphase/root tips through orcein and giemsa staining confirms the 2n = 48 chromosome number of the present cultivar of C. annuum (Fig. 1(b–d)). Somatic chromosomes are small in size and range from 2.32 to 4.56 μm; and total F% (TF%) was calculated as 42.12% (Table 1). Detailed karyomorphological studies (Fig. 1(g)) revealed 12 median (M), 24 nearly median (m), four submedian (Sm), four subterminal (St) chromosomes and four (sat) chromosomes with both primary and secondary constrictions. Total chromatin length was 85.07 μm, and the karyotype matched with Stebbins' 1A category (Table 1). Meiotic chromosomal analysis of 50 well-scattered diakinesis and 50 metaphase I plates from flower buds of D and R₀ plants confirmed the n = 24 haploid chromosome number of this cultivar. We obtained only 24 bivalent chromosomes (Fig. 1(e), (f)) and could not record any univalent or tetravalent condition. All the chromosomal data are presented in Table 1.

Table 1

Mitotic and meiotic details of Capsicum annuum L., a new cultivar

M, chromosome with median primary constriction; m, chromosome with nearly median primary constriction; Sm, chromosome with submedian primary constriction; St, chromosome with subterminal primary constriction; Sat, chromosome with both primary and secondary constriction; TCL, total chromatin length; L, long chromosome; S, small chromosome; TF% (total form percentage) = total short arm length × 100/total long arm+short arm lengths; KC, karyotype category; D, donor plants; R₀, micropropagated plants.

In columns 8 and 9, the results are mean ± standard error (calculated from five replicates).