Molecular and Cytogenetic Characterization of Triploid Somatic Hybrids between 'Shogun' Mandarin and Grapefruit.

Abstract

Somatic hybrids were produced hetween embryogenic callus protoplasts of 'Shogun' mandarin (Citrus reticulata Blanco, 2n = 2x = 1 8) and leaf protoplasts of grapefruit (C. paradisi Macf., 2n = 2x =18) by electrofusion. Hybridity of the two regenerated plants was confirmed by leaf characteristics and nuclear genorne analysis using RAPD and PCR RFLP. Flow cytometry and root tip cell count of the two hybrids revealed a chromosome number of 27. These triploid somatic hybrids on transfer to field showed norma] morphology and vigorous growth. Field performances as well as disease resistance are under investigation.

Utilization of intergeneric somatic hybrids as an index discriminating taxa in the genus Citrus and its related species

“…& M. Kell (2n=2x=18) were used in this study. The embryogenic callus of the 'Shogun' mandarin was induced from nucellar tissue and maintained by subculturing on Gellan gum-solidified MT basal medium (Murashige and Tucker 1969) containing 10 mg/l BA and 30 g/l sucrose at 25°C under continuous illumination (38 lmol m À2 s À1 ) for 3 years (Kunitake et al 2002).…”

“…Isolated protoplasts were fused by electrofusion according to Kunitake et al (2002) using a SSH-1 instrument (Shimadzu Somatic Hybridizer, Kyoto, Japan) equipped with a 0.8 ml volume FTC-03 type electrofusion chamber. Protoplasts were aligned into short chains by applying an alternating current of 120 V cm À1 at 1 MHz for 15 s. Fusion was induced by a direct current square pulse of 1.5 kV cm À1 for 30 ls.…”

“…Protoplasts of embryogenic calli and leaves were isolated and purified as described previously (Kunitake et al 2002), then washed twice with 0.6 M mannitol by centrifugation at 100 g for 5 min, and suspended in a fusion solution containing 1 mM CaCl 2 AE2H 2 O, 5 mM MES and 0.6 M mannitol at pH 5.7, with cell concentration adjusted to 1·10 6 /ml. Isolated protoplasts were fused by electrofusion according to Kunitake et al (2002) using a SSH-1 instrument (Shimadzu Somatic Hybridizer, Kyoto, Japan) equipped with a 0.8 ml volume FTC-03 type electrofusion chamber.…”

Production of intergeneric somatic hybrids between round kumquat (Fortunella japonica Swingle) and ?Morita navel? orange (Citrus sinensis Osbeck)

“…Protoplasts were isolated from embryogenic calli of 'Morita navel' and round kumquat according to the method of Kunitake et al (2002). Prior to protoplast isolation, embryogenic calli of both species were pretreated for 10 days by transferring to MT medium containing 50 g l 1 lactose and 2 g l 1 gellan gum to prevent bursting of protoplasts.…”

“…Electrofusion and protoplast culture Isolated protoplasts were fused by electrofusion according to Kunitake et al (2002). After fusion, protoplasts were cultured in MT medium containing 0.6 M sucrose and 2 g l 1 gellan gum, according to Ohgawara et al (1985).…”