Abstract
An efficient protocol to induce shoot buds regeneration in Citrus clementina cultivars (‘Monreal’, ‘SRA 63’ and ‘SRA 64’) by direct orgaNogenesis has been developed using cotyledons as explants. Cotyledons transversely cut in three segments and entire ones were cultured on Murashige and Skoog (1962) solidified medium containing vitamins, 500 mg l-1 malt extract, 50 g l-1 sucrose and supplemented with three different concentrations of BAP (8.8, 13.2 and 17.6 μM). In all three cultivars the entire cotyledons showed more shoot morphogenic potential than transversely cut ones and after 60 incubation days the optimum BAP concentration was 17.6 μM in ‘Monreal’ (50% ± 2.89% of frequency regeneration) and 13.2 μM in ‘SRA 63’ (33.33% ± 3.33%) and ‘SRA 64’ (25.93% ± 1.85%). In absence of BAP No mor-phogenesis occurred, demonstrating the absolute requirement of this hormone for shoots induction. The young shoots showed a regular growth in the culture tubes containing the basal medium without hormones, and the rooted plantlets survived after acclimatization. This protocol may find application in Citrus genetic improvement programs.
Highlights
Citrus clementina Hort. ex Tanaka is a very important crop because of its excellent organoleptic and easypeeling qualities
There are several studies on plant regeneration by organogenesis and embryogenesis from different types of explants in Citrus genus [8,9,10,11,12,13], but very little information is available about procedures for achieving regeneration from clementine mandarin: embryos were induced from calli of ovular tissue [14], aberrant plants were obtained by direct and indirect somatic embryogenesis from the nucellus of eight cultivars [7], and gynogenesis [15] and androgenesis [16,17,18] were studied in different cultivars
Regeneration in all genotypes only occurred in the presence of BAP (Figure 2), that has a significant effect (p ≤ 0.001) on the regeneration probability (Table 1(A)), while neither swelling nor morphogenic responses were noted when using the hormone-free basal medium
Summary
Citrus clementina Hort. ex Tanaka is a very important crop because of its excellent organoleptic and easypeeling qualities. In C. clementina, as in Citrus spp., bud mutations arise spontaneously [1,2,3,4] and growers select them to improve horticultural traits of economically interesting species; there is a high degree of interspecific sexual compatibility, so clementine is highly used in genetic improvement programs as mother plant to obtain new genotypes through hybridization by controlled inbreeding. There are several studies on plant regeneration by organogenesis and embryogenesis from different types of explants in Citrus genus [8,9,10,11,12,13], but very little information is available about procedures for achieving regeneration from clementine mandarin: embryos were induced from calli of ovular tissue [14], aberrant plants were obtained by direct and indirect somatic embryogenesis from the nucellus of eight cultivars [7], and gynogenesis [15] and androgenesis [16,17,18] were studied in different cultivars. In the last decade cotyledons and cotyledonary nodal regions were used as target tissues for transformation mediated by Agrobacterium [20]
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