Search

Five clones of sour orange (Citrus aurantium L.) showing significant morphological differences were selected from our germplasm collection and characterized both by genetic and leaf volatiles analysis. The genetic studies were undertaken by the use of molecular markers developed by PCR-based techniques (ISSR and RAPD), while the leaf essential oil patterns were obtained by chromatographic and mass spectrometric determination. Data obtained suggest that reasonably similar information can be achieved from the two techniques, supporting each other in characterization studies.

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16% (C. sinensis) or 10% (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50% after drying to 15% moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93% germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich-indirect-enzyme-linked immunosorbent assay (DASI-ELISA) in explants and embryogenic callus, but was not detested in any of the. plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.

A new method for rearing the citrus flower moth (Prays citri Mill.) (Lepidoptera, Yponomeutidae) on lemon [Citrus limon (L.) Burm.] callus is reported. In the present research callus (an undifferentiated mass of plant cells that can be grown under sterile conditions on an artificial medium in vitro) was induced from lemon stigma and style explants cultured on Murashige and Skoog (MS) medium supplemented with 500 mg l(-1) malt extract, 13.3 mu M 6-benzylaminopurine, and 146 mM sucrose. Also somatic embryogenesis and plant regeneration were obtained from the cultures of styles and stigmas of lemon. Adults were obtained from larvae on infested flowers collected in the field. Different oviposition substrates were used: white oval pearls (WOP), black oval pearls (BOP), rooted shoots (RS) of lemon obtained in vitro, and artificial flowers containing lemon callus (AF). Larvae were reared on lemon callus. Adults oviposited on RS, on WOP, and on AF. BOP were rejected as oviposition substrates. The flower moth reared on callus oviposited fertile eggs. In our tests P. citri completed three generations on callus or on callus plus shoots. In the latter case the larvae preferred callus and fed on shoots only after callus was completely eaten. The life cycle on callus at 23 +/- 1 degrees C lasted about 21 days. There were significant differences between oviposition substrates for what concerns the number of eggs laid. It was observed that females generally preferred WOP (about 25 eggs/female) to AF (about 20 eggs/female) or RS (about 12 eggs/female) as oviposition substrate. Nevertheless the percentage of eggs that developed into adults was higher when AF sealed with stretched Parafilm were used (about 70% of eggs developed into adults). The method of rearing P. citri with AF was labour-saving and the feeding substrate (callus) had less tendency to become mouldy or decompose than when WOP and RS were used. Since such a diet is available for the insect all year round and callus can be produced in unlimited quantity, it could be possible to obtain a mass production of this moth.

Minigrafting was used for rescue of tissue culture regenerants of the following four species of Citrus: sour orange (C, aurantium L, 'AA CNR 31'), sweet orange [C, sinensis (L,) Osb, 'Valencia Late'],lemon [C, lemon (L,) Burm, 'Femminello Comune'] and mandarin (C, deliciosa Tenore 'Tardivo di Ciaculli'), The grafting was carried out with different scion types including shoots, roots, inverted roots and somatic embryos, This material was obtained in vitro from embryogenic style-derived callus, Seedlings of open-pollinated sour orange (C, aurantium L,), Cleopatra mandarin (C, reshni Hort, ex Tan.) and 'Troyer' citrange [C, sinensis Osb. x Poncirus trifoliata (L,) Raf.] were used as rootstocks, Minigrafting of shoots, roots, inverted roots and embryos regenerated in vitro allowed successful rescue of these four species. Percentages of successful minigrafts ranged from 100 % (shoots) to 2.5 % (inverted roots). The probability of successful graft unions Increased with the age of the rootstock, The final mean canopy leaf area (120 days after grafting) ranged from 5.2 cm(2) ('Tardivo di Ciaculli' mandarin grafted on 6 month old Cleopatra mandarin) to 157.9 cm(2) ('Valencia Late' sweet orange grafted on 18-month-old Cleopatra mandarin). In this work we examined some of the variables which influenced minigrafting and we determined the efficacy of this method for rescue of in vitro regenerants of Citrus. This method is also suggested as a technique to produce a high percentage of viable plants from in vitro regenerants difficult to root.

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and underdeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg 1(-1) malt extract; 500 mg 1(-1) malt extract and 4.6 mu M kinetin; and 500 mg 1(-1) malt extract and 13.3 mu M 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1-3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg 1(-1) malt extract. Plants were successfully transferred to soil.

Callus induction, somatic embryogenesis and plant regeneration were obtained in Citrus limon (L.) Burm. (cv. Femminello) from cultures of pistil transverse thin cell layer explants [(t)TCLs]. Explants were cultured on two different media, based on Murashige and Skoog salts and vitamins, supplemented with 500 mg l-1 malt extract (MSI), or 500 mg l-1 malt extract and 13.3 ¼M 6-benzylaminopurine (MSII). Sucrose (146 mM) was used as carbon source. Somatic embryos appeared 3 months after culture initiation from stigma and style (t)TCLs; they were observed at the surface of the (t)TCL-derived callus. Although ovary (t)TCLs showed the highest callus formation, they never differentiated somatic embryos. Percentages of embryo formation from (t)TCLs incubated on MSI (13% and 2% for stigma and style, respectively) were lower than those from (t)TCLs incubated on MSII (36% and 7% for stigma and style, respectively). The embryogenic response of stigma (t)TCLs was usually higher than that of style (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies (57% and 62% for stigma- and style-derived somatic embryos, respectively). Keywords: Citrus limon; plant regeneration; somatic embryogenesis; thin cell layer

Five clones of sour orange (Citrus aurantium L.) showing significant morphological differences were selected from our germplasm collection and characterized both by genetic and leaf volatiles analysis. The genetic studies were undertaken by the use of molecular markers developed by PCR-based techniques (ISSR and RAPD), while the leaf essential oil patterns were obtained by chromatographic and mass spectrometric determination. Data obtained suggest that reasonably similar information can be achieved from the two techniques, supporting each other in characterization studies.

A new method for rearing the citrus flower moth (Prays citri Mill.) (Lepidoptera, Yponomeutidae) on lemon [Citrus limon (L.) Burm.] callus is reported. In the present research callus (an undifferentiated mass of plant cells that can be grown under sterile conditions on an artificial medium in vitro) was induced from lemon stigma and style explants cultured on Murashige and Skoog (MS) medium supplemented with 500 mg l -1 malt extract, 13.3 μM 6-benzylaminopurine, and 146 mM sucrose. Also somatic embryogenesis and plant regeneration were obtained from the cultures of styles and stigmas of lemon. Adults were obtained from larvae on infested flowers collected in the field. Different oviposition substrates were used: white oval pearls (WOP), black oval pearls (BOP), rooted shoots (RS) of lemon obtained in vitro, and artificial flowers containing lemon callus (AF). Larvae were reared on lemon callus. Adults oviposited on RS, on WOP, and on AF. BOP were rejected as oviposition substrates. The flower moth reared on callus oviposited fertile eggs. In our tests P. citri completed three generations on callus or on callus plus shoots. In the latter case the larvae preferred callus and fed on shoots only after callus was completely eaten. The life cycle on callus at 23 ± 1 °C lasted about 21 days. There were significant differences between oviposition substrates for what concerns the number of eggs laid. It was observed that females generally preferred WOP (about 25 eggs/female) to AF (about 20 eggs/female) or RS (about 12 eggs/female) as oviposition substrate. Nevertheless the percentage of eggs that developed into adults was higher when AF sealed with stretched Paratilm were used (about 70% of eggs developed into adults). The method of rearing P. citri with AF was labour-saving and the feeding substrate (callus) had less tendency to become mouldy or decompose than when WOP and RS were used. Since such a diet is available for the insect all year round and callus can be produced in unlimited quantity, it could be possible to obtain a mass production of this moth.