Fabio De Pasquale

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

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 undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 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 l-1 malt extract. Plants were successfully transferred to soil.

Somatic embryogenesis in Citrus from styles culture

Abstract Styles (including the stigma) of Citrus aurantium L. (cvs. ‘AA 12’, ‘AA 30’ and ‘AA31’), C. deliciosa Tenore (cvs. ‘Avana’ and ‘Tardivo di Ciaculli’), C. paradisi Macf. (cvs. ‘Marsh seedless’ and ‘Star Ruby’) and C. sinensis (L.) Osb. (cvs. ‘Bonanza’, ‘Brasiliano 92’, ‘Sanguinello’ and ‘Valencia’) were cultured for induction of somatic embryogenesis. Explants were excised from flower buds which were collected during full bloom, and cultured on Murashige and Skoog (MS) basal medium supplemented with 146 mM sucrose, 500 mg/l malt extract and 13.3 μM 6-benzylaminopurine (BAP) as well as the same medium without BAP. Callus development was observed from the style base 4 weeks after treatment initiation, and embryogenesis occurred 2–3 months later. Embryogenesis has been induced from the style-derived callus of all the cultivars tested except for the cultivars ‘Avana’ and ‘Star Ruby’. The best results for callus growth and embryo regeneration was obtained from explants of ‘Brasiliano 92’ cultured on medium containing BAP. Somatic embryos were isolated from callus and placed on MS medium supplemented with 146 mM sucrose, 500 mg/l malt extract and 0.27 μM α-naphthaleneacetic acid (NAA) where they formed entire plants. Two months later plants were successfully established in soil.

Micropropagation of Citrus

The genus Citrus is cultured in more than 100 countries making it one of the most important commercial fruit crops in terms of economic value and human nutrition (Barlass and Skene, 1986). Fresh fruits and juice are the most significant citrus products, but essential oils, pectin, and marmalade, as well as candied and dried rinds, also have commercial value (Barlass and Skene, 1986). In recent years, the importance of Citrus for the production of ornamental plants has increased considerably (De Pasquale and Carimi, 1998).

Somatic embryogenesis from styles of lemon (Citrus limon)

Lemon [Citrus limon (L.) Burm.] styles were treated with different growth regulators for induction of somatic embryos. Styles and stigmas were dissected from flowers and cultured on a Murashige and Skoog (MS) basal medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid and 13.3 μM 6-benzyladenine. Callus was induced from the style base 2 weeks after the treatment initiation, and embryos appeared 2 months later.

Effect of native arbuscular mycorrhizal fungi and Glomus mosseae on acclimatization and development of micropropagated Citrus limon (L.) Burm

Summary Arbuscular mycorrhizal fungi (AMF) have been successfully used to improve acclimatization, survival and growth of many micropropagated fruit species. In this work we tested the influence of the AM fungus Glomus mosseae (BEG 116) and that of a Sicilian native mixture of Glomus species, on the survival and growth of micropropagated lemon plants (Citrus limon (L.) Burm. ‘Zagara Bianca’) during the weaning phase to a final age of 16 months. Plantlets were obtained from embryogenic callus derived from style and stigma cultures. The native AM fungal mixture was obtained from the rhizosphere soil of a citrus grove (Sicily, Italy) and was found to be more infective than G. mosseae alone. It significantly increased plant height, root and shoot weight, leaf area, P content and shoot/root ratio at the end of the weaning phase, i.e. 17 weeks after in vivo transferring and inoculation. A severe growth depression was observed in plants inoculated with G. mosseae. During the early weaning phase, plant survival was reduced only by the native AMF mix inoculum that may still have been too rich in soil-borne pathogens; at the end of the experiment (69 weeks) plant survival was reduced by all the treatments except for the native AMF mix inoculum. A rhizosphere effect other than mycorrhizal was detected in the rhizosphere control inoculated with the sievate of the native AMF inoculum, in particular on root development. To our knowledge, this is the first report on mycorrhization of micropropagated citrus plants.

Somatic embryogenesis and plant regeneration from pistil transverse thin cell layers of lemon (Citrus limon)

Abstract 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 μM 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).

Rearing of Prays citri on callus derived from lemon stigma and style culture.

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 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.