Influence of rol genes in floriculture

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (Passiflora edulis Sims) genotypes

The propagation of many passion fruit species (Passiflora spp.) is done by seeds or sexual method, although asexual propagation, such stem cuttings, grafting or tissue culture is also possible and very useful in many cases. In this work, it was presented information on scientific and technological advances in sexual and asexual propagation of passion fruit, considering the different propagation methods and their applications. New demands for research and development activities are also related, given the wide range of passion fruit species and its potential for food, ornamental and medicinal use.

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Advances in passion fruit (Passiflora spp.) propagation

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.

Importance of co-cultivation medium pH for successful Agrobacterium -mediated transformation of Lilium × formolongi

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Agrobacterium-mediated transformation with rol genes of lilium longiflorum thunb.

The genus Passiflora, comprising about 500 species of vines, lianas and small trees, is the largest in the passion flower family (Passifloraceae). In the tropical and subtropical regions, several species are grown for their edible fruits (Passiflora edulis Sims.), while many others are cultivated in the warmer parts of the world, either outdoors or in the greenhouses, for their medicinal and ornamental value. Passiflora exhibits some unique floral traits, which are of particular interest to the floricultural market. With the aim to exploit the ornamental traits of some Passiflora species, a collection was settled at the CRA-FSO, in Sanremo. Tissue culture protocols were established for Passiflora hybrids 'Guglielmo Betto' and 'Manta', P. trifasciata Lemaire and P. foetida L. 'Hastata'. The in vitro regenerated plants were successfully acclimatized in the greenhouse. At flowering, P. 'Guglielmo Betto', P. 'Manta' and P. trifasciata plants, regenerated from tendrils, were similar to the mother plants concerning plant habit, flower shape, colour and form of leaves. P. foetida 'Hastata' plants which regenerated from immature-flowers showed morphological alterations in the flower and in the fruit involucral bracts. The reduced bracts left the fruit completely visible, conferring a new attractive phenotype. The modification is maintained in the cutting-derived plants.

In vitro plant regeneration as a tool to improve ornamental characters in Passiflora species.

Since 2006, a breeding programme for Hibiscus rosa-sinensis was established at CRA-FSO, Italy. The aim is to select specific cultivars suitable for pot plant production and well-adapted to the Mediterranean climate. A partial diallelic cross design (reciprocals without self-fecundations) was used to test crossing ability of the collected cultivars. However, only a limited number of cross combinations resulted in progenies with desirable characters. The first selected cultivars are presented. At the same time, we followed another approach to investigate whether desirable morphological modifications (plant size reduction for pot plant cultivation) could be obtained by transforming H. x rosa-sinensis with Agrobacterium rhizogenes. In vitro seedlings were used as sources of explants for the transformation experiments. Two A. rhizogenes strains (ATCC 15834 and NCPPB 1855) were used. Axenic hairy root cultures were established about 4 months after inoculation. Hairy roots grew vigorously on hormone free medium whereas normal roots did not. Transformed roots displayed a typical hairy root phenotype characterized by fast growth, high lateral branching and lack of geotropism. So far, after more than one year of cultivation, a clone of hairy root deriving from a cotyledon formed a friable yellowish callus at root node level and several adventitious buds are spontaneously regenerating from it.

New genotypes of Hibiscus × rosa-sinensis through classical breeding and genetic transformation.

RAPD analysis was used to determine the genetic similarity in wild species and cultivated varieties of Limonium genus and for verification of hybridity in progenies of interspecific crosses. Eleven wild species and ten commercial varieties were tested with ten selected RAPD primers. A total of 254 bands were scored and used for calculating genetic distances among entries. Two cultivars were grouped together with L. sinensis. The species L. bellidifolium, L. otolepis and L. caspia were clustered together. The largest cluster grouped five varieties and L. latifolium. The remaining three cultivars, belonging to the 'Altaica' group, resulted related to L. gmelinii. The wild species L. tataricum, L. perezii, L. sinuatum and L. longifolium were clearly separated from all of the other genotypes. Seven putative interspecific hybrids and their parents were characterised by means of nine selected RAPD primers. The putative parentage was confirmed for only two hybrids; the remaining five genotypes, on the basis of RAPD patterns, were identified as apomixis and/or self-pollinations. Our results demonstrated that RAPD analysis is a useful tool for the molecular characterization of Limonium genotypes. This simple technique may enable ornamental plant breeders to improve the efficiency of their breeding programs.

C. Bianchini

Papers

Agrobacterium-mediated transformation with rol genes of lilium longiflorum thunb.

In vitro plant regeneration as a tool to improve ornamental characters in Passiflora species.

New genotypes of Hibiscus × rosa-sinensis through classical breeding and genetic transformation.

Molecular analysis of limonium genus through RAPD markers