<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.     

Protoplast culture and plant regeneration of several species in the genus Dianthus

Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS Murashige and Skoog (1962) - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - FDA fluoroscein diacetate - NAA 1-naphthaleneacetic acid     

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l^?1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.     

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis

Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.     

Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.     

Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture

We have previously demonstrated that nestin-expressing multipotent hair follicle stem cells are located above the hair follicle bulge and can differentiate into neurons and other cell types in vitro. The nestin-expressing hair follicle stem cells promoted the recovery of pre-existing axons when they were transplanted to the severed sciatic nerve or injured spinal cord. We have also previously demonstrated that the whisker hair follicle contains nestin-expressing stem cells in the dermal papilla (DP) as well as in the bulge area (BA), but that their origin is in the BA. In the present study, we established the technique of long-term Gelfoam? histoculture of whiskers isolated from transgenic mice in which nestin drives green fluorescent protein (ND-GFP). Confocal imaging was used to monitor ND-GFP-expressing stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam? forming nerve-like structures. This new method of long-term histoculture of whiskers from ND-GFP mice will enable the extensive study of the behavior of nestin-expressing multipotent stem cells of the hair follicle.     

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l^–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l^–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed. Received: 24 February 1997 / Revision received: 10 July 1997 / Accepted: 28 July 1997