Elimination of bean seed-borne bacteria by thermotherapy and meristem culture

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Jasmonic acid promotes division of fern protoplasts, elongation of rhizoids and early development of gametophytes

The effects of jasmonic acid (JA) on spore germination, early gametophyte development and sporophytic protoplast culture of the fern Platycerium bifurcalum (Cav.) C. Chr. were investigated. JA and no influence on spore germination and primary rhizoid initiation, but significantly promoted early gametophyte development, which was evident from the longer primary rhizoids as well as the higher number of rhizoids and cells per gametophyte. Jasmonic acid (1 μ M ) also promoted the transition of gametophytes from a filamentous to a planar growth. Optimal primary rhizoid elongation and highest cell division activities in the gametophytes were observed at 0.01–1 μ M JA, while the highest number of rhizoids on gametophytes was obtained at 0.1–1 μ M JA. Jasmonic acid (0.01 μ M ) also stimulated initial protoplast divisions. Except for the experiment in which the effect of JA on germination was tested. JA concentrations exceeding 1 μ M did not promote cell elongation or cell division but were instead inhibitory. On the basis of these findings, we propose that JA may be involved in early stages of P. bifurcatum development.     

Micropropagation ofPinus sylvestris

Plantlet regeneration from axillary buds, induced on seedling apices ofPinus sylvestris, is described. The influence of medium containing 1, 5, 10 or 50 M BA on the initiation of buds on 3, 6 and 9-week-old seedlings was investigated. With higher BA concentrations the number of apices that formed buds and the total number of buds increased, wheres their length decreased. Soaking the explants for 2 h in 111 M BA and for 5 h in 44 M BA gave better results, best expressed in longer buds. After 30 days, axillary buds were separated and transferred to a medium with 2% sucrose. Separated buds exposed to far-red light during the first week elongated better in comparison with the control. 64% of shoots rooted after a 24 h period of treatment with 53.8 M NAA incorporated in 0.6% water agar and transferred to 1/16-strength Murashige & Skoog medium as modified by Cheng supplemented with 1% sucrose.     

Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)

The induction, origin, morphology, and ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C. Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%, 0.1%, 1%, or 2% sucrose. A low sucrose concentration (0.01%) and wounding of the adaxial side of the leaf significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes). Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both forms had the same ploidy level. On the basis of these findings, we propose a set of conditions which regularly and reproducibly induces apospory on most of the leaf explants of the fern P. bifurcatum.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Establishment of cell suspension cultures of yew (<Emphasis Type="Italic">Taxus × media</Emphasis> Rehd. and assessment of their genomic stability

Summary Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inibited growth while no effect on nuclear DNA content was noted.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).