The aim of this investigation was to establish the conditions for effective regeneration of adventitious shoots on leaf explants of apple dwarf rootstock P.59. A three stage procedure was used ? the induction on medium containing thidiazuron (TDZ) and -naphtalene acetic acid (NAA) for two weeks, then the regeneration on medium supplemented with 6-benzylaminopurine (BAP) and NAA and growth of adventitious shoots on medium with low concentration of BAP and indole-3-butyric acid (IBA). The youngest leaves from proliferating shoot cultures were taken as explants for adventitious regeneration. The effect of MS salt concentration and type of solidifying agent in the induction and regeneration media, and a type of cytokinin in the induction medium was studied. It was proved that the regeneration potential was higher when 1/2 MS salts, Gelrite and TDZ was used.
Bacterial contamination is quite frequent in plant tissue cultures although, theoretically, cultures have to be axenic. It is their ubiquity and adaptability to different conditions that enable bacteria to colonize also tissue cultures. Among them there are not only the typical endo- and egzophytic species connected with the plant kingdom, but also those which are common among people. The bacteria species isolated from plant tissue cultures were found to be vitropathogenic (pathogens facultative to in vitro explants), latent (pathogens not virulent in vitro), and cryptic (present in tissues but invisible). Some bacteria produce growth regulators, which can modify the morphogenetic mode of explants. They are all undesirable ones in cultures, but the explants contaminated with pathogenic species should be eliminated obligatorily. Various groups of bacteria, as well as the techniques of detecting, identifying and eliminating them, are briefly described.
The paper summarizes information on the raising up menace of ornamental and forest nurseries by Phytophthora spp. caused mostly by P. cinnamomi, P. citricola, P. citrophthora, P. cryptogea, P. ramorum. Some Phytophthora species are especially detrimental, but their identification is not possible on the basis of symptoms. To detect and identify pathogens in the plants (with- and without symptoms), soil and water, reliable tests are required. The survey on methods and techniques reported in the literature which can be used to establish such assays is given. The most needed are the tests based on DNA markers, especially when organized as micro-chips due to the large number of samples which can be screened in a short time.
Prequisites for successful transformation are developed protocols on efficient regeneration of adventitious shoots or somatic embryos. The most important works concerning regeneration, the objectives and achievements in rose transformation are discussed.
An efficient method for genetic Agrobacterium tumefaciens - mediated transformation of five cultivars Gerbera hybrida was established. The youngest leaves and shoot tips from proliferating in vitro cultures were co-cultivated with disarmed strain LBA 4404/pBI121 carrying the chimaeric genes nptII and gus. The inoculated explants were repeatedly cultured on regeneration medium with kanamycin and Biotaxym. After 9-10 months from 0.04 to 0.25 independently transformed shoots from one inoculated leaf and from 0.06 to 0.65 from one inoculated shoot were obtained, depending on cultivar and additional treatment. Transformed shoots accounted for 5.0 to 50.0% of all regenerated shoots. Additional wounding` of leaves before inoculation by squeezing and scratching off increased transformation efficiency. Better results for recalcitrant to regeneration cultivars were obtained if squeezed shoot tips were inoculated. Regenerating shoots were selected on kanamycin, screened for gus expression with X-gluc and tested by PCR for nptII gene. All together 162 transgenic shoots were obtained in cvs Amber, Boy, Ferrari, Mariola and Tamara, but only 24 relatively stable lines were established.
Bacterial contamination is a serious problem in plant tissue culture. In in vitro cultures, if bacteria are introduced, it is most frequently with the initial explants, but bacterial contamination can also come from the laboratory environment or from the staff themselves. Exogenous bacteria are easier to deal with, but endogenous bacteria remain problematic. Standard sterilization with ethanol, NaOCl or HgCl2 and with antibiotics can now be enriched with new components (NaDCC, AgNO3, nano-silver) or sanitation products (PPMTM, ProClin? 300, Biosept 33 SL, Vitrofural?, Dekaben). Some of these can be incorporated into a multiplication and rooting medium for one or more passages, if they are not phytotoxic to the plant explants. A special problem is presented by cryptic or viable but not cultivable bacteria which can be unable to multiply during many passages, but finally be disclosed in mass. The issue is, therefore, to find and apply tools for detection of different media and/or molecular markers. The above questions are discussed in the present paper based on the literature and results of our own study.
Long-term storage of shoots or nodal buds in vitro, in conditions which retard their growth, may be an alternative or supplementary method in the protection of plant resources and in the production of planting material for elite mother stocks. In this paper we describe the most important factors which influence the survival and further behaviour of stored explants.
Rosa indica 'Major' is a vegetatively propagated rootstock, valuable for greenhouse grown cultivars. In vitro propagation could help the supply of high quality plants for field stock plantations. Results from our experiments show that increase in the number of shoots >1 cm can be obtained through the use of 2,2 g/l Gelrite instead of Plant agar. Reduction of 6-benzylaminopurine concentration to 1,5 mg/l. FeEDDHA chelate did not improve the quality of the cultures. Rooting of shoots could be more effective when riboflavine is added to the auxin medium in a one-step procedure or when microcuttings are subcultured after root induction to auxin free medium.
A status of plants obtained from interspecific crosses of oriental lilly cultivar Marco Polo with Lilium henryi and L. henryi with cultivar Marco Polo was tested on the basis of morphology and C-band patterns of chromosomes. The positions of the secondary constrictions were found to be the best morphological markers. Also chromosomes of different C-band patterns from parental forms were chosen for hybrids identification. Four out of five plants cultivar Marco Polo x Lilium henryi and one out of four plants from reciprocal crosses were found to be true hybrids according to chromosomal markers.
Shoots of raspberry ?Mallig Seedling? originating from multiplication cultures were stored in vitro at 4C in the dark on a medium with 0,4 mg/l or without 6-benzylaminopurine (BAP), for 18 and 24 months. The cultures were transferred to the storage room immediately after the subculture or after one-week incubation in the growth chamber. A part of the shoots was cold-hardened for one week. Almost all the shoots survived 24 months of storage. The shoots stored on the medium containing BAP formed shoots during storage. Shoot multiplication was poor during the first passage, especially in the result of 24 months storage. Time of transfer and hardening did not influence multiplication capacity instantly.
We undertook an analysis of the genomic relationships between 15 isolates of Phytophthora ramorum Werres, de Cock et Man in't Veld, obtained from symptomatic plants growing in Polish ornamental nurseries, and 2 representatives of the European population and 3 of the North American population. Dendrograms were generated by UPGMA based on 786 amplification products obtained in ISSR-PCR reactions. The representatives of the European population and 13 of the 'Polish' isolates formed a common cluster. The other 2 ?Polish' isolates, which were found in 1998, and the 3 American representatives formed 2 separate clusters. There was no observed link between genomic distance on the basis of polymorphism and the origin of the isolates from plant species.
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