Occurrence of Latent Bacteria during Cryopreservation of Long-Term In Vitro Cultures of Coltsfoot, Tussilago farfara.

BACKGROUND: Long-term in vitro cultures of Tussilago farfara (L.), a traditional medicinal plant in Austria, had been stored at 14°C for over 20 years. The cultures were vigorous and showed no visual signs of bacterial presence. The transfer from growth regulator-free culture medium to medium containing kinetin and the increase of temperature from 14°C to 25°C for fast propagation led to the emergence of latent bacteria in all twelve accessions studied. OBJECTIVE: To investigate latent infections occurring during the development of a cryopreservation protocol of genetically interesting material using droplet-vitrification. MATERIALS AND METHODS: Two protocols for droplet-vitrification were tested using plant vitrification solutions (PVS) 2 and 3. The bacteria were isolated and identified using 16S rDNA analysis. Next, non-cryopreserved in vitro plantlets were acclimatized and transferred to the glasshouse. After 6 weeks, shoot tips were harvested from the pot plants, surface-sterilized and initiated into culture. Further, newly acquired achenes of Tussilago were surface-sterilized and germinated in vitro and seedlings checked for bacteria. RESULTS: The bacteria from the long-term cultures were isolated and identified as Luteibacter. Regeneration after cryopreservation using PVS3 was successful despite the continuing presence of Luteibacter. Luteibacter could no longer be detected in the newly-initiated in vitro material in subsequent tests and it was also not detected in the seedlings. CONCLUSION: Luteibacter withstood the cryopreservation procedure. Re-initiation of infected material may be an efficient alternative to antibiotic treatment to manage bacteria in micropropagation systems.

Micropropagation and Cryoconservation of the Endangered Plant Species Artemisia laciniata (Asteraceae).

BACKGROUND: Artemisia laciniata, mainly distributed in Siberia and Central Asia, is classified as critically endangered in Europe. OBJECTIVES: This study developed a protocol for its micropropagation and cryopreservation. MATERIALS AND METHODS: In vitro cultures from fresh seed and in vivo shoots were initiated. Micropropagation and cryopreservation protocols were developed. Bacteria detected after cryopreservation were investigated using 16S rRNA analysis. Genome size measurements of regenerated plants after cryopreservation using flow cytometry and carbon isotope measurements to evaluate stress status were also carried out. RESULTS: A. laciniata from both starting materials could be successfully propagated on MS medium with 0.5 uM BAP. Material initiated from in vivo shoots yielded lower regeneration percentages (16%) after cryopreservation than material generated from seed (57 and 63%) using the droplet-vitrification method and PVS3. Bacteria occurring after cryopreservation belonged to the genera Sphingomonas, Staphylococcus, Curtobacterium and Gordonia. There was no significant difference in the genome size and stress status between non-cryopreserved and cryopreserved plants. CONCLUSION: A. laciniata could be readily micropropagated and cryopreserved. No negative effects of cryopreservation on plant water use efficiency or on genetic stability were found.

Stability of cefepime in aqueous eye drops.

The aim of the studies was to determine HPLC the stability of cefepime in 1% and 5% buffered eye drops of developed formulary composition, which were stored for 30 days at the temperature of 4 degrees C and 20 degrees C, protected from light. Separation was performed on RP18 Gemini octadecylsilane column (250 mm x 4.6 mm, 5.0 microm) at a temperature of 25 degrees C. The mobile phase consisted of 0.015 M solution of sodium salt of pentane sulphonic acid brought to pH 4.0 with glacial acetic acid and 45% KOH solution and acetonitrile 94:6 w/w, with detection of 254 nm. The method was linear in the range of 12.6-125 microg/ml (R2 = 0.9996). The limit of detection (LOD) was 3 microg/ml and limit of quantification (LOQ) was 10 microg/ml. 10% degradation of cefepime in 1% and 5% buffered eye drops stored at the temperature of 4 degrees C, depending on the composition of the eye drops, occurred after 21-27 days in 1% eye drops and 18-21 days in 5% eye drops. In the eye drops, which were stored at the temperature of 20 degrees C, 10% degradation of cefepime took place on the third day of storage regardless of formulary composition of 1% and 5% drops. Cefepime stability lasting a couple of weeks in 1% and 5% solution allows extemporaneous preparation of buffered eye drops containing cefepime.

Physicochemical and microbiological properties as well as stability of ointments containing aloe extract (Aloe arborescens Mill.) or aloe extract associated to neomycin sulphate.

The aim of the study was to work out methods of quality assessment of ointments containing dry extract from fresh leaves of Aloe arborescens Mill. (Lilliaceae) and also of ointments containing both of dry extract and neomycin sulphate. The stability of the ointments, stored at 20 degrees C, was studied and the following criteria were considered: chromatographic analysis (TLC), pH of the ointments, the content of the substances in the dry extract converted to aloenin, the content of aloenin and aloin, anti-microbial activity of neomycin in the ointments, the size of the particles of the dry extract and of neomycin sulphate in the ointment suspension and the sterility of the ointments. After two years of storage at 20 degrees C, the ointments prepared with the anhydrous lipophilic base, did not change their physicochemical characteristics and neomycin in those ointments retained almost 100% of starting anti-microbial activity. Water or propylene glycol significantly decreased the stability of the biologically active substances of the dry extract in the ointments. Besides, in the ointments containing the dry extract and neomycin sulphate, the presence of water or propylene glycol induced degradation of the biologically active substances of the dry extract and a decrease in the anti-microbial activity of neomycin in the ointments. Considering the physicochemical and microbiological stability, the most advisable base for the ointments with aloe and neomycin sulphate was composed of white vaseline, liquid paraffin, solid paraffin, cholesterol.

Technology of eye drops containing aloe (Aloe arborescens Mill.--Liliaceae) and eye drops containing both aloe and neomycin sulphate.

Eye drops made of aloe are a sterile, aqueous extract of fresh leaves of Aloe arborescens Mill., containing necessary additives and neomycin sulphate. The aim of the studies was to establish the technology of eye drops containing biologically active aloe substances and those containing both chemical constituents of aloe and neomycin sulphate. Within the studies, the formulary content and the way of preparing eye drops were determined, criteria were defined and methods of qualitative assessment of drops were proposed. On the basis of the proposed analytical methods, the physicochemical and microbiological stability of the eye drops stored at a temperature of 20-25 degrees C was studied. As the criteria of qualitative assessment of the eye drops, the following analyses were considered: sterility, appearance of the eye drops (clarity), pH, osmotic pressure, density, viscosity, TLC analysis, content of aloenin and aloin, studies of anti-microbial activity of neomycin in the drops, and preservative efficiency of thiomersal in the eye drops. The studies showed that the additives such as: sodium chloride, benzalkonium chloride, chlorhexidine diacetate and digluconate, phenylmercuric borate and Nipagins M and P could not be used to prepare the eye drops because they were involved in pharmaceutical interactions with chemical constituents of aloe in the eye drops. The eye drops containing: aqueous extract of fresh leaves of aloe, boric acid, thiomersal, sodium pyrosulphite, disodium EDTA, beta-phenylethyl alcohol and neomycin sulphate, both freshly prepared and after two years of storage, met the requirements of the Polish Pharmacopoeia (PPh V) mentioned in the monograph Guttae ophthalmicae. They were sterile, clear, their osmotic pressure approximated the osmotic pressure of lacrimal fluid and they were characterized by appropriate pH. Aloenin in the drops was much more stable than aloin. Neomycin after two years of storage retained almost 98% of its starting antimicrobial activity which allows the conclusion that the biologically active aloe substances did not decrease the stability of neomycin in the drops. The preservation assay showed that thiomersal, both in the freshly prepared drops and after two years of storage, maintained antimicrobial activity, which was in accordance with PPh V.

Effect of spikelet position on rice anther culture efficiency.

The potential of anthers from different parts of the panicle to induce callus was investigated with the japonica rice variety Taipei 309. The results showed that the callusing abilities of anthers from different spikelet positions were significantly different. After plating 4483, 4496, 4348 anthers from the basal, middle and top parts, the percentage of anthers forming calli was 20% in the basal part, 12% in the middle part and 8% in the top part. The anthers of basal parts containing pollen at all uninucleate stages, including early, middle and late, showed higher callus induction frequency than those from middle and top parts. The green plantlet regeneration frequencies of top, middle and basal spikelets were around 18% in all three cases. From the results it would appear that anthers from the basal part of the panicle should be used in anther culture of rice in order to obtain higher efficiencies, and thereby optimise the usefulness of this technique in rice breeding programmes.