A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4 degrees C before use decreased frequency of GFP-expressing calli, for example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal 43 degrees C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at 43 degrees C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs were cooled at 25 degrees C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2 to 49.1%. The most optimal treatment, 43 degrees C for 3 min, cooling at 25 degrees C and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is several-fold higher than published frequencies.
HighlightsCrocus is a medicinally important plant and it is costliest spice of the world.An efficient microprapogation protocol of five Turkish Crocus species was developed.Crocus species: C. specious ssp. Specious, C. oliveri spp. Oliveri, C. pestalozzae, C. abantensis, and C. paschei..Different combinations and concentrations of auxins and cytokinins were used.Plant regeneration was developed via somatic embryogenesis.
Common mullein (Verbascum thapsus L.) is a medicinal plant readily found in roadsides, meadows and pasture lands and has been used to treat pulmonary problems, inflammatory diseases, asthma, spasmodic coughs, diarrhoea and migraine headaches. Although it has been used medicinally since ancient times, the popularity of common mullein has been increasing commercially for the past few years. Today, the dried leaves and flowers, swallow capsules, alcohol extracts and the flower oil of this plant can easily be found in health stores in the United States. The use of common mullein extracts in folk medicine begun recently to be supported by an increasing number of research studies. This paper thoroughly reviews all the scientific research related to Verbascum thapsus L. including plant tissue cultures and the biological properties of this plant.
A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/ IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l -1 KIN combined with 0.3 mg l -1 IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l -1 TDZ and 1.0 mg l -1 IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l -1 . Hardening off process resulted in a production of more than 80% healthy plantlets.
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