Gene amplification is prevalent in many eukaryotes and has been found linked to various phenomena such as ontogenesis, carcinogenesis, in vitro culturing, neoplasia and drug resistance. Earlier, we reported a novel B chromosome in Plantago lagopus L., which was found to have arisen as a result of massive amplification of 5S rDNA. In addition, the chromosome is also composed of 45S rDNA and transposable elements. While the importance of gene amplification cannot be underestimated, its mechanism of origin is still unclear. Therefore, the aim of the present study was to determine whether amplification can be reactivated in the novel B chromosome. For this purpose, in vitro culture was used as stress. Three modes of tissue culture, i.e., direct, indirect and somatic embryogenesis were used for raising in vitro cultures. The variations due to genetic and epigenetic mechanisms were assessed in regenerants using molecular techniques, namely, PCR-RFLP, SSAP and MSAP. The retrotransposon-based molecular markers were applied to detect the polymorphism within transposable elements of in vitro regenerated and mother plants. We detected the variations that may be due to genetic changes either because of element recombination or activation of transposable elements which can lead to increase in the copy number. MSAP analysis revealed the differences in the DNA methylation pattern of the regenerants derived from novel chromosome bearing mother plants. Some regenerated plants were associated with increase and decrease in DNA methylation of both internal and external cytosine of the CCGG sequence.
The seed husk of Plantago ovata known as psyllium (Isabgol) yields medicinally important mucilage. The amount of mucilage produced is about 25 % (by weight) of the total seed yield. In the present study, an attempt was made to increase the amount of mucilage through callus cultures of P. ovata. The first step involved establishment of callus cultures in P. ovata. Leaf explants from 10 to 20 day old seedlings were cultured on MS basal medium supplemented with various concentrations of different plant growth regulators. The highest rate of callus induction (89 %) was obtained on MS medium containing 0.5 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l -1 Kinetin. The mucilage content was estimated from the callus obtained in different media. The best mucilage production was obtained in the MS medium supplemented with 0.25 mg l -1 2,4-D and 0.25 mg l -1 thidiazuron. Significant differences with regard to the total mucilage content were recorded. Overall, the callus produced nearly five times more mucilage than the seeds. The present technology provides an alternative route to production of large quantities of mucilage without plants.The seed husk of Plantago ovata Forsk, is an effective laxative. Seeds of P. ovata release mucilage in water, which is an anionic polysaccharide of L-arabinose, D-xylose and Dgalacturonic acid [1], and is used to treat diverticulitis [2], constipation, diarrhoea, haemorrhoids, bladder problems, irritable bowel syndrome, high blood pressure, high cholesterol and type 2 diabetes [3]. Mucilage of plant origin is in high demand nowadays, and it is very difficult to fulfil the entire demand by field grown plants only. For this purpose, a striking and very promising alternative system for commercial exploitation is the plant tissue culture by using cell suspension culture systems [4]. However, so far any attempts to increase mucilage have not yielded positive results in P. ovata. Studies along these lines have been conducted in Plantago lanceolata where in media having different combinations of plant growth regulators (PGRs) have been used to obtain mucilage from callus [5]. It has been reported that callus cultures are relatively rich in mucilage which commonly make up between 8-10 and 0.2-3.7 % of dry weight in some plants [6,7]. To the best of our knowledge, there are no reports on in vitro mucilage production from the callus cultures of P. ovata. Therefore, the present study was aimed to explore the possibility of mucilage production through in vitro culture.Seeds of P. ovata were available in the collection center of School of Biotechnology, University of Jammu, Jammu, India. The seeds were sterilized first with 70 % V/V ethanol for 1 min, then with 5 % w/v sodium-hypochlorite solution for 20 min, finally washed five times with sterile distilled water. The sterilized seeds were aseptically germinated on an agar-solidified MS basal medium [8] and incubated at a temperature of 25 ± 1°C. Leaf segments (1.5 cm) were excised from 10 to 20 day-old seedlings and were used as explants ...
The present investigations were made attempting to develop a rapid, reliable, and reproduciblein vitroregeneration protocol forArtemisia absinthiumL., a medicinal plant of Kashmir Himalayas. Out of several auxin-cytokinin combinations tested, Murashige and Skoog’s (MS) medium supplemented with 0.5 mgL−12,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mgL−1kinetin (Kn) was found to be the best for the callus induction. On the other hand, 4.5 mgL−16-benzylaminopurine (BAP) and 0.5 mgL−11-α-naphthaleneacetic acid (NAA) in the medium resulted in maximum shoot induction from the callus. Similarly, BAP and NAA at a concentration of 1.5 mgL−1and 0.5 mgL−1, respectively, proved to be the best for the multiple shoot induction from nodal explants. Numerous shoots were obtained from nodal explants after third subculture.In vitrorooting was maximum on medium containing indole-3-butyric acid (IBA) at 0.5 mgL−1. The genetic stability of thein vitroraised plants ofArtemisia absinthiumwas assessed using the intersimple sequence repeat (ISSR) and sequence-specific amplification polymorphism (SSAP) molecular markers. Both markers were able to detect the somaclonal variations in the callus regenerated plants, while no variation was detected in the plants regenerated from the nodal explants. SSAP has been found to be more useful in detection of variability as compared to ISSR molecular marker. The results of present study concluded that the direct regeneration protocol will be useful for the production of true to type plants of this medicinally important plant. This will go a long way in reducing the pressure on the natural populations for the secondary metabolite production, especially for extraction of essential oils.
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