Direct differentiation of shoot buds from the collar region of hypocotyl segments of Coffea canephora was obtained on Murashige and Skoog (MS) medium supplemented with 40 lM silver nitrate (AgNO 3 ) and growth regulators indole-3-acetic acid (IAA) and N6 benzyladenine (BA). The highest response to shoot differentiation of 60% frequency and the maximum number of multiple shoots (2-3) per explant were obtained on MS medium containing 8.87 lM BA and 2.85 lM IAA. Apart from this, 70% of hypocotyl explants produced yellow friable embryogenic callus and also globular primary somatic embryos. Subsequent transfer onto the same medium induced secondary somatic embryogenesis. The micro-shoots, upon transfer to the same medium, in the following 6 weeks developed into 4-cm-long shoots with a single root. Further subculturing onto the same medium induced 4-5 roots in a 4-week period. The resulting plantlets were hardened and transferred to micro-pots containing sand:compost mixture (1:2), where 65% of them survived and resumed growth. By using optimal levels of AgNO 3 , it was possible to obtain effective direct organogenesis and embryogenesis. This system was used for genetic transformation using Agrobacterium tumefaciens. A stable transformation frequency of 2-5% was obtained when both types of explants, i.e., hypocotyl explants with collar region or hypocotyl explants without collar region, were co-cultivated with A. tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector. This is the first report of a hypocotyl collar region-based Agrobacterium-mediated transformation protocol for the economically important tropical plant C. canephora.
With the ever-growing concern of water deficit due to global climatic change, the drought and salinity stress response of plants is a major area of research. However, the effect of these stress on cup-quality of coffee especially, the accumulation of caffeine biosynthetic metabolites, has not been documented. This work studies the methylxanthines (7-methylxanthine, theobromine, caffeine and theophylline) contents in young leaves of coffee in response to PEG-6000 (1.5% and 15% w/v) induced drought and sodium chloride (20mM and 200mM) induced salinity stress. In general, both the stress reduced the caffeine content except for 20mM NaCl. 1.5% PEG reduced caffeine by 0.46 fold and 0.57 fold during first 24hr and 48hr of treatment, respectively; PEG at 15% caused a reduction by 0.36 fold only in the 48hr of treatment compared to untreated plants; and NaCl at 200mM caused a reduction of 0.26 fold and 0.47 fold in the first 24 and 48hrs of treatment, respectively. However 20mM NaCl augmented caffeine by 1.93 and 5.1 fold in the first 24 and 48hrs of treatment, respectively. The levels of caffeine subdued on the withdrawal of the stressor, affirmatively indicating the stress stimuli to be responsible for the observed changes in caffeine levels. The biochemical profile was supported by transcript expression of the caffeine biosynthetic NMT genes and the analysis of regulatory motifs of the promoters. The contents of upstream methylxanthines (7-methylxanthine and theobromine) and the degradation pathway (theophylline) indicate that salinity and drought might have a negative impact on biosynthesis of caffeine but accelerated the rate of caffeine degradation.
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