Samples (basal portions of shoots; 2–3 cm in length, 500 mg)
were harvested from the culture vessels in a random manner at an
interval of 20 days up to 140 days and immediately fixed in chilled
aqueous perchloric acid (PCA; 4.0%, v/v). Extraction and benzoylation
of PAs from samples were performed following the procedure
described by Kakkar and Nagar (1997) with slight modifications. In
brief,tissue from each stage was crushed with the help of prechilled
mortar and pestle in aqueous PCA (4.0%, v/v; 100 g l
−1 PCA). The
homogenates were kept at 4 ◦C for 1-h and then centrifuged at
15,000 × g (30 min, 5 ◦C). The supernatant was used as a source of
free PAs and PCA soluble conjugated PAs. The pellet contained PCA
insoluble PAs bound to macromolecules, like proteins and nucleic
acids (bound polyamines). A portion of the supernatant (5 ml) and
the pellet were hydrolysed with 6 N HCl for 18-h at 110 ◦C. The
hydrolysates were filtered, evaporated to dryness in vacuo at 45 ◦C
and re-suspended in 5 ml aqueous PCA (10%, v/v). The original
supernatant(free polyamines), HCl hydrolysed supernatant(conjugated
polyamines) and HCl hydrolysed pellet (bound polyamines)
were treated separately with insoluble PVP (50 mg ml−1 extract)
and stirred for 1-h to remove phenolics and other interfering compounds.
These were then filtered and 0.5–0.6 ml of HCl (0.01 N)
was used to clean the vials
Samples (basal portions of shoots; 2–3 cm in length, 500 mg)were harvested from the culture vessels in a random manner at aninterval of 20 days up to 140 days and immediately fixed in chilledaqueous perchloric acid (PCA; 4.0%, v/v). Extraction and benzoylationof PAs from samples were performed following the proceduredescribed by Kakkar and Nagar (1997) with slight modifications. Inbrief,tissue from each stage was crushed with the help of prechilledmortar and pestle in aqueous PCA (4.0%, v/v; 100 g l−1 PCA). Thehomogenates were kept at 4 ◦C for 1-h and then centrifuged at15,000 × g (30 min, 5 ◦C). The supernatant was used as a source offree PAs and PCA soluble conjugated PAs. The pellet contained PCAinsoluble PAs bound to macromolecules, like proteins and nucleicacids (bound polyamines). A portion of the supernatant (5 ml) andthe pellet were hydrolysed with 6 N HCl for 18-h at 110 ◦C. Thehydrolysates were filtered, evaporated to dryness in vacuo at 45 ◦Cand re-suspended in 5 ml aqueous PCA (10%, v/v). The originalsupernatant(free polyamines), HCl hydrolysed supernatant(conjugatedpolyamines) and HCl hydrolysed pellet (bound polyamines)were treated separately with insoluble PVP (50 mg ml−1 extract)and stirred for 1-h to remove phenolics and other interfering compounds.These were then filtered and 0.5–0.6 ml of HCl (0.01 N)was used to clean the vials
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