Approaches Plant materials The banana cultivar used in this study is the Cavendish subgroup with theMusa AAA genome. Banana plantlets have been propagated underneath a sterile tissue culture condition. Suckers had been made use of for multiplication and root ing by putting in plastic bags containing a growth medium. The medium for subculturing contains 1x Mura shige Skoog basal salt mixture, 3% sucrose, 7% agar, 4. 0 mg L one 6 benzylaminopurine, 0. five mgL one naphthlcetic acid, pH5. eight. The rooting medium is the similar as above except with 2. 0 mg L one 6 benzylaminopurine and 2. 0 mgL 1 naphthlcetic acid. The plantlets kinase inhibitor Tosedostat were grown in the 28 C development space by using a sixteen h 8 h light dark time period along with a light intensity of 5000 lux. Plantlets from the sealed bags were transferred to a greenhouse for 3 5 days and then re moved through the bags and grown hydroponically for 50 days within the medium containing MS salts.
Leaves, pseudostems, and roots were collected from individuals hydro ponically grown plants for RNA extraction. Floral tissues and banana fruits at different developmental phases have been collected selleck INK1197 in November, 2010 from a banana plantation area in Haikou, China. The tissues have been frozen in liquid nitrogen and stored in 80 C freezers till use. RNA extraction Total RNA was extracted from roots, pseudostems, leaves, floral organs, and building fruits individually working with a modified CTAB technique briefly described under. Two to five grams of tissues have been grounded in liquid nitrogen, as well as the powder was mixed with twenty mL CTAB buffer and incubated at 65 C for twenty min. The extract was mixed with 0.
6 volume of chloro kind by vortexing and span at 12000 g for 15 min at space temperature. The supernatant was transferred to a brand new tube and extracted with an equal volume of chloroform, as well as the supernatant was then mixed with 0. 5 volume of 12 M LiCl and incubated at 20 C for 2 hours. RNA was precipitated by centrifugation at 12000 g for 15 min at four C and also the pellet was re suspended in one mL 0. 2 M NaCl. The RNA remedy was extracted sequentially with an equal volume of water saturated phenol and chloro type. RNA was precipitated by mixing the answer with 3 volumes of ethanol and leaving on ice for 30 min be fore centrifugation at 14000 g for twenty min at four C. Right after washing the pellet with 75% ethanol, the RNA pellet was dissolved in 50 uL RNase totally free water. The good quality in the RNA samples was checked by using Agilent 2100 Bioana lyzer.