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The morphology of references mature anthers had been investigated with fluorescence stereo microscope and image was captured with a digital camera. The pollen grain number per anther was counted. In quick, anthers from CDK inhibitor mature flowers were collected and mixed ran domly, every time forty anthers were dissected and pollen grains had been suspended in 25 mL sterile water with four five drops of surfactant. The viability of mature pollen grains were evaluated by dying with 1% acetic acid magenta as well as 1% iodine potassium iodide solution. Just after staining for 5 min, pollen grains have been observed employing BX 61 fluores cence microscope and Pictures were captured with DP70 CCD digital camera procedure. A minimum of 1,000 pollen grains have been counted. These experiments were repeated three times.
The morphology of pollen grains was examined by scanning electron microscope.
For SEM, anthers at various developmental stages have been pre fixed with two. 5% glutaraldehyde in 0. 1 M sodium phosphate buffer for 24 h, dehydrated twice employing a gradient ethanol serial, then replaced ethanol with isopentyl acetate for twenty min. Following that, samples have been dried with crucial level drying method then sputtered coating with gold. Representative pictures were captured. RNA extraction and mRNA isolation The materials for RNA extraction have been sampled from a minimum of six independent plants, and mixed randomly. Complete RNA from flower samples at four phases had been extracted with modified Trizol strategy in accordance to. The RNA pellets were washed with 75% ethanol twice, dissolved in RNase free water and stored at ?80 C until finally use.
By mixing equal level of RNA with the four phases, RNA pools from each QS and EG have been established in parallel. Then mRNA was isolated from every in the RNA pools applying the Oligotex mRNA mini kit. The quality of RNA was established by Nanodrop 1000 spectrophotometer and 1. 2% agar ose gel electrophoresis. Suppression subtractive hybridization cDNA libraries building and cDNA inserts amplification Two micrograms of mRNA was utilized to synthesize cDNA for suppression subtractive hybridization. The SSH was performed with the PCR selectTM cDNA subtraction kit in accordance to the consumer manual. And the two forward and reverse SSH were conducted. For keep#RigosertibcDNA libraries building, two hybridizations were per formed followed by two rounds of PCR amplifications to enrich the wanted differentially expressed sequences. Then the second PCR amplified cDNAs have been purified and ligated in to the T A cloning vector pMD18 T overnight at 4 C.