| The modified genes of starch might change the amylose/amylopectin ratio in starch granule, producing starches with different amylose/amylopectin ratios. The characteristics of starch granules, such as amylose/amylopectin ratio, granule morphology features, granule diameter, crystallinity, have effects on starch processing properties such as starch paste clarity, hydrolysis, gel strength, retrogradation properties, rheology and so on, which can affect the starches’ application in starch industry. Therefore, it is important to investigate the structures, morphology, chemical composition of starches with different amylose/amylopectin ratios.In this thesis, LM (light microscopy), PLM (polarized light microscopy), SEM (scanning electron microscopy) and AFM (atomic force microscopy) were used to in situ observe the morphology features, KI/I2-staining results, polarization crosses, growth rings, blocklet structures of starch granules from different positions of five maize mutant kernels (WT, ae, GEMS-0067, wx ae, ae du) which had different amylose/amylopectin ratios; NIR (near infrared) and Raman spectroscopy were employed to analysis and discriminate different maize mutants; finally, the water absorption capacity, aging ability, crystallinity, linear chain and branched chain ratio of starches were investigated using IR (infrared) and Raman chemical imaging, IR imaging together with Principal Component Analysis (PCA) was also used to discriminate different maize mutants. Main contents and conclusions are as follows:(1) The KI/I2-staining endosperm starches from WT maize were dark blue, and the starches granules were spherical; the starches of ae mutant maize from kernel crown were fried egg-shaped and were stained to blue in center and pink outside, the granules were spherical or irregular spherical, this meant ae gene increased the heterogeneity of starch, which mainly occurred in the initial accumulation of starch; the starches of GEMS-0067mutant maize were stained to dark blue by KI/I2solution, they were rod-like, spherical, irregular spherical, fused, multiple, which meant the HAM gene could elongate the starch granule, increase the heterogeneity of starch morphology features; KI/I2-staining starches of wx ae mutant maize were pink and irregular spherical, but the diameters between the granules in cell center and beside the cell walls were very different; some starch granules of ae du maize mutant from crown and middle of the kernels were stained to blue, while some were pink, but granule diameters were homogeneous, indicating that the du gene increased the starch heterogeneity.(2) There were clear and bright polarized crosses in WT maize starch granules observed using polarized light microscopy, indicating that the crystalline was homogeneous, the whole granule was a spherocrystal; polarized results of ae maize starches were complex, there was a polarized cross in the center of typical fried egg-shaped starch granules, but the outside was black, some granules had two or more polarized crosses, which were caused by ae gene; GEMS-0067rod-like starch granules consisted of two or more polarization crosses, and the singe spherical granules had one bright cross, indicating the HAM and ae genes didn’t change the crystalline homogeneity; the polarization crosses in wx ae starch granules were abnormal, indicating that the crystallinity in wx ae maize was different from WT maize starches.(3) Observed using SEM, the surface of WT maize starch granules was rough, there were hilums in some granules; there was obvious boundary between the center and outside of typical ae fried egg-shaped starch granule, the blocklet structures were also obvious; the GEMS-0067starch granules were rod-like, there were hilums and blocklets in the granules; the wx ae starch granules were irregular spherical, the surface was rough, there were cavities and blocklets in the granules; lamellas and blocklets could be observed in ae du maize starches. Blocklets existed widely in maize starches with different amylose/amylopectin ratios.(4) It was easy to observe that there were obvious growth rings and blocklets in WT maize granules using AFM, and there were many small pits on the blocklet, which may be caused by starch amylase enzymolysis; the center in typical ae fried egg-like maize starches was higher than outside; there were growth rings in rod-like GEMS-0067maize starch granules; growth rings were only observed in the center of the wx ae maize granules; growth rings were also easily obtained from ae du maize starches. There were blocklets and small pits in the ae, GEMS-0067, wx ae and ae du starch granules. (5) AFM force curves illustrated that the blocklets near the hilum of starch granule arranged loosely, and obvious regularity couldn’t be found in other areas.(6) NIR and Raman spectroscopy with PCA can be used to discriminate different maize mutants. When five mutants were discriminated together by NIR spectroscopy, WT and ae maize blocks could be separated well, but the double mutants GEMS-0067, wx ae, and ae du couldn’t be separated from each other, which might be the results of the same ae gene. When every two mutants were discriminated by NIR spectroscopy, WT and ae, WT and wx ae, ae and wx ae could be separated well. When five mutants were discriminated together by Raman spectroscopy, all the floury and glassy blocks could be separated well, every mutant could be separated from others. The discrimination results of Raman spectroscopy was better than NIR spectroscopy, which could provide an effective and fast method for screening in maize breeding.(7) IR and Raman chemical imaging could be used to analyse the crystallinity among different granules/positions/mutants.The crystallinity in individual granules within different cells in different positions of WT maize kernels were homogenous, ae gene made higher crystallinity in ae maize, but linear chain and branched chain ratio and crystallinity were homogenous between the center and outside of ae fried egg-shaped starch granules, indicating that the blocklet structures or their directional arrangement were damaged in starch synthesis. Compared to WT maize starches, HAM and ae genes made GEMS-0067maize starches have higher crystallinity and increase the starch shape heterogeneity, but didn’t change the homogeneity of crystallinity, so the crystallinity in rod-like starches was still homogenous, wx ae maize starches had higher crystallinity than WT maize starches, and the crystallinity was homogenous, indicating wx and ae genes increased the crystallinity, but didn’t change linear chain and branched chain ratio. ae du maize starches had the lowest crystallinity among all the five mutants, du gene decreased the crystallinity.(8) IR imaging can be used to analyse starch water absorption capacity, starch aging abilities and protein content:1) wx ae maize starches had the strongest water absorption capacity. WT maize starches had homogenous crystalline state, so they were hard to absorb water relatively. The ae maize starches in middle and base of the kernels were easier to absorb water than WT maize starches. The GEMS-0067and ae du maize starches in middle and base of the kernels had different water absorption capacities comparing from ae maize starches.2) The aging abilities of WT, ae and GEMS-0067maize starches were nearly same, wx ae maize starches were hard to age. The ae du maize starches from the crown were easier to age than other two positions.3) In general, the starch content in WT maize was the highest, while the protein content was lowest, the starch granules were fully accumulated in WT maize kernels. Compared to WT maize, there were less starches and more proteins in ae mutant maize. The protein content in wx ae mutant maize was highest. Compared to ae maize, ae du maize kernels had more starches and less proteins. The starch and protein contents in crown and middle of GEMS-0067maize kernels were similar to ae maize.(9) Infrared chemical imaging can be used to discriminate different maize mutants with different amylose/amylopectin ratios, PCA results basing on special bands were better than those basing on whole band region. |
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