Studies On Morphology Of Several Leaf And Stem Mutants In Brassica Napus L

Mutants play an important role in crop genetic improvement and functional genomics researches. In this study, several leaf and stem mutants in Brassica napus were used as the experimental material for the research. Preliminary morphological and cytological studies of the mutants were conducted, including field traits observation, chlorophyll content determination, anatomical structure research of leaves, chloroplast ultrastructure observation and plant hormone treatment in mutants. Main results were as follows:1. Studies on chlorophyll-deficient mutants:NJ-CDM-1, NJ-CDM-2, NJ-CDM-3and NJ-CDM-4are four chlorophyll-deficient mutants obtained by EMSmutagenesis. The results of studies on these mutants were as follows:(1) Leaf phenotype of the mutants:Four mutants showed etiolation traits from seedling stage, and the degree of leaf etiolation deepened as the temperature decreases, and the leaves started to turn green at the blotting stage.NJ-CDM-1’s and NJ-CDM-2’s leaf turned back to green after bolting stage; NJ-CDM-3’s leaf turned to yellowish green after bolting stage; NJ-CDM-4’s leaf turn yellowish green after bolting stage and the plant type is dwarf. Four mutants showed obvious differences in leaf color during leaf development. The Chi a content of NJ-CDM-1, NJ-CDM-2, NJ-CDM-3and NJ-CDM-4are49.3%,44.7%,43.9%and39.1%of that in wild type for5leaf stages. The Chl b content of these mutants are29.0%,34.0%,30.7%and27.3%of that in wild type at5leaves stages. The ratio of Chl a/Chl b of mutants are1.722times,1.325times,1.423times and1.436times of that in wild type. In blotting stage, Chl a content of four mutants are49.9%,63.0%,38.5%and31.1%of that in wild type. Chl b content of four mutats are29.0%,34.0%,30.7%and27.3%of that in wild type. The ratio of Chl a/Chl b showed no difference between the mutant and the wild type plants. These results demonstrated that in the course of mutant leaves turning green, Chl b content has a more substantial increase than Chl a. Therefore, the chlorophyll content of four mutants are lower than that of the wild type, and the trait of chlorophyll-deficient mutant is related to its chlorophyll content, especially the chlorophyll b content. (2) Cytological observation:Anatomical observation of four chlorophyll-deficient mutants showed that compared to the wild type, the mesophyll cells of NJ-CDM-1were loosely arranged, starch grains can be observed. Palisade tissue was consisted of2-3layers cells but the cells are irregularly arranged. Spongy tissue have a larger cell gap. The mesophyll cells of NJ-CDM-2are mostly short column cells and the number of starch grains is lower. The number of palisade tissue and spongy tissue cell layers is more than the wild type. The mesophyll cells of NJ-CDM-3are mostly short column cells and loosely arranged. The number of starch grains in mutant is lower than the wild type. The mesophyll cells of NJ-CDM-4are mostly short column cells, the number of palisade tissue cell layers is more than the wild type, and no starch grains was found in cells. The common characteristics of four mutants in leaf anatomy is the mesophyll cells are mostly short column cells, the cells of palisade tissue and spongy tissue are loosely arranged. But the number of starch grains in NJ-CDM-1is more than in others.(3) Ultrastructure observation:The wild type showed regular-shaped chloroplast and the thylakoid membrane is highly differentiated. There are many grana and large starch grains in the chloroplast of the wild type. The NJ-CDM-1showed a spindle-shaped chloroplast arranged along the cell wall. The starch granules in chloroplasts of mutant are smaller and the thylakoid membranes differentiation is incomplete. The number of grana lamella is significantly lower than the wild type. The chloroplast structure of NJ-CDM-2is irregular, although the chloroplast thylakoid membrane differentiated compIetely.The number of chloroplast grana and grana lamella of the mutant are normal. However, there is only a small amount of starch grains but large plastoglobulus in the mutant. The chloroplast of NJ-CDM-3is deformity. Chloroplast thylakoid membrane is very few in number and has not differentiated. No grana lamellae and starch grains but large numbers of plastoglobuluswere found in the mutant. The chloroplast shape of NJ-CDM-4shows no significant difference with the wild type. There is large number of thylakoid membranes in the chloroplast, but not fully differentiated. Part of the thylakoid membranes was swollen. There are vesicles and plastoglobulusin the stromal, but no starch grains. These results showed that the chloroplast of four chlorophyll-deficient mutants has varying degrees of impairment, and the chloroplast structure is significantly different between each mutant.2. Morphology of down-curly leaf mutant M423:(1) Cytological observation: the mesophyll cells of M423are regularly arranged. It has small size and high density cells and more cell layers compared to the wild type. The cell density in the leaf tip is higher than other parts. Therefore, the down-curly leaf traits may be caused by over growth of the cells in the leaf tip.(2) Ultrasturcture observation:The chloroplast structure of M423is normal, but the chloroplast is shorter and thicker, less grana and thicker grana lamella were found in the chloroplast. It shows that the chloroplast structure of M423is deformity.3. Anatomical observation of hypocotyl in dwarf mutant M176treated by hormones:Seeds of the dwarf mutant M176were germinated in the MS medium with or without GA3, IAA and BR supplement in the light incubator to observe the hypocotyl growth. The hypocotyl of M176is shorter than the wild type without hormones treatment. Paraffin section observation of the hypocotyl showed that the cells of M176is shorter than that of the wild type. The hypocotyl cell length of M176was significantly increased by the hormone treatments, which is longer than that of the wild type plants. The hypocotyl of M176treated by GA3elongation sharpest, significantly longer than the wild type’s and GA3treated wild type’s hypocotyl. Thus, M176may be GA3-sensitive dwarf mutant.