Studies On The Cytological And Molecular Mechanism Referring Seedlessness Of 'Ougan' And Satsuma Mandarin

Male sterility is one of the main reasons for seedlessness in citrus. In the present study, cytological study was conducted on the microspore development of a CMS trait 'Guoqing No.1' satsuma, and several mitochondrial genes were cloned from 'Guoqing No.1' satsuma. By RT-PCR, Northern blot and RNA in situ hybridization, we researched the expression pattern of these mitochondrial genes during anther development. Morever, the seedless reasons of the mutant 'Ougan' mandarin and several other seedless citrus cultivars were investigated using cytology and cytochemical technologies. The main results were as follows:1. Pollen morphology and viability of the wide type and the mutant 'Ougan' mandarin were investigated. The shape of mature pollen grains and pollen grain number per anther from both wild type and mutant are similar. In the wide type, the pollen stainability of KI-I₂ and FDA staining was 84.5% and 71.4% respectively, but zero in the mutant. The results indicated that pollens abortion was the main reason due to seedlessness of the mutant 'Ougan' mandarin.By using technique of semi-thin section, an extensively cytological comparison study was conducted on the microspore development of both the wide type and the mutant 'Ougan' mandarin. The result indicated that almost all the tetrads of the mutant were abnormal. The mutant had heterogeneous microspore populations including monads, dyads, triads, tetrads and polyads in the same microsporangium. Moreover, tetrads of similar-sized microspores were rarely seen, and most of them consisted of microspores of different sizes from 10μm to 30μm. And all the tetrads of the wide type were normal. The results indicated that abnormal meiosis was the main reason due to pollens abortion of the mutant 'Ougan' mandarin.Cytological analysis of the wide type and the mutant 'Ougan' mandarin showed that abundant starch grains and lipids appeared in the wild type's pollen, but fewer amounts in the mutant. So we speculated that pollen grains of the mutant 'Ougan' mandarin cannot germinate by reason of nutrition shortage such as starch grains etc.2. Pollen viability and seed number of 'Guoqing No.1' satsuma were investigated. The results showed that the pollen was sterile with 35% pollen stainability and zero in vitro germination rate. In natural condition, each fruit only has one seed, but after pollinating with pollens of trifoliate orange, the seed number each fruit increased to four. The results showed that pollens abortion was the main reason due to seedlessness of 'Guoqing No.1' satsuma.By using technique of semi-thin section, an extensively cytological study was conducted on the microspore development of 'Guoqing No.1' satsuma. The results showed that the wholly developmental process could be tentatively divided into four stages, i. e. microsporocyte formation, microsporocyte meiosis, pollen formation, pollen abortion. The pollen abortion occurred first at the microsporocyte meiosis stage, but occurred mainly at the pollen abortion stage. The observation of meiosis revealed that there was only 16.9% abnormal chromosome behavior at metaphaseâ… stage and all chromosome behavior at anaphaseâ… stage was normal, the result demonstrated that pollen abortion occurred mainly after the microsporocyte meiosis stage. DNA damage of tapetal cells at the pollen abortion stage was detected by TUNEL assay in 'Guoqing No.1' satsuma. At the same time, DNA damage of microspores was detected from tetrad stage to pollen abortion stage. The results indicated that programmed cell death maybe occurred in the tapetal cells and microspores3. Synthetic oligonucleotides based on the mitochondrial gene conserved sequence were used to prime the synthesis and amplification of the specific fragments Citatp6, Citcob and Citcoxâ…¡by RT-PCR from 'Guoqing No.1' satsuma. Sequencing analysis showed that the deduced amino acid of Citatp6, Citcob and Citcoxâ…¡shared over 85%, 98% and 96% identities with homologic genes of other plants species respectively. Southern blot analysis showed that there existed more than one copies in the genome.RT-PCR and Northem blot analysis indicated that three mitochondrial genes Citatp6, Citcob and Citcoxâ…¡shared similar expression patterns; mRNA accumulated dramatically in flower buds at the microsporocyte meiosis stage, in leaves and was approximately equally abundant in all other tissues investigated in 'Guoqing No.1' satsuma. The Citcob gene was transcribed to produce a single mRNA, while the Citatp6 and Citcoxâ…¡genes produced two respectively. The results of in situ hybridization showed that the three mitochondrial genes shared similar expression patterns, consistent with the RT-PCR and northern blot results. Transcript levels were low in pollen mother cells, increased during meiosis, and accumulated to highest abundance during the tetrad stage, then decreased slightly in the early microspore stage and declined markedly during the mature microspore stage and resulted in abortion of pollen. The mRNA levels in tapetal cells were also temporally regulated during pollen development. Expression levels of all three mRNAs were low during the pollen mother cell stage, increased and reached a peak at the tetrad stage then suddenly decreased during the free microspore stage, finally becoming undetectable in surviving tapetal cells. These results demonstrated that the timing and cell-specificity of mitochondrial gene expression were differentially regulated during the development of stamens and pollen which may reflect the changing respiratory status of mitochondria during male gametophyte development, and led to male sterility of 'Guoqing No.1' satsuma.4. We compared four different fertility citrus cultivars, 'Glen' navel orange, 'Guoqing No.1' satsuma, 'Huanong Bendizao' tangerine and 'HB' pummelo by cytology and cytochemical methods. 'Glen' navel orange only produced sporogenous cell in its anther locule and no microspore mother cell formation, then anther aborted and no pollen production; the other citrus cultivars can produce mature pollens, but the pollen of 'Guoqing No.1' satsuma were sterile. Abundant starch grains accumulated in fertile pollens and abundant lipids were deposited at the surface of the fertile pollen wall, but few in sterile pollen. Among 'Glen' navel orange, 'Guoqing No.1' satsuma and 'Huanong Bendizao' tangerine, the activity of POD in anther before microsporocyte meiosis were the highest, but the activity of POD this stage in 'HB' pummelo was the lowest. The result indicated that POD isozyme was associated with citrus male sterility.