Studies On Cytological And Physiological Characteristics Of Pollen Abortion Of ’Niitaka’ (Pyrus Pyrifolia Nakai)

Pear （Pyrus） belongs to gametophytic self-incompatibility, and allogamy among somevarieties also occur incompatibility. So, the suitable pollination trees and artificalsupplementary pollination are employed to meet expectant yield and quality. Some malesterile varieties of pear in production, for example ’Niitaka’, ’Atagi’, ’whangkeumbae’, andso on, increase difficulties in choice of pollination tree and management. In order toconquer negative influence of male sterile cultivars, utilize potential cast of male sterileresource to solve pollen xenia effect and improve fruit quality, male sterile mechanism ofpear was investigated with male sterile ’Niitake’ and male fertile ’Housui’ （Pyrus pyrifolaiNakai）. In the present study, transmission electron microscope, fluorescent and opticalmicroscope and some modem analytic equipment were employed, and the technology offluorescence labeling and cyto-chemistry was introduced. Based on identification of malesterility of ’Niitaka’, pollen abortive stage and cytological characteristics, and distributionof fluorescent material were studied during pollen abortion. Moreover, Ca²⁺ andCa²⁺-ATPase in anther were located to indentify the relationship between their distributionand pollen abortion. In addtion, hormones and polyamines in floral bud were analyzed tofurther study physiological mechanism of male sterility in pear. Main results of presentstudy are as follows:1. Anther of ’Niitaka’ appeared shrinkage and whitening, and could normally take placedehiscence, but had a small quantity of pollen. There was a lack of pollen on transversesection of anther, or a few pollens were found on a few sections, but these pollens appearedlight dyeing, abnormal shape and lower germination rate. The microspore mother cell couldnormally occur meiosis and produce tetrad; Development of microspore was slow, and wallof microspore was thin and slowly thicken after microsporogenesis; Microspore adhered toeach other, or wall, cytoplasm and nucleus of microspore was disintegrated with microspore vacuolization, and two-called pollen was not observed. It was suggested that ’Niitaka’belong to male sterile cultivar of microspore degenerated form, pollen abortion of ’Niitaka’appeared from middle unimicrospore stage to late unimicrospore stage.2. Technology of paraffin section was used to observe anther development. There was notdifference of microspore mother and anther wall of ’Niitaka’ and ’Housui’ before meiosis,and anther wall were composed of epidermis, endothecium, middle layer and tapetum,microspore mother cell was surrounded by callose wall. Dyeing of microspore of ’Niitaka’was very light in tetrad, and was still light after callose wall disintegrated. Wall ofmicrospore slowly thickened and was very thin. Most microspores shnmk, deformed andappeared different size, cytoplasm was disintegrated, and nucleus disappeared. Microsporebecame hollow shell or debris with wall damaged and inclusion spilled. Tapetal cellappeared irregular dyeing at the stage of microspore just separated from tetrad, and othercomposition of anther wall disappeared difference between ’Niitaka’ and ’Housui’.3. Microsporocyte and male gametophyte of ’Housui’ had perfect mitochondria,integrated vacuole membrane and rich endoplasmic reticulum, ribosome and Golgi bodyunder transmission electron microscope. There were abundant vesicles in microspore attetrad. Callose wall which surrounded microsporocyte of ’Niitake’ appeared irregulardistribution, microsporocyte showed abnormal appearance, for example indiscerniblemitochondria structure, a small quantity of endoplasmic reticulum and so on; There were afew vesicle and some organdies of transparent membrane like mitochondria in microsporeof tetrad. Cytoplasm begun to disintegrate with vacuole membrane damaged, followingmitochondria, plastid and endoplasmic reticulum disintegrated, and nucleolus wasdegenerated. At last, there were some membrane bodies of concentric ring and debris ofcytoplasm and nucleus degenerated in microspore.4. Microspore of ’Housui’ could form perfect primexine at the tetrad stage, and couldrapid produce exine with male gametophyte development. Wall of mature pollen werecomposed of thin intine and exine including foot layer, baculum and tectum. Microspore of’Niitaka’ appeared a portion of primexine or primexine separated from membrane at thetetrad stage, and there were a small quantity of vesicle. Microspore showed lacksporopollenin in exine or a little sporopollenin on the surface of exine, so exine oftransparency or lack foot layer was formed. Although some microspores could formintegrated exine, exine was thinner than that of normal microspore. Two cultivars disappeared difference in callose wall of formation and disintegration, which surroundedmicrosporocyte and microspore at tetrad stage. Auto-fluorescent material of microsporewall gradually increased with male gametophyte development in ’Housui’, andauto-fluorescent material homogeneously distributed on wall of microspore except forcolporate at late microspore stage. There was not obvious difference in auto-fluorescentmaterial on wall of microspore between ’Housui’ and ’Niitaka’ at early microspore stage,microspore surface appeared transparency from portion to whole with auto-fluorescentmaterial decrease, and microspore deformed and adhered to each other, auto-fluorescentmaterial showed reticular distribution on the surface of some microspore which had beendisintegrated.5. Tapetal cell of ’Niitaka’ and ’Housui’ had abundant mitochondria and endoplasmicreticulum at microsporocyte stage; cytoplasm of tapetal cell shrunk with rnicrosporocytemeiosis of ’Niitaka’ and ’Housui’, lots of cavity was formed in intercellular space and innersurface of tapetum with tapetal cell shrinkage. Golgi body and vesicle gradually increasedin tapetal cell, endoplasmic reticulum became dilated cistamae. The number of excretivevesicle and endoplasmic reticulum with cistarae further increased in tapetal cell of ’Housui’,and tapetal cell excreted lots of ubish body for locule at tetrad stage. There was someabnormal appearance in tapetal cell of ’Niitaka’ at tetrad stage, including part organellesdegenerated, stacked endoplasmic reticulum, mitochondria of transparent edge and a lot ofvacuoles. Tapetal cell of ’Niitaka’ did not have perfect mitochondria and endoplasmicreticulum as tapetal cell of ’Housui’ had, and had deformed and indiscernible organelleswith microspore vacuolization; lipid body appeared polar distribution in tapetal cell of’Housui’ when tapetal cell disintegrated. Endoplasmic reticulum with cistarae and othermaterial appeared irregular distribution in tapetal cell of ’Niitaka’. Auto-fluorescentmaterial of tapetum appeared at microsporocyte, and fluorescent density strengthened withtapetal development in two cultivars. A mass of auto-fluorescent material which wasaccumulated on the surface of tapetal cell of ’Housui’ was released and regularly distributedon the surface of microspore. But auto-fluorescent material of ’Niitaka’ was less than thatof ’Housui’, irregularly distributed on the surface of microspore.6. The contents of iron （Fe）, zinc （Zn）, copper （Cu）, calcium （Ca）, magnesium （Mg） andpotassium （K） in basal bearing shoot and bearing shoot of ’Niitaka’ were lower than thoseof ’Housui’, especially the differences of the content of Ca and Zn in bearing shoot were even more between two cultivars, but the differences of the manganese （Mn） content wasnot significant. Mineral nutrition content in floral bud of ’Niitaka’ and ’Housui’ appeareddescendant trend from tetrad stage to late uninucleate microspore stage, and the contents ofFe、Zn、Cu、Mg and Ca of ’Niitaka’ was significantly lower than those of’Housui’, the Mncontent was significantly higher than those of ’Housui, the difference of K content was notsignificant.7. Potassium antimontate was used to locate Ca²⁺ in ’Niitaka’ and ’Housui’ anther. Theamount and distribution of calcium precipitates changed during pollen development andwere different between ’Niitaka’ and ’Housui’. The calcium precipitates accumulated on thesurface of microsporocyte of ’Niitaka’ and ’Housui’. Lots of calcium precipitates appearedon the surface of microspore of ’Housui’ at tetrad stage, but did not appeared on the surfaceof microspore of ’Niitaka’. The calcium precipitates gradually accumulated on thecytoplasmic membrane, vacuole membrane and exine of microspore of ’Housui’ aftercallose wall disintegrated, but the most calcium precipitates were distributed in cytoplasmand some organelles of microspore of ’Niitaka’. The calcium precipitates appeared on thesurface of tapetal cell of ’Housui’ at microsporocyte stage, and gradually increased withmicrosporogenesis and development, decreased at late uninucleate microspore stage. Thecalcium precipitates on the surface of tapetal cell of ’Niitaka’ were less than those of’Housui’ at tetrad stage, but were more than those of ’Housui’ at uninucleate microsporestage.8. Lead nitrate was used to locate Ca²⁺-ATPase in ’Niitaka’ and ’Housui’ anther. Theamount and distribution Ca²⁺-ATPase changed during pollen development and weredifferent between ’Niitaka’ and ’Housui’. Ca²⁺-ATPase did not appear on microsporocytemembrane and a small amount Ca²⁺-ATPase appeared some organelles membrane; theamount of Ca²⁺-ATPase on cytoplasmic membrane and intine of microspore graduallyincreased during microspore development, but was less in cytoplasm. A large amount ofCa²⁺-ATPase distributed on the cytoplasmic membrane, vacuole membrane and ubischbody of tapetal cell of ’Housui’ from microeporocyte stage, the amount of Ca²⁺-ATPasedecreased in disintegrating tapetum. The anthers were difference in Ca²⁺-ATPasedistribution as compared with the same kind of cells at the similar developmental stage. TheCa²⁺-ATPase did not appear on cytoplasmic membrane of microspore of ’Niitaka’ at tetradstage. The amount of Ca²⁺-ATPase on cytoplasmic membrane of microspore gradually increased, but was less than that of "Housui’; Ca²⁺-ATPase was distributed on cytoplasmicmembrane and ubisch body of tapetum of ’Niitaka’, and the amount of Ca²⁺-AYPase wassimilar between ’Niitaka’ and ’Housui’ except microsporocyte stage.9. Indole-3-acetic acid （IAA） content in floral bud of ’Housui’ was very significantlyhigher than that of ’Niitaka’ after tetrad stage. At the stage of pollen abortion, namely fromearly uninucleate pollen stage to late uninucleate pollen stage, IAA content of "Housui"was 3.70- 4.47 folds of ’Niitaka’. IAA deficiency of ’Niitaka’ might be one of reason thatleads to pollen abortion; IAA oxidase activity in floral bud in prophase of microsporedevelopment of ’Niitaka’ was significantly higher than that of ’Housui’. Change of PODactivity of both pear cultivars was very similar, and difference of POD activity was notsignificant, suggesting that POD was not key enzyme that leads to IAA deficiency.Abscisic acid （ABA） content of ’Niitaka’ was significantly higher than that of’Housui’ fromtetrad to binucleate stage.10. The variational range of contents of both free Putrescine （Put） and Spermidine （Spd）in ’Niitaka’ was small during floral bud development, whereas, variation of free Put andSpd content of ’Housui’ showed a peak from early uninucleate pollen stage to lateuninucleate pollen stage. The free Put and Spd contents in Housui were higher than those of’Niitaka’ at different developmental stage, especially at the stage of pollen abortion, thefree Put and Spd contents were 3-5 folds of ’Niitaka’. Free Spermine （Spm） content in’Niitaka’ was significantly lower than that of ’Housui’. The contents of perchloric acidcovalently soluble conjugated （PSCC） Put, Spd and Spm of ’Housui’ gradually increased,but the contents of PSCC Put, Spd and Spin in ’Niitaka’ showed a peak that did not appearin ’Housui’ during pollen abortion or ahead. The increase of PSCC polyamines in ’Niitaka’might result in the decrease of free polyamines and affect on pollen development.Variations of the content of perchloric acid covalently insoluble conjugated （PISCC）polyamine in two cultivars were similar. Total contents of different forms of Put, Spd andSpin did not have significant differences before pollen abortion, but were significant lowerin ’Niitaka’ than that of ’Housui’ after pollen abortion. The free Put, Spd and Spin were thepredominant forms in total contents in ’Housui’, however, the predominant forms in Niitakawere PSCC Put, Spd and Spm.In summary, the male sterile mechanism of ’Niitaka’ was well characterized atmorphologic, ultra-structural, tyro-chemical, physiological and biochemical level. The present research clarified the stage of abortion of male sterile material and its maincytological mechanism, and further found out its abortive mechanism by way of physiologyand cyto-ehemistry. All these studies provided the basis for further research of molecularmechanism of’Niitaka’ and the regulation of fertility expression.