Histology And In Situ Hormones Of Flower-bud Differentiation In Walnut

Walnut production is decided by the quality and quantity of flower-bud differentiation.While plant hormones are the important regulatory factors. Many previous studies have beendone about the relationship between plant hormones and flower-bud differentiation. Growthregulators were used to control flower-bud differentiation in production practice. However,even the most advanced method for the determination of hormones also need amount ofmaterial tissue and the result was the average of hundreds or thousands cells, but not accuratepositioning in a single cell or subcellular cell. At the same time, this method could not analysisthe transport and metabolic pathway of hormones accurately. Then we used new technology ofimmuno-colloidal gold localization to monitor the temporal and spatial pattern of IAA andABA and their transport during flower bud differentiation in the walnut (Juglans regia L.)cultivar Liaoning1of Chnia and Amigo from American. The HPLC-MS used to analysis thequantification of IAA and ABA for supporting role. The main results obtained are as follows:(1) The methods of morphology observation and tissue sections were used in staminateflower study. There is a close correlation among phenology, external morphology and internalanatomical structure. For the protandrous walnuts, as the staminate flowers beginning open, thetender and little bud wrapped with squama pasted in axils. Now in the microscope we can seethe staminate flower bud has entered squama differentiation. During shoot elongationphenology, the staminate flower was flat and covered with squama as the only externallyvisible portion of the catkin. The bract primordia formed from bottom to top and the floretprimordia formed at the base of bract. When fruit drop stage beginning, the length of catkinswas elongated, with catkins protruding from the squama and the floret, perianth, and stamenprimordia formed basipetally. The staminate catkins like pine cones and bracts’ color variedfrom yellow-green to brown. The whole catkins turned into stamens differentiation during theperiod of fruit grown quickly. When the fruit began to grow slowly, the length of catkin did not change evidently. The bracts were brown and each floret arranged tightly. The anthers began todifferentiation and lasted to the spring of next year. When turned into germination stage, thecatkin elongared and thicked, the perianth and anther became visible externally, pollen mothercells begin to differentiate in catkin. As shoots growing coming, the catkin elongated to10cmand the color of anther turned into yellow. As the anthers became matured, middle layercompressing and disintegrating, the tapetum dissoluted and degradated. At this point the entirebud differentiation completed, until flowering a new flower bud differentiation process willbegin.Each differentiation stage of protogynous walnut is the same as the protandrous walnut.However, there are also some differences, such as the time and process of the variousdifferentiation stages. The pistillate flower of protogynous walnut and the staminate flower ofprotandrous walnut opened at the same time. While the staminate flower differentiation is fallbehind the protandrous just staied at stamen differentiation period.(2) The distribution and variation of IAA was analysised using immuno-colloidal goldlocalization in staminate flower of walnut. In staminate flower the auxin signal was strongest inthe shoot apical meristem (SAM) during early differentiation of catkin; thus, the SAM may bea site of auxin production. When the floral organs began centralized differentiation, auxin wasdistributed mainly in the differentiating tissues. Our findings indicate that a high level of auxinmay strongly affect morphogenesis. Additionally, the tapetal and reproductive cells that ariseduring cellular specialization may be important for auxin production. The distribution of auxinwas centralized in germ pores at the pollen grain surface, indicating that a high level of auxininduces pollen germination.(3) The distribution and variation of ABA was monitored using immuno-colloidal goldlocalization in walnut pistillate and staminate flowers. During flower differentiation, highlevels of ABA were consistently indentified in pedicle, young leaf and vascular bundle,indicating there might be the important ABA source caused either by accumulation ororigination. Obvious changes in temporal and spatial pattern were observed during flower-bud differentiation. The differentiation of pistillate and staminate flower needed different level ofABA. The levels of ABA in SAM decreased obviously as the transition from leaf bud topistillate flower-bud. ABA preferentially localized at primordial cells. As the tissues (bract andperianth) became highly differentiated, ABA was significantly and uniformly decreased.However, during stamen development, ABA accumulation was instead restricted to cells thatare closely linked to the formation of reproductive cells. This direct evidence indicates that lowlevel ABA may be needed during pistillate flower induction and morphogenesis, instead, ABAmay strongly affect staminate flower differentiation.(4) We used immunogold localization and HPLC-MS to analysis the quantification of IAAand ABA during flower-bud differentiation in precocious walnut. The result indicated thatimmunogold localization is better than HPLC-MS, because it can respond the spatial andtemporal distribution of plant hormones and achieve the purpose of quantitative analysissimultaneously. To analysis the balance of auxin and abscisic acid, we found that the pistillateflower-bud differentiation requires a higher ratio of ABA/IAA, about1.9. However, thestaminate inflorescence differentiation may have little correlation with the balance of auxin andabscisic acid.(5) The variety ‘Amigo’ of precocious walnut planted in Henan Ruyang area was tested.The methods of pick leaves, remove the terminal bud and application of exogenous growthregulators were used. It is found that the secondary flower inhibited completely by removingterminal bud and the time of pick leaves will affect the occurrence of secondary flowers indifferent degrees. Pick leaves in April17th inhibited secondary flowers completely，while inMay7secondary flowering rate is21%. The auxin transport inhibitor (NPA) plays animportant role in promoting secondary flowers. The secondary flowering rate is65.6%and61.1%respectively.