Method Application of Leading Fluorescent Tracers into the Vascular Tissues of Ziziphus jujuba Mill cv. Lingwuchangzao Fruits

doi:10.13560/j.cnki.biotech.bull.1985.2020-1502

Abstract

Abstract:

The fruits of Ziziphus jujuba Mill cv. Lingwuchangzao in different developmental stages were used as experimental materials,the microstructure and change laws of vascular bundles in the fruits of different developmental stages can be clarified using paraffin section method. Based on these results and applying methods of leading fluorescent tracers into vascular tissues of Z. jujuba Mill cv. Lingwuchangzao fruits,the techniques of fluorescent dye tracer in living cell were used to explore the research methods of the unloading path of photosynthates in vascular bundles phloem in the Z. jujuba Mill cv. Lingwuchangzao fruit timely. The results showed that:1)The vascular bundles(especially the vascular bundles nearby endocarp)in the fruit was concentrated and abundant in the early bulking period,the vascular bundles were single or two back-to-back gathered together to be developing. In the rapid enlargement period fruit,the parenchyma cells in the mesocarp developed rapidly and arranged loosely,they gradually increased from the outer to the inner volume,some even arranged into a reticular structure,surrounded by a larger cavity,the number of vascular bundles decreased in the same visual field,and the vascular bundles were mostly two back-to-back,three or four gathered together to be developing. The cell cavities were further enlarged and the number increased in the pericarp in the coloring period,compared with the previous two stages,the number of vascular bundles was fewer in the same visual field,and formed multi-branch structures by multiple vascular bundles. Compared with the coloring period,the cavity space formed by parenchyma cells continued to increase,and the number of vascular bundles did not change significantly,multi-branch structures of multiple vascular bundles were slightly different in the maturation period fruit. 2)By using the techniques of fluorescent dye tracer in living cell,the fluorescent tracer carboxyfluorescein diacetate and plasma membrane penetrating reagent digitonin were led into the phloem of vascular bundles,and the fluorescent tracer Texas-Red were led into the vascular bundles xylem of Z. jujuba Mill cv. Lingwuchangzao fruit in different developmental stages,the fluorescence characteristics were observed by CLSM,the research methods of the phloem unloading path of photosynthates in Z. jujuba Mill cv. Lingwuchangzao fruit were gained,which provides scientific bases for photosynthates accumulation and quality regulation for Z. jujuba Mill cv. Lingwuchangzao fruit.

Key words: Ziziphus jujuba Mill cv. Lingwuchangzao, fruit, vascular tissues, fluorescent tracers, leading into

ZHANG Ying-cai, HUANG Yue, HAI Yuan, ZHANG Yuan, HU Ya-jie, ZHAO Meng-yi. Method Application of Leading Fluorescent Tracers into the Vascular Tissues of Ziziphus jujuba Mill cv. Lingwuchangzao Fruits[J]. Biotechnology Bulletin, 2021, 37(6): 295-304.

Figures/Tables 9

Fig.1 Photos showing the introduction method 1 of CFDA A:A cotton thread passes through the short branch phloem from up to down with a fine needle. B:Fix the Eppendorf tube with fine iron wire and keep it upright. C:Fix the cotton thread around the short branch and inject CFDA diluent into the Eppendorf tube with microinjector. D:The Eppendorf tube is closed with the lid and wrapped in silver paper

Fig.2 Photos showing the introduction method 2 of CFDA A:Intertwine the cotton dough around short branch near fruit stalk after rubbing with sandcloth. B:Inject CFDA into the cotton dough with a microinjector after partly sealing up the cotton dough with sealing film. C:Completely seal up the cotton dough with sealing film and applying vaseline on both ends. D:Completely wrap the sealed cotton dough with silver paper

Fig.3 Photos showing the simultaneous introduction meth-ods of CFDA and digitonin A:A cotton thread is passed through the phloem of short branch and the appropriate amount of EDTA into and out of the needle is dripped. B:Fix the Eppendorf tube with a fine iron wire and keep it upright,fix the cotton thread around the short branch,intertwine the cotton dough around the short branch near the Eppendorf tube after rubbing with sandcloth. C:Inject digitonin into the cotton dough with a microinjector after partly sealing up the cotton dough with sealing film,then completely sealing up. D:Inject CFDA diluent into the Eppendorf tube with a microinjector. E:The Eppendorf tube is closed with a lid rapidly after injecting CFDA diluent. F:Seal the cotton dough and completely wrap the Eppendorf tube with silver paper

Fig.4 Photos showing the introduction method of Texas-Red A:Photo showing the introduction of fruit stalk downward Texas-Red during the rapid enlargement period. B:Photo showing the introduction of fruit stalk downward Texas-Red during the maturation period

Fig.5 Vascular bundles characteristic of fruit during the early bulking period A:Vascular bundles of mesocarp. B:Vascular bundles and other structures of mesocarp. C:Endocarp and external vascular bundles. D:Vascular bundle structures of fruit X:Xylem. P:Phloem. VC:Vascular bundle cambium. VB:Vascular bundle. MC:Mucous cavity. CA:Cavity. ME:Mesocarp. EN:Endocarp. SE:Inner epidermis. Bar=50 μm. The same below

Fig.6 Vascular bundles characteristic of fruit during the rapid enlargement period A:Vascular bundles and other structures of mesocarp. B:Vascular bundle structures of fruit. C-D:Vascular bundle structures and branches of fruit

Fig.7 Vascular bundles characteristic of fruit during the coloring period A:Structures of mesocarp and vascular bundles in fruit. B:Branch structures of two vascular bundles of mesocarp. C:Branch structures of multiple vascular bundles of mesocarp. D:Structures connected by multiple vascular bundles

Fig.8 Vascular bundles characteristic of fruit during the maturation period A:Structures of mesocarp and vascular bundles in fruit. B:Vascular bundles structures of mesocarp. C:Branch structures of multiple vascular bundles. D:Branches of vascular bundles and length cutting and cross section structures

Fig.9 Fluorescence tracing map of vascular bundles in jujube fruit A1,A2,and A3 shows vascular bundles fluorography,the corresponding bright field,the overlaid pictures from fluorescence and bright field,respectively after CFDA was introduced into the phloem of the early bulking period fruit for 48 h. B1 andB2 shows vascular bundles fluorography when only CFDA and CFDA and digitonin introduced simultaneously into the phloem of the rapid enlargement period fruit after treatment with 48 h.C1 and C2 shows vascular bundles fluorography when only CFDA and CFDA and digitonin introduced simultaneously into the phloem of the coloring period fruit after treatment with 48 h. D1 and D2 shows vascular bundles fluorography when only CFDA and CFDA and Texas-Red were introduced simultaneously into the maturation period fruit(CF green fluorescence shows the phloem and Texas-Red red fluorescence shows the xylem). X:Xylem. P:Phloem. A1-A3,Bar=100 μm. B1,B2,C1,C2,D1,D2,Bar=250 μm

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