CN114279741B - Microscopic sampling method for Chinese narcissus organ morphogenesis stage - Google Patents
Microscopic sampling method for Chinese narcissus organ morphogenesis stage Download PDFInfo
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Abstract
The invention relates to the technical field of plant tissue sampling methods, in particular to a microscopic sampling method for a morphological establishment period of a Chinese narcissus organ. The microscopic sampling method provided by the invention comprises the following steps: firstly, removing buds and removing bracts, then sequentially removing the first round of the inflorescence, the second round of the inflorescence, the stamen or the petalous stamen, and finally removing the auxiliary crown-pistil-receptacle combination. The method for stripping the 1 st round of festoon before forming the auxiliary crown is as follows: under a stereoscopic microscope, the needle point of the dissecting needle is transversely placed at the joint of the tops of the first round of festoons, the tops of the festoons are pressed downwards with force along the joint of the festoons, and then the dissecting needle is inserted into the inside of the festoons with force outwards, so that the peeled first round of festoons is obtained. The method not only can well ensure the integrity and stripping accuracy of different flower organs, but also is simple to operate, does not need complicated instruments and operation steps, and is suitable for popularization and application in researches such as the development of Chinese narcissus and the morphological establishment of flower organs.
Description
Technical Field
The invention relates to the technical field of plant tissue sampling methods, in particular to a microscopic sampling method for a morphological establishment period of a Chinese narcissus organ.
Background
Flowers are the main reproductive organs of higher plants and are important basis for researching plant evolution and classification. The plant flower development process is a complex multi-step process that, together, results in the transformation of meristematic cells by a range of endogenous and exogenous factors, and initiates initiation of flower development. The differentiation process of the meristematic cells is as follows: the stem tip meristem is firstly converted into inflorescence meristem, then the inflorescence meristem is generated, finally, the inflorescence meristem is differentiated into various rounds of flower organs, and finally, the morphogenesis of various flower organs is completed. The dicotyledon flower structure is generally four-wheel concentric circles, and a typical flower structure is composed of several parts, namely sepals, petals, stamens and carpels, and flower organs such as a calyx, a corolla, a stamens group, a gynoecia group and the like are respectively formed, wherein the calyx and the corolla are collectively called a flower quilt. In recent years, genetic and biological researches of model plants such as arabidopsis thaliana, petunia, goldfish grass and the like promote the research progress of dicotyledon flower organs, and the research of the gene functions related to the dicotyledon flower organs provides strong evidence for the development of flower morphology. In the 90 s of the 20 th century, plant scientists have proposed the flower organ ABC model hypothesis for interpreting plant flower organ characteristics by systematic genetic analysis of homologous allotypic mutants in arabidopsis thaliana and goldfish grass. Recent reverse genetics studies have shown that two other types of homologous genes play an important role in regulating flower morphogenesis as well, namely, class D genes and class E genes, respectively, wherein class D genes are mainly involved in ovule development, while class E genes are involved in regulating the formation and development of all flower organs. The ABCDE model is understood from the protein level as a "tetramer model", i.e. a re-polymerization of homo-or heterodimeric proteins into tetramers involved in the transcriptional regulation of downstream target genes.
The genus narcissus is a perennial herb of the lycoris family (AMARYLLIDACEAE) with underground enlarged bulbs, she Xianxing, basal, surrounding the scape, known for its characteristic accessory crown morphology. The narcissus has rich flower color and pattern, and can be widely used in the fields of flower arrangement, bonsai, landscape, etc., and lectin component extracted from narcissus is used for treating Alzheimer disease and AIDS. The colchica existing variety 2 in excess, the international general colchica cultivar classification standard is formulated by the uk Royal gardening society (RHS), namely 12 horticultural classifications and 1 botanical classification, respectively: horn narcissus group (Trumpet Daffodil group), large cup narcissus group (Large-Cupped Daffodil group), small cup narcissus group (Small-Cupped Daffodil group), heavy petal narcissus group (Double Daffodil group), triple-core narcissus group (Triandrus Daffodil group), cyclamen narcissus group (Cyclamineus Daffodil group), clove narcissus group (Jonquilla Daffodil group), multi-flower narcissus group (Tazetta Daffodil group), red mouth narcissus group (Poeticus Daffodil group), apron narcissus group (Bulbocodium Daffodil group), schizocrown narcissus (Split Corona Daffodil group), poetry narcissus (Poetaz Daffodils group) and natural species (Daffodils distinguished solely by botanical name).
Natural distribution of 1 colchica in china-colchica in china (Narcissus tazetta l.var.chinensis Roem). The problem of chinese colchicine origin has been pending, mainly comprising two kinds of congress of mediterranean origin and chinese native origin, but most scholars consider chinese colchicine to be a variant of colchicine multiflorum (n.tazetta l.), whereas the search table compiled by the royalty zoo (RBG) in the united kingdom sets chinese colchicine as colchicine subspecies (WCSP, world Checklist of SELECTED PLANT FAMILIES). Chinese narcissus is known as one of the ten flowers in the traditional art, and is known to the public as its beautiful flower shape and unique flower fragrance. There are two commercial varieties of Chinese narcissus, single-petal 'Jin Zhanyin' and heavy-petal 'Yu exquisite'. 'Jin Zhanyin' calyx (sepal) petals, similar to petals (petal), 3 petals are alternately opposite and milky; the auxiliary crown (corona) is in a shape of a wine glass, is yellow and is adhered to the quilt tube; 6 stamens (stamen) and 3 stamens respectively; pistil (pistil) 1, chapped stigma 3, lower ovary. The 'exquisite' accessory crown and stamens are all valved, 12 split white are arranged in clusters, yellow split leaves are smaller, and white and yellow are alternate; having degenerated pistils. Most researchers believe that the heavy flap ' Yu exquisite ' is a cultivar of the single flap ' Jin Zhanyin. In addition, the Chinese narcissus 'Jin Zhanyin' naturally varies with 'golden triangle', 'golden edge', 'exquisite jade' naturally varies with 'exquisite green jade', and the like are reported, but the ornamental value and the commercialized application are weak.
In recent years, studies on the pattern, color and fragrance of narcissus have been increasing, but most of them have focused on cloning of homologous genes. There is currently no progress in whole genome sequencing due to the large genome of the Narcissus plant. At the transcriptome level, narcissus flower color studies continue to produce results, but there has been no substantial progress in comparison with individual reports on organ differentiation and organ development studies of narcissus flowers.
The structural origin of the colchicine accessory crown has been a hot spot for debate by researchers. Unlike 4 rounds of concentric circular flower structure to distinguish sepals and corolla, the narcissus has no obvious sepals, and 2 rounds of flower quilts have similar structures and are collectively called as flower quilts. Some scholars consider that the colchicine paracrown is a variant of the tongue or petal support leaf fusion or stamen developed by extension of the flower quilt. Another report suggests that paracrown is not related to stamens and develops from the perianth canal. Such disputes have been over a century. Phylogenetic data of different morphogenic origins indicate that the narcissus accessory cap has evolved multiple times in parallel in the lineage.
The colchicine paracrown is a colchicine plant marker phenotype, but the development mechanism and evolution process are poorly understood due to the limitations of sampling methods, and the paracrown and stamen-valved molecular mechanisms of 'exquisite' have not been fully revealed. The foundation of the research on flower development is a scientific and standard sampling method. In the above-mentioned research, the sampling method of flower development is concerned, the sampling position is the inflorescence total bud of differentiation stage, and the research on the morphogenesis mechanism of each flower organ is carried out, and it is necessary to sample each flower organ individually and carry out transcriptomics research.
The laser-assisted microdissection technology (Laser Microdissection, LMD) can rapidly and accurately separate and acquire single cell groups, even single cells under a microscope, and successfully solves the problem of heterogeneity of cells in tissues, and mainly comprises laser capture microdissection (laser capture microdissection, LCM), laser-assisted microdissection (LASERASSISTED MICRODISSECTION, LAM), pressure catapulting laser microdissection (laser microdissection and pressure catapulting, LMPC) and the like. There are studies reporting that combining LAM and ultra low RNA sequencing (RNA-seq) performed transcriptomic studies on epidermal cells of arabidopsis embryos. However, the laser micro-cutting technology has too high requirements on the operation skills of experimenters, is difficult to be practically applied, and has no application case in the narcissus plants at present. Therefore, the development of an accurate and efficient freehand microsampling method has important significance for researching the development and morphogenesis of the narcissus.
Disclosure of Invention
The invention aims to provide a microscopic sampling method for a Chinese narcissus organ in a morphogenesis stage, which can be used for sampling different tissue parts in the Chinese narcissus organ in the morphogenesis stage.
Specifically, the invention provides the following technical scheme:
The invention provides a microscopic sampling method for a Chinese narcissus organ morphogenesis stage, which comprises the following steps: firstly, removing buds and removing bracts, then sequentially removing the first round of the inflorescence, the second round of the inflorescence, the stamen or the petalous stamen, and finally removing the pistil-corolla combination or the auxiliary crown-pistil-corolla combination;
The method for stripping the 1 st round of festoon before forming the auxiliary crown is as follows: under a stereoscopic microscope, the needle point of the dissecting needle is transversely placed at the joint of the tops of the first round of festoons, the tops of the festoons are pressed downwards with force along the joint of the festoons, and then the needle point of the dissecting needle is inserted into the inner side of the festoons to be peeled off with force outwards, so that the peeled first round of festoons are obtained.
Because there is certain cross adhesion between 3 festoon tops of round 1, and little flower receptacle and the easy rupture of base junction, if peel by force, can lead to whole little flower tissue to drop from the inflorescence axle, can't realize the purpose of peeling off the sample of each flower organ to even if little flower tissue does not drop, tender festoon also very easily is torn, thereby leads to the festoon to remain and is difficult to peel off cleanly. The invention tries a plurality of stripping methods (including the treatment position of the dissecting needle, the force direction and the like), and finally determines the stripping method, and the method can well control the stripping force, avoid tissue damage, ensure the integrity and the accuracy of the obtained round 1 festoon quilt and can not damage the integrity of other flower organs. The whole stripping process is rapid and accurate, ensures the integrity of the genetic information, in particular the RNA information, of the test material as much as possible, and avoids intermixing with other tissues.
After the 1 st round of festoon is stripped, the 2 nd round of festoon is completely exposed, but the 2 nd round of festoon is more difficult to directly strip than the 1 st round of festoon and has smaller volume, the invention tries a plurality of stripping methods (including the treatment position of an anatomical needle, the force direction and the like), and finally determines the following stripping methods:
Under a stereoscopic microscope, an dissecting needle is inserted between the festoon and a stamen or a petalized stamen, force is applied outwards until the petalin tip of the festoon is downward even attached to a receptacle, the festoon is penetrated by the needle point of the dissecting needle, and the festoon is torn off by force applied by clockwise or anticlockwise rotation, so that the stripped 2 nd round festoon is obtained.
The method can well ensure the integrity of the obtained 2 nd round of festoon quilt, avoid residue and not damage the integrity of other flower organs.
When the flower is formed in the later stage of the morphogenesis, namely after the secondary crown is formed, the flower quilt of the 1 st round and the flower quilt of the 2 nd round are completely formed, the tissue structure is more compact, and the flower quilt of the 1 st round and the flower quilt of the 2 nd round are preferably peeled off by adopting the following method:
and transversely placing one forceps tip of the dissecting forceps at the joint of the top of the quilt, pressing the top of the quilt downwards with force along the joint of the quilt, clamping the inner side and the outer side of the quilt by the dissecting forceps, and tearing off with force downwards in an inclined manner to sequentially obtain the stripped quilt with the 1 st round and the stripped quilt with the 2 nd round.
After the auxiliary crown is built, the method is adopted to strip the flower quilts of the 1 st round and the 2 nd round, so that the efficiency of overall sampling can be improved.
After peeling off the outer 2 rounds of the inflorescence, the stamens or petalonized stamens were completely exposed. The invention discovers that different stripping methods are needed to be adopted respectively in the development period of different stamens or petalonized stamens to ensure better stripping effect, wherein the stamens or petalonized stamens is in a sphere primordium state at the initial stage of development, and can be stripped only by slightly picking up the stamens or petalonized stamens from outside to inside by using the tip of an anatomical needle. In the middle and later stages of stamen, the stamen is in a long column shape, 3 longitudinal grooves are formed on the inner side, and the outer side base is firmly connected with the flower stand; in the middle and later stages of the development of the petaloid stamen, the petaloid stamen is completely petaloid into a clustered petal-shaped structure, and the clustered petal-shaped structure is firmly connected with the flower receptacle. Both of the above cases require specific stripping tools and stripping means to ensure good stripping integrity and accuracy.
The method for peeling stamens or petaloid stamens at different developmental stages is as follows:
In the early stage of development of stamens or petioled stamens, the peeling method of the stamens or petioled stamens comprises the following steps: the dissected stamens or valved stamens are obtained by picking up from outside to inside with the tip of the dissecting needle.
In the middle and later stages of stamen development, the peeling method of the stamen or the valved stamen comprises the following steps: under a stereoscopic microscope, the forceps tips of the dissecting forceps are used for propping against two sides of the joint of the stamens or the valved stamens and the receptacle, and the stamens or the valved stamens are peeled off inwards by force.
After peeling off the stamen or the petalogenated stamen, the remaining pistil-receptacle assembly or the auxiliary crown-pistil-receptacle assembly, because the auxiliary crown does not continuously develop inside the receptacle between the perianth and the pistil, but eventually forms a ring-shaped or fragmented structure, and the pistil is in the central region of the floret tissue, the auxiliary crown and the pistil are difficult to peel off cleanly by bare hands, and therefore, the invention peels off the pistil, the receptacle or the auxiliary crown, the pistil and the receptacle as an assembly under a stereoscopic microscope.
Specifically, after peeling off the stamens or the petalous, under a stereoscopic microscope, the pistil-corolla combination or the auxiliary crown-pistil-corolla combination is peeled off with a force by puncturing the corolla oblique pressure with the tip of an dissecting needle.
In the above-described microscopic sampling method, the method of peeling off the bracts is as follows: under a stereoscopic microscope, along the edge of the flat bract, the two sides of the bract are lifted from bottom to top by using an dissecting needle, and the inner side of the bract is obliquely pressed by using the dissecting needle to exert downward force, so that the bract is completely torn off.
The method for peeling off the buds is as follows: the top is transversely cut and removed at the 1/2 position of the height of the narcissus bulb, 1 knife is longitudinally cut at least 1cm on the four sides of the outer side of the main flower bud, one knife is transversely cut at the lower part of the bulb tray after one side is put flat, finally a cuboid block is cut, and residual scales and leaves are peeled off piece by piece until the flat flower buds are completely exposed.
In the above-described microscopic sampling method, the peeled round 1 of the inflorescence, round 2 of the inflorescence, stamen or petalonized stamen or pistil-corolla combination or auxiliary crown-pistil-corolla combination is stored in liquid nitrogen.
Specifically, the stripped round 1 of the inflorescence, round 2 of the inflorescence, stamen or petioled stamen or auxiliary crown-pistil-corolla combination is picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
In the above-described microscopic sampling method, the force should be as slight as possible in the peeling of each flower organ so as not to damage the integrity of the flower organ.
As a preferable scheme of the invention, the microscopic sampling method of the Chinese narcissus organ morphogenesis stage is as follows:
(1) Removing the bud: cutting the top of the Narcissus bulb at 1/2 of the height, longitudinally cutting 1 knife at least 1cm of the four sides of the outer side of the main flower bud, putting one side of the main flower bud flat, cutting one knife at the lower part of the bulb disc in a transverse cutting manner, and finally cutting the main flower bud into cuboid blocks; and (5) stripping the residual scales and leaves piece by piece until the flat buds are completely exposed before sampling.
(2) Stripping off the bract: under a stereoscopic microscope, along the edge of a flat bract, using an dissecting needle to pick up two sides of the bract from bottom to top, using the dissecting needle to obliquely press the inner side of the bract to slightly exert downward force, completely tearing off the bract, using the dissecting needle tip to pick up the bract, and placing the bract into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(3) Stripping the festooned quilts of the 1 st round and the 2 nd round: the method for stripping the flower quilt of the 1 st round and the 2 nd round before the formation of the vice crown is as follows: under a stereoscopic microscope, transversely placing the needle point of the dissecting needle at the joint of the top of the first round of the festoon, slightly pressing the top of the festoon downwards along the joint of the festoon with force, inserting the needle point of the dissecting needle into the inner side of the festoon, slightly and forcibly peeling the inner side of the festoon outwards, and then quickly picking the festoon into a centrifuge tube floating on the surface of liquid nitrogen to quickly freeze; after the flower quilt of round 1 is peeled off, the flower quilt of round 2 is completely exposed, under a stereoscopic microscope, an dissecting needle is inserted between the flower quilt and a stamen or a petalized stamen, the force is applied outwards gently until the petaline tip of the flower quilt is downward even attached to a flower holder, the flower quilt is torn off by using the force applied by the gentle clockwise or anticlockwise rotation of the dissecting needle tip to penetrate the flower quilt, and then the flower quilt is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing;
after the formation of the auxiliary crown, the method for stripping the flower quilt of the 1 st round and the 2 nd round is as follows: and transversely placing one forceps tip of the dissecting forceps at the joint of the top of the quilt, pressing the top of the quilt downwards with force along the joint of the quilt, clamping the inner side and the outer side of the quilt by the dissecting forceps, and tearing off with force downwards in an inclined manner to sequentially obtain the stripped quilt with the 1 st round and the stripped quilt with the 2 nd round.
(4) Peeling off stamens or petalonized stamens: after the outmost 2 rounds of the inflorescences are stripped, stamens or valved stamens are completely exposed, wherein, in the early development stage of the stamens or valved stamens, the stamens or the valved stamens can be stripped by gently picking up from outside to inside by using the needle point of an dissecting needle, and the stamens or the valved stamens is picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing; in the middle and later stages of stamen or petalogenated stamen development, under a stereoscopic microscope, using dissecting forceps to prop against two sides of a joint of the stamen or petalogenated stamen and an receptacle, stripping the stamen or petalogenated stamen inwards by lightly applying force, and then rapidly picking the stamen or petalogenated stamen into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(5) Peeling the pistil-receptacle assembly or the accessory crown-pistil-receptacle assembly: after the stamens or the petalous stamens are peeled off, under a stereoscopic microscope, the needle tip of the dissecting needle is used for penetrating the receptacle, the pressure is lightly applied, the peeling can be realized, and then the combination body is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
The invention also provides application of the microscopic sampling method in the morphological establishment period of the Chinese narcissus organ in the differentiation and development analysis of the Chinese narcissus organ.
The invention has the beneficial effects that: the invention provides a microscopic rapid sampling method for different tissue parts in the morphological stage of a Chinese narcissus organ, which can well ensure the integrity and peeling precision of different flower organs, is simple and rapid to operate, does not need complicated instruments and operation steps, and is suitable for popularization and application in the practice of research on the morphological stage of the Chinese narcissus organ, the morphological stage of the flower organ and the like.
Drawings
FIG. 1 is a flow chart showing the operation of the method for microscopic sampling of flower organs at the initial stage of flower morphogenesis of 'Jin Zhanyin' in example 1 of the present invention, wherein P1 is the total inflorescence; p2, stripping off the bracts, wherein an arrow indicates the semi-stripped bracts; p3, stripping the 1 st round of festoon, wherein an arrow indicates the half stripped 1 st round of festoon; p4, stripping the 2 nd round of festoons, wherein an arrow indicates that the 2 nd round of festoons are not stripped yet; p5, peeling off the stamen primordia, wherein the arrow indicates early stage of development of stamen; p6, peeling off the pistil-flower stand combination; in the figure, the scale of P1 is 1.0mm, the scale of P4 is 0.2mm, and the scales of P2, P3, P5 and P6 are 0.5mm.
FIG. 2 is a flow chart showing the operation of the method for microscopic sampling of floral organs in the middle and late stage of 'Jin Zhanyin' floral morphogenesis in example 2 of the present invention, wherein P7, the 2 nd round of festoons are stripped, wherein the arrow indicates the half-stripped 2 nd round of festoons; p8, stamens to be stripped; p9, the accessory crown-pistil-receptacle combination to be peeled off; the scale bars in the figure are all 0.5mm.
FIG. 3 shows the morphology of part of the floral organs of examples 1 and 2 of the present invention, wherein P10, 'Jin Zhanyin' is the morphology of the round 1 quilt; p11, 'Jin Zhanyin' 2 nd round of festooned quilt morphology; p12, 'Jin Zhanyin' stage of mid-to-late stage stamen morphology; p13, 'Jin Zhanyin' joints of the middle and late developmental stamens and the receptacle, wherein the arrow indicates the joints of the stamens and the receptacle; in the figure, the P12 scale is 1.0mm, and the other scales are 0.5mm.
FIG. 4 is a flow chart showing the operation of the method for microscopic sampling of floral organs at the early stage of the morphological formation of the 'Yu exquisite' flower in example 3 of the present invention, wherein P14, the total inflorescence; p15, peeling off the bracts, wherein an arrow indicates the semi-peeled bracts; p16, stripping the 1 st round of festoon, wherein an arrow indicates the half stripped 1 st round of festoon; p17, stripping the 2 nd round of festoons, wherein an arrow indicates that the 2 nd round of festoons are not stripped yet; p18, peeling off the petalonic primordium, wherein the arrow indicates the petalonic stamen that has not been peeled off; p19, peeling off the pistil-flower stand combination; in the figure, the P14 and P15 scales are 1.0mm, and the other scales are 0.5mm.
FIG. 5 is a flow chart showing the operation of the method for microscopic sampling of floral organs in the middle and late stage of the 'exquisite jade' floral morphogenesis of example 4 of the present invention, wherein P20, the 2 nd round of festoons are stripped, wherein the arrow indicates the half stripped 2 nd round of festoons; p21, peeling off the petalonized stamens; p22, stripping the fragmented paracrown-pistil-receptacle combination; in the figure, the P20 and P21 scales are 0.5mm, and the P22 scales are 0.2mm.
FIG. 6 shows the morphology of the part of the flower organ of the 'exquisite' in examples 3 and 4 of the present invention, wherein P23 and P24 are the morphology of the 'exquisite' flower, and the early 3 rd and 4 th round of petal stamen, wherein the arrow of P23 indicates the characteristic structure of petal stamen; p25, 'exquisite' flower morphology builds middle and later stage petaloid stamen morphology; in the figure, the P23 and P24 scales are 0.5mm, and the P25 scale is 1.0mm.
FIG. 7 shows the failed sampling patterns of comparative examples 2 and 3 of the present invention, wherein P26, round 1, was crushed; p27, destroying the shape of the accessory crown structure in the stamen sampling process; in the figure, the scale P26 is 1.0mm and the scale P27 is 0.5mm.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a microscopic sampling method for different tissue parts of a flower organ in a morphological stage of Jin Zhanyin flowers of Narcissus chinensis, wherein when the stamen are in a primordial shape, the stamen are in a sphere shape or a cylinder shape and are not tightly combined with a receptacle, and the method specifically comprises the following steps: dissecting and stripping off bract, perianth, stamen and pistil-receptacle combination under a stereoscopic microscope, and quickly freezing and preserving each stripped tissue in liquid nitrogen, wherein the sampling steps are as follows:
(1) Removing the bud: cutting the top of the bulb at the position 1/2 of the height of the bulb, longitudinally cutting 1 knife at the position of at least 1cm on the four sides of the outer side of the main flower bud, cutting one knife at the position of the bulb disc after one side is flattened, and finally cutting into cuboid blocks; and (5) stripping the residual scales and leaves piece by piece until the flat buds are completely exposed before sampling.
(2) Stripping off the bract: under a stereoscopic microscope, along the edge of a flat bract, using an dissecting needle to pick up two sides of the bract from bottom to top, using the dissecting needle to obliquely press the inner side of the bract to slightly exert downward force, completely tearing off the bract, using the dissecting needle tip to pick up the bract, and placing the bract into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(3) Stripping the 1 st round of festooned quilt: under a stereoscopic microscope, the needle point of the dissecting needle is transversely placed at the joint of the top of the first round of festoon, the top of the festoon is slightly pressed downwards along the joint of the festoon, the needle point of the dissecting needle is inserted into the inside of the festoon to be slightly peeled outwards, and then the festoon is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen to be quickly frozen.
(4) Stripping the 2 nd round of festooned quilt: under a stereoscopic microscope, a dissecting needle is inserted between the festoon and the stamens, the dissecting needle is lightly forced outwards until the valve tip of the festoon is downward and even stuck on the receptacle, the festoon is torn off by lightly rotating the dissecting needle tip to the festoon clockwise or anticlockwise, and then the festoon is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen to be quickly frozen.
(5) Stripping stamen primordium: the stamens are in primordial shape, the stamens are in sphere or cylinder shape, and are not tightly combined with the receptacle, and the peeling method of the stamens is as follows: after the outer 2 rounds of festoons are stripped, the spherical or cylindrical stamen primordium is completely exposed, the stamen primordium can be stripped by slightly picking up the stamen primordium from outside to inside by using the tip of the dissecting needle, and the stamen primordium is picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(6) Stripping the "pistil-flower stand" combination: after the stamen primordium is stripped, the remaining pistil and receptacle tissues are used as an assembly, under a stereoscopic microscope, the receptacle is penetrated by a needle point of an dissecting needle and is lightly pressed and force is applied to strip, and then the 'pistil-receptacle' assembly is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
The method is used for separating each tissue part of the largest flower in the three-year-old commodity bulb flower development morphological stage inflorescence of the Chinese narcissus 'Jin Zhanyin', each obtained tissue part has higher integrity, each operation step is shown in figure 1, and the obtained partial flower organ morphology is shown in figure 3.
Example 2
The embodiment provides a microscopic sampling method for different tissue parts of a Chinese narcissus' Jin Zhanyin flower organ in a morphogenesis stage, wherein when a stamen is in a secondary crown in a middle and later stage of development, the tissue structure of the perianth is more compact, the stamen is in a long column shape, 3 longitudinal grooves are formed on the inner side, and the base on the outer side is more firmly connected with a receptacle, and the method is different from the method in the embodiment 1 only in the following steps:
(3) Stripping the 1 st round of festooned quilt: under a stereoscopic microscope, one forceps tip of the dissecting forceps is transversely placed at the joint of the tops of the flower quilts of the 1 st round, the tops of the flower quilts are slightly pressed downwards along the joint of the flower quilts with force, the inner side and the outer side of the flower quilts are clamped by the dissecting forceps to be torn downwards with force in an inclined mode, and then the flower quilts of the 1 st round are quickly picked into a centrifuge tube floating on the surface of liquid nitrogen to be quickly frozen.
(4) Stripping the 2 nd round of festooned quilt: under a stereoscopic microscope, the inner side and the outer side of the flower quilt are clamped by dissecting forceps and are torn off by obliquely downwards, and then the flower quilt of the 2 nd round is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(5) Peeling off stamens: after the two-wheel perianth at the outer side is stripped, the stamens with 3 longitudinal grooves at the inner side of the long column are completely exposed, under a stereoscopic microscope, the two sides of the joint of the stamens and the receptacle are propped by using the forceps tips of dissecting forceps, the stamens can be stripped by slightly exerting inward force, and then the stamens is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
(6) Stripping the "paracrown-pistil-flower receptacle" combination: after the stamen is stripped, the residual auxiliary crown, pistil and receptacle tissues are taken as the combination body, under a stereoscopic microscope, the split needle tip is used for penetrating the receptacle to be stripped by light oblique pressure, and then the combination body of the auxiliary crown, the pistil and the receptacle is quickly picked into a centrifuge tube floating on the surface of liquid nitrogen for quick freezing.
The method is used for separating each tissue part of the largest flower in the three-year-old commodity bulb flower development morphological stage inflorescence of the Chinese narcissus 'Jin Zhanyin', each obtained tissue part has higher integrity, each operation step is shown in figure 2, and the obtained partial flower organ morphology is shown in figure 3.
Example 3
The embodiment provides a microscopic sampling method for different tissue parts in a morphological stage of a flower organ of 'exquisite Yu' of a Chinese narcissus, wherein when a petaloid stamen is in a primordial shape, the petaloid stamen are in a sphere shape or a cochlea shape, are not tightly combined with a receptacle, and a fragmented accessory crown does not develop, and the method specifically comprises the following steps: microdissection and trace extraction of bract, perianth, petaloid stamen and pistil-receptacle combination, and then quick freezing and preservation by liquid nitrogen; this method differs from the method of example 1 only in that: in the step (5), the sampling tissue is a petaloid primordium.
The method is used for separating the tissue parts of the largest flowers in the inflorescence of the three-year-old commodity bulb flower development morphological stage of the Narcissus chinensis 'exquisite Yu' and the obtained tissue parts have higher integrity, the operation steps are shown in the figure 4, the obtained petalized stamen form is shown in the figure 6, and the 1 st round and the 2 nd round of inflorescences are highly similar to those of the P10 and P11 'Jin Zhanyin' inflorescences in the figure 3.
Example 4
The present embodiment provides a microscopic sampling method for different tissue parts of a Chinese narcissus 'exquisite-jade' flower organ in a morphogenesis stage, in which, when the petal stamens is in a middle-late stage of development, the petal stamens is completely folded inwards to form clusters, and is combined with a receptacle more tightly, and the difference between the method and the method of embodiment 2 is that: in the step (5), the sampling tissue is a valved stamen; in step (6), the sampled tissue is a "fragmented paracrown-pistil-receptacle combination".
The method is used for separating each tissue part of the largest flower in the inflorescence of the three-year-old commodity bulb flower development morphological formation period of the Narcissus chinensis 'Yu exquisite', each obtained tissue part has higher integrity, each operation step is shown in figure 5, and the obtained partial flower organ morphology is shown in figure 6.
Comparative example 1
The comparative example provides a microscopic sampling method of a1 st round of festoon in the initial stage of the morphological formation of a Chinese narcissus 'Jin Zhanyin' and 'Yu exquisite' flower organ, wherein the 1 st round of festoon and the 2 nd round of festoon are in primordial state or just morphological formation, wherein the 3 st round of festoon is provided with filiform substances at the valve tips, and when the festoon is directly stripped transversely or obliquely by an anatomical needle, a small flower stalk is separated from an inflorescence shaft due to uncontrollable force, so that sampling failure is caused.
Comparative example 2
The comparative example provides a microscopic sampling method of a1 st round of festoon in the middle and later stages of Chinese narcissus 'Jin Zhanyin' and 'exquisite jade' flower organ morphogenesis, wherein in the method, the 1 st round of festoon and the 2 nd round of festoon are morphogenic, the small festoon volume is enlarged, but filars still exist at the valve tips of the 3 rd round of festoon, and when an inflorescence basal part and a small flower stem are fixed with assistance of fingers and are directly peeled transversely or obliquely by an anatomical needle, the 1 st round of festoon cannot be peeled completely, so that the festoon tissue remains. The broken pattern of the 1 st round of the flower quilt is shown as P26 in fig. 7.
Comparative example 3
The comparative example provides a microscopic sampling method of stamens in the morphogenic stage of flower organs of 'Jin Zhanyin' narcissus in China. In the method, when the stamens is in a spherical or cylindrical primordial shape at the early stage of development, the tender water content of the tissue is extremely high, and the dissecting forceps are used for clamping the tissue to break the stamens so as to cause sampling failure; when the stamen are in the middle and late stages of development, the stamen are in a long column shape, and the stamen are cracked by themselves or the tissues of the accessory crowns are adhered by using the dissecting forceps to be peeled off transversely, so that the sampling is incomplete. Stamen sampling resulted in minor crown failure as shown at P27 in FIG. 7.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (2)
1. A microscopic sampling method for a Chinese narcissus organ morphogenesis stage, which is characterized by comprising the following steps: firstly, removing buds and removing bracts, then sequentially removing the first round of the inflorescence, the second round of the inflorescence, the stamen or the petalous stamen, and finally removing the pistil-corolla combination or the auxiliary crown-pistil-corolla combination;
The method for stripping the flower quilt of the 1 st round and the 2 nd round before the formation of the auxiliary crown is as follows: under a stereoscopic microscope, transversely placing the needle point of the dissecting needle at the joint of the top of the first round of festoon and downwards pressing the top of the festoon with force along the joint of the festoon, and then inserting the needle point of the dissecting needle into the inner side of the festoon to peel off with force outwards to obtain the peeled first round of festoon;
After the flower quilt of the 1 st round is stripped, under a stereoscopic microscope, an dissecting needle is inserted between the flower quilt and a stamen or a petalized stamen, force is applied outwards until the petaline tip of the flower quilt is downward even attached to a receptacle, the flower quilt is penetrated by the needle point of the dissecting needle, and the flower quilt is torn off by force applied by clockwise or anticlockwise rotation, so that the stripped flower quilt of the 2 nd round is obtained;
After the formation of the auxiliary crown, the method for stripping the flower quilt of the 1 st round and the 2 nd round is as follows: transversely placing one forceps tip of the dissecting forceps at the joint of the top of the quilt, pressing the top of the quilt downwards with force along the joint of the quilt, clamping the inner side and the outer side of the quilt by the dissecting forceps, and tearing off with force downwards in an inclined manner to sequentially obtain the stripped quilt with the 1 st round and the stripped quilt with the 2 nd round;
In the early stage of development of stamens or petioled stamens, the peeling method of the stamens or petioled stamens comprises the following steps: picking up from outside to inside by using the needle point of the dissecting needle to obtain a peeled stamen or a valved stamen;
In the middle and later stages of development of stamens or petioled stamens, the peeling method of the stamens or petioled stamens comprises the following steps: under a stereoscopic microscope, using forceps tips of dissecting forceps to prop two sides of a joint of the stamens or the petaloid stamens and the receptacle, and stripping the stamens or the petaloid stamens inwards by force;
After peeling off the stamens or the petaloid stamens, under a stereoscopic microscope, the pistil-corolla combination or the auxiliary crown-pistil-corolla combination is peeled off by using the oblique pressure of the anatomical needle to puncture the corolla with the needle point of the anatomical needle;
the method for peeling off the bracts comprises the following steps: under a stereoscopic microscope, along the edge of the flat bract, using an dissecting needle to separate two sides of the bract from bottom to top, using the dissecting needle to obliquely press the inner side of the bract to exert downward force, and completely tearing off the bract;
The method for peeling off the buds comprises the following steps: cutting the top of the Narcissus bulb at 1/2 height, longitudinally cutting at least 1cm of the four sides of the outer side of the main flower bud by 1 knife, cutting the lower part of the bulb tray by one knife after one side is flattened, finally cutting into cuboid blocks, and stripping the residual scales and leaves piece by piece until the flat flower buds are completely exposed;
The stripped round 1 comforter, round 2 comforter, stamen or petalonized stamen or pistil-corolla combination or vice crown-pistil-corolla combination is stored in liquid nitrogen.
2. The use of the microscopic sampling method of the morphological stage of the Chinese narcissus organ according to claim 1 in the analysis of the differentiation and development of the Chinese narcissus organ.
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