CN1552199A - Quick reproduction of Anzu flower by cell embryo induction - Google Patents
Quick reproduction of Anzu flower by cell embryo induction Download PDFInfo
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- CN1552199A CN1552199A CNA2003101097133A CN200310109713A CN1552199A CN 1552199 A CN1552199 A CN 1552199A CN A2003101097133 A CNA2003101097133 A CN A2003101097133A CN 200310109713 A CN200310109713 A CN 200310109713A CN 1552199 A CN1552199 A CN 1552199A
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Abstract
A fast reproduction method of Anzu flower by inducing somatic cells includes such steps as picking up the young stem of Anzu flower, cutting by 3-7 mm in length, inoculating in the inducing culture meidum under aseptic condition, culturing at 25-27 deg.C in dark condition for 40-50 days to induce embryo of somatic cell, and culturing at 25-27 deg.C under 2000 LX (12-16 hr per day) for 50-60 days. Its advantage is fast speed.
Description
Technical field
The present invention relates to the micropropagation of plants technology, be specially peace ancestral quirk blast and induce method for quickly breeding.
Background technology
An Zuhua originates in the Central and South America tropical rain forest, is the rare flower of introducing China in recent years.An Zuhua is gorgeous because of its flower shape uniqueness, pattern, bottle is inserted the characteristics that the life-span grow, blooms in the anniversary, ornamental value is high becomes the extremely top grade flower of people's welcome.In the trade of global tropical flowers, the sales volume of An Zuhua is only second to orchid, ranks second.An Zuhua both can be used for whole raising of cut-flower and had spent blue class, can be used for potted flower again and decorated.Owing to constantly cultivate the kind of spending more that makes new advances, the demand in its potted plant market just sharply rose in recent years.
Existing peace ancestral spends propagation method to mainly contain two kinds.A kind of is to carry out the specialization cultivation with seedling, and the sowing of this method utilization routine, offshoot carry out the An Zuhua breeding, and reproduction rate is extremely low, and the seed propagation required time is long, and easily produces variation.Another kind method is to utilize tissue culture to breed fast, is about to pacify the explant that the ancestral spends and cultivates in medium, produces callus by inducing, germinates, takes root, process such as implanting and cultivating breeds fast.Facts have proved that the peace ancestral of tissue culture propagation spends clone, mixing is planted all being better than greatly growing directly from seeds qualitatively and quantitatively.But because present peace ancestral spends the tissue culture propagation technology is to form regeneration plant by adventitious organogenesis, and its genetic stability is relatively poor relatively, and regeneration plant variability probability is higher relatively, and the breeding cycle is also longer relatively.And the method for utilizing the plant soma embryonal induction is carried out tissue-culturing rapid propagation to have inheritance relatively stable, the regeneration plant that formed regeneration plant variability forms less than adventitious organogenesis, and the breeding cycle is short, the characteristics that proliferative amount is big.Though the somatic embryo inducement method for quickly breeding is existing the application on other plant, the ancestral takes still manque at present report both at home and abroad in peace.
Summary of the invention
The present invention solves present still useless somatic embryo inducement method and breeds the problem that the peace ancestral spends fast, provides a kind of peace ancestral quirk blast to induce method for quickly breeding.
The present invention adopts following technical scheme to realize: peace ancestral quirk blast is induced method for quickly breeding, it comprises following steps: get the peace ancestral and spend young stem and young stem is cut into 3-7mm long, be linked in the inducing culture under the germ-free condition, temperature 25-27 ℃, dark culturing 40-50 days, change the somatic embryo that induces over to the form growth medium, temperature 25-27 ℃, see the light cultivation, light intensity 3000Lx, 12-16h/d cultivates and formed complete plant in 50-60 days; Described inducing culture based formulas is: 1/2MS+2.4-D1-2.5mg/L+TDZ0.05-0.5mg/L+ inositol 25-100mg/L+ casein 25-100mg/L+ sucrose 30-40g/L (2.4-D Chinese chemical name 2, the 4-dichlorphenoxyacetic acid, English chemical name 2,4-dichlorophenoxyacetic acid; TDZ Chinese chemical name plug benzene is grand, English chemical name Thidiazuron; The English chemical name Inositol of inositol; The English chemical name Casein of casein, acid hydrolysate); Described form grown cultures based formulas is: 1/2MS+KT0.05-0.5mg/L+TDZ0.05-0.5mg/L (KT Chinese chemical name 6-chaff aminopurine, English chemical name 6-Furfurylaminopurine).
Generally speaking, the growth course of somatic embryo (embryoid) is: cell is after dedifferentiation, and the p cell division forms small cell cluster, and passes through former embryonic stage, spherical embryonic stage, heart-shaped stage, pyriform embryonic stage and cotyledon period in turn, and then forms complete plant.Below by histocytology observed result in the peace ancestral quirk blast generating process is analyzed, prove that peace ancestral of the present invention spends quick-breeding method to meet the ordinary circumstance of somatic embryo development process, it is without tangible callus forming process, and its initialization mode belongs to significantly directly somatic embryo generation approach.
(1) the histocytology feature of young stem before the tissue culture
Young stem cross section ecto-entad is respectively before cultivating: ground floor is an epidermal area, marshalling, and regular shape is rectangle.Cell wall dyeing is darker, visible significantly cell nucleus.In the epidermis is 1-2 bed thickness horn cells, and thickened degree is not obvious.Cortical cell 6-10 layer is parenchyma cell, and volume is big, out-of-shape, the space between cells that tool is bigger, and cell nucleus is little, is distributed in cell edges, and the zone dyeing of whole cortex is more shallow.Cortex inside has 26 small bundles to distribute along concentric ring-shaped, and each vascular bundle has only 20 left and right sides cells to form, and phloem is in foreign side, and xylem belongs to collateral bundle interior side.No typical bundle sheath in the vascular bundle.Phloem, xylem are all grown.Conduit tool double wall, outer wall polygon, inner layer wall subcircular.The visible significantly bigger cell nucleus in phloem zone, and cytoplasm is denseer.Marrow is made up of 5 layers of parenchyma cell, and these cells are similar to cell in the cortex, and shape is justified (seeing Fig. 1, Fig. 2).
(2) young stem tissue starts the histocytology feature of division stage
The young stem of cultivating 5 days is observed discovery, and vascular bundle zone cell nucleus increases, and kernel is obvious, also occurs the cell nucleus that some have obvious kernel in the cortex.
The young stem of cultivating 8 days is observed discovery, middle part, cross section, vascular bundle zone cell nucleus comparatively dense, cell nucleus obviously becomes and becomes circle greatly, and kernel is positioned at cell nucleus central authorities.
The young stem of cultivating 11 days is observed discovery; cortex and a large amount of cells of vascular bundle intersection break; the vascular bundle endless belt separates with cortex with marrow, and pressing close to fascicular cortical cell has more starch granules accumulation, and there be not (Fig. 3, Fig. 4) in complete and independent vascular bundle structure.
Many big nucleuss appear in the screen casing cell peripheral in the vascular bundle, mainly are distributed near cortex one side, form the dense zonule of kytoplasm, and screen casing and these cells contacting are tight in the zone.These zonules are meristematic zones of cells,primordial, and this class cell should be preceding embryo decision cell, may be the initiator cells (Fig. 5, Fig. 6) of somatic embryo.
(3) tissue and cyto-architectural dynamic in the somatic embryo growth course
From cultivating back 21 days, found the many cells proembryo of different cell numbers.Proembryo and peripheral cell have obvious boundary, have heavy wall that proembryo is surrounded.Cell is little in the proembryo, but cell nucleus is very big.Cell structure around the proembryo is loose, and cell volume is big, seedless, wall thin, kytoplasm rare (Fig. 7).
Along with the increase of archiblast number, the arrangement of cell is tightr, and the shape of cell becomes polygon by subcircular, and can observe a large amount of chromosome behaviors this period.Along with the cell division of many cells proembryo, idiosome is expanded as circle gradually, forms globular embryo.
The state in embryo development procedure each period is seen in can be from Fig. 7 to Figure 12, is followed successively by early stage proembryos, globular embryo, heart-shape embryo, pyriform embryo, the cotyledonary embryos (scultellum embryo) of 8 cell proembryo, 32 cell proembryo, single suspensor.
Observed somatic embryo mainly is to form through interior generation approach, is to produce somatic embryo by the embryo decision cell in the vascular bundle in the stem.But the young stem of cultivating 34 days is observed, found an embryoid structure that outwards produces by young stem epidermal area.And, then keeping the structure similar to original stem at cross section other position.This embryoid clearly directly produces from the explant okioplast.Presentation of results when peace ancestral quirk blast is main mode with interior generation approach, is not got rid of exist (Figure 19) of outer generation approach yet.
(4) about the idiosome differentiating characteristic
In the idiosome idiophase, the position cell division that has in the idiosome is rapid, forms projection, becomes the scultellum embryo.Big space occurs between radicle district's idiosome and peripheral cell and be in isolated state (Figure 13) this moment.Organizing in the embryo begins differentiation, and the radicle district forms protoderm, its cell shape rule, marshalling, and easy and other tissue difference (Figure 14) is the cell mass between the fundamental meristem in it, cell nucleus distributes looser.Procambia is arranged in the cotyledon, and its cell is made longitudinal tensile strain, moves towards plumular axis from cotyledon, and the trend that forms continuous system is arranged, and this part regional kytoplasm is dense.Plumular axis is to the cell (Figure 15) that longitudinal tensile strain is also arranged between the radicle, and is similar to the procambia in the cotyledon.Along with the further growth of embryo, cotyledon continues elongation, can see that under high power lens (Figure 16) appears in the existing conduit in cotyledon district.
(5) morphogenesis phase feature
Mature embryo continue to be grown, form the plant blank and grow up to whole plant (Figure 17, Figure 18).
It is relatively stable that peace ancestral quirk blast of the present invention induces method for quickly breeding to have inheritance, the regeneration plant that formed regeneration plant variability forms less than adventitious organogenesis, breeding cycle is short, the characteristics that proliferative amount is big, this method carry out fast numerously taking place to shorten 50 days time than organ.Have the morphogenetic characteristic of the fertilized egg of recurrence in theory, have crucial meaning for gene expression and regulation and control in mechanism that discloses great theoretical questions such as cell differentiation, growth, form generation and zygotic embryo growth and the eukaryotic.The ancestral spends improvement, transgene receptor and screening mutant etc. that good vegetative propagation experimental system is provided for peace.
Description of drawings
Fig. 1 is the crosscut light micrograph of young stem before the tissue culture;
Fig. 2 is a young cauline bundle light micrograph before the tissue culture
Fig. 3 is the young stem light micrograph of cultivation after 11 days
Fig. 4 begins to divide light micrograph for the vascular bundle cell
Fig. 5 is the big nucleus light micrograph around the screen casing
Fig. 6 is embryo's generation initiator cell light micrograph
Fig. 7 is 8 cell proembryo light micrographs
Fig. 8 is the former embryo's light micrographs of 32 cells
Fig. 9 is the early stage proembryos light micrograph
Figure 10 is the globular embryo light micrograph
Figure 11 is a pyriform embryo light micrograph
Figure 12 is a peltate embryo light micrograph
Figure 13 is a radicle structure light micrograph
Figure 14 is a hypophysis epidermal area light micrograph
Figure 15 is a cotyledon district procambia light micrograph
Figure 16 is a cotyledon district catheter optical microphoto
Figure 17 is a mature embryo shape bulk optics microphoto
Figure 18 is a form generation light micrograph
Figure 19 is the outer embryoid light micrograph that takes place
Embodiment
Embodiment 1
Peace ancestral quirk blast is induced method for quickly breeding, it comprises following steps: get the peace ancestral and spend young stem and young stem is cut into 3mm long, be linked in the inducing culture 25 ℃ of temperature, dark culturing 40 days under the germ-free condition, change the somatic embryo that induces over to form grown cultures based formulas, temperature 25-27 ℃, see the light cultivation, light intensity 3000Lx, 12h/d cultivates and formed complete plant in 50 days; Described inducing culture based formulas is: 1/2MS+2.4-D1mg/L+TDZ0.05mg/L+ inositol 25mg/L+ casein 25mg/L+ sucrose 30g/L; Described form grown cultures based formulas 1/2MS+KT0.05mg/L+TDZ0.05mg/L.
The somatic embryo that induces can downcut from young stem and change the form growth medium over to; Less by the somatic embryo that induces in the young stem section, can change whole young stem section over to the form growth medium.
Embodiment 2
Peace ancestral quirk blast is induced method for quickly breeding, it comprises following steps: get the peace ancestral and spend young stem and young stem is cut into 7mm long, be linked in the inducing culture 27 ℃ of temperature, dark culturing 50 days under the germ-free condition, change the somatic embryo that induces over to form grown cultures based formulas, 27 ℃ of temperature are seen the light cultivation, light intensity 3000Lx, 16h/d cultivates and formed complete plant in 60 days; Described inducing culture based formulas is: 1/2MS+2.4-D2.5mg/L+TDZ0.5mg/L+ inositol 100mg/L+ casein 100mg/L+ sucrose 40g/L; Described form grown cultures based formulas 1/2MS+KT0.5mg/L+TDZ 0.5mg/L.
Embodiment 3
Peace ancestral quirk blast is induced method for quickly breeding, it comprises following steps: get the peace ancestral and spend young stem and young stem is cut into 5mm long, be linked in the inducing culture 26 ℃ of temperature, dark culturing 45 days under the germ-free condition, change the somatic embryo that induces over to form grown cultures based formulas, 26 ℃ of temperature are seen the light cultivation, light intensity 3000Lx, 14h/d cultivates and formed complete plant in 55 days; Described inducing culture based formulas is: 1/2MS+2.4-D1.8mg/L+TDZ0.27mg/L+ inositol 65mg/L+ casein 65mg/L+ sucrose 35g/L; Described form grown cultures based formulas 1/2MS+KT0.27mg/L+TDZ0.27mg/L.
Claims (1)
1, a kind of peace ancestral quirk blast is induced method for quickly breeding, it is characterized by: it comprises following steps: get the peace ancestral and spend young stem and young stem is cut into 3-7mm long, be linked in the inducing culture under the germ-free condition, temperature 25-27 ℃, dark culturing 40-50 days, change the somatic embryo that induces over to the form growth medium, temperature 25-27 ℃, see the light cultivation, light intensity 3000Lx, 12-16h/d cultivates and formed complete plant in 50-60 days; Described inducing culture based formulas is: 1/2MS+2.4-D1-2.5mg/L+TDZ0.05-0.5mg/L+ inositol 25-100mg/L+ casein 25-100mg/L+ sucrose 30-40g/L; Described form grown cultures based formulas is: 1/2MS+KT0.05-0.5mg/L+TDZ0.05-0.5mg/L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100361568C (en) * | 2005-05-12 | 2008-01-16 | 天津龙康泰生物技术有限公司 | Quick breeding method for inducing spheroidal embryo directly from tissue of anthurium |
| CN100424169C (en) * | 2005-01-31 | 2008-10-08 | 肖尊安 | Culture medium for anthurium andraeanum tissue culture and tissue culture seedling breeding method |
| CN102893868A (en) * | 2012-10-18 | 2013-01-30 | 广东省农业科学院花卉研究所 | Method for embryogenic cells of anthurium scherzerianum to quickly proliferate and regenerate plant |
| CN106212491A (en) * | 2016-04-02 | 2016-12-14 | 江苏辉丰农化股份有限公司 | A kind of plant growth regualting composition |
| CN111053034A (en) * | 2020-01-09 | 2020-04-24 | 上海市农业科学院 | Tissue culture method for generating anthurium embryoid through root tip induction |
-
2003
- 2003-12-19 CN CN 200310109713 patent/CN1235469C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424169C (en) * | 2005-01-31 | 2008-10-08 | 肖尊安 | Culture medium for anthurium andraeanum tissue culture and tissue culture seedling breeding method |
| CN100361568C (en) * | 2005-05-12 | 2008-01-16 | 天津龙康泰生物技术有限公司 | Quick breeding method for inducing spheroidal embryo directly from tissue of anthurium |
| CN102893868A (en) * | 2012-10-18 | 2013-01-30 | 广东省农业科学院花卉研究所 | Method for embryogenic cells of anthurium scherzerianum to quickly proliferate and regenerate plant |
| CN106212491A (en) * | 2016-04-02 | 2016-12-14 | 江苏辉丰农化股份有限公司 | A kind of plant growth regualting composition |
| CN106900730A (en) * | 2016-04-02 | 2017-06-30 | 江苏辉丰农化股份有限公司 | A kind of plant growth regualting composition |
| WO2017166566A1 (en) * | 2016-04-02 | 2017-10-05 | 江苏辉丰农化股份有限公司 | Plant growth regulating composition |
| CN111053034A (en) * | 2020-01-09 | 2020-04-24 | 上海市农业科学院 | Tissue culture method for generating anthurium embryoid through root tip induction |
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