CN101218894A - Embryogenesis and plant strain regeneration method for hybrid somatic cell of slash pine and cuban pine - Google Patents
Embryogenesis and plant strain regeneration method for hybrid somatic cell of slash pine and cuban pine Download PDFInfo
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Abstract
A method for somatic cell embryo generation and plant regeneration of a hybrid of a slash pine and a Caribbean pine is characterized in that the invention relates to a method which adopts a immature zygotic embryos of the hybrid of the slash pine and the Caribbean pine to carry out somatic cell embryo generation and plant regeneration and uses liquid and slide improved P6 mediums to alternatively combine for adjustment and control culture during the process of the somatic cell embryo generation and plant regeneration for a plurality of times and is appended with proper auxin combination. The invention provides a packaged technology for fast breeding the hybrid seedlings of the slash pine and the Caribbean pine industrializedly and can solve the current problem of short supply of the seedlings during the hybrid afforestation of the slash pin and the Caribbean pine.
Description
One, technical field
The invention belongs to the plant regeneration technical field of passing through tissue culture in the forestry, be specifically related to a kind of wet-land pine tree and pinus caribaea hybrid somatic embryo and take place and plant regeneration method.
Two, background technology
Wet-land pine tree (Pinus elliottii Englem) originates in southeastern US, pinus caribaea (Pinuscaribaea Morelet) originates in south American countries such as Cuba and Honduras, a small amount of introduction thirties in 20th century China, China introduces a fine variety wet-land pine tree and Cuba pinus caribaea in Guangdong Province's Zhanjiang region after the sixties.Wet-land pine tree is at the wide accommodation of China, and is more cold-resistant, can plant to Nanyang, Henan, Xinyang, some areas, the Shandong Peninsula; And pinus caribaea's cold resistance a little less than, regional culture often suffers freeze injury to the north of north latitude 23 degree.Do not see the report that has interspecific hybrid between two kinds under the native state.
Two trees introducing a fine variety are planted and all grow rapidly in China, and two kinds of plant flower synchronizations of introducing a fine variety, and before and after the eighties in 20th century, a spot of wet-land pine tree and pinus caribaea crossbreed have been cultivated in domestic beginning wet-land pine tree and pinus caribaea's crossbreeding research.Wet-land pine tree and pinus caribaea hybrid (being called for short the hybridization pine) demonstrate the more fast good characteristic of giving birth to than the parent, possess two parents and grow soon, form is perfectly straight, water-fast wet and impoverishment tolerant sand ground, and the characteristic that wind resistance is also stronger demonstrates the great potential of wet-land pine tree and pinus caribaea hybrid.But the hybrid seeding seed source is limited, and asexual cottage propagation is difficulty relatively, has limited applying of this elite hybrid.
The forest tissue culture technique have the breeding cycle short, breeding quantity is big, be subjected to natural surroundings influence little, be fit to advantage such as large-scale production.China's Plant Tissue Breeding research starting early is in international most advanced level in some fields after the seventies in last century.Over nearly 20 years, plant tissue culture technique has had great progress, wherein the tissue culture of woody plant has also obtained huge achievement, and is very rapid as aspects such as anther culture, stem apex and tender leaf cultivation, rataria cultivation, protoplast cultivation, somatic hybridization, screening mutant, detoxification cultivation, artificial seed progress.
But because the coniferous tree growth cycle is long, the regeneration difficulty is big, cell stage generation plant regeneration process complexity, and somatic embryo plant regeneration condition and frequency difference are bigger between different seeds, so progress is slow.
Present progress it has been generally acknowledged that coniferous tree cell stage generation plant regeneration process comprises following 4 key steps:
One, embryo callus subculture induces
Inducing the stable embryo callus with form generating ability is the basis that the body embryo takes place.In the coniferae, the key factor that influences embryonic callus induction comprises: the kind of the age of explant and physiological status, genotype, minimal medium, plant hormone and concentration, carbon source and temperature condition etc.Inducing generally containing the growth hormone of higher concentration of embryo callus subculture (as 10mg/L 2,4-D) and on the solid culture medium of the basic element of cell division (as 5mg/L BA) of higher concentration carried out.Tautorus etc. (1991) think that inducing of embryo callus subculture is that explant somatocyte is reset the process that it is grown direction, is converted into the dedifferentiation state by differentiation state.The starting material that coniferous tree can be selected the meristematic zone of cotyledon, upper and lower plumular axis, bud or root on egagametophyte, immature embryo, mature embryo, the children's aseptic seedling in age and the cotyledon somatic embryo of newly inducing takes place as embryo callus.But in general the tissue that differentiation degree is low helps inducing of embryo callus, and along with the growth and the maturation of tissue, inductivity reduces gradually.For the Pinus seeds, zygotic embryo is suitable for inducing of embryo callus most before the cotyledon, but can be subjected to strict restriction of growing season with this material usually as explant.In the case, generally consider the fully-developed explant is improved with cold treatment or hormone preliminary treatment the inductivity of embryo callus.
Minimal medium and genotype are to influence embryonic callus induction and morphogenetic two other key factor.Between different cultivars even between the same intravarietal different genotype, induce the ability that produces embryo callus that very big-difference is all arranged.In the tissue culture of coniferae, also do not set up a kind of medium at present as the MS medium that in the angiosperm tissue culture, is widely adopted.The MS medium is proved to be the improper tissue culture that is used for coniferae in many researchs.Therefore, the researcher improves the MS medium one after another, has also designed simultaneously some new minimal mediums, and wherein important have DCR, GD, BMS, LM, LP, MNCL, P6 and a SH etc.
Two, the subculture of embryo callus and propagation
The maintenance of embryo callus and propagation are carried out on the solid of usually identical with the medium of inducing embryo callus subculture in constituent but reduction hormone concentration (respectively reducing by 10 times usually) or the liquid nutrient medium.12～18 days subcultures once will shorten the subculture time when breeding on the liquid medium within when breeding on solid culture medium.Studies show that, as a rule, 2,4-D is the necessary condition that takes place of inductor blast but long-time the cultivation containing 2, on the medium of 4-D, easily causes the forfeiture of the ripe ability of somatic embryo.Thereby, replacing 2 with NAA in the multiplicative stage, 4-D more helps the generation of somatic embryo.Lulsdorf etc. (1993) think, it is identical but on the medium that osmotic pressure is higher that the embryo callus of propagation is transferred to composition, help forming sturdy late stage proembryos, and find in the research of dragon spruce and Picea mariana interiorly, compare with solid culture medium, the embryo callus growth rate under the liquid culture condition is faster.
Three, the growth of somatic embryo and maturation
The maturation of somatic embryo is the antimer blast has sprouting and early development on physiology and morphology a ability.At present, people have the ability of sprouting and early development on physiology and morphology to the plant soma embryo.At present, people understand very few to the mechanism of plant soma embryo maturation.From existing report, somatic embryo that can be ripe in early days in the somatic embryo shared ratio very low, and widely different between different genotype.But Robert etc. (1990) find, add the maturation that certain density ABA helps somatic embryo in the somatic embryo maturation medium.Robert etc. (1991) find that the degree of somatic embryo development depends on the concentration of ABA in the medium in the research of dragon spruce interiorly: in the medium during no ABA, and early stage somatic embryo stasi; Rising along with ABA concentration, a series of morphological change appears in somatic embryo, low concentration ABA less than 10 μ mol causes forming thin and weak bud shape body embryo, the intermediate concentration ABA of 10-20 μ mol causes forming precocious bipolar gonosome embryo, and the high concentration ABA of 30-40 μ mol causes the cotyledon body blast that forms stasi, enter resting stage.Under the condition of ABA+IBA, the synchronization degree of somatic embryo improves, and the quantity of mature embryo also obviously increases.It is generally acknowledged that the mechanism of action of ABA is to promote that starch, lipid and protein etc. store the accumulation of goods and materials in the cell, thereby promotes the maturation of somatic embryo.In addition, ABA ends cleavage polyembryony (Cleavage polyembryony) continuation generation in addition and suppresses the early ripe sprouting of somatic embryo.Because active carbon has adherent cell to produce the function of part hormone in metabolic waste and the medium, adds active carbon in this stage medium, can improve the effect that the cotyledonary embryos apical meristem is grown, and makes it more near zygotic embryo.
Four, the sprouting of somatic embryo and plant regeneration
Attree etc. (1993) think, the sprouting of somatic embryos of coniferous trees is meant the process of growing into regeneration plant after the somatic embryo maturation, and it comprises the elongation of root, cotyledon and plumular axis.The method that promotes somatic embryo to sprout is somatic embryo to be transferred to the hormone of no hormone or extremely low concentration, and its organic principle and osmotic pressure all reduce.Becwar etc. (1990) think that this transfer is the key that guarantees that somatic embryo is sprouted.They also find, the Norway spruce mature somatic embryo is inverted cultivation, can obtain the germination rate up to 56%.Somatic embryo induce and the maturation of somatic embryo is finished under dark and low light condition.But the sprouting of somatic embryo then needs illumination condition.After illumination on the germination medium was cultivated 2 months, major part was grown to all well-developed plantlet of root, plumular axis and cotyledon of high about 2cm.The sprouting of the somatic embryo of pinus caribaea (Pinus caribaea) (1992), pine (Pinusradiata) (1995), sugar pine (Pinus lambertiana) (1986) and Zhan Yesong (Pinus patula) pine genus plants such as (1993) is also finished under illumination condition.
The research report of relevant extensive needle pine somatic embryo regeneration plant field trial is few.Becwar etc. (1990) have studied 6 genotype Norway spruce growth of seedlings situations.Webster etc. (1991) have studied in 71 genotype ground dragon spruce somatic embryo regeneration plant at the growing state in field.They find that most of genotype somatic embryo regeneration plants are after the greenhouse transition, and transplanting survival rate is higher than 80%.David etc. (1989) have also obtained to be higher than 60% survival rate in the transplanting test of pinus caribaea's somatic embryo regeneration plant.Therefore, they think that the high-quality coniferous tree seedling that takes place from somatic embryo can be used for planting out and Chinese garden planting fully.
Tang Wei etc. (1997) think, although the morphogenetic research of coniferae tissue culture has obtained gratifying progress, remain in some problems in the research, specifically comprise:
(1) lacks the good morphogenetic tissue culture modular system of research;
(2) do not set up the minimal medium of the wider coniferae tissue culture of the scope of application;
(3) selection of explant still is confined to tender tissues such as zygotic embryo;
(4) induction frequency of embryo callus and somatic embryo in most seeds still the end reach medium level.
Since report Norway spruces such as Hakman in 1985 obtain acerose somatic embryo regeneration plant first, reported successively on more than the 30 kind of coniferous tree of abies (Abies), Larch (Larix), Picea (Picea), Pinus (Pinus), Pseudotsuga (Pseudosuga), Pseudolarix (Pseudolarix), Sequoia (Sequoia), Cunninghamia to have induced the somatic embryo generation.Nineteen ninety, Laine and David report pinus caribaea somatic embryo take place and plant regeneration, but inductivity is extremely low, not can manufacture the nursery stock of fine quality.The somatic embryo of Jain (1988) report over-wet land pine takes place and plant regeneration.
Have not yet to see the report of generation of wet-land pine tree and pinus caribaea hybrid somatic embryo and plant regeneration success.
Three, summary of the invention
The objective of the invention is, it is limited to hybridize seeds production of hybrid seeds seed source at current good wet-land pine tree and pinus caribaea, asexual cottage propagation is the present situation of difficulty relatively, seek the effective ways of generation of wet-land pine tree and pinus caribaea hybrid somatic embryo and plant regeneration, provide the complete set technology of industrialization fast breeding wet-land pine tree and pinus caribaea hybrid nursery stock, the problem in short supply of wet-land pine tree and pinus caribaea hybrid nursery stock in the solution current industrial afforestation.
Technical solution of the present invention is:
A kind of wet-land pine tree and pinus caribaea hybrid somatic embryo take place and plant regeneration method, it is characterized in that adopting the immature zygotic embryos of wet-land pine tree and pinus caribaea hybrid to carry out somatic embryo generation and plant regeneration, its somatic embryo generation and the main process of plant regeneration and condition are as follows:
A. the medium of somatic embryo induction period is improvement P6 medium, additional auximone 2,4-D 1-2mg/L, basic element of cell division 6-BA 1-2mg/L;
B. embryo head suspensor structure (ESM) the embryo culture fast breeding stage is adopted repeatedly liquid and the solid improvement P6 medium combinatorial regulation cultured method that alternates, additional plant hormone best of breed is 1-2mg/L 2,4-D, 0.5-1mg/L 6-BA and 0.5-1mg/L KT;
C. somatic embryo induction period, embryo head suspensor structure (ESM) embryo culture proliferation adjourning adopt liquid improvement P6 medium, additional 5～10mg/L abscisic acid (ABA) and 5～10mg/L gibberellin (GA
3);
D. the body embryonic development stage of ripeness of embryo head suspensor structure (ESM) embryo culture generation and somatic embryo are sprouted the stage employing solid improvement P6 medium of emerging, additional several amino acids, polyethylene glycol (PEG8000).
Explant sampling point, time control and pretreatment condition are: obtain immature embryo from kind of a tree cone, the optimal acquisition time is annual June; In order to help inducing of embryo callus subculture, the cone after the collection is placed in 4 ℃ of refrigerators and preserves more than 1 week after can using wet gauze, plastic sack parcel.
Operable 3 kinds of different improvement P6 culture medium prescriptions are as follows respectively:
Improvement P6 culture medium prescription 1 improvement P6 culture medium prescription 2
Improvement P6 culture medium prescription 3
| ?KNO 3?MgSO 4.7H 2O ?CaCl 2.2H 2O ?KH 2PO 4?KI ?H 3BO 3?ZnSO 4.7H 2O ?MnSO 4.4H 2O ?Na 2MoO 4.2H 2O ?CuSO 4.5H 2O ?CoCl 2.6H 2O ?FeSO 4.7H 2O ?Na2-EDTA | 1250mg/L 400mg/L 200mg/L 340mg/L 1mg/L 5mg/L 8mg/L 15.8mg/L 0.2mg/L 0.024mg/L 0.02mg/L 27.85mg/L 37.25mg/L | It is as follows that inositol nicotinic acid thiamine hydrochloride puridoxine hydrochloride glycine adds amino acid: L-proline L-asparagine L-arginine HCl L-alanine L-serine | 100mg/L 0.5mg/L 1.0mg/L 0.5mg/L 2.0mg/L 100mg/L 100mg/L 50mg/L 20mg/L 20mg/L |
The optimal medium of somatic embryo induction period is the third improvement P6 medium.
Four, description of drawings
The microphoto of state startup stage that Fig. 1 inducing for embryo callus subculture;
Fig. 2 observes embryo head suspensor structure microphoto startup stage of inducing for embryo callus subculture, a is an embryo head suspensor structure group among the figure, and b is the enlarged photograph of single embryo head suspensor structure;
A is the embryo callus subculture culture that induces among Fig. 3, and b is local enlarged photograph;
Fig. 4 is the callus culture of the nonideality that induces, is yellow or yellowish-brown, and the water content height can not continue differentiation and development;
A among Fig. 5, b, c are respectively the propagation embryo head suspensor structure group photo that 3 width of cloth immature embryo cultures are examined under a microscope;
A among Fig. 6, b are that two width of cloth mature embryo cultures are examined under a microscope no tangible embryo head suspensor structure photo;
The dysgonic photo of embryo callus subculture when Fig. 7 is 5000mg/L for inositol concentration;
A among Fig. 8, b are that two width of cloth show the photo of growing similar state in early stage with the nature zygotic embryo through the cells,primordial suspension culture that ABA adjusts;
Fig. 9 is the growth and the maturation germination situation of body embryo, and a, b are the growth photo of body embryo, and c, d are the maturation germination photo;
Figure 10 induces situation for zygotic embryo different development stage embryo callus subculture, and abscissa is expressed as all numbers that begin to gather cone at the beginning of 6 months, and ordinate is represented the embryo callus subculture inductivity;
Figure 11 induces situation for different medium embryo callus subcultures, and the inductivity of prescription 3 is higher relatively, but the total difference of 3 kinds of prescriptions is not very big.
Five, embodiment
Hybridize loose immature embryo, pick up from the Guangdong seed orchard, repeated for two years.Begin to gather cone from the beginning of June, gather once weekly, up to cone embryonic development early in August maturation.The cone of gathering is wrapped up in case the cone dehydration is wrapped with plastic sack with wet gauze, be placed in 4 ℃ of refrigerators and preserve (at least one week, low temperature helps inducing of embryo callus subculture).
Immature seed is removed kind of shell and interior exosper, earlier with 75% alcohol disinfecting 30s, aseptic water washing 3-5 time; Use 1: 10 84 thimerosals sterilization 8min then, aseptic water washing 3-5 time.Directly will be inoculated in the medium with the seed of endosperm.
With aforementioned 3 kinds of different improvement P6 medium, additional 1-2mg/L 2,4-D and 0.5-2mg/L BA, glutamine (Glu) 1000mg/L, caseinhydrolysate (CH) 500mg/L, inositol 1000mg/L, maltose 30g/L, quartzy agar 1.86g/L.
A. the embryo of hybridization pine is inoculated in the medium, the back discovery of 6 weeks, and the embryo callus that is derived by rataria is translucent, humidity ratio is big, viscosity, and the surface is thread (referring to accompanying drawing 1).Observe pencil embryo head suspensor structure (being called for short ESM) culture (referring to accompanying drawing 2) under body formula mirror, this ESM culture under the suitable culture condition, has the ability of continuous self duplication and the ability of organizator cell stage simultaneously.The callus that immature embryo is induced has two states: a kind of is embryo callus subculture, mainly results from the suspensor place, more, of light color white or white, translucent, the short texture (referring to accompanying drawing 3) of moisture; Another kind is the non-embryonic callus tissue, yellow of light color or yellow, and short texture, the surface is crystalloid, washiness (referring to accompanying drawing 4).
Can the developmental stage of zygotic embryo very crucial to realizing that the inducing of callus, propagation and body embryo form take place.The immature zygotic embryos inductivity of different development stage is widely different.Our experiments show that only have the beginning of June just to induce good embryo callus (referring to accompanying drawing 10) to the immature zygotic embryos of the middle ten days and the last ten days in June.Through microscopic examination, the embryo callus subculture that as seen derives has possessed embryo head suspensor cell basically, and tissue differentiation is good, compact structure (referring to accompanying drawing 5).And with the non-embryonic callus that mature embryo induces, examine under a microscope, major part is a round cell, does not have tangible institutional framework (referring to accompanying drawing 6A).Though in the process of the adjustment state in later stage, also can form embryo callus subculture, tangible embryo head suspensor tissue is arranged, its conversion ratio is extremely low.By the culture that mature embryo is induced, examine under a microscope a large amount of suspensor cells, but do not have the differentiation (referring to accompanying drawing 6B) of embryo head.And its state labile, brownization is dead gradually in the process of propagation.
Different carbon sources is to the influence of Plant Tissue Breeding.With maltose or lactose is that carbon source is cultivated the Agrostisstolonifera callus, and its seedling greening-rate reaches 76～93%,, exceed 4～36 times (Asano etc., 1994) than sucrose.Find also in the carrot suspension culture that maltose is better than sucrose (Verma etc., 1977).Flax culture sprouts with maltose to well; Long root is good (Millam etc., 1992) with sucrose then.The present invention adopts the isomorphism disaccharides as carbon source, replaces isomery disaccharides---sucrose, and is respond well.
The influence that the degree of inducing of different medium is induced embryo callus subculture is referring to accompanying drawing 11.
B. multiplicative stage of embryo callus subculture.Use first kind of minimal medium of improvement, adopt the process route of solid and liquid alternated cultivation.Solid culture medium adds 1mg/L2,4-D+1mg/L 6-BA+1000mg/L inositol, 1mg/L 2,4-D+0.5mg/L 6-BA+0.5mg/L KT+1000mg/L inositol, liquid nutrient medium adds 1mg/L 2,4-D+0.5mg/L 6-BA+0.5mg/L KT+5000mg/L inositol.Per 14 days subcultures of solid culture once, per 7 days subcultures of liquid culture are once.
Solid culture medium is used for ESM propagation and embryo's generating ability keeps, and liquid nutrient medium is mainly used in the fast breeding in the large-scale production process except that keeping the embryo sexual state, and its growth rate is about 1.5 times of solid culture.In different hormone combinations, the vegetative state of embryo callus subculture is different.The embryo callus subculture of in the environment consistent, growing with inducing culture, its structure is tight, and moisture is few, easy brownization, but its interior keeps good, and the embryo header structure is tight, grows complete (referring to accompanying drawing 5A-C); 6-BA is become the combination of 6-BA and KT, and the water content of its callus increases, and the structure of its callus is same to keep good, and just the callus state is more a little bit poorer a little than the former.Be in the combination of 1000mg/L at inositol but, its callus propagation is very fast, and brownization is slowly (referring to accompanying drawing 3A, B); And the inositol consumption increases under the situation of 5000mg/L, and its water content increases 1.5-2 doubly, and from the callus profile, structure is not tight, and the thorn-like projection reduces (referring to accompanying drawing 7).Microscopically is observed, and can see the callus structure degradation that its embryo head suspensor cell has more just derived.The performance of total its three kinds of combinations thinks that 1mg/L 2, the combination of 4-D+0.5mg/L 6-BA+0.5mg/L KT+1000mg/L inositol are fit to the maintenance of embryo callus subculture and the mode of propagation.
C. end embryo schizogenesis and inductor embryonic development stage.In the liquid nutrient medium, remove the growth hormone and the basic element of cell division, add 5-10mg/L abscisic acid (ABA) and 7.5mg/L gibberellin (GA
3).
Embryo callus subculture is through behind the enrichment culture, form the extremely complicated embryo sexual state of a composition: various types of proembryo in cotyledonary embryos period before existing not the entering, the cleavage polyembryony that the formation in period of preceding cotyledonary embryos is also arranged, do not controlled if do not continue, even the cotyledonary embryos that can obtain being shaped also can't guarantee its quality.Therefore liquid medium within uses 5-10mg/L high concentration ABA, ends the cleavage polyembryony process in the embryonal suspension culture.This stage embryonal suspension system propagation stops the beginning embryo development procedure.By the effect of ABA, end cleavage polyembryony, thereby make that proembryo can continue to grow, form globular embryo, and can make the synchronization better effects if of spheroidal embryo.
Experimental result shows, liquid A BA suspension through 3-4 week is adjusted, the state of embryo and callus all forms globular embryo basically, its embryo capitulum structure is tightr, it is big that volume becomes, become slender of suspensor cell and having closely simultaneously is with the preceding cotyledon period in the zygotic embryo growth course similar (referring to accompanying drawing 8).
D. somatic embryo stage of ripeness.In the solid culture medium, add 5-10mg/L abscisic acid ABA and 7.5mg/L gibberellin GA
3, 170g/L polyethylene glycol PEG800, active carbon 1g/L adds the several amino acids composition simultaneously, and two months subcultures are once.When forwarding solid culture to, notice that its medium moisture is not too many, in order to avoid have influence on the drying and other treatment that promotes in the somatic embryo generating process by liquid culture.
Adding ABA in the differentiation culture of somatic embryo can promote the somatic embryo maturation to break up at somatic embryos of coniferous trees significantly.The degree of somatic embryo development depends on the concentration of ABA in the medium: do not have or during low concentration ABA somatic embryo stasi, brownization death in the medium; When ABA concentration was increased to 5-10mg/L, certain metamorphosis took place in somatic embryo.On form, be transparence originally and be distributed in callus in the medium, can obviously observe its cell inclusion increases, and presents buff, and the distinguishable tissue that structure is arranged that goes out one by one comes (referring to accompanying drawing 9A, B).After cultivating one month, the formation that can be observed the 3-5mm cotyledonary embryos is (referring to accompanying drawing 9C, D).
Add certain density active carbon in the medium, the differentiation and the growth of somatic embryo had certain facilitation.Gupta etc. studies show that, add active carbon in containing the differential medium of ABA, have to improve the effect that the cotyledonary embryos apical meristem is grown, and it is grown more near zygotic embryo.
Osmotic pressure has the significant effects effect to the growth of coniferous tree later stage somatic embryo.Be used for improving its action effect of low-molecular-weight bleeding agent the earliest not as the bigger polyethylene glycol (PEG) of molecular weight.Attree etc. (1987) think that this mainly is because the method for water stress is sugar and the salt that adds high concentration under the low-molecular-weight material energy isolated culture condition.Yet resembling these low-molecular-weight materials of sucrose, salt and mannitol can be absorbed by symplast through cytoplasma membrane, and the osmotic value of cell is restored, and does not reach the purpose of water stress.Therefore just selected instead thing of the polyethylene glycol (PEG) of HMW and glucan for use, grown to produce long water stress, effect is better.
Somatic embryo is cultivated ripe after the formation of 3-5mm cotyledonary embryos, can use conventional cultural method to enter plant regeneration and grow seedlings the stage.
The invention provides the complete set technology of a kind of industrialization fast breeding wet-land pine tree and pinus caribaea hybrid nursery stock, can solve the problem in short supply of nursery stock in current wet-land pine tree and the pinus caribaea hybrid afforestation.Also for setting up the received coniferae tissue culture form emergence pattern of energy system, for the in-vitro propagate of multiple important coniferae, genetic transformation and breed improvement research provide important practical basis, can make the more societies that bring benefit to the mankind quickly of Developing Clonal Forestry at the early-stage.
Claims (3)
1. wet-land pine tree and pinus caribaea hybrid somatic embryo take place and plant regeneration method, it is characterized in that adopting the immature zygotic embryos of wet-land pine tree and pinus caribaea hybrid to carry out somatic embryo generation and plant regeneration, somatic embryo generation and the main process of plant regeneration and condition are as follows:
A. the medium of somatic embryo induction period is improvement P6 medium, additional auximone 2,4-D 1-2mg/L, basic element of cell division 6-BA 1-2mg/L;
B. embryo head suspensor structure (ESM) the embryo culture fast breeding stage is adopted repeatedly liquid and the solid improvement P6 medium combinatorial regulation cultured method that alternates, additional plant hormone best of breed is 1-2mg/L 2,4-D, 0.5-1mg/L 6-BA and 0.5-lmg/L KT;
C. somatic embryo induction period, embryo head suspensor structure (ESM) embryo culture proliferation adjourning adopt liquid improvement P6 medium, additional 5～10mg/L abscisic acid (ABA) and 5～10mg/L gibberellin (GA
3);
D. the body embryonic development stage of ripeness of embryo head suspensor structure (ESM) embryo culture generation and somatic embryo are sprouted the stage employing solid improvement P6 medium of emerging, additional several amino acids, polyethylene glycol (PEG8000).
2. wet-land pine tree as claimed in claim 1 and pinus caribaea hybrid somatic embryo take place and plant regeneration method, it is characterized in that explant sampling point, time control and pretreatment condition are:
A. obtain immature embryo from kind of a tree cone, the optimal acquisition time is annual June;
B. the cone after gathering is placed in 4 ℃ of refrigerators and preserves more than 1 week after wrapping up with wet gauze, plastic sack.
3. wet-land pine tree as claimed in claim 1 and pinus caribaea hybrid somatic embryo take place and plant regeneration method, it is characterized in that the optimal medium prescription of somatic embryo induction period is as follows:
KNO
3 1250mg/L
MgSO
4.7H
2O 400mg/L
CaCl
2.2H
2O 200mg/L
KH
2PO
4 340mg/L
KI 1mg/L
H
3BO
3 5mg/L
ZnSO
4.7H
2O 8mg/L
MnSO
4.4H
2O 15.8mg/L
Na
2MoO
4.2H
2O 0.2mg/L
CuSO
4.SH
2O 0.024mg/L
CoCl
2.6H
2O 0.02mg/L
FeSO
4.7H
2O 27.85mg/L
Na2-EDTA 37.25mg/L
Inositol 100mg/L
Nicotinic acid 0.5mg/L
Thiamine hydrochloride 1.0mg/L
Puridoxine hydrochloride 0.5mg/L
Glycine 2.0mg/L
It is as follows to add amino acid:
L-proline 100mg/L
L-asparagine 100mg/L
L-arginine?HCl 50mg/L
L-alanine 20mg/L
L-serine 20mg/L
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101327078A CN101218894A (en) | 2007-09-26 | 2007-09-26 | Embryogenesis and plant strain regeneration method for hybrid somatic cell of slash pine and cuban pine |
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| CN102369879A (en) * | 2010-08-10 | 2012-03-14 | 中国科学院植物研究所 | Succeeding preservation method for embryogenic callus of China fir and subculture medium used therein |
| CN102960249A (en) * | 2012-11-30 | 2013-03-13 | 通化师范学院 | In-vitro efficient seedling cultivation method synchronous in rooting and growing by utilizing tender stem segments of thuja koraiensis |
| CN104351051A (en) * | 2014-10-28 | 2015-02-18 | 毕节市中药研究所 | Intermediate propagation aseptic seedling of tender Taiwan calocedrus formosana leaves |
| CN104381131A (en) * | 2014-10-24 | 2015-03-04 | 北京林业大学 | Somatic embryogenesis and plant regeneration method for pinus tabulaeformis |
| CN107593454A (en) * | 2017-11-03 | 2018-01-19 | 南京林业大学 | A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method |
| CN107873512A (en) * | 2017-10-24 | 2018-04-06 | 南京林业大学 | Anti- pine nematode black pine somatic embryo regeneration plant high efficiency breeding technology |
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| CN111543325A (en) * | 2020-06-05 | 2020-08-18 | 江西农业大学 | Induction culture method and induction culture medium for slash pine embryonic callus |
| CN112352678A (en) * | 2020-11-13 | 2021-02-12 | 江西环境工程职业学院 | Tissue culture rapid propagation technology for slash pine seedlings |
| WO2021103165A1 (en) * | 2019-11-28 | 2021-06-03 | 南京林业大学 | Method for promoting somatic embryogenesis of cunninghamia lanceolata by using amino-oligosaccharin |
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| CN114788496A (en) * | 2022-04-07 | 2022-07-26 | 江苏省中国科学院植物研究所 | Method for inducing efficient somatic embryogenesis of larch through solid-liquid alternate culture |
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| CN102369879A (en) * | 2010-08-10 | 2012-03-14 | 中国科学院植物研究所 | Succeeding preservation method for embryogenic callus of China fir and subculture medium used therein |
| CN102960249A (en) * | 2012-11-30 | 2013-03-13 | 通化师范学院 | In-vitro efficient seedling cultivation method synchronous in rooting and growing by utilizing tender stem segments of thuja koraiensis |
| CN104381131A (en) * | 2014-10-24 | 2015-03-04 | 北京林业大学 | Somatic embryogenesis and plant regeneration method for pinus tabulaeformis |
| CN104351051A (en) * | 2014-10-28 | 2015-02-18 | 毕节市中药研究所 | Intermediate propagation aseptic seedling of tender Taiwan calocedrus formosana leaves |
| CN104351051B (en) * | 2014-10-28 | 2016-03-23 | 毕节市中药研究所 | A kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling |
| CN107873512A (en) * | 2017-10-24 | 2018-04-06 | 南京林业大学 | Anti- pine nematode black pine somatic embryo regeneration plant high efficiency breeding technology |
| CN107593454A (en) * | 2017-11-03 | 2018-01-19 | 南京林业大学 | A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method |
| CN110004176A (en) * | 2019-04-12 | 2019-07-12 | 东北林业大学 | The construction method of hybrid larch genetic conversion system |
| CN110004176B (en) * | 2019-04-12 | 2023-03-10 | 东北林业大学 | Construction method of hybrid larch genetic transformation system |
| WO2021103166A1 (en) * | 2019-11-28 | 2021-06-03 | 南京林业大学 | Method for promoting somatic embryogenesis of cunninghamia lanceolata by using spermidine |
| WO2021103165A1 (en) * | 2019-11-28 | 2021-06-03 | 南京林业大学 | Method for promoting somatic embryogenesis of cunninghamia lanceolata by using amino-oligosaccharin |
| CN113115706A (en) * | 2020-01-15 | 2021-07-16 | 东北林业大学 | Method for restoring and maintaining embryogenic property of embryogenic callus of larch |
| CN113115706B (en) * | 2020-01-15 | 2022-03-15 | 东北林业大学 | Method for restoring and maintaining embryogenic property of embryogenic callus of larch |
| CN111543325A (en) * | 2020-06-05 | 2020-08-18 | 江西农业大学 | Induction culture method and induction culture medium for slash pine embryonic callus |
| CN112352678A (en) * | 2020-11-13 | 2021-02-12 | 江西环境工程职业学院 | Tissue culture rapid propagation technology for slash pine seedlings |
| CN114788496A (en) * | 2022-04-07 | 2022-07-26 | 江苏省中国科学院植物研究所 | Method for inducing efficient somatic embryogenesis of larch through solid-liquid alternate culture |
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