CN107593454A - A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method - Google Patents
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method Download PDFInfo
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Abstract
The invention discloses a kind of anti-pine nematode masson pine somatic embryo generation and plant regeneration method, using anti-pine nematode masson pine immature zygotic embryos as material, through embryonic callus induction, the propagation of embryo callus and maintenance, differentiation and maturation and sprouting, and rooting induction, regeneration plant progress strong seedling culture and rooting culture are carried out to somatic embryo, finally obtain anti-pine nematode masson pine plant.The anti-pine nematode masson pine somatic embryo of the present invention occurs and plant regeneration method, using resistance masson pine immature zygotic embryos as material, regenerating system occurs for the preliminary resistance masson pine somatic embryo that establishes, antagonism masson pine embryonic callus induction optimizes, it further study the important steps such as somatic embryo propagation, differentiation, sprouting, Transplantation of Regenerated Plantlets domestication, disease-resistant masson pine suspension culture and the somatic cell embryogenesis regeneration plant of small lot are cultivated, good technical foundation has been established for the factorial praluction of resistance masson pine.
Description
Technical field
The invention belongs to plant tissue culture technical field, and in particular to a kind of anti-pine nematode masson pine somatic embryo fetal hair
Raw and plant regeneration method.
Background technology
Masson pine is that distribution area is most wide in China's coniferous species, widely used, producing level highest production fat afforestation township
Native seeds (Chen Bihua etc., 2010).Pine nematode is caused by pine wood nematode (Bursaphelenchus xylophilus)
A kind of destructive Pine diseases, have the characteristics that easy propagations, rapid onset, prevent and treat it is difficult.Pine occurs for masson pine forest in recent years
Nematodiasis causes serious harm in some areas.Therefore, seed selection is carried out to the excellent pine tree kind with anti-pine nematode,
Pine tree resistance quality is improved, is to preserve pine tree resource, fundamentally resists one of effective way of pine nematode (Wu little Qin
Deng 2009).- 2008 years 2001, China-Japan cooperation carried out " exploitation of masson pine pine wood nematode resistance breeding technology " project, and
In 2010-2015 implement to have carried out anti-pine nematode Pinus massoniana and resistance breeding technical research project (Xu six is first-class,
2013) high anti-masson pine family 68, high anti-clone 89, are filtered out.Due to the low still difficult satisfaction of resistance masson pine setting percentage
The demand of resistance breeding, and apply the method for tissue cultures to keep maternal merit, there is high genetic stability, profit
Bred in choiceness, can fast-propagation improved seeds, make seed improvement speed improve (iron of fine quality etc., 2010；Han Bing etc.,
2007；Oh et al., 2010).Somatic embryo occurs for organ occurs in tissue culture technology, has more excellent
Point is if produce the new individual more than structural integrity and quantity, while its reproductive speed is fast, regeneration rate is high, the development ways can shape
Into the regeneration plant that stability is strong, character is uniform, yield is high, most possibly realize that pine tree etc. has more long-living macrocyclic forest
Choiceness large-scale breeding (Gupta et al., 1993；Season, hole was multitudinous etc., and 2015).
At present, existing correlative study occurs for masson pine somatic embryo, but remain frequency of embryonic callus induction it is low, point
Change difficulty, germination rate is low, is difficult to the problems such as acquisition regeneration plant.
The content of the invention
Goal of the invention：For above-mentioned problems of the prior art, it is an object of the invention to provide a kind of anti-pine line
Parasitosis masson pine somatic embryo occurs and plant regeneration method, to establish out resistance masson pine suspension culture system and resist
Property masson pine somatic embryo regenerating system occurs, provide a feasible way for the quick large-scale breeding of disease-resistant masson pine, be
The factorial praluction of resistance masson pine lays the foundation.
Technical scheme：In order to realize foregoing invention purpose, the technical solution adopted by the present invention is：
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, comprises the following steps：
1) the immature cone of resistance masson pine is gathered between the 6-8 months, the collection same day is placed on 4 DEG C of refrigerators and saved backup；
Masson pine seed is sterilized, strips and obtains sterile egagametophyte as explant progress callus induction.
2) using LP, DCR as minimal medium, culture medium additional maltose 30g/L, inositol 1g/L, glutamine
450mg/L, acid hydrolyzed casein 500mg/L, MES 250mg/L, agar powder 6.24g/L；The explant of step 1) is cured
Injured tissue induces, Fiber differentiation 1~2 month；
3) embryo callus of induction is transferred in proliferated culture medium, liquid suspension culture and solid training is respectively adopted
Support two kinds of Reproduction methods and carry out squamous subculture；Fluid nutrient medium is using LP as minimal medium, addition 2,4-D 0.6mg/L, 6-BA
0.2mg/L, add maltose 30g/L, inositol 1g/L, glutamine 1g/L；Solid medium is using LP as minimal medium, addition
2,4-D 1mg/L, 6-BA 0.5mg/L, add maltose 15g/L, inositol 1g/L, glutamine 450mg/L, sour water solution junket egg
White 500mg/L, agar powder 6.24g/L；
4) embryo callus after Liquid Culture is transferred into using LP as minimal medium, adds 2mg/L ABA,
0.5mg/L GA3, 130g/L PEG8000,25g/L maltose, 1g/L activated carbons, 3.5g/L gels, carry out maturation culture；Again
The preferable somatic embryo of quality is transferred in germination medium, carries out sprouting culture；Germination medium is using 1/2LP as basic training
Base is supported, adds 20g/L maltose, 2g/L activated carbons, 8g/L agar；
5) the body embryo seedling after sprouting is transferred in the blake bottle equipped with strengthening seedling and rooting culture medium, promotes the root leaf of body embryo seedling
Further differentiation；Strengthening seedling and rooting culture medium addition 1mg/L IBA, 0.2mg/L NAA, adds using 1/4WPM as minimal medium
Sucrose 15g/L, inositol 0.1g/L, activated carbon 1g/L, carragheen 6.5g/L；
6) choose healthy and strong body embryo seedling to be tamed, the hardening by the way of half corkage+standard-sized sheet bottle；By the regeneration after domestication
Plant, transplant in sylvan life soil and soddy soil 2:In the matrix of 1 mixing.
In step 2), the formula of callus induction is：LP+2mg/L 2,4-D+1mg/L 6-BA.
In step 2), the formula of callus induction is：DCR+5mg/L 2,4-D+1mg/L 6-BA.
In step 3), bred using liquid suspension culture, maturation is carried out using solid culture in step 4).
In step 5), strong seedling culture base：1/4WPM+0.01mg/L brassinosteroid.
In step 6), height of seedling during transplanting is 5-7cm.
Described anti-pine nematode masson pine is resistance masson pine family horse 7-6 and and 15-1.
Beneficial effect：Compared with prior art, anti-pine nematode masson pine somatic embryo of the invention occurs and planted
Strain renovation process, using resistance masson pine immature zygotic embryos as material, tentatively establish the generation of resistance masson pine somatic embryo
Regenerating system, antagonism masson pine embryonic callus induction optimize, further study somatic embryo propagation, differentiation,
The important steps such as sprouting, Transplantation of Regenerated Plantlets domestication, disease-resistant masson pine suspension culture and the somatic embryo of small lot are cultivated
Embryogenesis regeneration plant, good technical foundation is established for the factorial praluction of resistance masson pine.
Through experiment, the results showed that：Resistance masson pine frequency of embryonic callus induction differs greatly under different hormone combinations, most
Up to 27.8%, minimum 0%.115 embryogenic cell lines are being induced, 22 are obtained altogether by the maintenance and propagation of half a year
Stable embryogenic cell line, callus survival rate are 19.1%.In embryo callus breeds maintenance process, in the culture that suspends
Growth coefficient and synchronize degree be all significantly higher than solid culture.And the cell line differentiation capability by the culture that suspends is stronger,
It is average that 350 body embryos can be differentiated per ware (per ware in addition 2mg/L ABA+130 g/LPEG8000 differential medium
Most multipotency breaks up 600 embryos), average germination rate is up to 89.6%.Root system can be smoothly only differentiated on a small quantity in germination process, not
The body embryo of root can smoothly be differentiated can advantageously promote it in addition 1mg/L IBA+0.2mg/L NAA root media
Take root.During strong seedling culture, it is weaker not add or add excessive brassinosteroid body embryo seedling growing way, and adds 0.01mg/L oil
Dish element lactones contributes to the growth of body embryo seedling.Body embryo seedling different size of transplantation of seedlings survival rate difference in rooting culture is larger,
5cm or so body embryo shoot survival percent is up to 90.3%.
It can be seen that：The inductivity of different hormone combinations induction of resistance masson pine embryo callus is different, using LP as basic training
Base is supported, adds 2mg/L 2,4-D and 1mg/L6-BA inductivities are up to 27.8%；The cell line after cultivating that suspends is more conducive to lure
Conductor embryo；The IBA and NAA of suitable concentration can promote body embryo seedling rooting；Regeneration plant is adding the strong of appropriate brassinosteroid
In seedling culture medium, can more preferably it grow；Treat seedling length to 5cm or so transplanting.
Brief description of the drawings
Fig. 1 is that somatic embryo breaks up situation result figure under different training methods；In figure, A：Solid multiplication；B-C：Solid increases
Grow differentiation；D：Liquid suspension is bred；E-F：Liquid Proliferation, Differentiation；
Fig. 2 is resistance masson pine somatic embryo forming process result figure；In figure, A：Callus embryo suspensor structure； B：Suspensor
Shrink；C：Suspensor continues to shrink, degenerated；D：Complete cotyledonary embryos, E：Maturation differentiation；F：Sprout；G：Complete body embryo seedling；H：Plumular axis,
Cotyledon development；
Fig. 3 is the influence result figure of brassinosteroid antagonism masson pine body embryo robust seedling seedling；In figure, A：It is not added with rape element
Lactones；B：0.01mg/L brassinosteroids；C：0.02mg/L brassinosteroids.
Embodiment
With reference to specific embodiment, the present invention is described further.
Embodiment 1
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, with resistance masson pine family horse
Exemplified by 7-6, process is as follows：
1) source of explant material and processing
Preserving garden in Anhui masson pine resistance candidate individual between the 2014-2015 6-8 months takes fixed point singling to gather in batches
The immature cone of resistance masson pine, determines 4 plants of select trees, 6 cones of every plant of collection, and each week collection once, gathers the same day
Be placed on 4 DEG C of refrigerators preserve 7 days it is standby.[Li Qingqing, Ye Jianren, Zhu Lihua .2012. black pine prematurities are waited with reference to Li Qingqing etc.
The somatic embryo of embryo occurs and plant regeneration forest-sciences, the ﹚ of 48 ﹙ 12:39-44] operating method, masson pine seed is disappeared
Poison, strip and obtain sterile egagametophyte (i.e. resistance masson pine immature zygotic embryos) as explant progress callus induction.
2) resistance masson pine embryonic callus induction
It is basic using LP, DCR using the immature seed in Anhui masson pine resistance candidate individual preservation garden as test material
Culture medium, culture medium additional maltose 30g/L, inositol 1g/L, glutamine 450mg/L, acid hydrolyzed casein 500mg/L,
MES 250mg/L, agar powder 6.24g/L.Fiber differentiation observes statistics callus induction rate after 1~2 month.Embryo callus
Total explant number × 100% of explant number/inoculation of inductivity=induce embryo callus.
As a result as shown in table 1, show：LP+2mg/L 2,4-D+1mg/L 6-BA are optimum proportioning, and inductivity is up to
27.8%, the callus status induced is preferable, is capable of the vegetative state of stable for extended periods of time, and occur in later stage body embryo
In can induce the preferable body embryo seedling of form.DCR+5mg/L 2, though 4-D+1mg/L 6-BA inductivities are relatively low, successfully lure
Derived embryo callus quality is preferable, its callus can long-term subculture propagation, embryo is kept, and in later stage body embryo
The complete body embryo seedling of a large amount of forms can be induced in generation.Find simultaneously, AgNO is added in inducing culture3It can promote
The induction of masson pine embryo callus, but the callus status induced is poor, and its callus induced is being bred
Easily browning in culture.
The influence of 1 different minimal mediums of table and hormone concentration antagonism masson pine callus induction
3) maintenance of embryo callus and propagation
Using the callus of induction as test material, two kinds of propagation sides of liquid suspension culture and solid culture are respectively adopted
Formula.Solid culture subculture cycle 15 days or so, liquid suspension culture subculture cycle 7 days or so.It is basic using LP during solid culture
Culture medium, addition 2,4-D 1mg/L, 6-BA 0.5mg/L, agar powder 6.24g/L, additional maltose 15g/L, inositol 1g/L, paddy
Glutamine 450mg/L, acid hydrolyzed casein 500mg/L, callus is directly gripped on solid medium, weighs initial inoculation
Quality with culture 15d days after callus quality.During liquid suspension culture, it is initial inoculum to take 1g callus, is put into new
Broken up in fresh fluid nutrient medium, shaking speed 100r.p.m, light culture is after 7 days, measure sedimentation cell volume (sediment
Cell volume, SCV) (Shi Kun etc., 2014).SCV assay method：The cell for the culture that suspended in conical flask is poured into scale
In test tube, 30min is stood at room temperature, after cell completely precipitation, reads the cell volume after precipitation.Fluid nutrient medium using LP as
Minimal medium, addition 2,4-D 0.6mg/L, 6-BA 0.2mg/L, additional maltose 30g/L, inositol 1g/L, glutamine
1g/L。
The embryo callus of induction is transferred in proliferated culture medium, carries out squamous subculture.Initial stage callus growth
Slowly, and only part cell line can enter stable vegetative state.Start coinduction and go out 115 embryogenic cell lines, pass through
The maintenance of half a year obtains 22 stable embryogenic cell lines with propagation altogether, and callus survival rate is 19.1%.
As a result it is as shown in table 2, it is found that the growth coefficient and synchronization degree for the culture that suspends all are significantly higher than solid culture.
Solid culture is employed during the Multiplying culture of callus for the present embodiment and the culture two ways that suspends.Solid culture week
Phase, for 15d subcultures once 15d growth coefficient was 2.845；And the general 7-9d of culture that suspends needs to change fresh medium, this
When growth coefficient reach 3.879, hence it is evident that higher than solid culture.Two kinds of training method contrasts, solid culture cycle length, are being maintained
It is time saving and energy saving on callus；But when callus needs amount reproduction, carries out factorial praluction, suspension culture is more suitable for.Together
When suspend culture single cells,primordial or cell mass synchronize degree it is also higher.
The influence that 2 different training methods of table are bred to embryo callus
| Training method | Cultivated days | Growth coefficient | Synchronization degree |
| Solid culture | 15 | 2.845±0.336 | It is low |
| Suspend culture | 7 | 3.879±0.105 | It is high |
4) the ripe differentiation and sprouting of resistance masson pine somatic embryo
Using the suspension cell line of foundation and the callus of solid culture as experiment material, transfer into using LP as basic culture
Base, addition 2mg/L ABA, 0.5mg/L GA3, 130g/L PEG8000,25g/L maltose, 1g/L activated carbons, 3.5g/L coagulates
Glue.Maturation culture observes the mature condition of somatic embryo after 60 days.
As a result find, after resistance masson pine callus is with different training method cultures, its differentiation capability has marked difference.
Embryo callus is using " solid culture-solid differentiation " mode culture (Fig. 1 A, B), the callus on differential medium
Slowly growth, after 2 months, there are a large amount of early stage proembryos (Fig. 1 B, C) occur on callus, body embryo can be formed on a small quantity, but
Body embryo deformity is serious.Embryo callus is cultivated using " Liquid Culture-solid differentiation " mode culture (Fig. 1 D, E) in differentiation
Callus growth is very fast on base, and after 2 months, callus can form a large amount of body embryos (Fig. 1 E, F), and body embryo state is good
It is good.
Cell after suspending and cultivating is inoculated into differential medium, suspension cell is eventually differentiated into body in 2 months
Blast.Cell mass during inoculation is made up of irregular embryo head and multiple elongated, translucent suspensors, and cell loose arrangement be (Fig. 2's
A).After differential medium is put into, 1 month or so, embryo head gradually protruded, and its multiple suspensor connected starts aggregation and collapses (Fig. 2
B).Suspensor continues to shrink afterwards, and embryo head gradually forms round and smooth " sub warhead " shape (Fig. 2 C).Also there is a small amount of embryo head non-round and smooth
Shape, but convex tip shaped, such blast can not form normal cotyledon in being developed in the later stage, can be only formed Embryos.In 2 wheat harvesting periods
Afterwards, cotyledon is formed gradually, and the cotyledon number of formation from 1 to 10, also further degenerate or disappear by number, suspensor, body embryo
Reach ripe (Fig. 2 D).Well-grown suspension cell is in differential medium, the inoculation 4mL cell suspending liquids per ware, 2
Highest can produce 600 body embryos (Fig. 2 E) per ware after month.
The preferable somatic embryo of quality is transferred in germination medium, carries out sprouting culture.Using 1/2LP as basic culture
Base, add 20g/L maltose, 2g/L activated carbons, 8g/L agar.After one month, the sprouting situation of body embryo is observed.
Choose well-developed body embryo to transfer in germination medium, by the sprouting of one month, somatic embryo plumular axis, son
Leaf grows simultaneously, and partly can smoothly take root (Fig. 2 G), and most root system developments are difficult (Fig. 2 F).Germination rate can reach
To 89.6%.
5) IBA combines the influence that antagonism masson pine body embryo is taken root with NAA
To induce the body embryo that differentiates as material, 1/4WPM is minimal medium, additional saccharose 15g/L, inositol 0.1g/L,
Activated carbon 1g/L, carragheen 6.5g/L, add various concentrations 0.5,1,1.5,2mg/L IBA, and 0.1,0.2mg/L
NAA, two factor randomized block experiments, after cultivating 50d, observe body embryo seedling growth conditions and situation of taking root.
Body embryo seedling after sprouting, which is transferred to, promotes the root leaf of body embryo seedling to enter in the blake bottle equipped with strengthening seedling and rooting culture medium
One step is broken up, and part body embryo seedling gradually sends out roots in blake bottle, turns into complete plant.
As a result (table 3) is shown, the increase of NAA concentration has been obviously promoted masson pine body embryo seedling rooting, body embryo seedling growth conditions
Also relatively preferably, thus 0.2mg/L NAA masson pine body embryo seedlings preferably take root.And when NAA is 0.2mg/L, with
The rise of IBA concentration, rooting rate are gradually reducing, while the growth conditions of body embryo seedling also difference.1mg/L IBA+
During 0.2mg/L NAA, body embryo seedling is higher, and plumular axis is relatively strong, and needle is vigorous.When IBA is 0.5mg/L and 2mg/L, body embryo seedling relatively thin
It is weak；And IBA, when being 1.5mg/L, body embryo seedling is thin and delicate, and needle is also relatively fewer.Therefore, 1mg/L IBA+0.2mg/L NAA are
Best of breed.
Table 3IBA and NAA antagonism masson pine body embryo rooting rates influence
Note：" +++ " body embryo seedling growing way is fine, and seedling is healthy and strong, and needle is vigorous in blackish green, and root system is good；" ++ " body embryo seedling is grown
Gesture is preferable, and seedling is more healthy and stronger, and vigorous needle is in peak green, and root system is good；"+" body embryo seedling growing way is poor, and seedling is thin and delicate, and needle is less,
Root system is poor；"-" body embryo seedling growing way is very poor, and seedling is thin and delicate, and needle does not almost grow, and root system is poor.
6) influence of brassinosteroid antagonism masson pine body embryo robust seedling seedling
The body embryo seedling differentiated using masson pine resistance family horse 7-6 inductions adds rape as material in strong seedling culture base
Plain lactones, set the concentration of brassinosteroid for 0,0.01,0.02mg/L, using 1/4WPM as minimal medium, additional saccharose
15g/L, inositol 0.1g/L, activated carbon 1g/L, carragheen 6.5g/L.24 DEG C of cultivation temperature, daily 16h illumination.Carry out strong sprout training
Support, observation statistics body embryo seedling growing state after 40d.
Complete body embryo seedling is transferred in using vermiculite and perlite as in the strong seedling culture base of matrix, body embryo seedling is in the culture
Further growth in base, loose due to perlite, the root system of body embryo seedling can preferably grow.
As a result show, appropriate brassinosteroid can promote the growth of body embryo seedling.At the place for being not added with brassinosteroid
In reason, although body embryo seedling is in peak green in further growth, needle, seedling is weaker, and root growth is more slowly (Fig. 3 A).When
When adding 0.02mg/L brassinosteroids, the growth of body embryo seedling is vigorous, and seedling is healthy and strong, and root growth is more slowly (Fig. 3 C).It is and oily
When dish element lactones is 0.01mg/L, not only needle is vigorous presents healthy and strong blackish green for body embryo seedling, and root growth is preferable, root life
Long very fast and lateral root is flourishing (Fig. 3 B).Therefore, 0.01mg/L brassinosteroids are added in strong seedling culture base, contribute to body
The growth of embryo seedling.
7) domestication and transplanting of resistance masson pine body embryo seedling
Healthy and strong body embryo seedling is chosen to be tamed.The hardening by the way of half corkage+standard-sized sheet bottle, that is, unclamp the bottle of tissue culture bottle
Lid, makes seedling in bottle progressively adapt to external environment condition, opens the lid of tissue culture bottle, finishing scouring seedling 4 days after 3 days completely.While hardening
Ensure indoor humidity, spray appropriate water to body embryo seedling if necessary.
The regeneration plant after domestication is transplanted in sylvan life soil and soddy soil 2:In the matrix of 1 mixing.Transplant in the latter moon
The humidity and temperature of nursery are kept, carries out and fosters work, survival rate is counted after 3 months.The strain that survival rate of plant=plant survives
The strain number * 100% of number/transplanting.
The Transplantation of Regenerated Plantlets after domestication to outdoor is transplanted in sylvan life soil and soddy soil 2:In the matrix of 1 mixing.Experiment
Different size of regeneration plant is have chosen, is found (table 4)：When body embryo seedling is too small, transplanting survival rate is very low, and only 34.7%；When
When height of seedling reaches 5 cm, survival rate highest, reach 90.3%；When height of seedling is more than 8 centimetres, transplanting survival rate is also higher,
Reach 82.9%, but because seedling is too high, body embryo seedling is thin and delicate, degree of lignification is low, lodging phenomenon easily occurs.
Influence of the horse 7-6 family body embryo seedling sizes of table 4 to transplanting survival rate
Embodiment 2
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, with resistance masson pine family and
Exemplified by 15-1, process is as follows：
1) processing of explant and the induction of embryo callus
Method is with example 1, and it is in best medium LP+2mg/L 2,4-D+1mg/L 6-BA, embryonic callus induction
Rate is up to 13.1%.
2) maintenance of embryo callus and propagation
Method can be stablized and breed with example 1, embryo callus.Carried out using solid culture and the culture two ways that suspends
Multiplying culture.
3) the ripe differentiation and sprouting of resistance masson pine somatic embryo
Method, using LP as minimal medium, adds 2mg/L ABA, 0.5mg/L GA with example 13, 130g/L
PEG8000,25g/L maltose, 1g/L activated carbons, in the maturation medium of 3.5g/L gels, a large amount of body cells can be differentiated
Embryo.
Using 1/2LP as minimal medium, 20g/L maltose, 2g/L activated carbons, the germination medium of 8g/L agar are added
In, germination rate 82.65%.
4) resistance masson pine body embryo seedling rooting strong sprout and transplanting
Method is the same as example 1, in addition 1mg/L IBA, 0.2mg/L NAA root media, rooting rate 70%.By
After strong seedling culture, transplant in farmland soil and soddy soil 2:In the matrix of 1 mixing, survival rate 80% after 3 months.
Embodiment 3
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, wide with resistance masson pine family
Exemplified by 42-1, process is as follows：
1) processing of explant and the induction of embryo callus and propagation
Method is with example 1, and it is in best medium LP+2mg/L 2,4-D+1mg/L 6-BA, embryonic callus induction
Rate is up to 6.8%.
2) maintenance of embryo callus and propagation
Method can be stablized and breed with example 1, embryo callus.Carried out using solid culture and the culture two ways that suspends
Multiplying culture.
3) the ripe differentiation and sprouting of resistance masson pine somatic embryo
Method, using LP as minimal medium, adds 2mg/L ABA, 0.5mg/L GA with embodiment 13, 130g/L
PEG8000,25g/L maltose, 1g/L activated carbons, in the maturation medium of 3.5g/L gels, somatic embryo can be differentiated.But
Quality of somatic embryo is poor, only successfully sprouts, is finally formed without regeneration plant on a small quantity.
Embodiment 4
A kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, with resistance masson pine family Chu
Exemplified by 7-1, process is as follows：
1) processing of explant and the induction of embryo callus
Method is with example 1, and it is in best medium LP+2mg/L 2,4-D+1mg/L 6-BA, embryonic callus induction
Rate is up to 2.2%.
2) maintenance of embryo callus and propagation
In breeding, the callus growth state of the family is poor, increasess slowly.And it is difficult to differentiate body cell
Embryo.
Claims (7)
1. a kind of anti-pine nematode masson pine somatic embryo occurs and plant regeneration method, it is characterised in that including following
Step：
1）The immature cone of resistance masson pine is gathered between the 6-8 months, the collection same day is placed on 4 DEG C of refrigerators and saved backup；To horse
Tail pine seed disinfection, strip and obtain sterile egagametophyte as explant progress callus induction；
2）Using LP, DCR as minimal medium, culture medium additional maltose 30g/L, inositol 1g/L, glutamine 450mg/L,
Acid hydrolyzed casein 500mg/L, MES 250mg/L, agar powder 6.24g/L；To step 1）Explant carry out callus lure
Lead, Fiber differentiation 1~2 month；
3）The embryo callus of induction is transferred in proliferated culture medium, liquid suspension culture and solid culture two is respectively adopted
Kind Reproduction methods carry out squamous subculture；Using LP as minimal medium, addition 2,4-D 1mg/L, 6-BA 0.5mg/L, agar powder
6.24g/L；
4）Embryo callus after propagation is transferred into using LP as minimal medium, adds 2 mg/L ABA, 1 mg/L GA3,
130g/L PEG8000,25g/L maltose, 1g/L activated carbons, 3.5g/L gels, carry out maturation culture；It is again that quality is preferable
Somatic embryo is transferred in germination medium, carries out sprouting culture；Germination medium adds using 1/2 LP as minimal medium
20g/L maltose, 2g/L activated carbons, 8g/L agar；
5）Body embryo seedling after sprouting, which is transferred to, promotes the root leaf of body embryo seedling to enter one in the blake bottle equipped with strengthening seedling and rooting culture medium
Step differentiation；Strengthening seedling and rooting culture medium prescription is 1 mg/L IBA+0.2 mg/L NAA；
6）Choose healthy and strong body embryo seedling to be tamed, the hardening by the way of half corkage+standard-sized sheet bottle；Regeneration after domestication is planted
Strain, transplant in sylvan life soil and soddy soil 2:In the matrix of 1 mixing.
2. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is, step 2）In, the formula of callus induction is：LP+2mg/L 2,4-D+1mg/L 6-BA.
3. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is, step 2）In, the formula of callus induction is：DCR+5mg/L 2,4-D+1mg/L 6-BA.
4. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is, step 3）In, bred using liquid suspension culture, step 4）It is middle that maturation is carried out using solid culture.
5. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is, step 5）In, strong seedling culture base：1/4WPM+0.01 mg/L brassinosteroids.
6. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is, step 6）In, height of seedling during transplanting is 5-7cm.
7. anti-pine nematode masson pine somatic embryo according to claim 1 occurs and plant regeneration method, it is special
Sign is that described anti-pine nematode masson pine is resistance masson pine family horse 7-6 and and 15-1.
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