CN103053420B - Method for in vitro regeneration of Feizixiao litchi variety - Google Patents
Method for in vitro regeneration of Feizixiao litchi variety Download PDFInfo
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Abstract
The invention discloses a method for in vitro regeneration of a Feizixiao litchi variety. The method comprises the following steps: step 1, taking the anther of the Feizixiao litchi variety as an explant for callus induction and culture, and screening to obtain embryogenic callus; the culture medium is added with 2, 4-D with final concentration of 3 mg/l, BA with final concentration of 0.5 mg/l, NAA with final concentration of 0.5 mg/l and sugar with final concentration of 30 g/L on the basis of a culture medium MS; step 2, carrying out somatic embryo induction and culture to the embryogenic callus to obtain a somatic embryo; the culture medium is added with NAA with final concentration of 0.1 mg/l, LKT with final concentration of 5 mg/l, inositol with final concentration of 100 mg/l and sucrose with final concentration of 50 g/L on the basis of the culture medium MS; step 3, carrying out maturation to the somatic embryo to obtain a mature embryo, culture mediums are MS, 50 ml/L coconut milk and 60 g/L sucrose; and step 4, carrying out regeneration culture to the mature embryo, the culture medium is added with sucrose with final concentration of 30 g/L and agar with final concentration of 7 g/L on the culture medium MS. The invention establishes a method for in vitro plant regeneration of a Feizixiao litchi variety, achieves the high induction rate, improves the production efficiency, reduces the cost and lays a good foundation for litchi variety improvement and biotechnology breeding.
Description
Technical field
The method that the present invention relates to ' cv. Feizixiao ' Litchi Varieties Regeneration in Vitro, concrete, the present invention relates to ' cv. Feizixiao ' Litchi Varieties from callus induction through body embryogenesis path, develop into the cultural method of complete Regeneration in Vitro plant.
Background technology
Lichee (Litchi chinensis Sonn.) is the main tropical south subtropics fruit tree of south China, in tropical agriculture is produced, occupies critical role, is the important component part in farmers' income source.Along with the raising day by day of people's living standard, also more and more higher to the pursuit of commodity value, the demand of accelerating lichee breeding technique and rearing new variety is extremely urgent.The virgin phase of lichee is long, genetic composition height heterozygosis, and traditional breeding technique improves lichee proterties and cultivation new variety are time-consuming, effort, difficult large, can not meet the needs of lichee industry development.And molecular cloning and transgenic technology are day by day ripe, adopt plant transgenic technology breeding to have the features such as orderly improvement, expansion breeding scope and shortening the breeding cycle.
Since the report lichee tissue culture such as nineteen eighty-three Fu Lianfang obtain whole plant, investigator has carried out Study on tissue culture successively, still, makes progress slower.The bottleneck of lichee biotechnology breeding is to fail to set up efficient genetic conversion system at present, is prerequisite and the basis that efficient genetic trasformation system is set up and set up lichee vitro Regeneration System.
Summary of the invention
The object of this invention is to provide a kind of method of Feizixiao Litchi kind Regeneration in Vitro.The method, by optimizing the component of callus inducing medium, selection and the concentration of the nutrient media components of somatic embryo inducement, particularly hormone, has been optimized culture condition simultaneously, has obtained very high callus induction rate and the induction efficiency of somatic embryo.
The method of lichee cv. Feizixiao kind Regeneration in Vitro provided by the present invention, comprises the steps:
1) taking lichee cv. Feizixiao kind flower pesticide as explant, explant is inoculated in to inducing culture and carries out induction of callus, screening obtains embryo callus, described callus inducing medium is that on the basis of MS substratum, to add final concentration be 2 of 3mg/L, on the 6-BA that 4-D, final concentration are 0.5mg/L, the NAA that final concentration is 0.5mg/L, the solid medium of sucrose that final concentration is 30g/L or the basis of MS substratum, adding final concentration is 2 of 1mg/L, the solid medium of the 6-BA that 4-D, final concentration are 1mg/L, the NAA that final concentration is 1mg/L, sucrose that final concentration is 30g/L, described callus inducing medium is preferably that on the basis of MS substratum, to add final concentration be 2 of 3mg/L, 4-D, final concentration is the 6-BA of 0.5mg/L, final concentration is the NAA of 0.5mg/L, final concentration is the sucrose of 30g/L and the agar that final concentration is 7g/L, after sterilizing, on the substratum of adjusting pH value to 5.8 acquisition or the basis of MS substratum, adding final concentration is 2 of 1mg/L, 4-D, final concentration is the 6-BA of 1mg/L, final concentration is the NAA of 1mg/L, final concentration is the sucrose of 30g/L and the agar that final concentration is 7g/L, after sterilizing, regulate the substratum of pH value to 5.8 acquisition, the condition of described induction of callus is the 23-27 DEG C of dark 15-30 days of cultivation.
2) to step 1) callus that obtains carries out somatic embryo inducement and cultivates and obtain somatic embryo; The substratum that described somatic induction is cultivated is on the basis of MS substratum, to add the solid medium that adds ZT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, sucrose that final concentration is 50g/L on KT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, the solid medium of sucrose that final concentration is 50g/L or the basis of MS substratum; The medium optimization that described somatic induction is cultivated is on the basis of MS substratum, to add the agar that sucrose that inositol+final concentration that KT that final concentration is 5mg/L, NAA+ final concentration that final concentration is 0.1mg/L are 100mg/L is 50g/L and final concentration are 10g/L, and sterilizing regulates the substratum of pH to 5.8 acquisition or is on the basis of MS substratum, to add the agar that ZT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, sucrose that final concentration is 50g/L and final concentration are 10g/L; The condition that described somatic induction is cultivated is the 23-27 DEG C of dark 30-45 days of cultivation;
3) to step 2) somatic embryo that obtains carries out ripe cultivation and obtains ripe somatic embryo; The solid medium of the coconut palm breast that the described ripe substratum of cultivating is 50ml/L, the sucrose that final concentration is 60g/L; The coconut palm breast that the described ripe medium optimization of cultivating is 50ml/L, sucrose and the final concentration that final concentration is 60g/L are 10g/L agar, and sterilizing, adjusts the substratum of pH to 5.8 acquisition; Described ripe condition of cultivating is temperature 23-27 DEG C, illumination cultivation.The Sucus Cocois obtaining after double gauze filters that coconut palm breast (or Coconut Juice) is poured out for fresh coconut.
4) to step 3) the ripe somatic embryo cultivation of regenerating that obtains, the substratum that regeneration is cultivated is on the basis of MS substratum, to add the solid medium that final concentration is the sucrose of 30g/L, be preferably that on the basis of MS substratum, to add sucrose and the final concentration that final concentration is 30g/L be 7g agar, sterilizing, adjusts the substratum of pH to 5.8 acquisition; The culture condition that regeneration is cultivated is temperature 23-27 DEG C, illumination cultivation.
In described method, described step 1) described in time of induction of callus be 15-30 days; Described step 2) described in somatic embryo inducement cultivate time be 30-45 days; Described step 3) described in the ripe time of cultivating be 25-35 days, preferably 30 days.Described step 4) described in regeneration cultivate time be 55-65 days, be preferably 60 days.
In described method, described step 3) and step 4) described in the intensity of illumination of illumination cultivation be 1800-2500Lx, 15-17 hour/day, be preferably 2000Lx, 16 hours/day.
In described method, described step 1) in also comprise the embryo callus obtaining carried out to succeeding transfer culture, the method of described succeeding transfer culture is that embryo callus is seeded in to subculture medium I and the upper alternate culture of subculture medium II, every 20-25d subculture once; Succeeding transfer culture condition, for being 23-27 DEG C, is secretly cultivated;
Wherein, subculture medium I is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the solid medium of the sucrose that 4-D, final concentration are 30g/L; Be preferably that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the agar that the sucrose that 4-D, final concentration are 30g/L and final concentration are 7g/L, sterilizing, the substratum of adjust pH to 5.8 acquisition; Subculture medium II is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the K T that 4-D, final concentration are 0.5mg/L, the AgNO that final concentration is 5mg/L
3, the final concentration sucrose that is 30g/L solid medium, be preferably that on the basis for MS substratum, to add final concentration be 2 of 1mg/L, the K T that 4-D, final concentration are 0.5mg/L, the AgNO that final concentration is 5mg/L
3, the final concentration sucrose that is 30g/L and final concentration be 7g/L agar, sterilizing, the substratum of adjust pH to 5.8 acquisition.The number of times of described succeeding transfer culture is 2-4 time, select faint yellow, Friable embryogenic callus and carry out subculture.
Technique scheme of the present invention has obtained excellent effect.Component by optimization callus inducing medium of the present invention, selection and the concentration of the nutrient media components of somatic embryo inducement, particularly hormone have been optimized culture condition simultaneously, have obtained very high callus induction rate and the induction efficiency of somatic embryo.Choosing Litchi Varieties ' cv. Feizixiao ' flower pesticide is that explant is seeded in callus inducing medium (MS+3mg/L 2,4-D+0.5mg/L BA+0.5mg/L NAA+30g/L sugar+7g/L agar) on secretly cultivate, callus induction rate reaches 92.86%; The embryo callus of screening is seeded in to the upper dark 30d of cultivation of body embryonal induction substratum (MS+0.1mg/L NAA+5mg/L KT+100mg/L inositol+50g/L sucrose+10g/L agar) left and right, can obtains the globular embryo (every fresh gram of callus induction somatic embryo reaches 489) of high score rate.Select that form size is basically identical, leukoplast somatic embryo is seeded to maturation medium (MS+50ml/L coconut palm breast+60g/L sucrose+10g/L agar, illumination cultivation; Leukoplast somatic embryo after maturation is inoculated in to plant regeneration substratum (MS+30g/L sucrose+7g agar) illumination cultivation 60d left and right, can obtains complete Regeneration in Vitro plant.
The present invention set up ' cv. Feizixiao ' Litchi Varieties from callus induction through body embryogenesis path, develop into the method for complete Regeneration in Vitro plant, capture the difficult problem of setting up of lichee vitro Regeneration System, and obtain very high inductivity, improve production efficiency, reduce cost, for Litchi Varieties improvement and biotechnology breeding are had laid a good foundation.
Brief description of the drawings
Fig. 1 is the callus (a) of initial induction in ' cv. Feizixiao ' Litchi Varieties vitro Regeneration System process of establishing; The embryo callus (b) of screening; The somatic embryo (c) of induction; Ripe somatic embryo (d); Regeneration in Vitro plant (e); The regeneration plant (f) of transplanting.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is quality percentage composition.
The material using in following embodiment:
2,4-D: purchased from Yuan Ju bio tech ltd, Shanghai, lot number 081223, (2,4 dichlorophenoxyacetic acid)
6-BA: purchased from Bai Ao bio tech ltd, Shanghai, lot number 090601, (6-benzyl aminopurine)
KT: purchased from Bai Ao bio tech ltd, Shanghai, lot number 080623, (6 chaff aminopurine)
NAA: purchased from Bai Ao bio tech ltd, Shanghai, lot number 090219, (a-naphthylacetic acid)
ZT: purchased from Yuan Ju bio tech ltd, Shanghai, lot number 091016, (anti-zeatin)
TDZ: purchased from Yuan Ju bio tech ltd, Shanghai, lot number 100312, (thidiazuron)
Method and the effect thereof of embodiment 1, Litchi Varieties ' cv. Feizixiao ' Regeneration in Vitro
One, the preparation of outer value body and inoculation
Material: Litchi Varieties ' cv. Feizixiao ' phase bud that shows money or valuables one carries unintentionally, take from December, 2009 in Danzhou City, Hainan Province Tropical Crop Variety Resource Institute of Chinese Academy of Tropical Agricultural Sciences lichee field gene bank.
Method: tap water rinses the bud 30min winning, on aseptic operating platform by 70% alcohol-pickled sterilization 30s for pretreated bud, after put into 0.1%HgCl
2the 8min that sterilizes in solution, finally uses autoclaving aseptic water washing 5-6 time, obtains sterilizable material.After flower pesticide being transferred to gently from bud with scalpel and tip tweezers, as explant, this explant is used for being seeded on callus inducing medium secretly to be cultivated.
Two, callus induction and screening
1, the preparation of tissue culture used medium
Callus inducing medium: taking MS as minimum medium, adopt Orthogonal Design Method Design, add 2, the agar that the sucrose that 4-D (final concentration be 0,1,2 or 3mg/L), 6-BA (final concentration be 0,0.2,0.5 or 1mg/L), KT (final concentration be 0,0.5,1 or 2mg/L), NAA (final concentration be 0,0.2,0.5 or 1mg/L), final concentration are 30g/L and final concentration are 7g/L, Medium's PH Value is 5.8 (wherein, four kinds of concrete final concentration combinations of hormone are as shown in table 1).
Wherein, MS substratum is conventional substratum, can commercial sources buy, also can be according to following composition and concentration, and oneself prepares MS minimum medium: moiety and the concentration thereof of MS minimum medium are as follows: macroelement: 1900mg/L saltpetre (KNO
3), 1650mg/L ammonium nitrate (NH
4nO
3), 170mg/L potassium primary phosphate (KH
2pO
4), 370mg/L magnesium sulfate heptahydrate (MgSO47H
2o), 440mg/L Calcium dichloride dihydrate (CaCl
22H
2o); Trace element: 0.83mg/L potassiumiodide (KI), 6.2mg/L boric acid (H
3bO
3), 22.3mg/L tetra-water manganous sulfates (MnSO44H2O), 8.6mg/L Zinc Sulphate Heptahydrate (ZnSO47H
2o), 0.25mg/L Sodium orthomolybdate (Na2MoO
42H
2o), 0.025mg/L cupric sulfate pentahydrate (CuSO
45H2O), 0.025mg/L CoCL2 6H2O (CoCl
26H
2o); Molysite: 37.25mg/L disodium ethylene diamine tetraacetate (Na
2eDTA), 27.85mg/L iron vitriol (FeSO
47H
2o); Organic composition: 100mg/L inositol, 2mg/L glycine, 0.1mg/L vitamin (VB1), 0.5mg/L pyridoxine hydrochloride (VB6), 0.5mg/L nicotinic acid (VB3 or VPP) composition.MS minimum medium solvent is water.
2, method for inducing and cultivating: the Feizixiao Litchi kind flower pesticide (explant) that step 1 is obtained is inoculated on above-mentioned callus inducing medium, be under 25 ± 2 DEG C of conditions in temperature, the dark 15-30 days that cultivates, induces callus (in Fig. 1 a).Calculate callus induction rate, study the impact on induction of anther callus of different combination of regulators and concentration.
Result is as shown in table 1.As can be seen from Table 1, the best inducing culture of callus is MS+3mg/L 2, (on the basis of MS substratum, add final concentration is 2 of 3mg/L to 4-D+0.5mg/L 6-BA+0.5mg/L NAA+30g/L sucrose+7g/L agar, the agar that the 6-BA that 4-D, final concentration are 0.5mg/L, the NAA that final concentration is 0.5mg/L, sucrose that final concentration is 30g/L and final concentration are 7g/L, regulate the substratum of pH value to 5.8 acquisition, sterilizing), inductivity reaches 92.86%.Next is MS+1mg/L 2, (on the basis of MS substratum, add final concentration is 2 of 1mg/L to 4-D+1mg/L 6-BA+1mg/L NAA+30g/L sucrose+7g/L agar, the agar that the 6-BA that 4-D, final concentration are 1mg/L, the NAA that final concentration is 1mg/L, sucrose that final concentration is 30g/L and final concentration are 7g/L, regulate the substratum of pH value to 5.8 acquisition, sterilizing), inductivity reaches 88.24%.
3, succeeding transfer culture
The callus that step 2 is induced is through screening (selecting faint yellow, Friable embryogenic callus), obtains faint yellow, Friable embryogenic callus (in Fig. 1 b).Faint yellow, Friable embryogenic callus that screening is obtained carry out succeeding transfer culture, subculture method is: be seeded in the upper alternate culture of subculture medium I and subculture medium II, culture condition is identical with induction of callus described in step 2, every 20-25d subculture once, subculture 2-4 time, screening obtain faint yellow, grow vigorous, particle is tiny and Friable embryogenic callus.
Subculture medium I is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the agar that the sucrose that 4-D, final concentration are 30g/L and final concentration are 7g/L, sterilizing, adjust pH to 5.8.
Subculture medium II is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the K T that 4-D, final concentration are 0.5mg/L, the AgNO that final concentration is 5mg/L
3, the final concentration sucrose that is 30g/L and final concentration be 7g/L agar, sterilizing, adjust pH to 5.8.
The different combination of regulators of table 1 and concentration affect ' cv. Feizixiao ' Litchi Anther evoked callus
*: the callus of induction is mainly divided into 3 types.Press Lai Zhongxiong [Lai Zhongxiong. longan biological technical study. Fujian science tech publishing house, 2003.6] standard callus is classified.CI: white consolidation, quality is harder, is generally bulk; CII a: faint yellow, quality is hard, loose, particle is very thick, has many proembryo differentiation; CII b: faint yellow, quality is harder, loose, particle is thicker, has minority proembryo to mix; CIII: faint yellow, loose, particle is tiny.
Three, somatic embryo inducement
By faint yellow, particle is less, Friable embryogenic callus is seeded in and adds NAA (final concentration be 0,1mg/L), KT (final concentration be 3,5 or 7mg/L) or ZT (final concentration be 3,5 or 7mg/L) or TDZ (final concentration be 3,5 or 7mg/L), final concentration is on the MS substratum (medium pH 5.8) of the inositol of 100mg/L, sucrose that final concentration is 50g/L and the final concentration agar that is 10g/L, be under 25 ± 2 DEG C of conditions in temperature, dark cultivation, 30-45d; Above-mentioned concrete hormone combinations in substratum is in table 2.
Cultivate 45d statistics body embryo generation number (1 fresh gram is that the fresh weight of callus is 1 gram, callus induction body embryo number), study the impact on somatic embryo inducement of different combination of regulators and concentration.
Result shows, by body embryonal induction substratum faint yellow, that particle is less, Friable embryogenic callus is seeded in above-mentioned hormon proportioning, 30d left and right can obtain high score rate globular embryo (somatic embryo of induction, in Fig. 1 c).As can be seen from Table 2, somatic embryo induction optimum formula is: MS+5mg/L KT+0.1mg/LNAA+100mg/L inositol+50g/L sucrose+10g/L agar (is on the basis of MS substratum, to add the agar that sucrose that inositol+final concentration that KT that final concentration is 5mg/L, NAA+ final concentration that final concentration is 0.1mg/L are 100mg/L is 50g/L and final concentration are 10g/L, sterilizing regulates the substratum of pH to 5.8 acquisition), every fresh gram of callus induction somatic embryo reaches 489.Next is MS+5mg/L ZT+0.1mg/L NAA+100mg/L inositol+50g/L sucrose+10g/L agar (being to add the agar that ZT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, sucrose that final concentration is 50g/L and final concentration are 10g/L on the basis of MS substratum), and every fresh gram of callus induction somatic embryo is 344.
The different combination of regulators of table 2 and concentration are on Feizixiao Litchi body embryo differentiation impact
*: every fresh gram of callus induction body embryo number; Fresh gram refers to the fresh weight of callus, and unit is gram.
Four, the maturation of somatic embryo and plant Regeneration in Vitro
Select that form size is basically identical, leukoplast somatic embryo is seeded to maturation medium, 25 ± 2 DEG C of temperature, illumination cultivation (2000Lx, 16h/d).Cultivate the somatic embryo that about 30d just can develop into maturation of the same size (in Fig. 1 d).
Maturation medium is on the basis of MS substratum, to add the coconut palm breast that final concentration is 50ml/L (coconut palm breast or be called Coconut Juice, the Sucus Cocois that obtains after double gauze filters of pouring out for fresh coconut), final concentration is 60g/L sucrose and final concentration are 10g/L agar, sterilizing, adjusts the substratum of pH to 5.8 acquisition.
Leukoplast somatic embryo after maturation is inoculated in to plant regeneration substratum.Culture condition: pH value is 5.8,25 ± 2 DEG C of temperature, intensity of illumination 2000Lx, 16h/d.Cultivate 60d, visible volume somatic embryo is sprouted, and forms complete regenerated plant (in Fig. 1 e).Regeneration culture medium is that on the basis of MS substratum, to add sucrose and the final concentration that final concentration is 30g/L be 7g agar, and the substratum of pH to 5.8 acquisition is adjusted in sterilizing.
The hardening of uncapping 2-3 days, washes root, transplanting medium (peat soil: perlite: coconut palm chaff=1: 1: 1) is carried out after sterilization simultaneously, transplants, and the regeneration plant of transplanting is shown in f in Fig. 1.The regeneration plant of transplanting is regularly sprayed water, water, the MS macroelement mother liquor that initial stage (first 7 days) pouring dilution is 50 times, avoids being placed on strong illumination district, after seedling stalwartness, transplants to land for growing field crops.
Claims (4)
1. the method for lichee cv. Feizixiao kind Regeneration in Vitro, comprises the steps:
1) taking lichee cv. Feizixiao kind flower pesticide as explant, explant is inoculated in to callus inducing medium and carries out induction of callus, screening obtains embryo callus; Described callus inducing medium is that on the basis of MS substratum, to add final concentration be 2 of 3mg/L, on the 6-BA that 4-D, final concentration are 0.5mg/L, the NAA that final concentration is 0.5mg/L, the solid medium of sucrose that final concentration is 30g/L or the basis of MS substratum, adding final concentration is 2 of 1mg/L, the solid medium of the 6-BA that 4-D, final concentration are 1mg/L, the NAA that final concentration is 1mg/L, sucrose that final concentration is 30g/L; The condition of described induction of callus is the 23-27 DEG C of dark 15-30 days of cultivation;
2) embryo callus step 1) being obtained carries out somatic embryo inducement cultivation and obtains somatic embryo; The substratum that described somatic embryo inducement is cultivated is on the basis of MS substratum, to add the solid medium that adds ZT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, sucrose that final concentration is 50g/L on KT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, the solid medium of sucrose that final concentration is 50g/L or the basis of MS substratum; The condition that described somatic embryo inducement is cultivated is the 23-27 DEG C of dark 30-45 days of cultivation;
3) to step 2) somatic embryo that obtains carries out ripe cultivation and obtains ripe somatic embryo; The described ripe substratum of cultivating be maturation medium be on the basis of MS substratum, add the coconut palm breast that final concentration is 50ml/L, sucrose and the final concentration that final concentration is 60g/L is 10g/L agar, sterilizing, adjusts the substratum of pH to 5.8 acquisition; Described ripe condition of cultivating is temperature 23-27 DEG C, illumination cultivation 25-35 days;
4) ripe somatic embryo cultivations of regenerate step 3) being obtained, the substratum of the cultivation of regenerating is on the basis of MS substratum, to add the solid medium that final concentration is the sucrose of 30g/L; The culture condition that regeneration is cultivated is temperature 23-27 DEG C, illumination cultivation 55-65 days;
Described in described step 3) and step 4), the intensity of illumination of illumination cultivation is 2000Lx, 16 hours/day;
In described step 1), also comprise the embryo callus obtaining is carried out to succeeding transfer culture, the method for described succeeding transfer culture is that embryo callus is seeded in to subculture medium I and the upper alternate culture of subculture medium II, every 20-25d subculture once; Succeeding transfer culture condition, for being 23-27 DEG C, is secretly cultivated;
Wherein, subculture medium I is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the solid medium of the sucrose that 4-D, final concentration are 30g/L; Subculture medium II is that on the basis of MS substratum, to add final concentration be 2 of 1mg/L, the K T that 4-D, final concentration are 0.5mg/L, the AgNO that final concentration is 5mg/L
3, the final concentration sucrose that is 30g/L solid medium;
The number of times of described succeeding transfer culture is 2-4 time.
2. method according to claim 1, is characterized in that: described in described step 3), the ripe time of cultivating is 30 days.
3. method according to claim 1, is characterized in that: the time that described in described step 4), regeneration is cultivated is 60 days.
4. the special-purpose assorted substratum of lichee cv. Feizixiao kind Regeneration in Vitro, comprises callus inducing medium and somatic embryo inducement substratum;
Described callus inducing medium is that on the basis of MS substratum, to add final concentration be 2 of 3mg/L, on the 6-BA that 4-D, final concentration are 0.5mg/L, the NAA that final concentration is 0.5mg/L, the solid medium of sucrose that final concentration is 30g/L or the basis of MS substratum, adding final concentration is 2 of 1mg/L, the solid medium of the 6-BA that 4-D, final concentration are 1mg/L, the NAA that final concentration is 1mg/L, sucrose that final concentration is 30g/L;
On the basis that described somatic embryo inducement substratum is MS substratum, add the solid medium that adds ZT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, sucrose that final concentration is 50g/L on KT that final concentration is 5mg/L, NAA that final concentration is 0.1mg/L, inositol that final concentration is 100mg/L, the solid medium of sucrose that final concentration is 50g/L or the basis of MS substratum;
In described supporting substratum, also comprise ripe cultivation with substratum and regeneration cultivation substratum;
Described ripe cultivate with substratum be on the basis of MS substratum, add the coconut palm breast that final concentration is 50ml/L, sucrose and the final concentration that final concentration is 60g/L is 10g/L agar, sterilizing, the substratum of tune pH to 5.8 acquisition; It is on the basis of MS substratum, to add the solid medium that final concentration is the sucrose of 30g/L with substratum that described regeneration is cultivated.
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| CN105028176A (en) * | 2015-06-10 | 2015-11-11 | 广东省农业科学院果树研究所 | Rapid seedling selection method of litchi |
| CN106386479A (en) * | 2016-08-29 | 2017-02-15 | 杨胜 | Culture method of litchi tree seedlings |
| CN106376462A (en) * | 2016-08-29 | 2017-02-08 | 杨胜 | Culture method of lichees |
| CN110946073B (en) * | 2018-09-27 | 2021-04-16 | 东北林业大学 | Method for rapidly propagating fraxinus mandshurica embryonic cells |
| CN111084106B (en) * | 2020-01-10 | 2023-04-28 | 中国热带农业科学院环境与植物保护研究所 | In-vitro regeneration method of 'Dasyringa oblata' litchi variety |
| CN112616668B (en) * | 2020-12-30 | 2022-12-06 | 中国热带农业科学院环境与植物保护研究所 | In-vitro regeneration method for litchi variety Lingnan No. 15 |
| CN114223540B (en) * | 2021-11-12 | 2023-05-02 | 中国热带农业科学院环境与植物保护研究所 | Product containing amino acid composite liquid for in-vitro regeneration of litchi, method and application |
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