CN112616668B - In-vitro regeneration method for litchi variety Lingnan No. 15 - Google Patents
In-vitro regeneration method for litchi variety Lingnan No. 15 Download PDFInfo
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Abstract
The invention discloses a method for in vitro regeneration of a litchi variety Lingnan No. 15. It comprises the following steps: 1) Taking litchi anther as an explant, and performing callus induction culture in a callus induction culture medium to obtain embryonic callus; 2) Performing somatic embryo induction culture on the embryogenic callus in a somatic embryo induction culture medium to obtain a somatic embryo; 3) Carrying out maturation culture on the somatic embryos in a maturation culture medium to obtain mature somatic embryos; 4) And performing regeneration culture on the mature somatic embryos in a regeneration culture medium to obtain regenerated plants. The invention establishes a method for litchi to develop into a complete in vitro regeneration plant from callus induction through somatic embryogenesis, solves the problem that a litchi in vitro regeneration system is difficult to establish, obtains high somatic embryo induction rate and germination rate, improves production efficiency, reduces cost, and lays a good foundation for litchi variety improvement and biotechnology breeding.
Description
Technical Field
The invention relates to an in vitro regeneration method of litchi, in particular to an in vitro regeneration method of a litchi variety Lingnan No. 15.
Background
Litchi (lichchi chinensis Sonn.) is one of the important economic crops in China and plays an important role in the hot-zone agricultural production. Sea reclamation number 10, its name: xiang Wan, qiongshan No. 6, lingnan No. 15, mother tree Yongxing Zhen Ru Chong village in Xiongying district of Hainan province. The germplasm is stored in the research institute of tropical crop variety resources of the Chinese tropical agricultural academy of sciences and the research institute of tropical fruit trees of the agricultural academy of sciences in Hainan province. The tree has upright tree posture, semicircular crown, strong growth potential, nearly spherical fruit shape, 1.07 shape index of the fruit, super large fruit, 52.9g of average single fruit weight and 68g of maximum single fruit weight. The peel is red and green, the cracking sheets are large and convex, and the arrangement is irregular. The fruit flesh is crisp and tasty, the residue is less, the fruit flesh is fresh, sweet and slightly light, the juice is much, the juice does not overflow, the edible part is 39.2g, and the weight of the edible part accounts for 74.1 percent of the weight of the fruit. Flowering in the first and last days of 2 months, and ripening in the first and last days of 6 months (Hainan litchi germplasm resources, eds Chen Yeyuan, li Songgang, 11 months 1 st edition 2014, chinese agriculture Press, chapter 6, page 62, 7 th litchi single plant, lingnan No. 15). Generally, the development of litchi industry is seriously restricted by the problems of unstable flower setting and fruit setting, serious pest and disease damage, difficult preservation after picking and the like of litchi, and the cultivation of excellent litchi varieties is a fundamental way for solving the problems.
The genetic high heterozygosis of litchi and the sterility of partial variety limit the application of sexual hybridization in improving litchi variety. A new method and means can be provided for variety detoxification, rejuvenation, genetic improvement and rapid propagation through transgenic breeding, and a litchi in vitro regeneration technical system is the basis of transgenic breeding and rootstock asexual propagation.
Since the report of obtaining complete plants by litchi tissue culture in 1983, fu Lianfang and the like, there have been many reports on the research on the in vitro regeneration of litchi. At present, genotype dependence exists in litchi in-vitro regeneration, the somatic embryo generation frequency is low, more malformed embryos and somatic embryo height are not synchronous, the plant regeneration frequency is not high, and the method is a technical bottleneck for establishing efficient litchi in-vitro regeneration.
Disclosure of Invention
The invention aims to provide an in vitro regeneration method of litchi, and particularly provides an in vitro regeneration method of a No. 15 litchi variety.
The invention provides a method for in vitro regeneration of litchi, which comprises the following steps: 1) Taking litchi anther as an explant, and performing callus induction culture in a callus induction culture medium to obtain embryonic callus;
2) Performing somatic embryo induction culture on the embryogenic callus in a somatic embryo induction culture medium to obtain a somatic embryo;
3) Carrying out maturation culture on the somatic embryos in a maturation culture medium to obtain mature somatic embryos;
4) And performing regeneration culture on the mature somatic embryos in a regeneration culture medium to obtain regenerated plants.
In the step 1) of the method, the callus induction medium comprises 2,4-D, 6-BA and NAA;
in the method, the final concentrations of 2,4-D, 6-BA and NAA in the callus induction culture medium are 0.5-3 mg/L, 0.1-1 mg/L and 0.1-1 mg/L respectively.
In the invention, the callus induction culture medium is composed of MS culture medium, 2,4-D, 6-BA, NAA, sucrose and agar; the final concentrations of 2,4-D, 6-BA, NAA, sucrose and agar in the callus induction medium can be respectively 3mg/L, 0.5mg/L, 30g/L and 7g/L, and the pH value of the callus induction medium is 5.8.
In step 2) of the above method, the somatic embryo induction medium comprises NAA, KT, ZT, inositol, hydrolyzed milk protein and coconut milk;
in the above method, the final concentrations of the NAA, the KT, the ZT, the inositol, the hydrolyzed milk protein, and the coconut milk in the somatic embryo induction medium may be 0.1 to 1mg/L, 3 to 7mg/L, 2 to 7mg/L, 50 to 200mg/L, 300 to 500mg/L, and 50 to 100ml/L, respectively.
In the invention, the somatic embryo induction culture medium consists of an MS culture medium, NAA, KT, ZT, inositol, hydrolyzed milk protein, coconut milk, sucrose and agar; the final concentrations of the NAA, the KT, the ZT, the inositol, the hydrolyzed milk protein, the coconut milk, the sucrose and the agar in the somatic embryo induction culture medium can be 0.1mg/L, 3.5mg/L, 2.5mg/L, 100mg/L, 400mg/L, 100ml/L, 60g/L and 10g/L respectively, and the pH value of the somatic embryo induction culture medium is 5.8.
In the step 3) of the method, the mature culture medium comprises IAA, ABA and coconut milk;
and/or in the maturation medium, the final concentration of the IAA, the ABA and the coconut milk can be 0.1-1 mg/L, 0.5-2 mg/L and 50-100 ml/L.
In the invention, the mature culture medium consists of an MS culture medium, IAA, ABA, coconut milk, sucrose and agar; the final concentrations of IAA, ABA, coconut milk, sucrose and agar in the maturation medium can be 0.5mg/L, 1mg/L, 100ml/L, 60g/L, 10g/L, respectively, and the pH of the maturation medium is 5.8.
In step 4) of the above method, the regeneration medium comprises GA 3 ；
In the above step 4), the regeneration medium is the GA 3 Can be 0.5-2 mg/L.
In the invention, the regeneration medium is composed of MS medium, MS medium and GA 3 Sucrose and agar; the GA 3 The final concentrations of sucrose and agar are 1mg/L, 30g/L and 7g/L respectively, and the pH of the regeneration medium is 5.8.
In the method, the step 1) further comprises the step of subculturing the embryogenic callus;
the subculture method specifically comprises the following steps: and alternately culturing the embryonic callus on a subculture medium I and a subculture medium II, and subculturing once every 20-25 days.
In the above method, the subculture medium I comprises 2,4-D.
The subculture medium II comprises 2,4-D, KT and AgNO 3 。
In the above method, the final concentration of 2,4-D in the subculture medium I may be specifically 0.5 to 2mg/L.
In the invention, the subculture medium I consists of an MS culture medium, 2,4-D, sucrose and agar; the final concentration of 2,4-D, sucrose and agar in the subculture medium I can be 1mg/L, 30g/L and 7g/L, and the pH value of the subculture medium I is 5.8.
In the above method, the 2,4-D, the KT, the AgNO are in the subculture medium II 3 The final concentration of the compound is 0.5-2 mg/L, 0.5-2 mg/L and 3-7 mg/L respectively.
In the invention, the subculture medium II is prepared from MS culture medium, 2,4-D, KT and AgNO 3 Sucrose and agar; the 2,4-D, KT and AgNO 3 The final concentrations of the sucrose and the agar in the secondary culture medium II can be 1mg/L, 0.5mg/L, 5mg/L, 30g/L and 7g/L respectively, and the pH value of the secondary culture medium II is 5.8.
In any of the above methods, the MS medium may be a medium commonly used in the art, commercially available, or formulated by itself according to the following ingredients and concentrations:
the solvent of the MS culture medium is water, and the solutes and the concentrations thereof are respectively as follows: macroelements: 1900mg/L potassium nitrate (KNO) 3 ) 1650mg/L ammonium Nitrate (NH) 4 NO 3 ) 170mg/L potassium dihydrogen phosphate (KH) 2 PO 4 ) 370mg/L magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 O), 440mg/L calcium chloride dihydrate (CaCl) 2 ·2H 2 O); trace elements: 0.83mg/L potassium iodide (KI), 6.2mg/L boric acid (H) 3 BO 3 ) 22.3mg/L manganese sulfate tetrahydrate (MnSO) 4 ·4H 2 O), 8.6mg/L Zinc sulfate heptahydrate (ZnSO) 4 ·7H 2 O), 0.25mg/L sodium molybdate (Na) 2 MoO 4 ·2H 2 O), 0.025mg/L copper sulfate pentahydrate (CuSO) 4 ·5H 2 O), 0.025mg/L cobalt chloride hexahydrate (CoCl) 2 ·6H 2 O); iron salt: 37.25mg/L disodium edetate (Na) 2 EDTA), 27.85mg/L ferrous sulfate heptahydrate (FeSO) 4 ·7H 2 O); organic components: 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride (VB 1), 0.5mg/L pyridoxine hydrochloride (VB 6), 0.5mg/L niacin (VB 3) or niacinamide (VPP).
In the step 1) of the method, the callus induction culture conditions are as follows: dark culture is carried out for 45-60 days at 23-27 ℃.
In step 2) of the above method, the somatic embryo induction culture conditions are as follows: dark culture is carried out for 30-45 days at 23-27 ℃.
In step 3) of the method, the maturation culture conditions are as follows: culturing in dark at 23-27 deg.c for 50-65 days.
In step 4), the regeneration culture conditions are as follows: the illumination culture is carried out for 30 to 40 days at the temperature of between 23 and 27 ℃, the illumination intensity is 2000 to 5000Lux, and the illumination time can be 15 to 17 hours per day.
In the above method, the subculture conditions are as follows: culturing at 23-27 deg.c in dark.
In the above method, the number of subcultures may be 8 to 10, specifically 9.
Any of the above-mentioned media is used after the sterilization treatment.
The invention also provides a complete set of culture medium for litchi in vitro regeneration, which comprises the callus induction culture medium and/or the somatic embryo induction culture medium and/or the maturation culture medium and/or the regeneration culture medium and/or the subculture medium I and/or the subculture medium II.
The invention also provides any one of the following applications a 1) to a 4):
a1 Application of the complete set of culture medium in the in vitro regeneration culture of the litchi chinensis Sonn;
a2 Application of the complete set of culture medium in preparation of a product for in vitro regeneration culture of litchi;
a3 Application of the somatic embryo induction culture medium in improving the induction rate of the litchi somatic embryos;
a4 Application of the regeneration culture medium in improving the germination rate of the somatic embryos of the litchi.
In the above method of the present invention or the above set of culture media or the above application, the litchi variety is Lingnan No. 15.
The invention has the following advantages:
the invention establishes a method for inducing litchi varieties (such as litchi Lingnan No. 15 varieties) from calluses through somatic embryogenesis to develop into complete in vitro regeneration plants, solves the problem that an in vitro regeneration system of litchi is difficult to establish, obtains very high somatic embryo induction rate and germination rate, improves production efficiency, reduces cost, and lays a good foundation for litchi variety improvement and biotechnology breeding.
Drawings
FIG. 1 is a picture of the in vitro regeneration system of Lingnan No. 15 litchi variety. (a) The primary culture of anther induced callus in the process of establishing an in vitro regeneration system of No. 15 Lingnan litchi variety; (b) Establishing a stable embryogenic callus line for the in vitro regeneration system of the No. 15 Lingnan litchi variety through subculture in the process; (c) High-frequency somatic embryogenesis is established in the process of establishing an in vitro regeneration system for No. 15 Lingnan litchi varieties; (d) A somatic embryo of initial stem drawing in the process of establishing an isolated regeneration system for the Lingnan No. 15 litchi variety; (e) Expanding leaves of somatic embryos in the process of establishing an in vitro regeneration system for No. 15 Lingnan litchi varieties; (f) Establishing a regeneration plant for the Lingnan No. 15 litchi variety in-vitro regeneration system; (g) Establishing a regeneration plant with 2 leaves and 1 core of rhizome to be transplanted in the process of establishing an in vitro regeneration system of No. 15 Lingnan litchi variety; (h) The method is a regeneration plant transplanted in the process of establishing an isolated regeneration system of Lingnan No. 15 litchi variety.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
The sources of reagents and media involved in the following examples are as follows:
2,4-D (2,4-dichlorophenoxyacetic acid) is a product of Shanghai-sourced Biotech, inc. under the designation 081223.
6-BA (6-benzylaminopurine) is a product of Shanghai Bo' ao Biotech, inc. under the product number 090601.
NAA (alpha-naphthylacetic acid) is a product of Shanghai Boo Biotech, inc. with a product number of 090219.
KT (6-furfurylaminopurine) is a product of Bo' ao Biotechnology Ltd, shanghai, lot number 080623,
ZT (zeatin) is a product of Shanghai-derived Biotech Co., ltd., lot number 200113.
IAA (indole-3-acetic acid) is a product of Biotechnology engineering (Shanghai) Inc., having a Cat number CA13BA0040.
ABA (abscisic acid) is a product of a source leaf organism, with a cargo number S21F8J29658.
GA 3 (gibberellins) are products of the source leaf organisms, cat # S27S10P98642.
Coconut milk (or coconut juice) is coconut water obtained by filtering coconut juice poured from fresh coconuts with double-layer gauze.
The hydrolyzed milk protein is a product of biological engineering (Shanghai) GmbH, having a product number of BB07BA0326.
Inositol is a product of Shanghai-sourced bioscience, inc., with the product number of 071013.
M519 (MS culture medium) is a product of Beijing Western Mejie science and technology, inc., and has a product number of AAK0519209A.
Example 1 in vitro regeneration method of litchi variety Lingnan No. 15
1. Preparation of explants
Experimental materials: litchi chinensis, lingnan No. 15, was obtained from a germplasm resource garden of research institute of tropical crop variety, institute of tropical crop, academy of tropical agriculture science, oakhizhou, hainan, in 2019, at 3 months.
The experimental method comprises the following steps: washing the picked flower buds with tap water for 30min to obtain pretreated flower buds; then soaking the pretreated flower buds in 70% (volume fraction) alcohol solution for 30s on a sterile operating table, sterilizing, and adding 0.1% (mass fraction) HgCl 2 Sterilizing in the solution for 8min, and washing with autoclaved sterile water for 5-6 times to obtain sterile flower bud. The anther is gently pulled out of the sterile bud with a scalpel and a sharp-pointed forceps to serve as an explant, and the explant is used for being inoculated on a callus induction culture medium for dark culture.
2. Callus induction and subculture
1. Preparation of callus induction culture medium
The callus induction culture medium is prepared by adding 2,4-D with final concentration of 3mg/L, 6-BA with final concentration of 0.5mg/L, NAA with final concentration of 0.5mg/L, sucrose with final concentration of 30g/L and agar with final concentration of 7g/L on the basis of MS culture medium, adjusting pH to 5.8, and sterilizing.
Wherein, the MS culture medium is a common culture medium, can be purchased from commercial sources, and can be prepared according to the following components and concentrations: the solvent of the MS culture medium is water, and the solutes and the concentrations thereof are respectively as follows: macroelements: 1900mg/L potassium nitrate (KNO) 3 ) 1650mg/L ammonium Nitrate (NH) 4 NO 3 ) 170mg/L potassium dihydrogen phosphate (KH) 2 PO 4 ) 370mg/L magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 O), 440mg/L calcium chloride dihydrate (CaCl) 2 ·2H 2 O); trace elements: 0.83mg/L potassium iodide (KI), 6.2mg/L boric acid (H) 3 BO 3 ) 22.3mg/L manganese sulfate tetrahydrate (MnSO) 4 ·4H 2 O), 8.6mg/L Zinc sulfate heptahydrate (ZnSO) 4 ·7H 2 O), 0.25mg/L sodium molybdate (Na) 2 MoO 4 ·2H 2 O), 0.025mg/L copper sulfate pentahydrate (CuSO) 4 ·5H 2 O), 0.025mg/L cobalt chloride hexahydrate (CoCl) 2 ·6H 2 O); iron salt: 37.25mg/L disodium edetate (Na) 2 EDTA), 27.85mg/L ferrous sulfate heptahydrate (FeSO) 4 ·7H 2 O); organic components: 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride (VB 1), 0.5mg/L pyridoxine hydrochloride (VB 6), 0.5mg/L niacin (VB 3) or niacinamide (VPP).
2. Induced culture
Inoculating anther (explant) of litchi variety Lingnan No. 15 obtained in the first step to callus induction medium obtained in the second step-1, culturing in dark at 25 + -2 deg.C for 45-60 days, inducing and screening to obtain embryogenic callus (fig. 1 (a) - (b)).
3. Subculture
And selecting light yellow loose embryonic callus, inoculating the light yellow loose embryonic callus to a subculture medium I and a subculture medium II for alternate culture, wherein the culture conditions are the same as those of the callus induced culture in the step two-2, subculturing once every 20-25 days for 8-10 times, and screening to obtain the light yellow loose embryonic callus with vigorous growth and fine particles.
The subculture medium I is a culture medium obtained by adding 2,4-D with a final concentration of 1mg/L, sucrose with a final concentration of 30g/L and agar with a final concentration of 7g/L to an MS culture medium, adjusting the pH value to 5.8 and sterilizing.
The subculture medium II is prepared by adding 2,4-D with final concentration of 1mg/L, KT with final concentration of 0.5mg/L, and AgNO with final concentration of 5mg/L on the basis of MS culture medium 3 Sucrose with a final concentration of 30g/L and agar with a final concentration of 7g/L, adjusting the pH value to 5.8, and sterilizing to obtain the culture medium.
3. Somatic embryo induction culture
1. And (3) respectively inoculating the faint yellow, vigorous-growing, fine-particle and loose embryogenic callus obtained in the second step into somatic embryo induction culture media containing different growth regulators shown in the table 1 to perform somatic embryo induction, and researching the influence of the different growth regulators on the induction of the somatic embryos.
The somatic embryo induction medium shown in Table 1 is a medium obtained by adding NAA (final concentration of 0.1 mg/L), KT (final concentration of 3, 5 mg/L), ZT (final concentration of 2, 5 mg/L), inositol (final concentration of 100 mg/L), lactoprotein hydrolysate (final concentration of 400 mg/L), coconut milk (final concentration of 100 ml/L), sucrose (final concentration of 60 g/L) and agar (final concentration of 10 g/L) to MS medium as a basic medium, adjusting pH to 5.8, and sterilizing.
The culture conditions were as follows: culturing at 25 + -2 deg.C in dark for 30-45 days.
The number of somatic embryos generated was counted at 45 days of culture (1 fresh gram, i.e., 1 gram of fresh weight of callus, number of callus-induced somatic embryos).
The results show that: the pale yellow, vigorously growing, fine-grained and loose embryogenic callus is inoculated to somatic embryo induction medium containing different combinations of growth regulators for induction culture, and somatic embryos with high differentiation rate (induced somatic embryos, fig. 1 (c)) can be obtained after about 30 days of culture. As can be seen from Table 1, the T7 somatic embryo induction medium induces the largest number of somatic embryos, and each fresh gram of callus induces 480 somatic embryos with the highest germination rate. Secondly, inducing culture medium for T5 somatic embryos, wherein 434 somatic embryos are induced for each fresh gram of callus. Therefore, the T7 somatic embryo induction culture medium is determined to be the optimal somatic embryo induction culture medium in vitro regeneration, and the specific formula is as follows: MS +0.1mg/L NAA +5mg/L ZT +100mg/L inositol +400mg/L lactoprotein hydrolysate +100ml/L coconut milk +60g/L sucrose +10g/L agar.
TABLE 1 Effect of different growth regulators on the in vitro regeneration of "Linnan No. 15" litchi
Table 1. Table: the number of the callus induced somatic embryos is per gram; fresh gram means the fresh weight of the callus, and the unit is gram. Different letters in Table 1 indicate significant differences (P < 0.05) in the Duncan New Complex Difference test.
4. Maturation of somatic embryo and in-vitro regeneration culture of plant
1. Selecting white somatic embryos with basically consistent shape and size from the somatic embryos obtained in the third step, inoculating the white somatic embryos to a maturation culture medium, and performing dark culture at the temperature of 25 +/-2 ℃ for about 60 days to develop mature somatic embryos with consistent size.
The maturation culture medium is prepared by adding IAA with a final concentration of 0.5mg/L, ABA with a final concentration of 1mg/L, coconut milk with a final concentration of 100ml/L, sucrose with a final concentration of 60g/L and agar with a final concentration of 10g/L on the basis of MS culture medium, adjusting pH to 5.8, and sterilizing.
The condition of the mature culture is that the temperature is 23-27 ℃, the culture is carried out in the dark, and the culture time is 50-65 days.
2. White somatic embryos of substantially uniform morphology and size were selected from the mature somatic embryos obtained in step 1, and inoculated into a regeneration medium for R3 to be cultured, thereby obtaining regenerated plants (FIG. 1 (d), (e)).
The regeneration medium shown by R3 is MS culture medium as basic culture medium, and GA with final concentration of 1mg/L is added 3 Sucrose with a final concentration of 30g/L and agar with a final concentration of 7g/L, adjusting pH to 5.8, and sterilizing to obtain the culture medium.
The regeneration culture condition is that the temperature is 25 +/-2 ℃, the illumination culture (the illumination intensity is 2000Lux, the illumination time is 16 hours/day) is carried out, and the culture time is 30-40 days.
The germination rate of somatic embryos was counted after 40 days of culture in regeneration medium. Somatic embryo germination rate = number of germinated somatic embryos/total number of somatic embryos inoculated into regeneration medium.
The results are shown in Table 1. As can be seen from the table: the somatic embryo germination rate of the T7 regeneration culture medium is highest, so that the T7 culture medium is determined to be the optimal culture medium for inducing somatic embryos in vitro regeneration, and the specific formula is as follows: MS +0.1mg/L NAA +5mg/L ZT +100mg/L inositol +400mg/L lactoprotein hydrolysate +100ml/L coconut milk +60g/L sucrose +10g/L agar.
After the mature somatic embryos were cultured in the R3 regeneration medium for 40 days, it was observed that the somatic embryos germinated by stem shoots to form whole regenerated plants (FIG. 1 (f)).
3. Transplanting the regenerated seedling with more than 2 leaves, wherein the transplanted regenerated plant is shown as g and h in figure 1. Cleaning the culture medium, transplanting the culture medium into a sterilized substrate, and hardening seedlings by 3000Lux weak light (25 +/-2) DEG C and over 90% air humidity. After two new canes of leaves grow and are aged, the seedlings can be transferred to a greenhouse with 75% shading degree for hardening seedlings, and finally the seedlings are transferred to the field for planting.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A method for in vitro regeneration of litchi comprises the following steps: 1) Taking litchi anther as an explant, and performing callus induction culture in a callus induction culture medium to obtain embryonic callus;
in the step 1), the callus induction culture medium comprises 2,4-D, 6-BA and NAA; in the callus induction culture medium, the final concentrations of 2,4-D, 6-BA and NAA are 0.5-3 mg/L, 0.1-1 mg/L and 0.1-1 mg/L respectively;
2) Performing somatic embryo induction culture on the embryonic callus in a somatic embryo induction culture medium to obtain a somatic embryo;
in the step 2), the induction culture of the somatic embryos comprises the following components: MS +0.1mg/L NAA +5mg/L ZT +100mg/L inositol +400mg/L lactoprotein hydrolysate +100mL/L coconut milk +60g/L sucrose +10g/L agar;
3) Performing maturation culture on the somatic embryos in a maturation culture medium to obtain mature somatic embryos;
the maturation culture medium consists of an MS culture medium, IAA, ABA, coconut milk, sucrose and agar; the final concentrations of the IAA, the ABA, the coconut milk, the sucrose and the agar in the mature culture medium are respectively 0.5mg/L, 1mg/L, 100mL/L, 60g/L and 10g/L, and the pH of the mature culture medium is 5.8;
4) Carrying out regeneration culture on the mature somatic embryos in a regeneration culture medium to obtain regenerated plants;
the regeneration culture medium comprises MS culture medium, MS culture medium and GA 3 Sucrose and agar; the GA 3 The final concentrations of the sucrose and the agar are 1mg/L, 30g/L and 7g/L respectively, and the pH value of the regeneration culture medium is 5.8;
the litchi variety is Lingnan No. 15.
2. The method of claim 1, wherein: the step 1) also comprises the step of carrying out subculture on the embryogenic callus;
the subculture method specifically comprises the following steps: alternately culturing the embryonic callus on a subculture medium I and a subculture medium II, and subculturing once every 20-25 days;
the subculture medium I comprises 2,4-D;
the subculture medium II comprises 2,4-D, KT and AgNO 3 ；
And/or the final concentration of 2,4-D in the subculture medium I is 0.5-2 mg/L;
the 2,4-D, the KT, the AgNO in the subculture medium II 3 The final concentrations of the compounds are respectively 0.5-2 mg/L, 0.5-2 mg/L and 3-7 mg/L.
3. The method according to claim 1 or 2, characterized in that: in the step 1), the callus induction culture conditions are as follows: dark culture is carried out for 45 to 60 days at a temperature of between 23 and 27 ℃;
and/or, in the step 2), the somatic embryo induction culture conditions are as follows: dark culture is carried out for 30-45 days at 23-27 ℃;
and/or, in step 3), the mature culture conditions are as follows: culturing in dark at 23-27 deg.c for 50-65 days;
and/or, in the step 4), the regeneration culture conditions are as follows: illumination culture is carried out for 30 to 40 days at the temperature of between 23 and 27 ℃, the illumination intensity is 2000 to 5000Lux, and the illumination time is 15 to 17 hours per day;
and/or, the subculture conditions are as follows: dark culture at 23-27 deg.c;
and/or the number of subcultures is 8-10.
4. A set of media for ex vivo regeneration of litchi comprising the callus induction medium of any one of claims 1-3, the somatic embryo induction medium, the maturation medium, the regeneration medium, the subculture medium I and the subculture medium II.
5. Any one of the following a 1) -a 2):
a1 Use of the set of culture media of claim 4 in the ex vivo regeneration culture of litchi chinensis Sonn;
a2 Use of the set of culture media according to claim 4 for the preparation of a product for the ex vivo regenerative culture of litchi;
the litchi variety is Lingnan No. 15.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011607637.9A CN112616668B (en) | 2020-12-30 | 2020-12-30 | In-vitro regeneration method for litchi variety Lingnan No. 15 |
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