CN1915002A - Method for inducing embryo callus of larch, and dedicated culture medium - Google Patents

Abstract

A method for inducing the embryonic callus with high inducing effect and its special culture medium are disclosed. Said special culture medium DCR contains proportionally VB1, VB6, nicotinic acid, inositol, glutamine, hydrolytic casein, 2,4-dichlorophenoxyacetic acid, 6-BA, cane sugar and agar.

Description

Method for Inducing Embryogenic Callus of Larix Plants and Its Special Medium

technical field

The invention relates to a method for inducing embryogenic callus of larix plants and a special culture medium thereof.

Background technique

At present, a variety of angiosperms have been successfully propagated in vitro. However, the asexual reproduction of pines and fir plants is much more difficult than other species. For example, the induction of embryogenic tissues mostly comes from young tissues (such as immature embryos). ); the maturation and germination of somatic embryos strictly depend on the genotype of explants; the induction rate and conversion rate of seedlings are low, so further research is still needed. The induction of embryogenic callus of pine and fir plants is very sensitive to various components and physical and chemical conditions of the medium. So far, there is no effective induction method and effective medium for the embryogenic callus of pine and fir plants, resulting in somatic cells. Studies on embryogenesis and organogenesis are relatively lagging behind. Adjusting the medium formula of forest callus and improving the induction method are obviously of great significance for asexual reproduction such as somatic embryogenesis and organogenesis, as well as the protection of the ecological environment.

Japanese larch (Larix leptolepis) is native to Japan, with a height of up to 30 meters and a diameter at breast height of 1 meter. Through breeding, Japanese larch has been distributed in the Heilongjiang to Yangtze River basins in my country. It has the characteristics of fast growth, straight trunk, and strong adaptability. This species can be used as a green tree species for roads, railways and farmland shelterbelts. It is the main timber tree species in forestry production, and it is also an important paper-making raw material. Japanese larix can be propagated by seeds, but the seeds are strong and have obvious harvests in poor years, and there may be no harvest in poor years, which cannot meet the needs of large-scale seedling cultivation. The development of asexual forestry is an important way to create fast-growing and high-yielding timber. The use of tissue culture to expand the propagation of excellent tree species has more advantages and potential than conventional asexual cuttings. It is not restricted by factors such as seasons and the number of materials taken, and is convenient for intensive and factory production. It has important practical significance and application value to use tissue culture technology to accelerate reproduction to meet the needs of forestry production and environmental protection.

Contents of the invention

The object of the present invention is to provide a method for inducing callus of larch plants and a special culture medium thereof.

The special medium for inducing callus of larix plants provided by the present invention is a DCR medium containing the following components: vitamin B ₁ (VB ₁ ) 0.4-0.6 mg/L, vitamin B ₆ (VB ₆ ) 0.4- 0.6mg/L, Niacin 0.4-0.6mg/L, Inositol 80-120mg/L, Glutamine 300-500mg/L, Casein hydrolysate (CH) 400-600mg/L, 2 , 4-dichlorophenoxyacetic acid (2,4-D) 0.5-4mg/L, 6-benzylaminopurine (6-BA) 0.4-0.6mg/L, sucrose 29.5-30.5g/L and agar 6.5-7.5 g/L.

The special medium for inducing larch callus provided by the present invention is preferably a DCR solid medium containing the following components: 0.5 mg/L vitamin B ₁ (VB ₁ ), 0.5 mg/L vitamin B ₆ (VB ₆ ) , 0.5mg/L niacin, 100mg/L inositol, 400mg/L glutamine (Glutamine), 500mg/L hydrolyzed casein (caseinhydrolysate, CH), 2mg/L 2,4-D, 0.5mg/L 6 - BA, 30 g/L sucrose and 7.0 g/L agar.

The pH value of the special culture medium for inducing larch callus is 5.7-5.9, preferably 5.8.

The DCR medium is composed of macroelements, trace elements and iron salts, wherein macroelements: 340mg/L KNO ₃ , 556mg/L CaNO ₃ ·4H ₂ O, 400mg/L NH ₄ NO ₃ , 170mg/L KH ₂ PO ₄ , 85mg/LCaCl ₂ ·2H ₂ O, 370mg/L MgSO ₄ ; trace elements: 6.2mg/L HBO ₃ , 8.6mg/L ZnSO ₄ ·7H ₂ O, 22.3mg/L MnSO ₄ ·H ₂ O , O.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.83mg/L KI, O.25mg/LCuSO ₄ ·5H ₂ O, 0.025mg/L CoCl ₂ ; iron salt composition: 5.57g/L FeSO ₄ , 7.45g/L _Na2EDTA .

The method for inducing callus of Larix genus plant provided by the present invention is to induce and cultivate explants on the above medium.

The explants are seed immature embryos, which are obtained according to the following method: picking cones in the stage of pre-cotyledon zygotic embryos, refrigerating at a low temperature of 4-6°C for 26-30 days, and extracting from the cones Remove the seeds, and detach the young embryos from the seeds.

The induction culture condition is 23-27° C., cultured in dark conditions protected from light.

The induction culture time is 26-32 days, preferably 28-30 days.

The method and the special culture medium of the invention are suitable for inducing callus of all Larix genus plants, especially for inducing embryogenic callus of Japanese larch, North China larch, Changbai larch and the like.

Sucrose and other organic components contained in the special medium for inducing larch callus of the present invention are the main nutrients for callus induction and growth, and maintain the solid state environment of the medium with agar 6.5-7.5g/L; The combination of 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5-4mg/L + 6-benzylaminopurine (6-BA) 0.4-0.6mg/L, the higher concentration of 2,4-D has It is conducive to the induction of callus; in addition, the special medium with pH5.7-5.9 provides the necessary slightly acidic environment for the induction and growth of embryogenic callus. The method of the present invention combines the use of the special medium to induce the embryogenic callus of Larix genus plants, the induction effect is good, and the induction rate reaches 71.4%, which brings great convenience for future research on somatic embryogenesis and organogenesis. It has great practical application value.

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1. Induction of embryogenic callus of Japanese larch and its effect.

1. Preparation of induction medium

Induction medium: DCR medium is used as the basic medium, and its composition is DCR + vitamin B ₁ (VB ₁ ) 0.5 mg/L + vitamin B ₆ (VB ₆ ) 0.5 mg/L + niacin 0.5 mg/L + inositol 100 mg/L + Glutamine (Glutamine) 400mg/L+ hydrolyzed casein (casein hydrolysate, CH) 500mg/L+2,4-D2mg/L+6-BA 0.5mg/L+ sucrose (surcose) 30g/L+ agar (agar) 7g/ L, the pH value of the culture medium is 5.8.

Among them, the composition of the DCR medium is: macroelements: 340mg/L KNO ₃ , 556mg/L CaNO ₃ 4H ₂ O, 400mg/L NH ₄ NO ₃ , 170mg/L KH ₂ PO ₄ , 85mg/L CaCl ₂ . 2H ₂ O, 370mg/L MgSO ₄ ; trace elements: 6.2mg/L HBO ₃ , 8.6mg/L ZnSO ₄ ·7H ₂ O, 22.3mg/L MnSO ₄ ·H ₂ O, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.83mg/L KI, 0.25mg/L CuSO ₄ ·5H ₂ O, 0.025mg/L CoCl ₂ ; iron salt composition: 5.57g/L FeSO ₄ , 7.45g/L Na ₂ EDTA.

2. Induction of Japanese larch callus

1. After the immature Japanese larch cones from the embryo development to the pre-cotyledon stage of the zygotic embryo stage are plucked, they are stored cold for 30 days at a low temperature of 4°C, then the seeds are taken out from the cones, and the seeds are mixed with 70% (volume percentage content) ) alcohol infiltration for 1min, rinsed with sterile water for 3 times, then put into 0.1% (mass percentage) mercuric chloride solution for sterilization for 5-10min, rinsed with sterile water for three times, the young embryos (cotyledons) Prezygotic embryo) as an explant, the explant is transferred in the Erlenmeyer flask (40ml culture medium) that the induction medium of step 1 is housed, and the number of explants in each Erlenmeyer flask is 6-8 and is placed in the dark In an environment with a temperature of 25°C, the culture was induced for 28 days. The callus formation was observed, and the callus induction rate was counted (callus induction rate=number of explants producing callus/number of inoculated explants). At the same time, the induction medium prepared by replacing the DCR basic medium in the induction medium prepared in step 1 with MS basic medium was used as a control, and the other culture methods were the same.

The results are shown in Table 1. DCR is more frequently induced in the basic medium than MS. Experiments show that the DCR induction effect in the basic medium is better, and the induction frequency reaches 71%.

Table 1. Effects of different basal media on callus induction frequency of Japanese larch basic medium Number of immature embryos inoculated (pieces) The number of wounds produced (pieces) Induction frequency (%) DCR 63 44 71.0 MS 44 14 31.8

2. Prepare the induction medium according to the method of step 1, wherein, except that the concentration of 2,4-D is 0.0mg/L, 0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, , other ingredients are all identical with the induction medium composition of step 1, carry out Japanese larch callus induction culture according to the method for step 1, observe the callus formation situation, count callus induction rate (callus induction rate=production The number of callus explants/the number of inoculated explants) to compare the effects of different concentrations of 2,4-D on the induction frequency. The results are shown in Table 2, showing that 2mg/L2, 4-D+0.5mg/L 6-BA hormone combination is the best combination and concentration for inducing larch immature embryos to produce embryogenic callus, and its callus induction frequency was 71.4%.

Table 2. Effects of different 2,4-D concentrations on callus induction frequency of Japanese larch 2,4-D concentration (mg/L) 6-BA concentration (mg/L) Number of immature embryos inoculated (pieces) The number of wounds produced (pieces) Induction frequency (%) 0.0 0.5 85 0 0.0 0.5 0.5 125 55 44.0 1.0 0.5 130 80 61.5 2.0 0.5 105 75 71.4 4.0 0.5 85 35 41.2

Claims (10)

1. A special medium for inducing callus of larix plants, which is a DCR medium containing the following components: vitamin B ₁ 0.4-0.6mg/L, vitamin B ₆ 0.4-0.6mg/L, niacin 0.4 -0.6mg/L, inositol 80-120mg/L, glutamine 300-500mg/L, hydrolyzed casein 400-600mg/L, 2,4-dichlorophenoxyacetic acid 0.5-4mg/L, 6-benzyl Aminopurine 0.4-0.6mg/L, sucrose 29.5-30.5g/L and agar 6.5-7.5g/L. 2. The medium according to claim 1, characterized in that: the special medium for inducing callus of larch plants is a DCR solid medium containing the following components: 0.5 mg/L vitamin B ₁ , 0.5mg/L vitamin _B6 , 0.5mg/L niacin, 100mg/L inositol, 400mg/L glutamine, 500mg/L hydrolyzed casein, 2mg/L 2,4-dichlorophenoxyacetic acid, 0.5mg /L 6-benzylaminopurine, 30g/L sucrose and 7.0g/L agar. . 3. The medium according to claim 1 or 2, characterized in that the pH value of the special medium for inducing embryogenic callus of Larix genus plants is 5.7-5.9. 4. A method for inducing embryogenic callus of Larix genus plants, comprising inducing and culturing the explants on the special medium as claimed in claim 1 or 2 or 3. 5. The method according to claim 4, characterized in that the explants are immature seed embryos. 6. The method according to claim 5, characterized in that the explant is a seed embryo developed to a prezygotic embryo of the cotyledons. 7. The method according to claim 6, characterized in that: the immature embryos of the seeds are obtained according to the following method: picking cones in the stage of precotyledonous zygotic embryos, and refrigerating at a low temperature of 4-6°C for 26-30 days , remove the seeds from the cones, and detach the young embryos from the seeds. 8. The method according to any one of claims 4-7, characterized in that: the induction culture condition is 23-27°C, dark culture; the induction culture time is 26-32 days, preferably 28-30 days sky. 9. The method according to claim 8, wherein the larch plant is Japanese larch, North China larch or Changbai larch. 10. The method of claim 9, wherein said larch is Japanese larch.