Method for inducing callus of potato "kangsin 13"

Abstract

The present application belongs to the technical field of plant cell culture, and relates to a method for inducing callus of potato 'Kexin 13'. The method comprises the following steps: (1) taking potato 'Kexin 13' explants and transferring them to callus induction medium to form callus, wherein the induction medium is based on MS medium and supplemented with 6-benzylaminopurine, kinetin and 2,4-dichlorophenoxyacetic acid, and has a pH of 5.8-6; (2) taking the callus formed in step (1) and transferring it to differentiation medium to form adventitious buds, wherein the differentiation medium is based on MS basic medium and contains 6-benzylaminopurine, kinetin, gibberellin and myo-inositol, and has a pH of 5.8-6. Through induction culture and differentiation culture, the callus of potato 'Kexin 13' has a high induction rate and differentiation rate.

Description

Technical Field

[0001] This invention belongs to the field of plant cell culture technology and relates to a method for inducing callus tissue of potato “Kexin 13”. Background Technology

[0002] Potato (Solanum tuberosum L.), also known as potato, is a tetraploid herbaceous plant belonging to the Solanaceae family and the Solanum genus that completes germination, growth, flowering, and fruiting in one year. Potatoes have the advantages of rich nutrition, strong adaptability, and high yield. "Kexin 13" potato is a variety bred from Mira as the base material through two generations of self-pollination. Its original pedigree number was S9-9-(12). This potato variety is a mid-to-late maturing variety, with approximately 97 days from emergence to maturity. It is characterized by large and uniform tubers, with large and medium-sized tubers accounting for about 77%. It has excellent disease resistance, including resistance to potato leafroll virus, moderate resistance to late blight, and resistance to field allergy to heavy mosaic virus. Fresh potatoes are rich in vitamin C, starch, and reducing sugars. Efficient and stable potato plant regeneration technology is the premise and foundation for the establishment of genetic transformation technology, rapid acquisition of superior seedlings, and screening of mutants. Summary of the Invention

[0003] The technical problem solved by this invention is to provide a method for inducing callus tissue of potato "Kexin 13" and a stable and efficient potato plant regeneration technology. Through induction culture and differentiation culture steps, and by rationally selecting and scientifically proportioning different components in the culture medium, the callus tissue of potato "Kexin 13" has a high induction rate and differentiation rate.

[0004] The present invention will solve the above problems through the following technical solutions:

[0005] This invention provides a method for inducing callus tissue from potato variety "Kexin 13", comprising the following steps:

[0006] (1) Potato explants of “Kexin 13” were transferred to callus induction medium for induction culture to form callus. The induction medium was based on MS basal medium containing 1.9-2.1 mg·L⁻¹. -1 6-Benzylaminopurine, 0.3-0.5 mg / L -1 Kinetin and 0.5-1 mg / L -1 2,4-Dichlorophenoxyacetic acid, pH 5.8-6;

[0007] (2) Take the explants from the callus tissue formed in step (1) and transfer them to a differentiation medium for callus tissue differentiation culture to form adventitious shoots. The differentiation medium is based on MS basal medium, which contains 1.9-2.1 mg·L⁻¹. -16-benzylaminoadenine, 2.9-3.1 mg·L -1 Kinetin, 0.4-0.6 mg·L -1 Gibberellin and 0.1 g·L -1 Myo-inositol, pH 5.8-6.

[0008] Further, in step (1), the explant is one of stem segments and microtuber slices; preferably stem segments.

[0009] Further, in step (1), the explant is subjected to sterilization and disinfection treatment, and the sterilization and disinfection step comprises: washing and soaking the explant with 70-80 wt% ethanol solution for 20-30 s, then soaking the explant with 8-10 wt% NaClO solution for 7-8 min for disinfection, and washing with sterilized water.

[0010] Further, in step (1), the induction culture conditions include illumination of 1500-2500 Lx and temperature of 22-26℃; preferably illumination of 2000 Lx and temperature of 24℃.

[0011] Further, in step (1), the induction culture time is 15-25 days; preferably 20 days.

[0012] Further, in step (2), the differentiation culture conditions include illumination of 1500-2500 Lx and temperature of 22-26℃; preferably illumination of 2000 Lx and temperature of 24℃.

[0013] Further, in step (2), the differentiation culture time is 15-25 days; preferably 20 days.

[0014] Further, it further comprises step (3): after the adventitious buds differentiated and sprouted in step (2), the sprouts are transferred to a rooting culture medium for inducing rooting.

[0015] Further, the rooting culture medium is based on MS basic medium, and contains 0.9-1.1 mg·L -1 α-naphthaleneacetic acid.

[0016] The present application has the beneficial effects that the present application provides a method for inducing callus of potato "Kexin 13", and establishes a stable and efficient potato plant regeneration technology. Through the induction culture and differentiation culture steps, reasonable selection and scientific proportioning of different components in the culture medium make the callus of potato "Kexin 13" have a high induction rate and differentiation rate. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : Photos of induction of callus in Examples 1-2.

[0018] Figure 2: Figure of Example 3-4 inducing callus formation.

[0019] Figure 3 : Figure of Comparative Example 1-2 inducing callus formation.

[0020] Figure 4 : A is Figure of Example 1 inducing callus differentiation; B is Figure of Comparative Example 3 inducing callus differentiation.

[0021] Figure 5 : Figure of Example 1 inducing seedling rooting. DETAILED DESCRIPTION

[0022] The application will be further described in conjunction with the specific embodiments and drawings, obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0023] The application provides a callus induction culture method of potato "Kexin 13", comprising the following steps:

[0024] (1) Taking potato "Kexin 13" explants to be grafted into a callus induction culture medium to form callus by induction culture, wherein the induction culture medium is based on MS basic medium, and contains 1.9-2.1 mg·L -1 6-benzylaminopurine (hereinafter referred to as 6-BA), 0.3-0.5 mg·L -1 kinetin (hereinafter referred to as KT) and 0.5-1 mg·L -1 2,4-dichlorophenoxyacetic acid (hereinafter referred to as 2,4-D), and the pH is 5.8-6;

[0025] (2) Taking the callus explants formed in step (1) to be grafted into a differentiation culture medium to form adventitious buds by callus differentiation culture, wherein the differentiation culture medium is based on MS basic medium, and contains 1.9-2.1 mg·L -1 6-BA, 2.9-3.1 mg·L -1 KT, 0.4-0.6 mg·L -1 gibberellin (hereinafter referred to as GA3) and 0.1 g·L -1 myo-inositol, and the pH is 5.8-6.

[0026] In a preferred embodiment of the application, in step (1), the explants are stem segments.

[0027] In a preferred embodiment of the application, in step (1), the explants are microtuber slices.

[0028] In a preferred embodiment of the present application, in step (1), the explant is an explant that has been thoroughly disinfected. Disinfection is mainly to kill the microorganisms attached to the surface of the explant without damaging the explant.

[0029] In a preferred embodiment of the present application, in step (1), the explant is an explant that has been washed with a 75 wt% ethanol solution for 30 s, then disinfected by immersing the explant in an 8 wt% or 10 wt% NaClO solution for 7 or 8 min, and washed with sterile water for 2 or 3 times.

[0030] In a preferred embodiment of the present application, in step (1), the induction culture conditions are a light intensity of 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 Lx, and a temperature of 22, 23, 24, 25, or 26 °C.

[0031] In a preferred embodiment of the present application, in step (1), the induction culture conditions are a light intensity of 2000 Lx and a temperature of 24 °C.

[0032] In a preferred embodiment of the present application, in step (1), the induction culture time is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days.

[0033] In a preferred embodiment of the present application, in step (1), the induction culture time is 20 days.

[0034] In a preferred embodiment of the present application, in step (1), the concentration of 6-BA in the induction medium is 1.9 mg·L -1 , 2.0 mg·L -1 , or 2.1 mg·L -1 .

[0035] In a preferred embodiment of the present application, in step (1), the concentration of KT in the induction medium is 0.3 mg·L -1 , 0.4 mg·L -1 , or 0.5 mg·L -1 .

[0036] In a preferred embodiment of the present application, in step (1), the concentration of 2,4-D in the induction medium is 0.5 mg·L -1 , 0.6 mg·L -1 , 0.7 mg·L -1 , 0.8 mg·L -1 , 0.9 mg·L -1 , or 1.0 mg·L -1.

[0037] In a preferred embodiment of the present application, in step (1), the induction medium is based on MS basal medium comprising 2 mg·L -1 6-BA, 0.4 mg·L -1 KT and 0.5 mg·L -1 2,4-D.

[0038] In a preferred embodiment of the present application, in step (1), the pH of the induction medium is 5.8, 5.9 or 6.0.

[0039] In a preferred embodiment of the present application, in step (2), the differentiation culture conditions are a light intensity of 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 or 2500 Lx, and a temperature of 22, 23, 24, 25 or 26 °C.

[0040] In a preferred embodiment of the present application, in step (2), the differentiation culture conditions are a light intensity of 2000 Lx and a temperature of 24 °C.

[0041] In a preferred embodiment of the present application, in step (2), the differentiation culture time is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days.

[0042] In a preferred embodiment of the present application, in step (2), the differentiation culture time is 20 days.

[0043] In a preferred embodiment of the present application, in step (2), the concentration of 6-BA in the differentiation medium is 1.9 mg·L -1 , 2.0 mg·L -1 or 2.1 mg·L -1 .

[0044] In a preferred embodiment of the present application, in step (2), the concentration of KT in the differentiation medium is 2.9 mg·L -1 , 3.0 mg·L -1 or 3.1 mg·L -1 .

[0045] In a preferred embodiment of the present application, in step (2), the concentration of GA3 in the differentiation medium is 0.4 mg·L -1 , 0.5 mg·L -1 or 0.6 mg·L -1 .

[0046] In a preferred embodiment of the present application, in step (2), the concentration of myo-inositol in the differentiation medium is 0.1 g·L -1 , 0.2 g·L -1 , or 0.3 g·L -1 .

[0047] In a preferred embodiment of the present application, in step (2), the pH of the differentiation medium is 5.8, 5.9, or 6.0.

[0048] In a preferred embodiment of the present application, in step (2), the differentiation medium is based on MS basal medium, which contains 2 mg·L -1 6-BA, 3 mg·L -1 KT, 0.5 mg·L -1 GA3, and 0.1 g·L -1 myo-inositol.

[0049] In a preferred embodiment of the present application, further comprising, after the adventitious buds in step (2) are differentiated into seedlings, the seedlings are transferred to a rooting medium to induce rooting, and the rooting medium is based on MS basal medium, which contains 0.9-1.1 mg·L -1 α-naphthaleneacetic acid (hereinafter referred to as NAA).

[0050] The present application will be further described below in conjunction with examples and comparative examples.

[0051] It should be noted in advance that the explants selected in the specific embodiments are all taken from the vigorous seedlings of "Kexin No. 13" potato at 4 weeks of age.

[0052] The method for obtaining the above-mentioned explants is as follows:

[0053] (1) Stem segment: using a sterilized scissors, a stem segment of 0.5-1.5 cm without growth points is cut and laid flat on the callus induction medium.

[0054] (2) Leaf blade: a leaf blade is cut, the petiole is cut off, and using a sterilized scalpel, 2-4 incisions are made vertically to the leaf veins, and the leaf blade is placed on the callus induction medium with the leaf surface facing down.

[0055] (3) Microtuber slice: using a sterilized scalpel, the two ends of the microtuber are cut off, the microtuber skin is cut off to avoid the growth points on the skin affecting the subsequent test results, and then the microtuber is cut into slices of 2-4 mm thick.

[0056] The above-mentioned explants are washed and soaked in a 75 wt% ethanol solution for 30 s, then the explants are soaked in an 8 wt% NaClO solution for 7 min for sterilization, washed with sterilized water for 3 times, and the water is absorbed with a high-temperature sterilized filter paper.

[0057] Example 1:

[0058] This embodiment provides a method for inducing and culturing callus tissue of potato "Kexin 13", including the following steps:

[0059] (1) Potato explants of “Kexin 13” were transplanted into callus induction medium for induction culture to form callus tissue; the culture conditions were light intensity of 2000 Lx, temperature of 24℃, and culture time of 20 days; the induction medium was MS medium supplemented with 2 mg·L⁻¹. -1 6-BA + 0.4 mg·L -1 KT+0.5mg·L -1 2,4-D; The explants are stem segments, with 120 explants transferred onto each culture medium;

[0060] (2) All explants from the callus tissue formed in step (1) were transferred to differentiation medium for callus tissue differentiation culture to form adventitious shoots. The culture conditions were: light intensity 2000 Lx, temperature 24℃, and culture time 20 days. The differentiation medium was MS medium supplemented with 2 mg·L⁻¹. -1 6-BA + 3 mg·L -1 KT+0.5mg·L -1 GA3+0.1g·L -1 Inositol.

[0061] Example 2:

[0062] This embodiment provides a method for inducing and culturing callus tissue of potato "Kexin 13". The difference from Embodiment 1 is that in step (1), the induction medium is MS medium supplemented with 2 mg·L⁻¹. -1 6-BA + 0.4 mg·L -1 KT+1.0mg·L -1 2,4-D.

[0063] Example 3:

[0064] This embodiment provides a method for inducing and culturing callus tissue of potato “Kexin 13”, which differs from Embodiment 1 in that: in step (1), the explant is a miniature potato chip.

[0065] Example 4:

[0066] This embodiment provides a method for inducing and culturing callus tissue of potato "Kexin 13". The difference from Embodiment 3 is that in step (1), the induction medium is MS medium supplemented with 2 mg·L⁻¹. -1 6-BA + 0.4 mg·L -1 KT+1.0mg·L -12,4-D.

[0067] Comparative Example 1:

[0068] The difference between this comparative example and Example 1 is that the induction medium is MS medium supplemented with 2 mg·L-1 2,4-D. -1 6-BA + 0.4 mg·L-1 KT -1 KT + 0.1 mg·L-1 2,4-D -1 2,4-D.

[0069] Comparative Example 2:

[0070] The difference between this comparative example and Example 1 is that the explant is leaf.

[0071] Comparative Example 3:

[0072] The difference between this comparative example and Example 1 is that the differentiation medium does not contain myo-inositol.

[0073] The results of callus induction of Examples 1-4 and Comparative Examples 1-2 are shown in Table 1, wherein the induction rate is the ratio of the amount of explants successfully inducing callus in step (1) to the total amount of explants in step (1).

[0074] Table 1: Results of callus induction of Examples 1-4 and Comparative Examples 1-2

[0075]

[0076] The above results show that the induction rate and quality of callus are related to the hormone ratio, and different explant materials also have an impact. The callus of Examples 1 and 2 grows vigorously and densely, and is mostly yellow-green and light yellow, and has good growth conditions in the subsequent culture stage, with no obvious browning, and the callus rate is more than 95%. The medium of Comparative Example 1 also has a high callus rate, but compared with Examples 1 and 2, the callus induction period is long, and obvious browning occurs in the later stage.

[0077] Under the same medium conditions, the stem callus rate is more than 95%, and the callus grows densely and vigorously, which is the best explant material. The microtuber callus rate is 100%, and the callus grows densely and well, but due to the growth point on the surface of the microtuber, it will affect the test, and the material needs to be carefully treated to prevent the growth point from sprouting too early and affecting the test. The callus rate of leaf is low, because the leaf itself is more fragile than the stem, and after multiple treatments, the leaf will wither and die, so the leaf is difficult to operate in practice, resulting in a low callus induction rate.

[0078] The results of callus differentiation of Example 1 and Comparative Example 3 are shown in Table 2, wherein the differentiation rate is the ratio of the amount of successfully differentiated adventitious buds in step (2) to the total amount of explants in step (2).

[0079] Table 2: Results of callus differentiation of Example 1 and Comparative Example 3

[0080]

[0081] The induction rate of the callus without inositol is similar to that of the callus with inositol, and the above results show that, under the same culture time, the presence of inositol can promote the differentiation and growth of adventitious buds, thereby shortening the cultivation period. The presence of inositol can promote the growth of plants and the differentiation of adventitious buds, and improve the differentiation rate. Without inositol, the growth of plants is significantly slower, and the differentiation rate of adventitious buds is lower.

[0082] After the adventitious buds of the above Example 1 are differentiated and grow to 2-4 cm, they are transferred to a rooting medium to induce rooting, wherein the rooting medium is based on MS basic medium and contains 1 mg·L -1 NAA, and the culture conditions are: light intensity of 2000 Lx and temperature of 24°C. As shown in Figure 5 the transferred explants grow vigorously, and the roots and stems are developed.

[0083] It can be understood that the above specific examples are further illustrations of the present application, and are not used to limit the protection scope of the present application. All other decorations and modifications obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

Claims

1. A method for inducing rapid propagation of potato 'Kexin 13', characterized in that, Includes the following steps: (1) Potato explants of "Kexin 13" were transferred to callus induction medium for induction culture to form callus. The induction medium was based on MS medium supplemented with 1.9-2.1 mg·L⁻¹. -1 6-Benzylaminopurine, 0.3-0.5 mg / L -1 Kinetin and 0.5-1 mg / L -1 2,4-Dichlorophenoxyacetic acid, pH 5.8-6; the explant is either a stem segment or a miniature potato chip; (2) Take the explants from the callus tissue formed in step (1) and transfer them to the differentiation medium for callus tissue differentiation culture to form adventitious shoots. The differentiation medium is based on MS medium supplemented with 1.9-2.1 mg·L⁻¹. -1 6-Benzylaminopurine, 2.9-3.1 mg·L -1 Kinetin, 0.4-0.6 mg / L -1 Gibberellin and 0.1 g·L -1 Inositol, pH 5.8-6.

2. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, In step (1), the explants are sterilized and disinfected. The sterilization and disinfection steps include: washing and soaking with 70-80wt% ethanol solution for 20-30 seconds, then soaking the explants in 8-10wt% NaClO solution for 7-8 minutes for disinfection, and then washing thoroughly with sterile water.

3. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, In step (1), the induction culture conditions are 1500-2500 Lx light and 22-26℃ temperature.

4. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, In step (1), the induction culture time is 15-25 days.

5. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, In step (2), the differentiation culture conditions are 1500-2500 Lx light and 22-26℃ temperature.

6. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, In step (2), the differentiation culture time is 15-25 days.

7. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 1, characterized in that, It also includes step (3): after the adventitious buds described in step (2) differentiate into seedlings, they are then transferred to a rooting medium to induce rooting.

8. The method for inducing rapid propagation of potato variety "Kexin 13" according to claim 7, characterized in that, The rooting medium was based on MS medium, containing 0.9-1.1 mg·L⁻¹. -1 α-Naphthaleneacetic acid.

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