CN111374053B

CN111374053B - Method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots

Info

Publication number: CN111374053B
Application number: CN202010254891.9A
Authority: CN
Inventors: 熊欢; 毛小雪; 邹锋; 袁德义; 王荣洁; 唐窈
Original assignee: Central South University of Forestry and Technology
Current assignee: Central South University of Forestry and Technology
Priority date: 2020-04-02
Filing date: 2020-04-02
Publication date: 2022-05-10
Anticipated expiration: 2040-04-02
Also published as: CN111374053A

Abstract

The invention relates to a method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots. The method comprises the following steps: s1, taking down the fresh leaves of the castanea henryi tissue culture seedlings and stabbing the fresh leaves; and S2, transferring the punctured fresh leaves into a second MS culture medium for inoculation, and culturing in the dark and then in the light to generate adventitious roots, wherein the second MS culture medium further comprises sucrose, agar, IBA and zeatin. The method is not affected by seasons, and can be used for annual culture production; the adventitious roots are directly generated through the leaves, callus induction is not needed, the culture time is shortened, and the cost is favorably reduced.

Description

Method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots.

Background

Castanea henyi (Castanea henyi) is a chestnut plant of Fagaceae (Fagaceae), is one of woody grain tree species unique in China, and is called 'iron stem crops'. Castanea henryi is mainly distributed in tropical hilly and mountainous areas of south asia of Qinling mountains of China, including Zhejiang, Fujian, Hunan, Jiangxi, Sichuan, Guizhou and the like. The castanea henryi nuts are rich in starch, fragrant, sweet and delicious, and are excellent forest health-care food. The bark and hull of Castanea henryi contains tannin, and can be used for extracting tannin extract; the root can be used as a medicine, the decoction can be used for treating sore toxicity by external washing, and the adventitious root can be used as a sustainable resource for being used as a medicine. The method for developing the production of the castanea henryi can not only guarantee the food safety of China, but also green barren mountains, and realize the green economic growth of mountain areas.

At present, the propagation of the castanea henryi seedlings is mainly carried out by seedling sowing, cuttage, grafting and other modes, but the growth period is generally longer, and meanwhile, the propagation is easily influenced by factors such as external environmental conditions and the like. The totipotency of the cells can be utilized to regenerate plants through tissue culture, so that the propagation coefficient is high, the material selection and propagation can be carried out at any growth stage, conditions are provided for fine variety propagation and industrial seedling culture, and the method is gradually applied to forestry production practice.

At present, most of reports about the culture of the castanea henryi tissues include the establishment of a castanea henryi embryo regeneration system, the induction of castanea henryi callus, the research of different explant disinfection treatment methods and the like, the castanea henryi leaves contain substances such as tannin, polyphenol and the like, the problems of easy browning, low multiplication coefficient, difficult rooting and the like exist, and the research on the induction of adventitious roots of the castanea henryi tissue culture seedling leaves is not reported. Therefore, the direct induction of the adventitious roots by the leaves has important significance for the researches such as the promotion of castanea henryi tissue culture, the construction of a genetic transformation system and the like.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the technical problem of how to directly generate adventitious roots by castanea henryi through leaves.

In order to solve the technical problem, the invention provides a method for inducing the leaves of the tissue culture seedlings of castanea henryi to generate adventitious roots.

The invention provides a method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots, which comprises the following steps:

s1, taking down the fresh leaves of the castanea henryi tissue culture seedlings and stabbing the fresh leaves;

and S2, transferring the punctured fresh leaves into a second MS culture medium for inoculation, and culturing in the dark and then in the light to generate adventitious roots, wherein the second MS culture medium further comprises sucrose, agar, IBA and zeatin.

Preferably, before step S1, the method further comprises the step of inoculating the seed embryo of the castanea henryi into a first MS culture medium to culture to obtain a tissue culture seedling with a seedling age of 25-65 d, wherein the first MS culture medium further comprises sucrose and agar, and the pH of the first MS culture medium is 5.80; the concentration of sucrose in the first MS culture medium is 25-30g/L, and the concentration of agar is 6.0-6.5 g/L.

Preferably, the pricking of the fresh tender leaves in step S1 further comprises cutting the fresh tender leaves into pieces.

Preferably, in step S1, the fresh tender leaves are cut into blocks by:

taking out the blade midrib by using a blade, and dividing the blade midrib into first small blocks;

and/or, cutting the veins in the leaf with a blade to divide the mesophyll into second pieces.

Preferably, in step S1, the leaflet midvein is divided into the first pieces perpendicular to the leaflet midvein.

Preferably, in step S1, the first small block has a length of 1 cm; and/or the size of the second small block is 1.5cm x 1.0 cm.

Preferably, in step S2, the concentration of sucrose in the second MS culture medium is 30g/L, the concentration of agar is 6.5g/L, the concentration of IBA is 1.0-2.0mg/L, and the concentration of zeatin is 5-10 μm/L.

Preferably, in step S2, the fresh leaves after being pricked are transferred to the second MS medium for inoculation, and the dark culture is performed for 5-7 days, and then the light culture is performed for 20-30 days to generate adventitious roots.

Further, the seed embryo of the castanea henryi is obtained by the following steps: sterilizing semen Castaneae with 75% ethanol for at least 90s, and washing with sterile water for at least 3 times; sterilizing with 0.1% mercuric chloride solution for no less than 9min, washing with sterile water for no less than 5 times, peeling semen Castaneae, washing with tap water for no less than 20min, sterilizing on a clean bench, and cutting off part of cotyledon with sterile blade to obtain the embryo.

Compared with the prior art, the invention has the advantages that: the method is not affected by seasons, and can be used for annual culture production; adventitious roots are directly generated through the leaves, callus induction is not needed, the culture time is shortened, the cost is reduced, and technical references are provided for researches and production applications of chinquapin germplasm resource preservation, establishment of a genetic transformation system, mycorrhizal symbiosis early signal identification and the like.

Drawings

The features and advantages of the present invention will be more clearly understood by reference to the accompanying drawings, which are illustrative and not to be construed as limiting the invention in any way, and in which:

a, B, C and D in FIG. 1 are graphs of the adventitious roots results obtained by the four treatment methods 1, 2, 3 and 4 in example 1, respectively.

A and B in FIG. 2 are graphs showing the results of rooting induction of the leaves of the tissue-cultured seedlings of 25 to 35d and 55 to 65d in example 2.

FIG. 3 shows the tissue culture seedlings of Castanea henryi with the top cut off with scissors after the leaves were inoculated.

FIG. 4 is a schematic view of four blade processing modes according to the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The specific embodiment provides a method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots, which comprises the following steps:

1) sterilizing semen Castaneae with 75% ethanol for at least 90s, and washing with sterile water for at least 3 times; sterilizing the Castanea henryi seeds with 0.1% mercuric chloride solution for at least 9min, washing the Castanea henryi seeds with sterile water for at least 5 times, peeling the Castanea henryi seeds, washing with tap water for at least 20min, sterilizing again on an ultraclean workbench, and cutting off partial cotyledons with a sterile blade to obtain embryo;

2) inoculating the seed embryo of the castanea henryi to a first MS culture medium subjected to high-temperature sterilization, placing the first MS culture medium in a light culture chamber for culture to obtain a culture seedling with the seedling age of 25-65 d, wherein the first MS culture medium further comprises 25-30g/L of sucrose and 6.0-6.5g/L of agar, and the pH value of the first MS culture medium is 5.80; the temperature of the light culture room is 27 +/-1 ℃, the light period is 14/10h (namely 14h of light in 24h of a day, 10h of dark for treatment, and the like), and the light intensity is 50molm^-2s^-1。

3) Taking off the fresh leaves of the castanea henryi cultivation seedlings, cutting the fresh tender leaves into blocks and stabbing the fresh leaves; the manner of cutting the fresh tender leaves into blocks comprises the following steps:

taking out the blade midrib by using a blade, and dividing the blade midrib into first small blocks with the length of 1cm by being vertical to the blade midrib; preferably, the midrib components of the leaves are divided into three equal parts;

alternatively, the mesophyll of the leaf is cut with a blade to divide the mesophyll into second pieces of 1.5cm by 1.0 cm.

4) And transferring the punctured fresh leaves into a second MS culture medium, inoculating the fresh leaves into a dark culture medium for 5-7 days, and then transferring the fresh leaves into an illumination culture medium for 20-30 days to generate adventitious roots, wherein the second MS culture medium further comprises 25-30g/L of sucrose, 6.0-6.5g/L of agar, 1.0-2.0mg/L of IBA and 5-10 mu m/L of zeatin. 1.0-2.0mg/L IBA promotes rooting, and 5-10 mu m/L zeatin promotes callus proliferation.

5) Cutting off the top end of the cultured Castanea henryi seedling after inoculating leaves with a pair of scissors, and culturing in a light culture chamber to provide explant material for the next leaf to induce adventitious roots.

In the present invention, the first MS medium or the second MS medium contains the following components: KNO₃1900mg/L，NH₄NO₃ 1650mg/L，KH₂PO₄ 170mg/L，MgSO₄.7H₂O 370mg/L,CaCl₂.2H₂O 440mg/L,KI 0.83mg/L,H₃BO₃ 6.2mg/L,MnSO₄.4H₂O 22.3mg/L,ZnSO₄.7H₂O 8.6mg/L,Na₂MoO₄.2H₂O 0.25mg/L,CuSO₄.5H₂O 0.025mg/L,CoCl₂.6H₂O 0.025mg/L,Na₂EDTA 37.25mg/L,FeSO₄.7H₂27.85mg/L of O, 100mg/L of inositol, 2mg/L of glycine, 0.1mg/L of thiamine hydrochloride, 0.5mg/L of pyridoxine hydrochloride and 0.5mg/L of nicotinic acid.

To further illustrate the methods set forth in the detailed description of the invention, specific examples are set forth below.

Example 1

A method for inducing castanea henryi tissue culture seedling leaves to directly generate adventitious roots comprises the following steps:

s1, culturing the castanea henryi tissue culture seedlings: peeling semen Castaneae, washing with tap water for 20min, sterilizing with 75% ethanol for 90s on a clean bench, and washing with sterile water for 3 times; then sterilizing with 0.1% mercuric chloride solution for 9min, and washing with sterile water for 5-6 times. On the filter paper after the high temperature sterilization, a part of cotyledons of the seeds was excised with a sterile blade to obtain embryos of 1cm × 0.6cm × 0.6cm in size. Inoculating to the first stage after high-temperature sterilizationIn an MS medium. Placing the culture medium in a light culture room for culturing for 25-35 days. The temperature of the illumination culture room is 27 +/-1 ℃; the illumination period is 14/10h, and the illumination intensity is 50mol^-2s^-1. The culture media all contain 30g/L of sucrose and 6.5g/L of agar, and the pH value is 5.80.

S2, inducing adventitious roots of castanea henryi leaves: selecting the tissue culture seedlings of castanea henryi with strong growth and seedling age of 25-35d, taking down healthy and tender leaves on a superclean bench by using sterilized tweezers, wherein the processing mode of the leaves is shown in table 1, referring to fig. 4, a knife tip is used for pricking wounds on explants, and the abaxial surfaces of the leaves are sequentially inoculated in an MS induction culture medium (namely a second MS culture medium). The leaves were inoculated, cultured for 7 days under dark culture conditions, and then cultured for 30 days under light conditions to grow adventitious roots, and the results are shown in FIG. 3. The temperature of the illumination culture room is 27 +/-1 ℃; the illumination period is 14/10h, and the illumination intensity is 50mol^-2s^-1. The culture media all contain 30g/L of sucrose, 6.5g/L of agar, 1.0mg/L of IBA and 5 mu m/L of zeatin, and the pH value is 5.80.

According to the formula:

adventitious root induction rate (%) (number of explants inducing adventitious roots/total number of inoculated explants) × 100%;

the rooting number (strips/plant) is the total rooting number/the number of explants with adventitious roots induced;

average root length (cm) — total root length/total root number of the induced adventitious roots;

the results are shown in Table 2.

TABLE 1 different treatment modalities for the leaves

Treatment number	Blade processing mode
		1	Taking off the leaves of the tissue culture seedling with tweezers without cutting
2	Taking out midrib vein of leaf with blade, and dividing into small pieces of 1.0cm in length
		3	Cutting off mesophyll with blade to divide mesophyll into small pieces of 1.5cm × 1.0cm
4	The blade is divided into three parts which are more equal by the blade which is vertical to the midrib of the blade

TABLE 2 rooting percentage, number of roots and length of leaves treated in different ways

Treatment number	Rooting percentage of leaves (%)	Root number (strip/plant)	Average root length (cm)
				1	29.89	3.59	2.98
2	32.61	2.33	2.05
				3	39.29	5.27	1.67
4	15.56	2.36	2.81

Example 2

s1, culturing the castanea henryi tissue culture seedlings: peeling semen Castaneae, washing with tap water for 20min, sterilizing with 75% ethanol for 90s on a clean bench, and washing with sterile water for 3 times; and then sterilizing for 9min by using 0.1% mercuric chloride solution, and washing for 5-6 times by using sterile water. On the filter paper after the high temperature sterilization, a part of cotyledons of the seeds was excised with a sterile blade to obtain embryos of 1cm × 0.6cm × 0.6cm in size. Inoculating into the first MS culture medium after high-temperature sterilization. Placing the culture medium in a light culture room for culturing for 25-35 days. The temperature of the illumination culture room is 27 +/-1 ℃; the illumination period is 14/10h, and the illumination intensity is 50mol^-2s^-1. The culture media all contain 30g/L of sucrose and 6.5g/L of agar, and the pH value is 5.80.

S2, inducing adventitious roots of castanea henryi leaves: selecting the healthy and robust castanea henryi sterile tissue culture seedlings with the seedling age of 25-35d in the step S1 and the healthy and robust castanea henryi sterile tissue culture seedlings with the seedling age of 55-65d obtained in the step S3, taking down healthy and young leaves on an ultraclean workbench respectively by using sterilized tweezers, pricking wounds on the leaves by using a tool nose of a sterile blade, and sequentially inoculating the leaves in an MS induction culture medium (namely a second MS culture medium) with the abaxial surfaces of the leaves facing downwards. After inoculating the leaves, placing the leaves under the dark culture condition for culturing for 7d, placing the leaves under the illumination condition for continuously culturing for 30d, and then growing adventitious roots. The temperature of the illumination culture room is 27 +/-1 ℃; the light period is 14/10h,illumination intensity of 50 mol-²s^-1. The culture media all contain 30g/L of sucrose, 6.5g/L of agar, 1.0mg/L of IBA and 5 mu m/L of zeatin, and the pH value is 5.80.

S3, processing the castanea henryi tissue culture seedlings: and (3) cutting off the top ends of the castanea henryi tissue culture seedlings with the seedling ages of 25-35d and inoculated with the leaves in the step S2 by using scissors, and placing the castanea henryi tissue culture seedlings in an illumination culture room for culturing for 30 d.

The formula in example 1 was used for the calculation, and the results are shown in Table 3.

TABLE 3 rooting percentage, rooting number and root length of leaves of different seedling ages

Miao age (d)	Rooting percentage of leaves (%)	Root number (strip/plant)	Average root length (cm)
				25-35	31.82	2.88	5.89
55-65	29.23	3.89	2.24

And (4) analyzing results:

as shown in Table 2, the four ways have better rooting rate and rooting number by combining the different leaf inoculation ways, and the rooting rate and the rooting number of the leaves of the treatment group 3 are higher than those of other treatment groups, wherein the rooting rate of the leaves is 39.29%, the total rooting number is the most, and the effect is good.

As shown in Table 3, the results obtained by directly inducing rooting by using the leaves of the tissue culture seedlings with different seedling ages in the two treatment groups are not very different, so that the utilization rate of the leaf resources of the tissue culture seedlings of castanea henryi is improved, and the production cost is reduced.

Other beneficial effects are as follows:

1) the method has the advantages that the process of directly inducing the adventitious roots by taking the castanea henryi tissue culture seedling leaves as the explants is not more than 37 days, the growth period is short, the growth and development are fast, the callus induction stage is not needed, the culture time is greatly shortened, and the production cost is effectively reduced.

2) The method has the advantages of simple required reagent and low consumption; the used leaf material has low requirement on leaf age, greatly increases the source of the material, is economic and sustainable, and simultaneously ensures that the biological source of the explant is single and the genetic background is consistent.

3) The method is cultured in an artificial controllable environment, and the direct generation of the adventitious roots by the induction of the leaves can provide technical reference for the research related to the culture of the castanea henryi tissues.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

Claims

1. A method for inducing castanea henryi tissue culture seedling leaves to generate adventitious roots is characterized by comprising the following steps:

s2, transferring the punctured fresh leaves into a second MS culture medium for inoculation, and culturing in the dark firstly and then in the light to generate adventitious roots;

wherein the second MS medium consists of the following components: KNO₃1900mg/L、NH₄NO₃1650mg/L、KH₂PO₄170mg/L、MgSO₄·7H₂O370mg/L、CaCl₂·2H₂O440mg/L、KI0.83mg/L、H₃BO₃6.2mg/L、MnSO₄·4H₂O22.3mg/L、ZnSO₄·7H₂O8.6mg/L、Na₂MoO₄·2H₂O0.25mg/L、CuSO₄·5H₂O0.025mg/L、CoCl₂·6H₂O0.025mg/L、Na₂EDTA37.25mg/L、FeSO₄·7H₂O27.85mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, sucrose 25-30g/L, agar 6.0-6.5g/L, IBA 1.0.0-2.0 mg/L and zeatin 5-10 μm/L.

2. The method according to claim 1, wherein before step S1, the method further comprises inoculating the seed embryo of castanea henryi into a first MS culture medium, and culturing to obtain a tissue culture seedling with a seedling age of 25-65 days;

wherein the pH of the first MS medium is 5.80 and the first MS medium consists of: KNO₃1900mg/L、NH₄NO₃1650mg/L、KH₂PO₄170mg/L、MgSO₄·7H₂O370mg/L、CaCl₂·2H₂O440mg/L、KI0.83mg/L、H₃BO₃6.2mg/L、MnSO₄·4H₂O22.3mg/L、ZnSO₄·7H₂O8.6mg/L、Na₂MoO₄·2H₂O0.25mg/L、CuSO₄·5H₂O0.025mg/L、CoCl₂·6H₂O0.025mg/L、Na₂EDTA37.25mg/L、FeSO₄·7H₂O27.85mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, sucrose 25-30g/L, agar 6.0-6.5 g/L.

3. The method as claimed in claim 1, wherein the stabbing of the fresh tender leaves in step S1 further comprises cutting the fresh tender leaves into pieces.

4. The method as claimed in claim 3, wherein in step S1, the fresh tender leaves are cut into blocks by the method comprising:

alternatively, the mesophyll is divided into second pieces by cutting the midrib of the leaf with a blade.

5. The method of claim 4, wherein in step S1, the leaflet midvein is divided into the first pieces perpendicular to the leaflet midvein.

6. The method according to claim 4, wherein in step S1, the first piece has a length of 1 cm; and/or the size of the second small block is 1.5cm x 1.0 cm.

7. The method according to claim 1, wherein in step S2, the fresh leaves after being pricked are transferred to the second MS culture medium for inoculation, and the dark culture is performed for 5-7 days, and then the light culture is performed for 20-30 days to generate adventitious roots.

8. The method according to claim 2, characterized in that said embryo of castanea henryi is obtained by the following steps: sterilizing semen Castaneae with 75% alcohol for 90s, and washing with sterile water for 3 times; sterilizing with 0.1% mercuric chloride solution for 9min, washing with sterile water for 5 times, peeling semen Castaneae, washing with tap water for 20min, sterilizing again on a clean bench, and cutting off part of cotyledon with sterile blade to obtain embryo.