CN115152627B

CN115152627B - Tissue culture and rapid propagation method for Nandina domestica Brisson

Info

Publication number: CN115152627B
Application number: CN202210674334.1A
Authority: CN
Inventors: 马广莹; 周琴; 朱开元; 詹书侠; 周媛
Original assignee: Zhejiang Institute Of Landscape Plants And Flowers
Current assignee: Zhejiang Institute Of Landscape Plants And Flowers
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-02-28
Anticipated expiration: 2042-06-15
Also published as: CN115152627A

Abstract

The invention discloses a tissue culture and rapid propagation method of Nandina domestica Thunb of lemon yellow, which comprises the following steps: 1) Selection, treatment and sterility of explants; 2) Multiplication of aseptic seedlings; after the explant is cultured in the dark for 5 days, the explant is placed under an LED lamp, the light dark culture time is 18/6 hours, the culture temperature is 24 ℃, if no pollution exists after 10 days, the explant is considered to be sterilized successfully, the explant is immediately transferred to a culture medium with the number (1) of MS +0.6-0.7 mg/L BA +8g/L agar +30g/L sucrose and the pH value of 5.9 for culture for 30-35 days, the fasciculate multiplication buds are cut and not truncated, transferring all the materials to a culture medium (No. 2MS +0.2-0.3 mg/L BA +8g/L agar +30g/L sucrose) with pH adjusted to 5.9, continuing culturing, and after culturing for 40-45 days, rooting the proliferated bud short section, or inoculating the proliferated bud short section into the culture medium (No. 1) again for proliferation, wherein the culture time on the culture medium (No. 1) is not more than 35 days; 3) And (5) rooting culture of the proliferated seedlings. The method obviously reduces the pollution rate, the propagation coefficient reaches about 15 or even 20 on average, the production cost is obviously reduced, and the variety popularization of the Nandina domestica Brisson can be powerfully promoted.

Description

Tissue culture and rapid propagation method for Nandina domestica Brisson

Technical Field

The invention relates to a tissue culture and rapid propagation method of Nandina domestica Thunb.

Background

Lemon yellow Nandina domestica (B)Nandina domestica'Lemon-Lime') is a novel nandina species which is evergreen in all seasons, tall and straight in plant shape, verdant in leaf and very beautiful. The most remarkable characteristic of the variety is that the new leaves are yellow and green in the cool seasons such as winter and spring, are very striking, and are a new excellent variety which is popular in the garden market. As Nandina domestica belongs to short shrubs and has poor branching, the Nandina domestica cannot be subjected to large-scale propagation in modes of cuttage, grafting, sowing and the like from the viewpoint of production cost, and can only be subjected to tissue cultureThe propagation is expanded, the tissue culture technical effects of different varieties of Nandina domestica have larger difference, and particularly, the difference is obvious in the aspect of propagation coefficient, so that the development of a mature and reliable tissue culture system has great significance for propagation of the Limonia flava Nandina seedlings and is vital to industrial development.

Before the invention, some enterprises and scientific research institutions adopt a tissue culture mode to carry out tissue culture research and production on the Nandina domestica Brisson, including earlier stage research of the unit, the culture medium formula proposed in the prior report has a general multiplication coefficient of about 2-6 times in a 60-day production period, and under the existing tissue culture technology, the Nandina domestica Brisson seedling price of the lemon is higher, the seedling popularization is limited, if the tissue culture propagation efficiency can be obviously improved through technical improvement, the tissue culture propagation efficiency can be greatly improved, and the tissue culture method can bring positive influence on the popularization of the variety.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a tissue culture and rapid propagation method of Nandina domestica Brisson.

The technical scheme for realizing the purpose of the invention is as follows:

a tissue culture and rapid propagation method of Nandina domestica Thunb of lemon yellow comprises the following steps:

1) Selection, treatment and sterility of explants;

using pesticide azoxystrobin (25% of active ingredient, suspending agent) and pesticide (46% of active ingredient, water dispersible granule), soaking the vigorous top tender stem of Nandina domestica with cut leaves in 1000 times of water solution 2 days before sampling for 5 seconds, and cutting the top tender stem for 3 cm after 2-3 days for aseptic treatment; in the aseptic treatment, 0.1% mercuric chloride solution is adopted for disinfection for 15 minutes, the sterilized water is exchanged and washed for 10 minutes, the explant is cut into 1 cm long and is placed in MS minimal medium, and dark culture is carried out for 5 days for subsequent treatment;

2) Multiplication of aseptic seedlings;

after the explant is cultured in the dark for 5 days, the explant is placed under 2 18W LED lamps, the light dark culture time is 18/6 hours, the culture temperature is 24 ℃, if no pollution exists after 10 days, the explant is considered to be sterilized successfully, and then the explant is transferred to a culture medium (1) with MS +0.6-0.7 mg/L BA +8g/L agar +30g/L sucrose and pH 5.9 for culture for 30-35 days, the clustered proliferation buds are cut, transferring the whole to a culture medium (2) of MS +0.2-0.3 mg/L BA +8g/L agar +30g/L sucrose and pH regulation 5.9 without cutting, continuously culturing until the culture lasts for 40-45 days, and then cutting the proliferated buds into roots, or cutting the proliferated buds into the culture medium (1) again for proliferation, wherein the culture time on the culture medium (1) is not more than 35 days;

3) And (5) rooting culture of the proliferated seedlings.

In the method, the rooting of the proliferated seedling adopts a culture medium (3): 1/2MS +0.15 mg/L IBA +1.2 mg/L NAA +8g/L agar +30g/L sucrose, pH 5.9.

The invention has the beneficial effects that:

according to the technical scheme provided by the invention, firstly, the explant can be subjected to a sterile stage, the pollution rate is obviously reduced, and meanwhile, the propagation coefficient of the tissue culture seedling of the Nandina domestica Brisson can be averagely up to about 15, even 20 in a propagation stage within a 60-day growth period, so that the propagation coefficient is greatly improved compared with that of the conventional Nandina domestica Brisson, and the production cost is obviously reduced. The advantages of the invention are comprehensively considered, and the variety popularization of the lemon yellow nandina can be powerfully promoted.

Drawings

FIG. 1 is a graph showing the proliferation of a seedling in a flask at 35 days in Medium No. 5 in example 2.

FIG. 2 is a view showing the abduction of a No. 5 medium seedling in a flask for 35 days in example 2.

FIG. 3 is a 35-day-old culture of the cultured seedling on Medium No. 2 in example 2.

Detailed Description

The invention is further illustrated below with reference to the figures and examples.

Example 1: the influence of the sampling mode and pretreatment on the present invention

1) Experimental materials: the experiment is carried out by adopting 5-year strong and uniform-specification potted seedlings of the Nandina domestica Brisson. The experimental group treatment method is that about 3 cm of tip tissue of the Nandina domestica with vigorous growth is selected, 3 days before sampling, leaves are removed, and stems are reserved.

2) The experimental method comprises the following steps: the stems are soaked for 5 seconds by using 3 pesticide combinations, namely (1) No. treatment chlorothalonil (75% powder) 1000-time liquid, (2) No. treatment azoxystrobin (25% suspending agent) 1000-time liquid, (3) No. treatment (46% water dispersible granules) 1000-time liquid, (4) No. treatment azoxystrobin + 1000-time liquid, (5) No. treatment chlorothalonil + azoxystrobin 1000-time liquid, and (6) No. treatment chlorothalonil + 1000-time liquid. The control group was the young top treated with clear water. After 3 days, the stems were cut, washed with clear water, placed in a sterilized empty bottle, washed first with 75% alcohol for 30 seconds, then quickly soaked in 0.1% mercuric chloride solution for 15 minutes, while repeatedly shaken. Repeatedly washing the treated branches with sterile water for about 15 minutes, draining, cutting into small segments of about 1 cm, and inserting into a sterilized MS culture medium according to the growth direction for dark culture. After 5 days, the culture is carried out normally under the condition of 2 18-watt LED lamps, the light-dark culture time is 18/6 hours, and the temperature is 24 ℃. The contamination was counted after 5 days and 10 days. The treatments were performed with 30 explants each, and the contamination rate was counted twice 5 days and 10 days (excluding browned dead explants).

3) Experimental results and conclusions:

TABLE 1 evaluation of the Sterilization Effect of Nandina domestica, lemon yellow, on different pretreatments

Treatment method	5 days of pollution rate	10 days of pollution rate
			Clear water control	60%	90%
Chlorothalonil No. 1	56.7%	83.3%
			Azoxystrobin No. 2	53.3%	60%
No. 3 kocide	60%	63.3%
			Azoxystrobin No. 4 + fluazinam	26.7%	26.7%
Chlorothalonil No. 5 + azoxystrobin	36.7%	50%
			Chlorothalonil No. 6 and fluazifop	33.3%	53.3%

And (4) conclusion: through statistical analysis of different treatment modes, the method finds that the contamination rate of explants can be remarkably reduced when the bactericide is mixed and used in common bactericide compared with the single bactericide, wherein the azoxystrobin + in 1000 times of solution can be treated by killing, the contamination rate can be controlled within 26.7%, and the pollution prevention effect is the best. It is generally believed that no contamination occurred during 10 days of culture and the probability of contamination of explants will be very low. In this example, experiments show that the mixed drug is the best explant pretreatment method of the present invention.

Example 2: influence of the bottle seedling proliferation formula on success or failure of the invention

1) Experimental materials: the experimental basic material is aseptic lemon yellow nandina bottle seedlings. The basic culture medium is a commercially available MS culture medium, the hormone is 6-BA, and agar powder, sucrose and the like are additionally needed. Each formulation was 30 bottles with 10 independent stem segments inserted per bottle.

2) The experimental method comprises the following steps: dividing the sterile bottle seedlings into two groups, and performing enrichment culture on one group according to culture mediums No. 1 to No. 3 respectively, wherein the formula is MS +6-BA (0.2 mg/L,0.25mg/L,0.3 mg/L) + agar 8g/L + sucrose 30g/L, the ph is adjusted to be about 5.9, 2 LED lamps with 18 watts are used, the light and dark culture time is 18/6 hours, the temperature is 24 ℃, the culture is performed for 60 days, and the proliferation rate is counted; another group was cultured by intensive proliferation in the medium (4) - (6) according to the recipe MS +6-BA (0.6 mg/L,0.65 mg/L,0.7 mg/L) + agar 8g/L + sucrose 30g/L, ph adjusted to about 5.9, 2 LED lamps of 18W, light and dark culture time 18/6 hours, and temperature 24 ℃. Transferring into culture medium (2) after 30-35 days for culture, wherein the formula is MS +6-BA 0.25mg/L + agar 8g/L + sucrose 30g/L, and the ph is adjusted to about 5.9. After 60 days, the proliferation rate was counted as the number of shoots produced at the end divided by the number of shoots initially inoculated.

3) And (4) experimental conclusion:

TABLE 2 Effect of different proliferation modes on the proliferation and vitrification of Nandina domestica, lemon yellow

And (4) conclusion: the optimal concentration of 6-BA in the first group of proliferation culture medium is 0.25mg/L, and the proliferation multiple in 60 days is about 3.2 on average; the second group, proliferation medium, although requiring one transfer, has a proliferation rate as high as 19.6, far exceeding that of the first group. Therefore, the invention obviously improves the multiplication factor of the Nandina domestica, namely the lemon yellow, through the mode of early-stage intensive culture and one-time later-stage transfer, and greatly improves the production efficiency. The application of the technology has great significance for seedling production. Of course, if the culture time of the invention in the No. 5 culture medium exceeds 35 days, the vitrification ratio of the bottle seedling is greatly increased, and the subsequent proliferation multiple is affected, therefore, the conclusion of this embodiment is particularly emphasized that the seedling on the No. 5 culture medium needs to be transferred to the No. 2 culture medium in about 35 days, so as to better highlight the technical advantages of the invention.

FIG. 1 is a graph showing the proliferation of a seedling in a flask at 35 days in Medium No. 5 in example 2; wherein, the arrow only indicates the proliferation bud seen from the front, and the sight-line sheltering part cannot be displayed.

FIG. 2 is a view showing the abduction of a No. 5 medium bottle seedling in example 2 at 35 days; wherein, the arrow only indicates the proliferation bud seen from the front, and the sight-line sheltering part cannot be displayed.

FIG. 3 is a 35-day presentation of the culture of a cultured seedling on Medium No. 2 in example 2; wherein the arrows indicate proliferating shoots, with an average of 2-3.

Example 3: influence of rooting medium formula on success of the invention

1) Experimental materials: the propagation time is more than 40 days. The type of the culture medium, (1) the culture medium is 1/2MS +0.15 mg/L IBA +0.2 mg/L NAA +8g/L agar +30g/L sucrose, and the pH is adjusted to 5.9; (2) the type of the culture medium is 1/2MS +0.15 mg/L IBA +8g/L agar +30g/L cane sugar, and the pH is adjusted to 5.9; (3) the medium is 1/2MS +0.2 mg/L NAA +8g/L agar +30g/L sucrose, pH is adjusted to 5.9. (3) The medium is 1/2MS +8g/L agar +30g/L sucrose, pH is adjusted to 5.9.2 pieces of 18 watt LED lamps, culturing in dark for 18/6 h at 24 deg.C.

2) The experimental method comprises the following steps: taking out the bottle seedlings in the proliferation state, cutting the bottle seedlings into stem segments within 1 cm in a short time under the aseptic condition, inserting the stem segments into a rooting culture medium according to the growth polarity, treating 30 bottles each with 10 plants in each bottle, and counting the average number and the average length of root systems after 40 days. 3) The experimental results are as follows:

TABLE 3 Effect of different rooting media on rooting of Nandina domestica, lemon yellow

Rooting culture medium	Average root number (number)	Average root length (cm)
			1	5.3	0.52
2	4.6	0.31
			3	4.8	0.47
4	3.1	0.26

And (4) conclusion: by adopting the rooting culture medium formula provided by the invention, the average root number of the Nandina domestica, namely lemon yellow, can reach 5.3 in 40 days, the average root length can reach 0.52 cm, and seedlings can emerge after about 50 days of culture. And the formula using single rooting hormone is not beneficial to the seedling hardening and survival in the later period because the root system quantity is less or the root system is slender. The experiment proves that the optimal rooting formula suitable for Nandina domestica, lemon yellow, is 1/2MS +0.15 mg/L IBA +0.2 mg/L NAA +8g/L agar +30g/L sucrose, and the pH is adjusted to 5.9. The formula ensures that the bottle seedlings have short and thick root systems and more roots in reasonable time, and is favorable for the success rate of seedling hardening.

The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A tissue culture and rapid propagation method of Nandina domestica Thunb is characterized by comprising the following steps:

1) Selection, treatment and sterility of explants;

the pesticide azoxystrobin and the pesticide kozak are used for killing, 1000 times of water solution is used for soaking the vigorous growing top tender stem of the nandina domestica without leaves for 5 seconds 2 days before sampling, and 3 cm of the top tender stem is cut after 2-3 days for aseptic treatment; in the aseptic treatment, 0.1% mercuric chloride solution is adopted for disinfection for 15 minutes, the sterilized water is exchanged and washed for 10 minutes, the explant is cut into 1 cm long and is placed in MS minimal medium, and dark culture is carried out for 5 days for subsequent treatment;

2) Multiplication of aseptic seedlings;

after the explant is cultured in the dark for 5 days, the explant is placed under 2 18W LED lamps, the light dark culture time is 18/6 hours, the culture temperature is 24 ℃, if no pollution exists after 10 days, the explant is considered to be sterile successfully, and then the explant is transferred to a culture medium with (1) MS +0.6-0.7 mg/L BA +8g/L agar +30g/L sucrose and pH 5.9 for culture for 30-35 days, the clustered proliferating buds are cut and not truncated, and the clustered proliferating buds are completely transferred to a culture medium with (2) MS +0.2-0.3 mg/L BA +8g/L agar +30g/L sucrose and pH adjusted to 5.9 for continuous culture, after the explant is cultured for 40-45 days, the proliferating buds are truncated to root, or the proliferating buds are again truncated into the culture medium with (1) for proliferation, and the culture time on the culture medium with (1) cannot exceed 35 days;

3) Rooting culture of the proliferated seedling;

the rooting of the proliferated seedling adopts a culture medium No. 3: 1/2MS +0.15 mg/L IBA +1.2 mg/L NAA +8g/L agar +30g/L sucrose, pH 5.9.