CN104054549B - The method of rich No. 6 tissue-culturing rapid propagations of a kind of apple variety cigarette - Google Patents

Abstract

The invention discloses a method for tissue culture and rapid propagation of apple variety Yanfu No. 6. The method for tissue culture and rapid propagation of apple variety Yanfu No. 6 adopts the medium of MS+6-BA0.5mg/L+IBA0. 1mg+PVP500mg/L medium primary culture, subculture treated with MS+6-BA0.5mg/L+IBA0.1mg/L, rooting culture with rooting medium 1/2MS+IBA1.0mg/L, Using pure sawdust to harden seedlings and transplant them; realized the rapid propagation of the apple variety Yanfu No. 6, which not only ensured a high survival rate, but also retained the excellent characteristics of the variety, providing convenience for the development and further expansion of the apple industry methods and ways.

Description

A method for tissue culture and rapid propagation of apple variety Yanfu No. 6

technical field

The invention belongs to the technical field of apple seedling cultivation, in particular to a method for tissue culture and rapid propagation of apple variety Yanfu No. 6.

Background technique

The apple industry occupies a very important position in China's agricultural economy. In 2013, China's apple planting area was 2.253 million hectares, with an output of 31.7 million tons. It accounts for 1/2 of the world's total production. At present, my country's apple production model has gradually changed from the traditional low-efficiency canopy orchard model to the modern low-stock dense planting intensive and high-efficiency production and cultivation model, and gradually transformed from the world's largest apple production country to a strong apple industry. Apple seedlings are the basis of apple production and cultivation, and the quality of seedlings directly affects the growth, flowering, fruiting, stress resistance, disease resistance and lifespan of apple trees. After the seedlings are infected by the virus, the normal cell proliferation of the tree will be destroyed. In severe cases, it will affect the normal physiological mechanisms such as the growth potential, fruit quality and yield of fruit trees. This has become a hidden danger for the development of China's apple industry. The next period of time will be a critical period for the large-scale replacement of apples in our country. How to provide support and guarantee from the perspective of seedlings has become a problem worthy of great attention. Yanfu 6 is an excellent bud change of Changfu 2. Compared with the parent plant Changfu No. 2, Yanfu No. 6 has short-branched buds, early fruiting at the young tree stage, high early yield, large fruit size, and good coloring, and is loved by many fruit farmers. The invention establishes a method for rapid propagation of apple variety Yanfu No. 6 through tissue culture and rapid propagation, which is of great significance for producing high-quality short-branch apple seedlings and has broad market prospects.

Contents of the invention

The purpose of the embodiment of the present invention is to provide a method for tissue culture and rapid propagation of apple variety Yanfu No. 6, aiming to provide a method for tissue culture and rapid propagation for the cultivation of apple seedlings in my country, so as to speed up the large-scale replacement of apples in my country.

The embodiment of the present invention is achieved in this way, a method for tissue culture and rapid propagation of apple variety Yanfu No. 6, the method for tissue culture and rapid propagation of apple variety Yanfu No. 6 comprises the following steps:

Step 1: Use the apple variety Yanfu No. 6 as the material, select a mother tree that grows vigorously and has no pests and diseases, take 1-year-old branches in March, and place them in a greenhouse at 25°C for hydroponics. Each liter of water contains 30g of sucrose, and it is changed every 3 days Water and cut off the old cut. When the buds on the hydroponic annual branches grow to 1.5-2.0cm, cut the buds, remove the unfolded leaves, wash them under tap water for 6-8 hours, and treat them with 75% alcohol for 8 seconds. 0.1% mercury chloride treatment for 7 minutes;

Step 2: After the disinfection treatment, wash with an ultrasonic cleaner for 3 minutes, then rinse with sterile water for 3 to 4 times, and inoculate the treated shoots into MS+6-BA0.5mg/L+IBA0.1mg+PVP ( Polyvinylpyrrolidone) 500mg/L medium for culture, add 30g sucrose and 8g agar per liter of medium, and then place it under the conditions of light 16h/d, light 2000lux, and 26°C;

Step 3, inoculate the obtained Yanfu No. 6 sterile explants into MS+6-BA0.5mg/L+IBA0.1mg/L agar 8g/L, and carry out subculture in the culture medium of light 2000lux, and add 30g sucrose, 8g agar;

Step 4, select the vigorous growth obtained by subculture, the height is greater than 1.5cm, and the buds with a growth time of 20 days are inoculated in 1/2MS+IBA1.0mg/L (sucrose 30g/L, agar 8g/L) for rooting culture Carry out rooting culture in the base;

Step 5: When the root length of the tissue cultured seedling reaches 2cm, move it outdoors to shade and harden the seedling for about 10 to 12 days, then open the bottle of the culture container, open the bottle and harden the seedling under natural light for 2 to 4 days, and remove the rooted seedling from the Take it out from the culture medium, soak it in a 0.1% carbendazim solution for 5 minutes, clean the excess culture medium at the root, and finally rinse it with water for 2 to 3 times, and then transplant it into a nutrient bowl with pure sawdust.

Further, in step five, the management method of transplanted seedlings is: after transplanting, the rooted seedlings are managed under the shade net, and the first week is sprayed with 0.1% carbendazim solution, and the second week is sprayed with water every day Once, keep the transplanting substrate moist and airy. After the rooted seedlings sprout new shoots, they can be removed from the shade net.

The method for tissue culture and rapid propagation of apple variety Yanfu No. 6 provided by the present invention adopts the culture medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L for primary culture, and the survival rate is 30.77%. , which is conducive to the establishment of Yanfu No. 6 tissue culture seedlings; subculture with MS+6-BA0.5mg/L+IBA0.1mg/L treatment, the multiplication coefficient is as high as 5.64; rooting medium 1/2MS+IBA1 .0mg/L rooting culture and the rooting rate reached 45.8%; using pure sawdust for seedling hardening and transplanting, the survival rate reached 75%; repeated subculture of Yanfu No. 6 variety, rooting culture and transplanting can be done in a relatively In a short period of time, the rapid propagation of the virus-free seedlings of the apple variety Yanfu No. 6 is realized. The excellent characteristics of the Yanfu No. 6 variety are preserved through tissue culture technology, which is conducive to the preservation of the excellent Yanfu No. 6 variety, provides efficient and convenient breeding technology for the virus-free seedlings of Apple Yanfu No. 6, and promotes the further development of the apple industry .

Description of drawings

Fig. 1 is a flow chart of the method for tissue culture and rapid propagation of apple variety Yanfu No. 6 provided by the embodiment of the present invention.

detailed description

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The application principle of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

As shown in Figure 1, the method for tissue culture rapid propagation of apple variety Yanfu No. 6 of the embodiment of the present invention comprises the following steps:

S101: Using the apple variety Yanfu No. 6 as the material, select a mother tree that grows robustly and is free from diseases and insect pests. Take 1-year-old branches in March and place them in a greenhouse at 25°C for hydroponics. Each liter of water contains 30g of sucrose, and the water is changed every 3 days And cut off the old notch. When the buds on the hydroponic annual branches grow to 1.5-2.0 cm, cut the buds, remove the expanded leaves, wash them under tap water for 6-8 hours, and treat them with 75% alcohol for 8 seconds, 0.1 % Mercury chloride treatment for 7 minutes;

S102: After disinfection treatment, wash with an ultrasonic cleaner for 3 minutes, then rinse with sterile water for 3 to 4 times, inoculate the treated shoots into MS+6-BA0.5mg/L+IBA0.1mg+PVP (poly Vinylpyrrolidone) 500mg/L medium for culture, add 30g sucrose and 8g agar per liter of medium, and then place it under the conditions of light 16h/d, light 2000lux, and 26°C;

S103: Inoculate the obtained Yanfu No. 6 sterile explants into the optimal medium for treatment, that is, MS+6-BA0.5mg/L+IBA0.1mg/L agar 8g/L, followed by medium with light of 2000lux For subculture, add 30g sucrose and 8g agar per liter;

S104: Select the shoots obtained by subculture that grow robustly, have a height greater than 1.5cm, and have a growth time of 20 days, and inoculate them in 1/2MS+IBA1.0mg/L (sucrose 30g/L, agar 8g/L) rooting medium rooting culture in

S105: When the root length of the tissue cultured seedling reaches 2cm, move it outdoors to shade for about 10 to 12 days, then open the bottle of the culture container, open the bottle and harden the seedling under natural light for 2 to 4 days, and remove the rooted seedling from the cultured Take it out from the base, soak it in a 0.1% carbendazim solution for 5 minutes, and clean the excess medium at the root, and finally rinse it with water for 2 to 3 times, and then transplant it into a nutrient bowl with pure sawdust.

In step S105, the management method of the transplanted seedlings is: after transplanting, the rooted seedlings are managed under the shade net, and the first week is sprayed with 0.1% carbendazim solution, and the second week is sprayed with water once a day. Keep the cultivation medium moist and ventilated. After the rooted seedlings sprout new shoots, they can be removed from the shade net.

Concrete steps of the present invention are as follows:

Step 1, primary culture

The apple variety Yanfu No. 6 was used as the material, and the mother tree with strong growth and no pests and diseases was selected. In March, the 1-year-old branches were taken and placed in a greenhouse at 25°C for hydroponics (each liter of water contained 30g of sucrose), and the water was changed every 3 days and cut. Remove the old cuts, when the buds on the hydroponic annual branches are 1.5-2.0 cm, cut the buds, remove the unfolded leaves, rinse under tap water for 6-8 hours, treat with 75% alcohol for 8 seconds, 0.1% liters Mercury treatment for 7 minutes, after disinfection treatment, wash with an ultrasonic cleaner for 3 minutes, and then rinse with sterile water for 3 to 4 times, inoculate the treated shoots into MS+6-BA0.5mg/L+IBA0.1mg/L+ Culture in the medium of polyvinylpyrrolidone (PVP) with different concentrations (0, 0.3, 0.5, 0.7, 1.0g/L), add 30g sucrose and 8g agar per liter of medium, and connect 3 buds in each Erlenmeyer flask , 15 bottles per treatment, and then cultured under the conditions of light 16h/d, light 2000lux, and 26°C. After 14 days, the pollution rate, browning rate, and survival rate were investigated respectively. It was concluded that the primary culture of Yanfu No. 6 was the most suitable. The culture medium is MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L (sucrose 30g/L, agar 8g/L);

Step two, subculture

Inoculate the obtained sterile explants to subculture lines containing different concentrations of 6-BA, add 30g sucrose per liter, 8g agar, 3 plants per bottle, 10 bottles for each treatment, repeat 3 times, and the Yanfu No. 6 The optimal medium is, namely, MS+6-BA0.5mg/L+IBA0.1mg/L (agar 8g/L, light 2000lux);

Step 3, Rooting and Culture

Select the vigorous growth obtained by subculture, the plants with a height greater than 1.5cm, the buds with a growth time of 20 days, and inoculate the rooting plants treated with 1/2MS+different concentrations of IBA (0, 1.0, 0.9, 1.0, 1.1mg/L). Carry out rooting culture in cultivation, inoculate 3 in every bottle, each process 10 bottles, repeat 3 times, obtain the most suitable rooting medium of Yan Fu No. 6 and be 1/2MS+IBA1.0mg/L (sucrose 30g/L, agar 8g/L);

Step 4, seedling hardening and transplanting

When the root length of the tissue-cultured seedlings reaches 2cm, move them outdoors to shade for about 10-12 days, then open the bottle of the culture container, open the bottle and harden the seedlings under natural light for 2-4 days, and remove the rooted seedlings from the culture medium. Take it out, put it into a 0.1% carbendazim solution and soak it for 5 minutes, and clean the excess culture medium at the root, and finally rinse it with water for 2 to 3 times, and then transplant it to different substrates with the same capacity for treatment (pure sawdust, sawdust + substrate, pure substrate, substrate+perlite) in the nutrient bowl, the management mode of transplanting seedling is: the rooted seedling is managed under the shade net, adopts concentration to be that 0.1% carbendazim solution is sprayed in the first week, In the second week, spray water once a day to keep the transplanting substrate moist and ventilated. After 14 days, the survival rate of hardened seedlings transplanted was counted, and the survival rate of hardened seedlings transplanted with pure sawdust was the best.

Principle of the present invention is further illustrated by following experiments and data:

1. Refer to Table 1 for the effects of different concentrations of PVP treatment on the growth of explants;

Table 1 Effects of different concentrations of PVP treatment on the growth of explants

Note: The same letter in the same column indicates no significant difference (P>0.05), and different letters indicate significant difference (P<0.05). The data are Mean±SE (mean±standard error); the same below.

2. Effects of different 6-BA concentration treatments on the subculture of Yanfu 6:

Table 2 Effects of different 6-BA concentrations on the subculture of Yanfu 6

basic medium 6-BA(mg/L) IBA(mg/L) multiplication factor MS 0 0.1 1.25±0.06c MS 0.3 0.1 1.41±0.09c MS 0.4 0.1 2.78±0.11b MS 0.5 0.1 5.64±0.22a MS 0.6 0.1 4.32±0.14a

3. Effects of different IBA concentration treatments on the rooting of Yanfu No. 6:

Table 3 Effects of different IBA concentration treatments on rooting of Yanfu No. 6

4 Effects of different hardening substrates on the growth of rooted seedlings:

Table 4 Effects of different cultivation substrate treatments on the growth of transplanted seedlings

Substrate treatment Transplanting Survival Rate of Yanfu No.6 sawdust 75.00±4.23a sawdust + substrate 67.50±2.42b matrix 55.00±1.32c

matrix + perlite 57.50±5.32c

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (1)

1. a method for rich No. 6 tissue-culturing rapid propagations of apple variety cigarette, is characterized in that, the method for rich No. 6 tissue-culturing rapid propagations of this apple variety cigarette comprises the following steps:

Step one, with No. 6, apple variety cigarette richness for material, cleans 3min with supersonic wave cleaning machine, then uses aseptic water washing 3 ~ 4 times, be inoculated into by the tender shoots handled well in the medium of polyvinylpyrrolidone 500mg/L and cultivate; Often liter of medium adds 30g sucrose, 8g agar, is then placed on illumination 16h/d, illumination 2000lux, cultivates under 26 DEG C of conditions;

Step 2, is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg/L by the aseptic explant of acquisition, carries out squamous subculture, adds 30g sucrose in often liter of medium, 8g agar under illumination 2000lux condition;

Step 3, select the robust growth that squamous subculture obtains, be highly greater than the plant of 1.5cm, growth time is bud of 20 days, is inoculated in 1/2MS+IBA1.0mg/L root media and carries out culture of rootage, often liter of medium supplemented 30g sucrose, 8g agar;

Before step one is with supersonic wave cleaning machine cleaning, with No. 6, apple variety cigarette richness for material, the healthy and strong elite stand without damage by disease and insect of growth selection, in clip in March 1 year raw branch, water planting is carried out in the greenhouse placing 25 DEG C, water planting is that every premium on currency contains 30g sucrose, change a water every 3d and cut the old clip of water planting branch, when bud pumping on the annotinous branch of water planting is 1.5 ~ 2.0cm, clip tender shoots, removes and launches blade, under running water, rinse 6 ~ 8h, with 75% ethanol postincubation 8s, 0.1% mercuric chloride process 7min;

After the culture of rootage step of step 3, plantlet in vitro root length reaches 2cm, move on to outdoor to shelter from heat or light hardening 10 ~ 12 days, then opened by culture vessel bottleneck, carry out uncork hardening after 2 ~ 4 days under natural daylight, seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary for root is cleaned up, finally rinse 2 ~ 3 times with clear water, in the nutritive cube be then transplanted to pure sawdust;

The way to manage of transplanted seedling is: seedling of taking root after transplanting manages under shade net, within first week, concentration is adopted to be that 0.1% carbendazim solution is sprayed, once, keep transplanting medium moistening ventilative, namely offspring pumping to be generated shifts out shade net after going out sprouting to the sprinkling irrigation of second week use every day water.