CN104082152A - Tissue culture and rapid propagation method for clematis ranunculoides - Google Patents

Abstract

The invention provides a method for tissue culture and rapid propagation of clematis ranunculus. After obtaining aseptic raw materials, inducing buds, cultivating adventitious buds, rooting culture, plant hardening and transplanting, clematis ranunculus plants are finally obtained. The method can rapidly reproduce clematis ranunculus, has a short seedling cultivation period, can quickly obtain clematis ranunculus plants, has a survival rate of over 90%, has a high reproduction coefficient, is not restricted by external conditions, can be carried out in all seasons, and saves the cost of raising seedlings. The ground area can form a large number of excellent test-tube seedlings in a short period of time, and can be produced on a large scale.

Description

A method for tissue culture and rapid propagation of buttercup clematis

technical field

The invention relates to a method for tissue culture and rapid propagation of plants, in particular to a method for tissue culture and rapid propagation of clematis ranunculus, belonging to the technical field of plant tissue culture.

Background technique

Buttercup clematis ( Clematis ranunculoides Franch . ) is a herbaceous vine of Ranunculaceae, distributed in Southwest China. The flowers are dense, purple-red, and the flowering period is from September to October, which coincides with the National Day and Mid-Autumn Festival. It has high ornamental value and can be used as potted flowers or climbing green plants. At the same time, the whole herb is used as medicine to treat various diseases. Therefore, clematis ranunculus is a medicinal ornamental plant with great development value.

However, in the prior art, most of the clematis ranunculus grows and breeds naturally, and there is no publicly reported method for tissue culture and rapid propagation of clematis ranunculus. In order to efficiently and quickly obtain this clematis ranunculus with medicinal value, it is necessary to develop the technology of tissue culture and rapid propagation of clematis ranunculus, so as to realize the short period of seedling cultivation of clematis ranunculus and obtain the plants of clematis ranunculus quickly. At the same time, it maintains the advantages of good traits of the female parent.

Contents of the invention

In order to solve the long natural breeding cycle of clematis ranunculus, which is difficult to meet the needs of medicinal use, the present invention provides a method for tissue culture and rapid propagation of clematis ranunculus, so as to realize the short breeding cycle of clematis ranunculus, so that people can quickly obtain Buttercup clematis plant.

The present invention is accomplished through the following technical solutions: a method for tissue culture and rapid propagation of Buttercup clematis, which is characterized in that the steps are as follows:

(1) Cut the young shoots of Buttercup clematis, wash them with water for 1 hour, then soak them in ethanol with a volume concentration of 75% for 25-35 seconds, then soak them in mercury chloride with a concentration of 1‰ for 5-10 minutes, and then wash them with sterile water After 3-5 times, blot the surface water dry, cut the base of the shoots into 1cm, and inoculate them on the bud induction medium: MS+6-BA 1.5-2.5mg/L+NAA 0.4-0.6mg/L+AgNO ₃ 2.5 ～3.5mg/L, cultivated under the conditions of temperature 22～26℃, light intensity 1500～2000Lx, relative humidity 65～75%;

(2) When the bud base of the young shoots in step (1) begins to expand and yellow-green protrusions appear, continue to cultivate for 20-25 days and then obvious callus will appear, and then continue to culture for 30-35 days until the callus grows small bud;

(3) Excision step (2) The callus with buds is inoculated on the adventitious bud proliferation medium: MS+BA 1.5～2.5mg/L+NAA 0.04～0.06mg/L, at a temperature of 22～26°C, Cultivate under the conditions of light intensity of 1500-2000Lx and relative humidity of 65-75%, so that the adventitious buds proliferate until the adventitious buds grow to 2-3cm;

(4) Take the adventitious bud plants with a height of 2-3cm in step (3) and transfer them into the rooting medium: 1/2MS+IBA 0.4-0.6mg/L+NAA 0.9-1.1mg/L +AC 0.1～0.25g/L, rooting is induced under the conditions of temperature 22～26℃, light intensity 1500～2000Lx, relative humidity 65～75%, and after 30 days, the base of the plant seedlings differentiates into white root roots base until the root primordium grows to 3 to 5 cm to obtain rooted seedlings;

(5) Take the vigorous rooting bottle seedlings in step (4) and remove the sealing film indoors for 3-8 days, wash the roots after taking them out, transplant them in a greenhouse for 25-35 days, and then transplant them outdoors and dry them. Appropriate fertilizer and water management is given to obtain buttercup clematis plants.

Compared with the prior art, the present invention has the following advantages and effects: the method can rapidly propagate clematis ranunculus, the seedling raising cycle is short, and clematis ranunculus plants can be obtained quickly, the survival rate is as high as more than 90%, the reproduction coefficient is high, and no Restricted by external conditions, it can be carried out in all seasons, saves the area of seedling cultivation, can form a large number of excellent test tube seedlings in a short period of time, and can carry out large-scale production.

Detailed ways

The present invention will be further described below in conjunction with the examples.

Example 1

(1) Cut the young shoots of Buttercup clematis, wash them with water for 1 hour, soak them in ethanol with a volume concentration of 75% for 25 seconds, and soak them with mercury chloride with a concentration of 1‰ for 5 minutes, and then wash them with sterile water Rinse 3 times and blot the surface water dry, cut the base of the shoots into 1cm, the contamination rate is 92%, and then inoculate on the bud induction medium: MS+6-BA 1.5mg/L+NAA 0.5mg/L+AgNO ₃ 3mg/L, cultured at a temperature of 24°C, a light intensity of 1500Lx, and a relative humidity of 70%;

(2) When the bud base of the tender shoots in step (1) begins to expand and yellow-green protrusions appear, the obvious callus tissue will appear after continuing to culture for 20 days, and then continue to culture for 30 days until the callus grows small buds, and induces rate 64%;

(3) Excision step (2) The callus with buds is inoculated on the adventitious bud proliferation medium: MS+BA 1.5mg/L+NAA 0.05mg/L, at a temperature of 26°C, a light intensity of 1800Lx, and a relative Under the condition of 75% humidity, culture is carried out to make the adventitious buds proliferate until the adventitious buds grow to 2-3cm, and the proliferation rate of 20 days is 6.8 times;

(4) Take the adventitious bud plants with a height of 2-3cm in step (3) and transfer them into the rooting medium: 1/2MS+IBA 0.5mg/L+NAA 1mg/L+AC 0.1g/ L, inducing rooting under the conditions that the temperature is 26°C, the light intensity is 1500x, and the relative humidity is 65%. After 30 days, the base of the plant seedlings differentiates into white root primordium until the root primordium grows to 3-5cm, and the root primordium is obtained. Root seedlings, the rooting rate is 35%;

(5) Take the vigorous rooting bottle seedlings from step (4) and remove the sealing film indoors for 5 days, wash the roots after taking them out, transplant them in a plastic greenhouse for 30 days, and then transplant them outdoors and give appropriate fertilizer and water After management, clematis buttercup plants were obtained, with a survival rate of 92%.

Example 2

(1) Cut the young shoots of Buttercup clematis, wash them with water for 1 hour, soak them in ethanol with a volume concentration of 75% for 30 seconds, soak them in mercuric chloride with a concentration of 1‰ for 8 minutes, and then wash them with sterile water for 4 times before inhaling. Dry the surface moisture, take the base of the shoot and cut it into 1cm, the pollution rate is 32.5%, and then inoculate it on the bud induction medium: MS+6-BA 2mg/L+NAA 0.4mg/L+AgNO ₃ 3.5mg/L, in The temperature is 22°C, the light intensity is 1700Lx, and the relative humidity is 65%;

(2) When the bud base of the tender shoots in step (1) begins to expand and yellow-green protrusions appear, the obvious callus tissue will appear after continuing to culture for 22 days, and then continue to culture for 33 days until the callus grows small buds, and induces rate 83%;

(3) Excision step (2) The callus with buds is inoculated on the adventitious bud proliferation medium: MS+BA 2mg/L+NAA 0.04mg/L, at a temperature of 24°C, a light intensity of 1500Lx, and a relative humidity Under the condition of 70%, the adventitious buds are multiplied until the adventitious buds grow to 2-3 cm, and the multiplication rate of 20 days is 11.5 times;

(4) Take the adventitious bud plants with a height of 2-3cm in step (3) and transfer them into the rooting medium: 1/2MS+IBA 0.4mg/L+NAA 1.1mg/L+AC 0.15g /L, under the condition that the temperature is 24°C, the light intensity is 1700Lx, and the relative humidity is 70%, rooting is induced. After 30 days, the base of the plant seedlings differentiates into white root primordium until the root primordium grows to 3-5cm, and the root primordium is obtained. Rooting seedlings, the rooting rate is 60%;

(5) Take the vigorously grown rooted bottle seedlings in step (4) and remove the sealing film indoors for 3 days, wash the roots after taking them out, transplant them in a plastic greenhouse for 35 days, and then transplant them outdoors and give appropriate fertilizer and water After management, clematis buttercup plants were obtained, with a survival rate of 90.2%.

Example 3

(1) Cut out the young shoots of Buttercup clematis, wash them with water for 1 hour, then soak them in ethanol with a volume concentration of 75% for 35 seconds, then soak them in mercury chloride with a concentration of 1‰ for 10 minutes, then wash them with sterile water for 5 times and suck them. Dry the surface moisture, cut the base of the shoots into 1cm, the pollution rate is 0, and then inoculate on the bud induction medium: MS+6-BA 2.5mg/L+NAA 0.6mg/L+AgNO ₃ 2.5mg/L, in The temperature is 26°C, the light intensity is 2000Lx, and the relative humidity is 75%;

(2) When the bud base of the tender shoots in step (1) begins to expand and yellow-green protrusions appear, then continue to cultivate for 25 days and then appear obvious callus tissue, and then continue to culture for 35 days until the callus grows small buds and induces rate 53%;

(3) Excision step (2) The callus with buds is inoculated on the adventitious bud proliferation medium: MS+BA 2.5mg/L+NAA 0.06mg/L, at a temperature of 22°C and a light intensity of 2000Lx, relative The humidity is 65% under the condition of cultivating to make the adventitious buds proliferate until the adventitious buds grow to 2-3cm, and the proliferation rate in 20 days is 5.1 times;

(4) Take the adventitious bud plants with a height of 2-3cm in step (3) and transfer them into the rooting medium: 1/2MS+IBA 0.6mg/L+NAA 0.9mg/L+AC 0.25g /L, the temperature is 22 ℃, the light intensity is 2000Lx, and the relative humidity is 75% to induce rooting. After 30 days, the base of the plant seedlings differentiates into white root primordium until the root primordium grows to 3 ~ 5cm. Rooted seedlings, the rooting rate is 78.3%;

(5) Take the vigorous rooted bottle seedlings from step (4) and remove the sealing film indoors for 8 days, wash the roots after taking them out, transplant them in a plastic greenhouse for 25 days, and then transplant them outdoors and give appropriate fertilizer and water After management, clematis buttercup plants were obtained, with a survival rate of 95.2%.

Claims (1)

1. a method for rapid propagation of clematis buttercup tissue culture, characterized in that through the following steps: (1) Cut the young shoots of Buttercup clematis, wash them with water for 1 hour, then soak them in ethanol with a volume concentration of 75% for 25-35 seconds, then soak them in mercury chloride with a concentration of 1‰ for 5-10 minutes, and then wash them with sterile water After 3-5 times, blot the surface water dry, cut the base of the shoots into 1cm, and inoculate them on the bud induction medium: MS+6-BA 1.5-2.5mg/L+NAA 0.4-0.6mg/L+AgNO ₃ 2.5 ～3.5mg/L, cultivated under the conditions of temperature 22～26℃, light intensity 1500～2000Lx, relative humidity 65～75%; (2) When the bud base of the young shoots in step (1) begins to expand and yellow-green protrusions appear, continue to cultivate for 20-25 days and then obvious callus will appear, and then continue to culture for 30-35 days until the callus grows small bud; (3) Excision step (2) The callus with buds is inoculated on the adventitious bud proliferation medium: MS+BA 1.5～2.5mg/L+NAA 0.04～0.06mg/L, at a temperature of 22～26°C, Cultivate under the conditions of light intensity of 1500-2000Lx and relative humidity of 65-75%, so that the adventitious buds proliferate until the adventitious buds grow to 2-3cm; (4) Take the adventitious bud plants with a height of 2-3cm in step (3) and transfer them into the rooting medium: 1/2MS+IBA 0.4-0.6mg/L+NAA 0.9-1.1mg/L +AC 0.1～0.25g/L, rooting is induced under the conditions of temperature 22～26℃, light intensity 1500～2000Lx, relative humidity 65～75%, and after 30 days, the base of the plant seedlings differentiates into white root roots base until the root primordium grows to 3 to 5 cm to obtain rooted seedlings; (5) Take the vigorous rooting bottle seedlings in step (4) and remove the sealing film indoors for 3-8 days, wash the roots after taking them out, transplant them in a greenhouse for 25-35 days, and then transplant them outdoors and dry them. Appropriate fertilizer and water management is given to obtain buttercup clematis plants.