CN109287490B

CN109287490B - Method for rapidly propagating high-quality seedling of aquatic plant water soft-shelled turtle and application

Info

Publication number: CN109287490B
Application number: CN201811441591.0A
Authority: CN
Inventors: 周长芳; 张宸荣; 潘璠; 仲昭朝
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2021-07-30
Anticipated expiration: 2038-11-29
Also published as: CN109287490A

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for rapidly propagating aquatic plant water soft-shelled turtle high-quality seedlings and application. Aiming at the large-scale propagation of the water soft-shelled turtles under the indoor artificial condition, the method is not limited by natural environment factors, sterile female parents are obtained by multiple decontamination and disinfection methods, and the high-efficiency propagation expansion of cluster buds and the induction of developed root systems are realized by the adjustment of MS, sucrose and hormone contents. The technology has low cost and quick response, and can produce healthy and strong detoxified water soft-shelled turtle high-quality tissue culture seedlings in a short time in batches. The obtained seedlings can be applied to indoor aquarium ornamental scenery, outdoor wetland park construction, large-scale wetland ecological restoration engineering and the like, and can effectively relieve the current problem of scarcity of the water turtle seedling market in China.

Description

Method for rapidly propagating high-quality seedling of aquatic plant water soft-shelled turtle and application

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for rapidly propagating aquatic plant water soft-shelled turtle high-quality seedlings and application.

Background

Hydrochis dubia (Bl.) Back is a perennial floating plant of the genus Hydrochis of the family Hydrochidaceae, with leaves floating, which also emerge from the water surface when dense, and grow in standing water ponds and marshes. The water turtle has strong water quality purification capacity, and the market demand of the plant seedlings is extremely large along with the implementation of a large number of wetland ecological restoration projects in China in recent years. In addition, the water turtle plant, especially the leaves thereof, has high ornamental value, the leaves are emerald green and are heart-shaped or round, and the back of the leaves is provided with a raised honeycomb air storage tissue. 3 pieces of water turtle petal, white. The plant also has a high position in the design of small family aquatic animal ornamental scenery and large wetland park waterscape.

Because the seeds of the water soft-shelled turtles are difficult to collect, the seedlings required by the existing market are mainly obtained by the dormant buds and natural division propagation, the collection of related seed sources is limited by seasons, the obtained seedlings are often uneven in size, the labor cost is high, and the harvesting efficiency is extremely low. Especially, under the condition that the existing wetland habitat is largely lost, the wild water turtle seed source is difficult to find. In addition, as the wild soft-shelled turtles are easy to be attacked by diseases and insect pests, the leaves of the wild soft-shelled turtles are provided with insect spots, so that the ornamental effect is influenced when the wild soft-shelled turtles are directly transplanted into a landscape water body and an aquarium, pathogenic bacteria are easy to spread, and the safety of other animals and plants in the water body is harmed.

So far, no research and report about culture of virus-free seedlings of the water turtles and large-scale artificial propagation of high-quality seedlings of the water turtles are seen at home and abroad.

Disclosure of Invention

The purpose of the invention is: provides a tissue culture rapid propagation method and application for producing high-quality seedlings of soft-shelled turtles in a large scale by utilizing indoor artificial conditions.

The technical scheme of the invention is as follows:

the invention provides a rapid propagation and application method of water turtle high-quality seedlings, which comprises the following steps:

(1) obtaining detoxified female parent

Collecting a small amount of water turtle plants from a wild natural wetland, shearing stem segments with tillering buds, soaking and washing in a washing solution for 0.5-2 h, and then washing with tap water. Soaking in 70-75% alcohol for 3min, and adding NaClO solution containing 0.2-1% available chlorine or 1-5% H₂O₂Soaking in disinfectant to sterilize for 10 s-5 min or HgCl in 0.05-1%₂And soaking and disinfecting the mixture for 1-30 min by using a disinfectant, and thoroughly rinsing the mixture by using sterile water. The material is further cut, arranged and inoculated into a triangular flask filled with a sterilized culture solution I, sealed by a breathable sealing film and placed on a special tissue culture frame for culture. The average illumination intensity is maintained to be 1000-5000 lx, the illumination is carried out for about 8-12 h every day, and the indoor temperature is 20-30 ℃. After one month, selecting a material with new buds growing and no bacteria growth, namely the detoxified female parent of the water turtle. The culture solution (i) is: 1/2-1 times of MS, 10-40 g/L of sucrose, 0.5-3.0 mg/L of cytokinin KT or 6-BA and 0.1-1.0 mg/L of auxin IAA or IBA, and adjusting the pH to be 5.6-6.2.

(2) Amplification of Cluster shoots

And (3) taking the female parent of the detoxified water turtle obtained in the step (1), and transferring the female parent of the detoxified water turtle into a sterilized culture solution on a sterile operating platform. And (3) putting the triangular flask back to the original tissue culture frame for culturing for 15-30 d, taking out the triangular flask when the cluster buds of the water soft-shelled turtles basically grow to the whole flask, cutting every 1-3 buds into separate flasks, and continuing to propagate. The culture solution II comprises: 1/2-1 times of MS, 10-40 g/L of sucrose, 0.5-3.0 mg/L of cytokinin KT or 6-BA, 0.1-1.0 mg/L of auxin IAA or IBA, and 5-10% of coconut milk, and adjusting the pH to be 5.6-6.2.

(3) Induction of rooting and Whole plants

And (3) cutting and separating 1-2 buds of the water turtle cluster buds obtained in the step (2) on a sterile operating platform, and transferring the water turtle cluster buds into a sterilized culture solution. And (5) putting the triangular flask back to the original tissue culture frame for culturing for 15-30 d, and waiting for the water turtles in the flask to root and form complete seedlings. The culture solution (c) is: 1/4-1 times of MS, 7.5-30 g/L of sucrose, 0.01-0.5 mg/L of auxin NAA or IBA, and adjusting the pH value to be 5.6-6.2.

(4) Strong seedling and transplanting

And (3) taking out the water turtle seedlings obtained in the step (3) from the triangular flask, if the water turtle seedlings are to be applied to an indoor aquarium, firstly washing the water turtle seedlings with sterile water, deionized water or tap water to remove residual culture solution on the surface of the material according to the sterile requirement of the aquarium cultivation, and then putting the tissue culture seedlings into the aquarium under the sterile or aseptic condition. If the method is to be applied to outdoor large-scale projects such as wetland park construction or wetland ecological restoration, the tissue culture seedlings are firstly washed by tap water, the MS and hormone residues are removed, then the tissue culture seedlings are transferred into an open hydroponic box containing diluted Hoagland nutrient solution, and the open hydroponic box is placed in a greenhouse or outdoor strong seedlings for 2-4 weeks. In the early stage, if the outdoor illumination is too strong, the sunlight around 1/2 can be blocked by a sunshade net and the like. The tissue culture seedlings can completely adapt to natural light after 3-7 days. The large-scale transplanting of the water turtle tissue culture seedlings can adopt two modes. One is directly thrown into natural water to enable the water to float and grow, and the water can be enclosed to prevent the water from floating at the initial stage. The second method is that the seeds are firstly planted in a substrate which takes pond sludge, peat soil and the like as main components and then are moved into a shallow water body, so that the planting is faster, but the water level depth is not more than 1m during the initial planting.

The invention has the beneficial effects that:

(1) obtaining the female parent of water turtle. The water turtle seedlings collected in the field have a large amount of germs and pollutants, and are directly put into an aquarium to be cultured, so that the infection risk is easily brought to other organisms such as ornamental fishes and the like in the aquarium, and the water quality is deteriorated. Even if the method is used for outdoor wetland landscaping or wetland ecological restoration, the colonization effect of the water turtles is often influenced due to germ pollution. The technology firstly adopts mild detergent for immersion cleaning, primarily removes pollutants, and then uses ethanol + NaClO and H₂O₂、HgCl₂And the disinfectant is disinfected in two steps to achieve the effect of thorough sterilization. Through the disinfection process, the aseptic rate of the water turtle material reaches more than 50 percent, wherein the aseptic material is cultured in MSThe regeneration rate in the nutrient solution I is more than 50 percent. The newly generated plumule is the detoxified female parent for the subsequent expanding propagation of the soft-shelled turtle.

(2) Establishes the rapid propagation technology of the high-quality seedling of the water turtle. On the basis of the female parent for water turtle detoxification, the technology directly induces a large amount of cluster buds of water turtle through a culture solution II, and the crescent bud propagation rate can reach more than 1: 30. When a complete seedling is needed, the new bud is cut off and transferred into a culture solution to induce rooting, and a complete plant is formed, wherein the rooting rate reaches more than 95%. The technology does not need large area and space, the used reagent raw materials are conventional chemical products, the cost is low, and the large-scale industrial production is convenient.

(3) The invention is not limited by the field natural environment, and can provide high-quality virus-free seedlings of the water turtles for the indoor aquarium ornamental scenery at any time in any season; and high-quality water turtle seedlings with consistent specifications can be provided for large-scale projects such as wetland park construction, wetland ecological restoration and the like in batches under the condition of lack of natural resources.

The method is not influenced by the natural phenological laws of plants, has low input cost, high propagation efficiency, robust seedlings and uniform specification, and can realize annual factory batch production. The obtained water soft-shelled turtle high-quality seedlings can meet the requirements of large-batch floating-leaf plant seedlings in wetland park construction and wetland ecological restoration engineering, and can also meet the requirements of high-specification detoxified floating-leaf ornamental plants in indoor ornamental aquarium scenery.

The specific implementation mode is as follows:

example 10 water soft-shelled turtles were harvested from the mature south lake of Jiangsu in 11 months earlier than 1.2015, and 20 tender stems with buds were soaked in detergent for 1 hour and then washed with tap water for 2 hours. Soaking in 70% ethanol for 20s in ultra-clean room, and soaking in 0.2% HgCl₂Soaking for 30min, and thoroughly washing with sterile water. Inoculating into culture solution containing 1/2MS +15g/L sucrose +2.0mg/L KT +0.5mg/L IAA and pH 5.60, and culturing. During the period, the illumination intensity is maintained at 2000lx, the illumination is carried out for 8h every day, and the indoor temperature is about 25 ℃. The sterility of the batch was found to be 60% after 1 month, with 8 of the sterile material regenerating the detoxified sprouts. Taking 4 detoxified sprouts, inoculating 1/2MS +15g/L sucrose +2.0mg/L KT +1.0mg/L IAA + 10% coconut milk, and adjusting pH to 5.75, inducing cluster buds in the culture solution II. After 2 transfers, all shoots were split and transferred to sterile medium (③ 6.05 pH) containing 1/4MS +7.5g/L sucrose +0.01mg/L NAA. And after another 1 month, all the materials are taken out to obtain about 4000 strong tissue culture seedlings of the water soft-shelled turtles with developed root systems. 1000 strains of the strain are taken, and washed by sterile water to remove residual culture solution on the surface, and the strain is used for family ornamental aquarium scenery. The rest tissue culture seedlings are washed by tap water and then transferred into a 400L plastic water tank, and 1/10Hoagland nutrient solution is given to strengthen the seedlings. The plastic water tank is stored in a glass greenhouse, the illumination is about 30-70% of outdoor natural light, and the temperature is maintained at about 25 ℃. After 20 days of strong seedlings, the further grown water turtle tissue culture seedlings are directly floated in the river course of the campus of our school for wetland landscape construction. In the batch of the water turtle tissue culture seedlings, the survival rate of the seedlings for viewing scenery in the aquarium is 100 percent, and the pollution phenomenon does not occur; the seedlings used in the campus landscape wetland are successfully established and bloomed in summer in the current year.

Example 5 Hydrocotyle sinensis was harvested in Xianlin lake of Nanjing in 10 months in 2.2016, and 10 young stems with shoot points were collected, soaked in detergent for 30min, and then washed with tap water for 30 min. Soaking in 75% ethanol for 1min, soaking in commercial bleaching agent (NaClO solution) diluted at a ratio of 1:10 for 2min, and thoroughly washing with sterile water. Inoculating into culture solution containing 1/2MS +15g/L sucrose +2.0 mg/L6-BA +0.5mg/L IBA, and culturing at pH 5.82. The conditions of the culture room are that the illumination intensity is 1500lx, the illumination is 10h every day, and the indoor temperature is about 25 ℃. After 1 month 6 of the batch were found sterile, of which 5 regenerated sterilized shoots. The detoxicated plumule is inoculated into culture medium containing MS 1 times, cane sugar 30g/L, 6-BA 3.0mg/L, IBA 1.0mg/L, coconut juice 5% and pH 6.05 to induce cluster bud. After 3 times of transfer, all cluster buds are cut, a small amount of seeds are removed, the cluster buds are transferred to a culture solution II, and the rest are transferred to a sterile culture solution III containing 1/2MS, 15g/L of cane sugar, 0.5mg/L of NAA and the pH value is 6.12. And taking out the rooted material after 25 days, washing the rooted material by tap water, and transferring the rooted material into 1/2Hoagland nutrient solution to strengthen seedlings. And after 20 days, counting to obtain more than 10 million strong tissue culture seedlings of the water soft-shelled turtles. The batch of tissue culture seedlings is applied to the purification of water quality in the south lake wetland park of the everlasting maturity of Jiangsu. Wherein about 2000 healthy tissue culture seedlings are firstly planted in pond sludge matrix and then are moved into a pond shallow water area with the water depth of about 50 cm. The rest tissue culture seedlings are directly thrown into the enclosed river channel. And observing the field survival condition of the water turtle tissue culture seedlings after 2 weeks, and finding that the seedlings of the two transplanting modes are recovered to grow and the coverage of the water turtles in the dense areas exceeds 80 percent.

Claims

1. A method for rapidly propagating high-quality seedlings of water turtles and application thereof are characterized by comprising the following steps:

(1) obtaining detoxified female parent

Collecting a small amount of water turtle plants from a wild natural wetland, shearing stem segments with tillering buds, washing the stem segments in a washing solution for 0.5 to 2 hours, washing the stem segments clean with tap water, soaking the stem segments in 70 to 75 percent ethanol in an ultraclean working chamber for no more than 3 minutes, and changing the stem segments into a NaClO solution containing 0.2 to 1 percent of available chlorine or 1 to 5 percent of H₂O₂Soaking in disinfectant to sterilize for 10 s-5 min or HgCl in 0.05-1%₂Soaking and disinfecting with a disinfectant for 1-30 min, thoroughly rinsing with sterile water, further cutting and finishing the material, inoculating the cut and finished material into a triangular flask filled with a sterilized culture solution I, sealing the triangular flask with a breathable sealing film, placing the sealed triangular flask on a special tissue culture frame for culture, maintaining the average illumination intensity of 1000-5000 lx, illuminating for 8-12 h every day at the indoor temperature of 20-30 ℃, and picking out a material with new buds growing and no bacterial growth, namely a water turtle detoxified female parent after one month, wherein the culture solution I is as follows: 1/2-1 times of MS, 10-40 g/L of sucrose, 0.5-3.0 mg/L of cytokinin KT or 6-BA and 0.1-1.0 mg/L of auxin IAA or IBA, and adjusting the pH value to be 5.6-6.2;

(2) amplification of Cluster shoots

Taking the female parent of the water turtle detoxication obtained in the step 1, transferring the female parent of the water turtle detoxication to a sterilized culture solution II on a sterile operating platform, putting the triangular flask back to the original tissue culture frame for culturing for 15-30 d, taking the female parent of the water turtle detoxication out when cluster buds of the water turtle basically grow up to the whole bottle, cutting every 1-3 buds into bottles, and continuing to propagate, wherein the culture solution II is as follows: 1/2-1 times of MS, 10-40 g/L of sucrose, 0.5-3.0 mg/L of cytokinin KT or 6-BA, 0.1-1.0 mg/L of auxin IAA or IBA and 5-10% of coconut juice, and adjusting the pH to be between 5.6 and 6.2;

(3) induction of rooting and Whole plants

Taking the cluster buds of the water soft-shelled turtles obtained in the step (2), cutting and separating every 1-2 buds on an aseptic operation table, transferring the cluster buds into a sterilized culture solution III, putting the triangular flask back to an original tissue culture frame for culturing for 15-30 d, and waiting for the water soft-shelled turtles in the flasks to root and form complete seedlings, wherein the culture solution III is as follows: 1/4-1 times of MS, 7.5-30 g/L of sucrose and 0.01-0.5 mg/L of auxin NAA or IBA, and adjusting the pH value to 5.6-6.2;

(4) strong seedling and transplanting

Taking out the water turtle seedlings obtained in the step (3) from a triangular flask, if the water turtle seedlings are to be applied to an indoor aquarium, firstly washing the water turtle seedlings with sterile water, deionized water or tap water to remove residual culture solution on the surface of the materials according to the sterile requirement of the aquarium cultivation, then putting the tissue culture seedlings into the aquarium under the sterile or bacteria condition, if the water turtle seedlings are to be applied to wetland park construction or wetland ecological restoration outdoor large-scale engineering, washing the tissue culture seedlings with tap water, removing MS and hormone residues, then transferring the tissue culture seedlings into an open water culture box containing diluted Hoagland nutrient solution, placing the open water culture box in a greenhouse or an outdoor strong seedling for 2-4 weeks, if the outdoor light is too strong in the initial stage, blocking 1/2 sunlight by using a sunshade net, after 3-7 days, enabling the tissue culture seedlings to completely adapt to natural light, and adopting two modes for large-scale transplanting of the water turtle tissue culture seedlings, wherein one mode is that the water seedlings are directly thrown into a natural water body to enable the water body to float and grow, the enclosure is used for preventing the floating of the soil in the initial stage, and the soil is firstly planted into a substrate which takes pond sludge and peat soil as main components and then is moved into a shallow water body, so that the planting is faster, but the water level depth is not more than 1m in the initial planting process.

2. The tissue culture and rapid propagation method of water soft-shelled turtle high-quality seedlings according to claim 1, applied to indoor aquarium ornamental scenery or outdoor wetland park construction or large wetland ecological restoration engineering.