阅读说明：本技术 一种促进北美冬青组培苗生根方法 (Method for promoting rooting of ilex verticillata tissue culture seedlings ) 是由 陈庆生 张敏 周鹏 黄婧 宋倩云 于 2019-10-07 设计创作，主要内容包括：本发明涉及一种北美冬青组培微插穗直接生根方法,其主要是解决现北美冬青组培苗生根存在培养程序多、成活率低、周期长等的技术问题。包括以下步骤：(1)瓶苗选择；(2)炼苗；(3)组培苗瓶外扦插；(4)扦插后管理。本发明建立了有效的北美冬青组培苗瓶外生根技术,优化了其组培快繁体系。该技术无需经过组培瓶内生根程序,生根和驯化同时进行,25~30d幼苗基部长出大量根系。2个月后得到健壮的生根苗。与传统瓶内生根相比,育苗周期缩短1个月以上,生根率大于93%,移栽成活率大于95%,能够降低生产成本40%左右,节省组培空间50%以上,对于北美冬青组培集约化生产具有重要意义。(The invention relates to a method for directly rooting tissue culture micro cuttings of ilex verticillata, which mainly solves the technical problems of multiple culture procedures, low survival rate, long period and the like existing in the rooting of tissue culture seedlings of ilex verticillata. The method comprises the following steps: (1) selecting bottle seedlings; (2) hardening seedlings; (3) cuttage is carried out outside a tissue culture seedling bottle; (4) and (5) managing after cuttage. The invention establishes an effective ilex verticillata tissue culture seedling ex-vitro rooting technology and optimizes a tissue culture and rapid propagation system thereof. The technology does not need a rooting procedure in a tissue culture bottle, rooting and domestication are carried out simultaneously, and a large number of root systems grow on the base of the seedling within 25-30 d. After 2 months, robust rooted seedlings are obtained. Compared with the traditional bottle rooting, the seedling culture period is shortened by more than 1 month, the rooting rate is more than 93%, the transplanting survival rate is more than 95%, the production cost can be reduced by about 40%, the tissue culture space is saved by more than 50%, and the method has important significance for tissue culture intensive production of the ilex verticillata.)

1. A method for directly rooting tissue culture micro-cuttings of ilex verticillata is characterized by comprising the following steps:

(1) selecting bottle seedlings: selecting good-growing ilex verticillata tissue culture seedlings with the height of about 8-10 cm and strong branches, and carrying out hardening and domestication;

(2) hardening seedlings: firstly, placing ilex verticillata tissue culture seedlings in a seedling hardening chamber, wherein the seedling hardening chamber is mainly based on scattered light, the illumination intensity is 3000-;

(3) tissue culture seedling out-of-bottle cuttage: taking out the tissue culture seedlings from the bottle, cleaning the culture medium adhered to the plant base, shearing off the culture medium according to a single plant, and removing the yellow damaged leaves at the bottom; cutting each plant into 3-4 cm stem segments with buds, dipping in rooting water before cuttage, wherein the cuttage depth is 1-1.5 cm, and the substrate for cuttage is perlite; water is sprayed at intervals in the cutting process to ensure that the tissue culture seedlings are not withered;

(4) managing after cuttage: and (3) watering thoroughly after the cuttage is finished, covering a thin film on the seedlings, culturing for 1-2 weeks, removing the film, watering at regular intervals, applying a foliar fertilizer and a bactericide, growing a large number of root systems on the base parts of the seedlings within 25-30 d, obtaining robust North American winter seedlings after 2 months, and transplanting the seedlings into a nutrition pot for planting.

2. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: in the step (3), the rooting water adopts ABT rooting powder No. 1, the concentration is 800-1000 ppm, the dipping time is 3-5 seconds, and the base part of the tissue culture seedling is soaked by 1-2 cm.

3. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: the preparation method of each liter of rooting aqueous solution comprises the following steps: dissolving 500 mg ABT rooting powder No. 1 in a small amount of ethanol, and metering to 1L with clear water.

4. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: and (4) selecting a plastic seedling raising plate as the cuttage container in the step (3), wherein the matrix occupies the volume of the seedling raising plate 3/4.

5. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: treating the peat obtained in the step (3) with 800 times of carbendazim for disinfection; 800 times of carbendazim is as follows: 1 g of carbendazim with the concentration of 50% is diluted by 800 mL of water.

6. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: the culture temperature in the step (4) is controlled to be 20-25 ℃, the humidity is kept above 85%, the light intensity is 1500-2000 lx, and the illumination time is 16 h/d.

7. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: in the step (4), the foliar fertilizer is a mixture of flowers and bambol which are different and are used alternately, and the bactericide is 0.1 percent of carbendazim and chlorothalonil.

8. The method for directly rooting tissue culture micro cuttings of ilex verticillata according to claim 1, wherein the method comprises the following steps: watering and applying the foliar fertilizer and the bactericide at regular intervals, namely watering once every three days, spraying the foliar fertilizer once every two weeks, and spraying the bactericide once every month.

Technical Field

The invention relates to a method for breeding ilex verticillata, in particular to a method for rapid ex vitro rooting of tissue culture seedlings of ilex verticillata.

Background

North American holly root (Ilex verticillata) Is a plant of the genus ilex of the family Aquifoliaceae, and the red fruit on the head of the branch shows more joyful and gorgeous in the morning after the leaves fall off in winter, is a famous branch cutting tree species and is named as red bean in the market name. The red beans can be listed in the festival atmosphere of the New year's day, Christmas, sweet-and-night festival, valentine's day and the like from the end of 11 months to around the New year's day, and the market acceptance in China is very high. At present, the North America holly cutting production is still in the preliminary stage, and China needs to import a large amount of branches every year.

Scholars at home and abroad have carried out a great deal of research work on the aspects of ilex verticillata germplasm resource collection and excellent variety breeding, and cuttage and tissue culture are the most main modes for fine variety breeding. The early-stage research of the subject group finds that the ilex verticillata belongs to tree species which are difficult to root, the rooting is slow, the seedling rate of cutting propagation is lower than 50 percent, and a large number of clone seedlings cannot be obtained in a short period. Therefore, tissue culture has become an important means for the rapid propagation of the variety of ilex verticillata, wherein the rooting of the test-tube plantlet is a necessary process for the rapid propagation of the tissue. At present, the traditional bottle rooting is still adopted for the test-tube plantlet rooting of the ilex verticillata, and the problems of high production cost, long seedling growing period (60 days are needed for rooting and transplanting in the bottle), low transplanting survival rate (the root system of the seedling with the roots in the tissue culture bottle is weak and the activity is weak, and the transplanting survival rate is lower than 70 percent) and the like exist. Therefore, how to improve the rooting and transplanting efficiency of the tissue culture seedlings becomes an important research direction for the large-scale breeding of the ilex verticillata.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a method for tissue culture of ilex verticillata and direct rooting of micro cuttings, which combines tissue culture with cutting propagation, compared with rooting in a bottle, the technology can not only reduce the steps of one-time aseptic operation, simplify the tissue culture procedure, improve the culture space, but also reduce the production cost, improve the rooting efficiency and provide technical support for large-scale rapid propagation of the good variety of ilex verticillata; meanwhile, compared with common cutting, the meristem capability of the micro cutting slips cultured by the tissue is enhanced after the tissue culture and the rejuvenation, and the rooting capability of the cutting slips can be effectively improved.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme that the method for promoting the rooting of the ilex verticillata tissue culture seedling comprises the following steps:

(1) selecting bottle seedlings: selecting good-growing ilex verticillata tissue culture seedlings with the height of about 8-10 cm and strong branches, and carrying out hardening and domestication;

(2) hardening seedlings: firstly, placing ilex verticillata tissue culture seedlings in a seedling hardening chamber, wherein the seedling hardening chamber is mainly based on scattered light, the illumination intensity is 3000-;

(3) tissue culture seedling out-of-bottle cuttage: taking out the seedlings from the bottle, cleaning the culture medium adhered to the plant base, shearing off the seedlings according to a single plant, and removing the yellow damaged leaves at the bottom; cutting each plant into 3-4 cm stem segments with buds, dipping in rooting water before cuttage, wherein the cuttage depth is 1-1.5 cm, and peat is used as a substrate for cuttage; water is sprayed at intervals in the cutting process to ensure that the seedlings are not withered;

(4) managing after cuttage: watering thoroughly after cuttage is finished, controlling the water content to be about 80%, supporting a small shed above seedlings by using a preservative film, culturing for 1-2 weeks, then removing the shed, watering once every three days, spraying foliar fertilizer once every two weeks, and spraying bactericide once a month. And (5) growing a large number of roots at the base of the seedling for 25-30 d. After 2 months, robust North American winter green seedlings are obtained and transplanted into a nutrition pot for planting.

Preferably, the rooting water in the step (3) adopts ABT rooting powder No. 1 with the concentration of 800-. The preparation method of the rooting agent solution per liter comprises the following steps: dissolving 800-1000 mg ABT rooting powder with a small amount of ethanol, and metering the volume to 1L with clear water;

preferably, the cuttage container in the step (3) is a plastic seedling raising plate, and the matrix occupies the volume of the seedling raising plate 3/4.

Preferably, the peat in the step (3) is treated and sterilized by 800 times of carbendazim; 800 times of carbendazim is as follows: 1 g of carbendazim with the concentration of 50% is diluted by 800 mL of water.

Preferably, the culture temperature in the step (4) is controlled to be 20-25 ℃, the humidity is kept above 85%, the light intensity is 1500-2000 Lux, and the illumination time is 16 h/d.

Preferably, the foliar fertilizer in the step (4) is a mixture of flowers and bambol, and the bactericide is 0.1% of carbendazim and chlorothalonil.

The invention adopts the tissue culture micro-cutting direct rooting technology to carry out rooting test, combines the tissue culture with the cutting propagation, can not only reduce the steps of one-time aseptic operation, simplify the tissue culture procedure, improve the culture space, but also reduce the production cost and improve the rooting efficiency, compared with the in-bottle rooting, the technology not only can reduce the steps of one-time aseptic operation, simplify the tissue culture procedure, improve the culture space, but also can reduce the production cost, thereby improving the rooting efficiency; meanwhile, compared with common cuttage, the meristem capability of the micro cutting slips cultured by the tissue is enhanced after the tissue culture and the rejuvenation, the rooting capability of the cutting slips can be effectively improved, the rooting rate of tissue culture seedlings can be obviously improved, and the survival rate is also obviously improved after the transplanting due to the developed root system, so that the problem of tissue culture industrialized seedling rooting is solved.

The invention combines years of research and production experience, summarizes the bottle-outside rooting technology of the tissue culture seedlings of the ilex verticillata, compared with bottle-inside rooting, the technology is a propagation method combining rapid propagation in a bottle and a cutting technology, after secondary propagation seedlings are treated, the seedlings are directly transplanted and rooted in a bacteria-bearing and autotrophic environment, the rooting and domestication of test-tube seedlings are effectively combined, the tissue culture procedure can be simplified, the production cost is reduced by more than 40%, the tissue culture space is saved by more than 50%, the tissue culture seedling culture period is shortened by more than 1 month, and the rooting efficiency is improved; meanwhile, compared with common cutting, the meristem capability of the micro cutting slips cultured by the tissue is enhanced after the tissue culture and the rejuvenation, the rooting capability of the cutting slips can be effectively improved, the rooting rate and the survival rate after transplantation of tissue culture seedlings can be obviously improved, and the method has important technical significance for optimizing a tissue culture and rapid propagation system of the ilex verticillata.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides a referable technology and method for rapid ex vitro rooting of ilex verticillata tissue culture seedlings, and has the following advantages:

1. compared with the conventional tissue culture method, the method saves the rooting link in the bottle, shortens the seedling culture period by more than 1 month, and has simple and convenient operation and strong practicability;

2. in the invention, rooting and domestication are carried out simultaneously, the rooting rate is more than 98%, the root system is developed, the seedling grows strongly, the transplanting survival rate is more than 95%, and the seedling consistency is good;

3. the ex vitro rooting technology can reduce the production cost by about 40 percent, saves the tissue culture space by more than 50 percent, and has important significance for improving the tissue culture industrialization level of the ilex verticillata.

Detailed Description

The following will describe in detail embodiments of the present invention with reference to specific examples: