阅读说明：本技术 一种通过胚拯救获得重瓣萱草杂交后代的方法 (Method for obtaining filial generation of hemerocallis sempervirens through embryo rescue ) 是由 陈芬 马丽 刘博� 蔡卫佳 王昊 于 2021-10-18 设计创作，主要内容包括：本发明提供一种通过受精幼胚获得重重瓣萱草杂种后代的方法属于植物组织培养技术的植物再生技术领域,其方法是通过取人工去雄授粉9d的胚珠采用离体培养的方式进行胚拯救,具体步骤为：(1)授粉,(2)取胚珠,(3)诱导培养,(4)继代培养,(5)生根培养,(6)移栽；本发明弥补了重瓣萱草杂交结实率低下的问题,避免了外界环境的不确定性对成活率的影响,同时也缩短了育种周期,可运用于重瓣萱草杂交育种工作。(The invention provides a method for obtaining a hybrid progeny of hemerocallis fulva by fertilizing a young embryo, which belongs to the technical field of plant regeneration of plant tissue culture technology, and the method comprises the following steps of carrying out embryo rescue by taking ovules which are artificially emasculated and pollinated for 9d in an in vitro culture mode: (1) pollinating, (2) taking ovules, (3) performing induction culture, (4) performing subculture, (5) performing rooting culture, and (6) transplanting; the method solves the problem of low fruiting rate of Hemerocallis fulva hybrid, avoids the influence of uncertainty of external environment on survival rate, shortens breeding period, and can be applied to Hemerocallis fulva hybrid breeding work.)

1. A method for obtaining filial generation of Hemerocallis sempervirens by embryo rescue is characterized by comprising the following steps:

(1) pollination: selecting a well-developed hemerocallis fulva variety as a parent material, wherein the parent material is obtained by the following steps of 8: emasculation, pollination and branding are carried out at a ratio of 00-8: 30;

(2) taking ovules: picking fruits pollinated for 9d, putting the fruits into a container, adding detergent into the container, washing for 30 min under running water, washing for 30s in 75% alcohol on a super-clean workbench after washing, finally soaking for 10min in 0.1% mercuric chloride solution, washing for 3 times by sterile water, removing peel, and peeling ovary wall to obtain ovules;

(3) inducing callus: inoculating ovule into MS with pH of 5.8, sucrose 9%, 6-BA 0.2mg/L, No. II culture medium 0.02mg/LNAA, and Shannon 0.1%, and placing into incubator to induce callus;

(4) culturing adventitious buds: transferring the mixture to a culture medium of MS with the pH value of 5.8, 3 percent of cane sugar, 2.0mg/L6-BA, 0.1mg/LNAA and 0.1 percent of Shannon type II after 30 to 40 days, and then putting the mixture into an incubator to induce adventitious buds;

(5) rooting induction: after 30 days, selecting adventitious buds with good growth vigor and about 5cm, transferring the adventitious buds to a rooting culture medium 1/2MS +0.2mg/L IBA +0.1mg/L NAA +0.1% Shannon type II, and putting the adventitious buds into an incubator to induce rooting;

(6) transplanting: acclimatizing the well-developed aseptic seedlings of the root system in an indoor environment for 5d, taking out the aseptic seedlings, cleaning attachments, transplanting the aseptic seedlings to peat with a perlite ratio of 7: 3, covering a sunshade net in the hole tray, culturing for 8 days in a greenhouse environment with the temperature of 25-28 ℃ and the humidity of about 80%, gradually opening the sunshade net, and performing conventional management.

2. The method according to claim 1, wherein the parents selected in step (1) are of the Hemerocallis sempervirens variety of the same flowering stage.

3. The method for obtaining filial generation of Hemerocallis sempervirens through embryo rescue according to claim 1, wherein the culture medium in step (3), step (4) and step (5) is 5g/L agar.

4. The method for obtaining filial generation of Hemerocallis sempervirens through embryo rescue according to claim 1, wherein the culture conditions in the culture box in the steps (3), (4) and (5) are as follows: the temperature is 25 ℃, the photoperiod is 12h/d, and the illumination intensity is 1500-2500 Lx.

5. The method according to claim 1, wherein the step (3) is performed in a culture medium of MS +9% sucrose +0.2mg/L6-BA +0.02mg/LNAA +0.1% Shannon type II, wherein 0.1% Shannon type II can effectively inhibit the contamination of endophytes.

Technical Field

The invention relates to plant regeneration through a plant tissue culture technology, and relates to a method for obtaining a Hemerocallis sempervirens hybrid progeny through immature embryo rescue.

Background

The hemerocallis fulva is a perennial herb of hemerocallis of Africaceae, and the breeding center of the hemerocallis fulva is in the United states at present, and the breeding of hemerocallis fulva varieties is relatively late in China. The variety of hemerocallis registered by the American hemerocallis society is more than 8 thousands, wherein the number of the hemerocallis registered by the national hemerocallis society is not more than 50, and the result is that the fruiting rate of the filial generation of hemerocallis is low to a great extent. The hemerocallis nepalensis is used as a variety of wild hemerocallis nepalensis, stamens of the hemerocallis nepalensis are petal-shaped, and the hemerocallis nepalensis has higher ornamental value than that of a single-petal hemerocallis nepalensis, but the maturing rate of the hemerocallis nepalensis is far inferior to that of the single-petal variety due to the underground pollen activity of the hemerocallis nepalensis, and in order to relieve the problem, the method is continuously tested on the basis of the theoretical basis of embryo rescue after hybridization so as to obtain more fertile hybrid progeny. The post-hybrid embryo rescue is an effective method for overcoming immature embryo abortion, plays an extremely important role in plant breeding work, and is widely applied to many species, so that the invention provides a method for obtaining a Hemerocallis fulva hybrid progeny by utilizing immature embryo rescue.

Disclosure of Invention

In order to overcome the problems, the invention provides a method for obtaining the hybrid progeny of the hemerocallis fulva through fertilization of immature embryos, the method makes up the defect of low fruiting rate of the hemerocallis fulva hybrid, avoids the great influence of uncertainty of the external environment on the survival rate, shortens the breeding period, and can be applied to the hemerocallis fulva hybrid breeding work.

The invention is realized by the following technical scheme: a method for obtaining filial generation of Hemerocallis sempervirens by embryo rescue is characterized by comprising the following steps:

(1) pollination: selecting a well-developed hemerocallis fulva variety as a parent material, wherein the parent material is obtained by the following steps of 8: emasculation, pollination and branding are carried out at a ratio of 00-8: 30;

(2) taking ovules: picking fruits pollinated for 9d, putting the fruits into a container, adding detergent into the container, washing for 30 min under running water, washing for 30s in 75% alcohol on a super-clean workbench after washing, finally soaking for 10min in 0.1% mercuric chloride solution, washing for 3 times by sterile water, removing peel, and peeling ovary wall to obtain ovules;

(3) inducing callus: inoculating ovule into MS with pH of 5.8, sucrose 9%, 6-BA 0.2mg/L, No. II culture medium 0.02mg/LNAA, and Shannon 0.1%, and placing into incubator to induce callus;

(4) culturing adventitious buds: transferring the mixture to a culture medium of MS with the pH value of 5.8, 3 percent of cane sugar, 2.0mg/L6-BA, 0.1mg/LNAA and 0.1 percent of Shannon type II after 30 to 40 days, and then putting the mixture into an incubator to induce adventitious buds;

(5) rooting induction: after 30 days, selecting adventitious buds with good growth vigor and about 5cm, transferring the adventitious buds to a rooting culture medium 1/2MS +0.2mg/L IBA +0.1mg/L NAA +0.1% Shannon type II, and putting the adventitious buds into an incubator to induce rooting;

(6) transplanting: acclimatizing the well-developed aseptic seedlings of the root system in an indoor environment for 5d, taking out the aseptic seedlings, cleaning attachments, transplanting the aseptic seedlings to peat with a perlite ratio of 7: 3, covering a sunshade net in the hole tray, culturing for 8 days in a greenhouse environment with the temperature of 25-28 ℃ and the humidity of about 80%, gradually opening the sunshade net, and performing conventional management.

As a further optimization of the scheme, the parents selected in the step (1) are hemerocallis sempervirens variety in the same flowering phase.

As a further optimization of the scheme, the culture media in the step (3), the step (4) and the step (5) are all 5g/L agar.

As a further optimization of the scheme, the culture conditions in the culture box in the step (3), the step (4) and the step (5) are as follows: the temperature is 25 ℃, the photoperiod is 12h/d, and the illumination intensity is 1500-2500 Lx.

As a further optimization of the scheme, the step (3) is carried out in a culture medium of MS +9% of cane sugar +0.2mg/L6-BA +0.02mg/LNAA +0.1% of shannon type II, wherein 0.1% of shannon type II can effectively inhibit endophyte pollution.

In the invention, fruits in the container are washed by running water, and the container is added with the detergent, so that the exogenous bacteria pollution can be effectively inhibited.

The invention preferably summarizes the optimal technical parameters through the following comparison tests:

selection of embryos at different developmental stages: selecting embryos of 6, 9, 12, 15, 18 and 21d in different development periods after pollination respectively, picking up the embryo pollinated for 9d, adding detergent into a container, washing for 30 min under running water, washing for 30s in 75% alcohol on a super clean bench, soaking for 10min by using 0.1% mercuric chloride solution, washing for 3 times by using sterile water, removing peel, and peeling off ovary wall to obtain ovules; inoculating ovule into MS +9% sucrose +0.2mg/L6-BA +0.02mg/LNAA +0.1% Shannon type II culture medium with pH of 5.8-6.0, and inducing callus in an incubator with 25 deg.C, light cycle of 12h/d and illumination intensity of 1500-2500 Lx; 15. 18d embryos can be successfully induced to bud, seed abortion usually occurs about 10d after pollination in the actual hybridization process, and embryo rescue carried out 15 d and 18d after pollination has no practical significance.

Selection test of induction medium: taking the hybrid Hemerocallis fulva ovule fertilized for 9d, selecting MS culture medium from the basic culture medium, adding two hormones of 6-BA and NAA, setting the 6-BA at four levels of 0.1mg/L, 0.2mg/L, 0.3 mg/L and 0.4mg/L, setting the NAA at four levels of 0.01 mg/L, 0.02mg/L, 0.03 mg/L and 0.04 mg/L, and showing that the optimum concentration is 0.2mg/L6-BA +0.02 mg/LNAA.

Selection test of adventitious bud induction medium: the sterile seedling successfully inducing the callus is taken as a material, MS culture medium is selected as basic culture medium, two hormones of 6-BA and NAA are added, the 6-BA is set to four levels of 1.0mg/L, 2.0mg/L, 3.0mg/L and 4.0mg/L, the NAA is set to four levels of 0.1mg/L, 0.2mg/L, 0.3 mg/L and 0.4mg/L, and the result shows that 2.0mg/L6-BA +0.1mg/LNAA is the most suitable concentration.

According to the technical scheme provided by the invention, the method for obtaining the hybrid progeny of the hemerocallis fulva through the fertilization of the immature embryos makes up the defect of low fruiting body of the hemerocallis fulva hybrid embryo, avoids the great influence of uncertainty of the external environment on the survival rate, shortens the breeding period, and can be applied to the hybridization breeding work of the hemerocallis fulva.

Detailed description of the invention

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any feature disclosed in this specification (including any accompanying claims, abstract) may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.

Examples

A method for obtaining filial generation of Hemerocallis sempervirens by embryo rescue is characterized by comprising the following steps:

(1) pollination: selecting a well-developed hemerocallis fulva variety as a parent material, wherein the parent material is obtained by the following steps of 8: emasculation, pollination and branding are carried out at a ratio of 00-8: 30;

(2) taking ovules: picking fruits pollinated for 9d, putting the fruits into a container, adding detergent into the container, washing for 30 min under running water, washing for 30s in 75% alcohol on a super-clean workbench after washing, finally soaking for 10min in 0.1% mercuric chloride solution, washing for 3 times by sterile water, removing peel, and peeling ovary wall to obtain ovules;

(3) inducing callus: inoculating ovule into MS with pH of 5.8, sucrose 9%, 6-BA 0.2mg/L, No. II culture medium 0.02mg/LNAA, and Shannon 0.1%, and placing into incubator to induce callus;

(4) culturing adventitious buds: transferring the mixture to a culture medium of MS with the pH value of 5.8, 3 percent of cane sugar, 2.0mg/L6-BA, 0.1mg/LNAA and 0.1 percent of Shannon type II after 30 to 40 days, and then putting the mixture into an incubator to induce adventitious buds;

(5) rooting induction: after 30 days, selecting adventitious buds with good growth vigor and about 5cm, transferring the adventitious buds to a rooting culture medium 1/2MS +0.2mg/L IBA +0.1mg/L NAA +0.1% Shannon type II, and putting the adventitious buds into an incubator to induce rooting;

(6) transplanting: acclimatizing the well-developed aseptic seedlings of the root system in an indoor environment for 5d, taking out the aseptic seedlings, cleaning attachments, transplanting the aseptic seedlings to peat with a perlite ratio of 7: 3, covering a sunshade net in the hole tray, culturing for 8 days in a greenhouse environment with the temperature of 25-28 ℃ and the humidity of about 80%, gradually opening the sunshade net, and performing conventional management.

As a further optimization of the scheme, the parents selected in the step (1) are hemerocallis sempervirens variety in the same flowering phase.

As a further optimization of the scheme, the culture media in the step (3), the step (4) and the step (5) are all 5g/L agar.

As a further optimization of the scheme, the culture conditions in the culture box in the step (3), the step (4) and the step (5) are as follows: the temperature is 25 ℃, the photoperiod is 12h/d, and the illumination intensity is 1500-2500 Lx.

As a further optimization of the scheme, the step (3) is carried out in a culture medium of MS +9% of cane sugar +0.2mg/L6-BA +0.02mg/LNAA +0.1% of shannon type II, wherein 0.1% of shannon type II can effectively inhibit endophyte pollution.

While the basic teachings of the present invention have been described, numerous extensions and variations will be apparent to those of ordinary skill in the art. As the present invention disclosed in the specification may be embodied in other specific forms without departing from the spirit or general characteristics thereof, and it is noted that some of these specific forms have been set forth, the embodiments disclosed in the specification should be considered as illustrative and not restrictive. The scope of the invention is indicated by the appended claims, rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.