CN116784230A - Method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls - Google Patents

Abstract

The application discloses a method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls, and belongs to the field of biotechnology breeding. Hybridization is carried out by adopting artificial supplementary pollination, and bagging and isolation are carried out; peeling young embryo 40-45 days after pollination; placing the peeled young embryo on an MS culture medium to culture into a seedling; transplanting tissue culture seedlings, and changing the pot after transplanting survival; the seed balls are harvested when the leaf blight of the seedling reaches 90-95% next year. The application can greatly reduce the risk of incapability of obtaining iris gratifolia seeds caused by uncertain environmental factors by utilizing embryo culture means, and compared with the common sowing technology, the method remarkably shortens the growth time of iris gratifolia from seeds to transplantable seedlings, and meanwhile, the peripheral diameter of the harvested offspring seed balls is larger. Therefore, the method greatly shortens the breeding time of the iris lilacina, accelerates the breeding process of new varieties, and has important significance for promoting the development of the iris lilacina industry in China.

Description

Method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls

Technical Field

The application belongs to the field of biotechnology breeding, and particularly relates to a method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls.

Background

The Iris (Iris×holllandica) is the most widely cultivated and applied type of Iris, and has a natural flowering period of 4 months and a group flowering period of about 30 days, and can be used for various application forms such as cut flowers, flower fields, flower environments, forests and the like. In the bulblet flower commercial variety ball produced in the Netherlands, the cultivation area of the Netherlands iris is in the front, and most of the bulblet flower commercial variety ball is used for cut flower production. The first introduction of the cultivated iris Netherlands in the last 90 th century in China, but the application and breeding research of the iris Netherlands are less, and the fresh cut flowers sold on the market almost depend on import or seed ball import planting production. Along with the continuous development of social economy, the flower industry has become an important component part of national economy in China, plays an important role in national ecological civilization strategy and country plain strategy, the requirements of the lotus iris as high-grade cut flowers in China are increased year by year in recent years, and compared with other imported bulbous flowers, the lotus iris bulbs are light in degradation, can be continuously planted for many years after one time of introduction, does not need to purchase bulbs year by year, and therefore has wide market prospect.

However, there are few and few commercial varieties of iris Netherlands with independent intellectual property rights in China at present, and the reason for this is mainly because most of the iris Netherlands have female or male sterile characteristics, which results in difficult crossbreeding.

However, based on the massive introduction of foreign elite varieties and adaptive cultivation, we found that certain specific hybrid combinations can be matured through massive crossing sets. However, due to environmental factors such as excessive rain or insect damage during seed production, mature hybrid seeds may not be harvested. In addition, even if the harvested seeds are transplanted after common sowing, the circumference diameter of the seed balls is small by the next year when the underground seed balls are harvested, and the long breeding cycle time can prevent the pace of the innovation of the germplasm of the domestic iris.

The embryo culture specifically means that the development condition of the young embryo is improved artificially, and nutrition necessary for the development of the young embryo is provided so as to promote the young embryo to grow into a plant. However, no related research report exists in the iris Netherlands.

Disclosure of Invention

The application aims to provide a method for rapidly obtaining iris Netherlands offspring seedlings and excellent seed balls.

The application successfully shortens the time from the seeds to the seedlings of the iris liliflora by utilizing the embryo culture technology, and in addition, the peripheral diameter of the seed balls collected by the seedlings cultured by the embryo is obviously larger than that of the seed balls generated by common seeding seedlings, so that the use of the technology greatly shortens the breeding period of the iris liliflora, improves the breeding efficiency and quality, and promotes the innovation progress of the germplasm of the iris liliflora in China.

Furthermore, the result is started in the middle and last ten days of the hybrid of the iris, tender fruits of the iris are found to be eaten by insects during the fruiting period, in addition, if the fruits are mildewed due to excessive rainwater, mature seeds are finally obtained, the risk of the seeds not being obtained due to external uncertain environmental factors is greatly reduced by using an embryo culture method, the peripheral diameter of the hybrid offspring seed balls obtained through the embryo culture technology is obviously larger than that of the seed balls generated by common seeding seedlings, and the technology is used for greatly accelerating the innovation process of the germplasm of the iris in China.

In order to achieve the aim of the application, the application adopts the following technical scheme:

a method for rapidly obtaining offspring seedlings and excellent seed balls of iris Netherlands comprises the following steps:

(1) Parent material preparation: selecting definite hybrid combined parent varieties capable of obtaining seeds, and performing commercial ball autumn planting to ensure sufficient fertilizer and water;

(2) Artificial pollination hybridization: when the female parent flowers and the anther of the stamen is immature and scattered, all stamen, flag petals and vertical petals of the female parent are cut off, and only three eclosion stigmas are left and isolated by a sulfuric acid paper bagging; when female parent pistil is mature, pollen of male parent is fertilized, bagging and isolation are continued after pollination, and in order to improve the pollination success rate, pollination is repeated once again for 2-3 days after the first pollination is finished, and the parchment paper can be taken down when the stigma is in a desiccation and wilting state after the second pollination is finished for about one week; and pollination is preferably performed in the morning when the weather is clear;

(3) Peeling young embryo: after 40-45 days from the first hybridization pollination, shearing the expanded capsules, flushing the surfaces of the capsules with clear water, spraying 75% alcohol on the surfaces of the capsules, and then placing the capsules in an ultra-clean workbench for sterilization by ultraviolet rays for 30min; peeling off capsules under the aseptic condition of an ultra-clean workbench after ultraviolet sterilization is finished, taking out expanded seeds, scratching seed coats by using dissecting needles, and extruding young embryos;

(4) Culturing young embryo in vitro: inoculating the young embryo obtained in the step (3) onto an MS culture medium under the aseptic condition for culture, wherein the culture conditions of a tissue culture room are as follows: culturing at 22-24deg.C in the light period of 14 h/10 h darkness with 4000-6000Lux until seedlings root;

(5) Transplanting seedlings: taking out the seedlings with root length more than 3cm and height more than 4cm in the step (4) from the culture medium, cleaning the roots, transplanting the seedlings into a seedling tray, transplanting the culture medium into sterilized vermiculite, covering a tray cover for moisturizing after the seedlings are watered thoroughly, placing the seedlings in a climatic chamber for culturing, uncovering the tray cover for about one week, and transplanting the seedlings for changing the pot after the seedlings survive; and the seedlings after transplanting and changing the pot are continuously placed in a climatic chamber for culture in the early stage, and are normally watered and managed when the seedlings are moved outdoors in the late 11 months;

(6) Seed ball harvesting: the seed balls can be harvested when the leaves on the upper parts of the iris of the Netherlands wilt next year.

Optionally, in the step (5), transplanting and changing the pot after the seedlings survive, and the specific operation is as follows:

after the root system of the seedling grows out of the bottom of the tray, transplanting the seedling together with the cultivated vermiculite matrix into a big basin; wherein, the volume ratio of the matrix in the basin is 1:1:1, turf, nutrient soil and vermiculite.

Further, the culture conditions of the artificial climate box in the step (5) are as follows: the illumination time is 16h/d, the temperature is 23+/-1 ℃ during illumination, the temperature is 18+/-1 ℃ during darkness, and the illumination intensity is 20000-25000Lux.

Optionally, the above-ground leaves of the iris tectorum in the step (6) are withered, namely, the seed balls are harvested when withered and yellow leaves account for 90-95% of the total leaf amount.

It is worth to say that, the green leaves can also carry out photosynthesis when the harvest is too early, and the growth of the seed balls is affected; if the harvesting is too late, the blades decay and the lower seed balls decay when the harvesting is too much in rainy days. When the green leaves only occupy 5-10%, the photosynthesis has smaller accumulation effect on the nutrition matters of the seed balls and can avoid the rot of the seed balls, so that the method is suitable for harvesting the seed balls.

Compared with the prior art, the application has the following beneficial effects:

the application adopts artificial supplementary pollination to carry out hybridization and bagging isolation; peeling young embryo 40-45 days after pollination; placing the peeled young embryo on an MS culture medium to culture into a seedling; transplanting tissue culture seedlings, and changing the pot after transplanting survival; the seed balls are harvested when the leaf blight of the seedling reaches 90-95% next year. The application can greatly reduce the risk of incapability of obtaining iris gratifolia seeds caused by uncertain environmental factors by utilizing embryo culture means, and compared with the common sowing technology, the method remarkably shortens the growth time of iris gratifolia from seeds to transplantable seedlings, and meanwhile, the peripheral diameter of the harvested offspring seed balls is larger. Therefore, the method greatly shortens the breeding time of the iris lilacina, accelerates the breeding process of new varieties, and has important significance for promoting the development of the iris lilacina industry in China.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present application, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the pest damage to Iris Netherlands fruit.

FIG. 2 shows the culture (A-C) and transplantation (D-E) of Iris Netherlands embryos.

FIG. 3 shows the seed balls produced by the cultured seedlings of Iris Netherlands (left) and the seed balls produced by the normal sown seedlings (right).

Detailed Description

The following will clearly and completely describe the technical solutions in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples of the present application, unless otherwise specified, was performed using conventional testing methods in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically mentioned in the present application are those commonly used by those skilled in the art.

In the description of the present application, it should be understood that the terms "medium," "upper," "lower," "ascending," "descending," "vertical," "face," "top," "bottom," "inner," "outer," and the like indicate or are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the present application and simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present application.

Numerous specific details are set forth in the following examples in order to provide a better understanding of the present application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.

On the premise of no conflict, the technical features disclosed by the embodiment of the application can be combined at will, and the obtained technical scheme belongs to the disclosure of the embodiment of the application.

The iris fruit in the following examples was collected from iris resource nursery of the academy of sciences of China, jiangsu province, and the test tools were all obtained from conventional products commercially available or commonly used in laboratories.

Example 1:

(1) Parent material preparation: selecting Red Ember and Tiger Eye as parents, and planting commodity balls in autumn to ensure sufficient fertilizer and water;

(2) Artificial pollination hybridization: when female parent Red Ember flowers and anther of stamen is immature and scattered, cutting off all stamen, flag petals and vertical petals of the female parent, only leaving three eclosion stigmas and isolating by using a sulfuric acid paper bagging; when female parent pistil is mature, pollen of male parent Tiger Eye is fertilized, bagging and isolation are continued, pollination is repeated once again 2-3 days after the first pollination is finished, and when the stigma is in a desiccation wilting state after the second pollination is finished for about one week, the sulfuric acid paper can be removed; the pollination time is 9-10 am, and the day of pollination is clear;

(3) Peeling young embryo: after 40 days from the first hybridization pollination, shearing the expanded capsules, flushing the surfaces of the capsules with clear water, spraying 75% alcohol on the surfaces of the capsules, and then placing the capsules in an ultra-clean workbench for sterilization by ultraviolet rays for 30min; peeling off capsules under the aseptic condition of an ultra-clean workbench after ultraviolet sterilization is finished, taking out expanded seeds, scratching seed coats by using dissecting needles, and extruding young embryos;

(4) Culturing young embryo in vitro: inoculating the young embryo obtained in the step (3) to an MS culture medium for culture under the aseptic condition, wherein the culture conditions of a tissue culture room are as follows: culturing at 22-24deg.C in the light period of 14 h/10 h darkness with 4000-6000Lux until seedlings root;

(5) Transplanting seedlings: when the seedlings grow to the root length of more than 3cm and the height of more than 4cm, taking out from the culture medium, cleaning the roots, transplanting the roots into a seedling tray, transplanting the culture medium into sterilized vermiculite, covering a plug tray cover for moisturizing after watering thoroughly, and culturing in a climatic chamber under the following culture conditions: the illumination time is 16h/d, the temperature is 23+/-1 ℃ during illumination, the temperature is 18+/-1 ℃ during darkness, and the illumination intensity is 20000-25000Lux. And uncovering the hole tray cover after 7 days, and transplanting the seedling root system and the cultivated vermiculite matrix in a big basin after the seedling root system grows out of the bottom of the hole tray. The substrate in the basin is turf: nutrient soil: vermiculite = 1:1:1 (V: V: V). The seedlings after transplanting and pot changing are continuously placed in a climatic chamber for cultivation in the early stage, and are moved outdoors for 20 days for normal watering management;

(6) Seed ball harvesting: and harvesting the seed balls when the wilted leaves of the overground parts of the iris seedlings reach 90-95% in the next year.

The hybrid offspring seedlings are cultured to a height of >4cm by calculating Red EmberxTiger Eye embryo, which takes about 50 days, and the seed balls planted next year after transplanting are shown in figure 3 (left), and the circumference diameter is about 4.2cm.

Example 2:

similar to the procedure of example 1, the difference is that: the steps (2) and (3) are specifically as follows:

(2) Artificial pollination hybridization: hybridization procedure was the same as in example 1 using Tiger Eye as female parent and Red Eye as male parent;

(3) Peeling young embryo: 45 days from the first cross pollination, the expanded capsules were cut off, the remainder of the procedure being the same as in example 1.

The time required for the cultivation of the hybrid offspring seedlings to grow to a height of >4cm by the Tiger eye×Red embryo is calculated to be about 55 days, and the diameter of the circumference of the seed ball which is grown next year after the transplanting is about 5.4cm. Comparative example 1:

steps (1), (2) and (6) are the same as in example 1, the remaining steps being as follows:

(3) And (3) seed collection: harvesting the capsules produced by hybridization after the capsules become yellow and mature, putting the capsules into a nylon mesh bag, hanging the nylon mesh bag at a ventilation position for drying, carefully pulling the capsules after drying to take out seeds, and selecting the filled seeds to be put into a fine nylon bag and hanging the nylon mesh bag at a drying position for later use;

(4) Sowing: soaking hybrid seeds of iris Netherlands for 30min by using a thousandth carbendazim aqueous solution, and then washing off carbendazim powder on the surfaces of the seeds by using clear water for 2-3 times. Then placed on wet filter paper and incubated at room temperature for about 2 days. Sowing the water-saturated iris seeds in a plastic basin, filling a layer of plastic woven bag at the bottom of the plastic basin, then putting yellow sand with the thickness of about 5cm, and finally dispersing and spreading the iris hybrid seeds on the yellow sand and covering the yellow sand by about 2cm. The well sown iris seeds are watered thoroughly and then put into an artificial climatic chamber with illumination time of 16h/d, the temperature of 23 ℃ plus or minus 1 ℃ during illumination and the temperature of 18 ℃ plus or minus 1 ℃ during darkness and illumination intensity of 20000-25000Lux until seedlings emerge.

(5) Transplanting seedlings: when the seedling height is more than 4cm, carefully taking out the seedling and transplanting the seedling to turf: nutrient soil: vermiculite = 1:1:1 (V: V: V) in a mixed matrix, and culturing in a climatic chamber at the early stage. And (5) moving the plant outdoors for 11 months and 20 days, and carrying out normal watering management.

The hybrid offspring seedlings of the comparative example Red EmberxTiger Eye harvested and commonly sown are calculated to have a height of >4cm and take about 70 days, and the seed balls planted next year after transplanting are shown in fig. 3 (right), and the circumference diameter is about 2.8cm.

Comparative example 2:

steps (1) and (2) are the same as in example 2, and steps (3) to (6) are the same as in comparative example 1.

The hybrid offspring seedlings of the comparative example Tiger eye×Red Ember, which were commonly sown after seed collection, were calculated to take about 73 days to grow to a height >4cm, and the diameter of the seed ball after transplanting was about 3.0cm at the next year.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (6)

1. A method for rapidly obtaining offspring seedlings and excellent seed balls of iris Netherlands is characterized by comprising the following steps:

(1) Parent material preparation: selecting a hybrid combined parent variety with seeds obtained definitely, and planting commodity balls in autumn to ensure sufficient fertilizer and water;

(2) Artificial pollination hybridization: when female parent flowers and anther of stamen is immature and scattered, all stamen, flag and vertical petals of the female parent are cut off, only three eclosion stigmas are left and isolated by a sulfuric acid paper bagging, when female parent pistil is mature, pollen of male parent is fed, and bagging and isolation are continued after pollination. In order to improve the pollination success rate, the secondary pollination can be performed 2-3 days after the first pollination is finished;

(3) Peeling young embryo: after the step (2) of hybridization pollination, shearing off the capsule after the capsule is expanded, flushing the surface with clear water and spraying 75% alcohol on the surface of the capsule; sterilizing with ultraviolet rays for 30min; peeling off capsules under the aseptic condition of an ultra-clean workbench after ultraviolet sterilization is finished, taking out expanded seeds, scratching seed coats by using dissecting needles, and extruding young embryos;

(4) Culturing young embryo in vitro: inoculating the young embryo obtained in the step (3) to an MS culture medium for culture under the aseptic condition, wherein the culture conditions of a tissue culture room are as follows: culturing at 22-24deg.C in the light period of 14 h/10 h darkness with 4000-6000Lux until seedlings root;

(5) Transplanting seedlings: taking out seedlings with good rooting in the step (4) and leaves growing to the middle upper part of the tissue culture bottle from a culture medium, cleaning roots, transplanting the seedlings into a seedling tray, covering a hole tray cover for moisturizing after the seedlings are watered thoroughly, culturing the seedlings in a climatic chamber, and transplanting the seedlings for changing pots after the seedlings survive;

(6) Seed ball harvesting: and harvesting the seed balls when the leaves on the upper parts of the iris of the Netherlands wither in the next year.

2. The method of claim 1, wherein in step (3), the young embryo is taken from a capsule grown for 40-45 days after the first cross pollination.

3. The method according to claim 1, wherein in step (5), the root length of the seedling to be transplanted in the tissue culture flask is >3cm and the height is >4cm.

4. A method according to claim 1 or 3, wherein in step (5), the tissue culture seedling transplanting culture medium is sterilized vermiculite, and the culture conditions in the artificial climatic chamber are as follows:

the illumination time is 16h/d, the temperature is 23+/-1 ℃ during illumination, the temperature is 18+/-1 ℃ during darkness, and the illumination intensity is 20000-25000Lux.

5. The method according to claim 4, wherein in the step (5), the seedlings are transplanted and the pot is replaced after survival, and the specific operation is as follows:

after the root system of the seedling grows out of the bottom of the tray, transplanting the seedling together with the cultivated vermiculite matrix into a big basin; wherein, the volume ratio of the matrix in the basin is 1:1:1, turf, nutrient soil and vermiculite.

6. The method according to claim 1, wherein in the step (6), the seed balls are harvested when the upper leaves of the iris of the Netherlands wilt, i.e., the withered and yellow leaves account for 90 to 95% of the total leaves.