A method for quickly obtaining progeny seedlings and excellent bulbs of Iris hollandica

Abstract

The present invention discloses a method for quickly obtaining offspring seedlings and excellent bulbs of Dutch iris, and belongs to the field of biotechnology breeding. Artificial assisted pollination is used for hybridization and bagging isolation; young embryos 40-45 days after pollination are stripped; the stripped young embryos are placed on MS culture medium and cultured into seedlings; tissue culture seedlings are transplanted and transplanted and repotted after survival; bulbs are harvested when the leaves of the seedlings wither to 90-95% in the following year. The present invention utilizes embryo culture means to not only greatly reduce the risk of Dutch iris seeds being unavailable due to uncertain environmental factors, but also significantly shorten the growth time of Dutch iris from seeds to transplantable seedlings compared to ordinary sowing techniques, and the circumference of the offspring bulbs harvested is larger. Therefore, this method greatly shortens the breeding time of Dutch iris, accelerates the breeding process of new varieties, and is of great significance to promoting the development of my country's Dutch iris industry.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnological breeding, and more specifically, relates to a method for rapidly obtaining offspring seedlings and excellent bulbs of Iris × hollandica. Background Art

[0002] Iris × hollandica is a bulbous flower of the genus Iris in the family Iridaceae. It is one of the most widely cultivated and applied types among bulbous irises. Its natural flowering period is in April, and the population flowering period is about 30 days. It can be used in various application forms such as potted cut flowers, flower seas, flower fields, flower borders, and under forests. Among the commercial bulbs of bulbous flowers produced in the Netherlands, the cultivation area of Iris × hollandica ranks among the top, and most of them are used for cut flower production. China first introduced and cultivated Iris × hollandica in the late 1990s, but there is relatively little research on the application and breeding of Iris × hollandica. Almost all the fresh cut flowers sold on the market rely on imports or bulb imports for planting and production. With the continuous development of the social economy, the flower industry has become an important part of China's national economy and plays an important role in the national ecological civilization strategy and rural revitalization strategy. As a high-grade cut flower, the domestic demand for Iris × hollandica has been increasing year by year in recent years. Compared with other imported bulbous flowers, the bulbs of Iris × hollandica degenerate less severely. Once introduced, they can be continuously planted for many years without the need to purchase bulbs every year. Therefore, it has broad market prospects.

[0003] However, at present, there are few Iris × hollandica varieties with independent intellectual property rights in China, and there is basically no commercial cultivation. The main reason is that most Iris × hollandica varieties have the characteristics of female or male sterility, which makes cross-breeding difficult.

[0004] However, on the basis of a large number of introductions of foreign excellent varieties and their adaptive cultivation, through a large number of cross combinations, we found that certain specific cross combinations can produce seeds. However, due to uncertain external environmental factors, such as excessive rain or insect pests during the seed-setting period, mature hybrid seeds cannot be harvested. In addition, even if the harvested seeds are sown normally and transplanted, the bulb circumference of the underground bulbs harvested the following year is very small. The long breeding cycle will hinder the pace of germplasm innovation of Iris × hollandica in China.

[0005] Embryo culture specifically refers to artificially improving the development conditions of young embryos, providing the necessary nutrients for the development of young embryos, and thus promoting the growth of young embryos into plants. However, there is no relevant research report on this technology for Iris × hollandica at present. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for rapidly obtaining offspring seedlings and excellent bulbs of Iris × hollandica.

[0007] It should be noted that the present invention has successfully shortened the time from seed to seedling of Dutch iris by using embryo culture technology. In addition, the circumference of the bulbs harvested from the seedlings obtained by embryo culture in the following year is significantly larger than that of the bulbs produced by ordinary sowing seedlings. Therefore, the use of this technology will greatly shorten the breeding cycle of Dutch iris, improve the breeding efficiency and quality, and promote the germplasm innovation process of domestic Dutch iris.

[0008] Furthermore, Dutch iris begins to bear fruit in the middle and late April after hybridization. During the fruiting period, it is found that the young fruits of Dutch iris will be eaten by insects. In addition, if there is too much rain, the fruits will also mold, ultimately resulting in the failure to harvest mature seeds. The use of the embryo culture method will greatly reduce the risk of failure to harvest seeds caused by these uncertain external environmental factors. And the circumference of the hybrid offspring bulbs obtained by embryo culture technology is significantly larger than that of the bulbs produced by ordinary sowing seedlings. The use of this technology will greatly accelerate the germplasm innovation process of domestic Dutch iris.

[0009] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for quickly obtaining Dutch iris offspring seedlings and excellent bulbs, comprising the following steps:

[0011] (1) Preparation of parental materials: Select the parental varieties of the hybridization combination that can obtain seeds and are clearly defined, plant the commercial bulbs in autumn, and ensure sufficient fertilizer and water;

[0012] (2) Artificial pollination and hybridization: When the female parent blooms and the stamens of the anthers are not yet mature and shed pollen, cut off all the stamens, standard petals and drooping petals of the female parent, leaving only three stigmas in the shape of a feather and isolate them with sulfuric acid paper bags; When the pistil of the female parent is mature, pollinate it with the pollen of the male parent. After pollination, continue to isolate it with a bag. To improve the pollination success rate, repeat the pollination once 2-3 days after the first pollination. About one week after the second pollination, when the stigma shows a state of water loss and wilting, the sulfuric acid paper can be removed; And pollination should be carried out in the sunny morning;

[0013] (3) Embryo extraction: 40-45 days after the first cross-pollination, cut off the swollen capsule, rinse the surface with clean water, spray 75% alcohol on the surface of the capsule, and then place it in a laminar flow hood for 30 minutes of ultraviolet sterilization; After the ultraviolet sterilization is completed, open the capsule under sterile conditions in the laminar flow hood, take out the swollen seeds, cut the seed coat with a dissecting needle, and squeeze out the young embryo;

[0014] (4) In vitro culture of young embryos: Under sterile conditions, inoculate the young embryos obtained in step (3) onto MS medium for culture. The culture conditions in the tissue culture room are: temperature 22-24°C, photoperiod 14h light / 10h darkness, light intensity 4000-6000 Lux, and culture until the seedlings take root;

[0015] (5) Seedling transplantation: Take out the seedlings with root length > 3 cm and height > 4 cm in step (4) from the culture medium, wash the roots, and transplant them into a seedling tray. The transplanting medium is sterilized vermiculite. After watering thoroughly, cover the tray with a lid to keep moisture, and place it in an artificial climate chamber for cultivation. Open the tray lid after about one week. After the seedlings survive, transplant and repot them. And the seedlings after transplanting and repotting are continuously placed in the artificial climate chamber for cultivation in the early stage, and then moved outdoors in late November for normal watering management;

[0016] (6) Bulb harvesting: The bulbs can be harvested when the above-ground leaves of Dutch iris wither in the following year.

[0017] Optionally, in step (5), the specific operation of transplanting and repotting after the seedlings survive is as follows:

[0018] After the roots of the seedlings grow out of the bottom of the seedling tray, transplant them into a large pot together with the cultured vermiculite substrate. Among them, the substrate in the pot is a mixed substrate of peat, nutrient soil and vermiculite with a volume ratio of 1:1:1.

[0019] Furthermore, the cultivation conditions in the artificial climate chamber in step (5) are: light time 16 h / d, temperature 23°C ± 1°C during the light period, temperature 18°C ± 1°C during the dark period, and light intensity 20000 - 25000 Lux.

[0020] Optionally, in step (6), the above-ground leaves of Dutch iris withering means harvesting the bulbs when the withered and yellowed leaves account for 90 - 95% of the total leaf amount.

[0021] It should be noted that if harvested too early, the green leaves can still carry out photosynthesis, affecting the growth of the bulbs; if harvested too late, in case of excessive rain, the leaves will rot, which will cause the lower bulbs to rot. When the green leaves only account for 5 - 10%, their photosynthesis has a relatively small effect on the accumulation of bulb nutrients and can avoid bulb rot. Therefore, it is a more suitable time to harvest the bulbs.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses artificial assisted pollination for hybridization and bag isolation; dissect the young embryos 40 - 45 days after pollination; culture the dissected young embryos on MS medium to grow into seedlings; transplant the tissue-cultured seedlings and repot them after transplanting and survival; harvest the bulbs when the leaves of the seedlings wither by 90 - 95% in the following year. The present invention uses embryo culture means, which can not only greatly reduce the risk that Dutch iris seeds cannot be obtained due to uncertain environmental factors, but also significantly shorten the growth time of Dutch iris from seeds to transplantable seedlings compared with ordinary sowing techniques. At the same time, the harvested offspring bulbs have a larger circumference. Therefore, this method greatly shortens the breeding time of Dutch iris, accelerates the breeding process of new varieties, and has important significance for promoting the development of the Dutch iris industry in China. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0025] Figure 1 The fruits of Iris hollandica are damaged by pests.

[0026] Figure 2 Embryo culture (A - C) and transplantation (D - E) of Iris hollandica.

[0027] Figure 3 The bulbs produced by embryo - cultured seedlings of Iris hollandica (left) and the bulbs produced by ordinary sown seedlings (right). Detailed Description of the Embodiments

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be interpreted as superior to or better than other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, the conventional test methods in the art are adopted. It should be understood that the terms described in this application are only used to describe specific embodiments and are not used to limit the content disclosed in this application.

[0030] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs; other test methods and technical means not specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0031] In the description of the present invention, it should be understood that the orientation or positional relationships indicated by the terms "middle", "upper", "lower", "rise", "fall", "vertical", "surface", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0032] To better illustrate the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that this application can still be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. that are well-known to those skilled in the art are not described in detail in order to highlight the gist of this application.

[0033] On the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of this application.

[0034] It should be noted that the Dutch iris fruits in the following embodiments are collected from the iris resource nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, and the test utensils are all purchased on the market or are conventional products commonly used in the laboratory.

[0035] Example 1:

[0036] (1) Preparation of parental materials: Select Red Ember and Tiger Eye as parents, plant the commercial bulbs in autumn, and ensure sufficient fertilizer and water.

[0037] (2) Artificial pollination and hybridization: When the female parent Red Ember blooms and the stamens have not matured and shed pollen, cut off all the stamens, standard petals and drooping petals of the female parent, leaving only three stigmas and isolate them with sulfuric acid paper bags; when the pistil of the female parent is mature, pollinate it with the pollen of the male parent Tiger Eye, and continue to isolate it with a bag after pollination. Repeat pollination once 2-3 days after the first pollination. About one week after the second pollination, when the stigma shows a state of water loss and wilting, the sulfuric acid paper can be removed; the pollination time is from 9 to 10 am, and the weather is sunny on the pollination day.

[0038] (3) Immature embryo extraction: 40 days after the first cross-pollination, cut off the swollen capsule, rinse the surface with clean water, spray 75% alcohol on the surface of the capsule, and then place it in a laminar flow hood for 30 minutes of ultraviolet sterilization; after the ultraviolet sterilization is completed, open the capsule under sterile conditions in the laminar flow hood, take out the swollen seeds, pierce the seed coat with a dissecting needle, and squeeze out the immature embryo.

[0039] (4) In vitro culture of immature embryos: Under sterile conditions, inoculate the immature embryos obtained in step (3) onto MS medium for culture. The culture conditions in the tissue culture room are: temperature 22-24 °C, photoperiod 14 h light / 10 h dark, light intensity 4000-6000 Lux, and culture until the seedlings take root.

[0040] (5) Seedling transplantation: When the seedlings grow to a root length > 3 cm and a height > 4 cm, they are taken out of the culture medium, the roots are washed, and then transplanted into a seedling tray. The transplanting medium is sterilized vermiculite. After thoroughly watering, the tray lid is covered to keep moisture, and it is placed in an artificial climate chamber for cultivation. The cultivation conditions are as follows: light duration 16 h / d, temperature during light period 23°C ± 1°C, temperature during dark period 18°C ± 1°C, light intensity 20000 - 25000 Lux. After 7 days, the tray lid is removed. When the roots of the seedlings grow out of the bottom of the tray, they are transplanted into a large pot together with the cultured vermiculite substrate. The substrate in the pot is peat: nutrient soil: vermiculite = 1:1:1 (V:V:V). The seedlings after transplanting and pot-changing are initially continued to be cultured in the artificial climate chamber and moved outdoors on November 20th, with normal watering management;

[0041] (6) Bulb harvesting: The bulbs are harvested when 90 - 95% of the leaves of the Dutch iris seedlings above the ground wither in the second year.

[0042] It is calculated that it takes about 50 days for the seedlings of the embryo culture hybrid offspring of Red Ember × Tiger Eye to grow to a height > 4 cm. The bulbs produced in the following year after transplantation are as attached Figure 3 (left), with a circumference of about 4.2 cm.

[0043] Example 2:

[0044] Similar to the steps of Example 1, the difference lies in: steps (2) and (3), and the specific operations are as follows:

[0045] (2) Artificial pollination and hybridization: Using Tiger Eye as the female parent and Red Ember as the male parent, the hybridization process is the same as in Example 1;

[0046] (3) Immature embryo extraction: 45 days after the first cross-pollination, the swollen capsule is cut off, and the remaining operations are the same as in Example 1.

[0047] It is calculated that it takes about 55 days for the seedlings of the embryo culture hybrid offspring of Tiger Eye × Red Ember to grow to a height > 4 cm. The circumference of the bulbs produced in the following year after transplantation is about 5.4 cm. Comparative Example 1:

[0048] Steps (1), (2), and (6) are the same as in Example 1, and the remaining operating steps are as follows:

[0049] (3) Seed collection: When the capsules produced by cross-fertilization turn yellow and mature, they are harvested and taken back to the laboratory, placed in a nylon mesh bag and hung in a ventilated place to dry. After drying, the fruit pods are carefully opened to take out the seeds, and the plump seeds are selected and packed in a fine nylon bag and hung in a dry place for standby;

[0050] (4) Sowing: Soak the hybrid seeds of Iris hollandica in a carbendazim aqueous solution at a concentration of one-thousandth for 30 minutes, then rinse off the carbendazim powder on the seed surface with clean water for 2 - 3 times. Then place them on a moist filter paper and culture at room temperature for about 2 days. The water-saturated Iris hollandica seeds are sown in a plastic pot. A layer of plastic woven bag should be placed at the bottom of the plastic pot, and then about 5 cm thick yellow sand is put in. Finally, the hybrid seeds of Iris hollandica are scattered and spread flat on the yellow sand and covered with about 2 cm of yellow sand. The sown Iris hollandica seeds are watered thoroughly and then placed in an artificial climate chamber with a light duration of 16 h / d, a temperature of 23℃ ± 1℃ during the light period, a temperature of 18℃ ± 1℃ during the dark period, and a light intensity of 20000 - 25000 Lux until emergence.

[0051] (5) Seedling transplantation: When the seedling height > 4 cm, the seedlings can be carefully taken out and transplanted into a mixed substrate of peat: nutrient soil: vermiculite = 1:1:1 (V:V:V). In the early stage, continue to culture them in an artificial climate chamber. Move them outdoors on November 20th and manage normal watering.

[0052] It is calculated that the hybrid offspring seedlings of the control group Red Ember × Tiger Eye need about 70 days to grow to a height > 4 cm after sowing the harvested seeds. The bulbs formed in the following year after transplantation are as shown in the attached Figure 3 (right), with a circumference of about 2.8 cm.

[0053] Control Group 2:

[0054] Steps (1) and (2) are the same as those in Example 2, and steps (3) - (6) are the same as those in Control Group 1.

[0055] It is calculated that the hybrid offspring seedlings of the control group Tiger Eye × Red Ember need about 73 days to grow to a height > 4 cm after sowing the harvested seeds. The circumference of the bulbs formed in the following year after transplantation is about 3.0 cm.

[0056] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapidly obtaining offspring seedlings and excellent bulbs of Iris hollandica, characterized in that, it comprises the following steps: (1) Preparation of parental materials: Select Red Ember and Tiger Eye as parents, plant the commercial bulbs in autumn, and ensure sufficient fertilizer and water; (2) Artificial pollination and hybridization: When the female parent Red Ember blooms and the stamens' anthers are not mature and do not shed pollen, cut off all the stamens, standard petals and drooping petals of the female parent, leaving only three stigmas and isolate them with sulfuric acid paper bags; When the pistil of the female parent is mature, pollinate it with the pollen of the male parent Tiger Eye. After pollination, continue to isolate with a bag. Repeat pollination once 2-3 days after the end of the first pollination. About one week after the end of the second pollination, when the stigma shows a dehydrated and wilted state, remove the sulfuric acid paper; The pollination time is from 9 to 10 am, and the weather is sunny on the pollination day; (3) Immature embryo extraction: 40-45 days after the first cross-pollination, cut off the swollen capsule, wash the surface with clean water and spray 75% alcohol on the surface of the capsule; Then sterilize with ultraviolet light for 30 min; After the ultraviolet sterilization is completed, open the capsule under sterile conditions in a clean bench, take out the swollen seeds, scratch the seed coat with a dissecting needle, and squeeze out the immature embryo; (4) In vitro culture of immature embryos: Under sterile conditions, inoculate the immature embryos obtained in step (3) onto MS medium for culture. The culture conditions in the tissue culture room are: temperature 22-24°C, photoperiod 14 h light / 10 h darkness, light intensity 4000-6000 Lux, and culture until the seedlings take root; (5) Seedling transplantation: When the seedlings grow to a root length > 3 cm and a height > 4 cm, take them out of the medium, wash the roots and transplant them into a seedling tray. The transplanting medium is sterilized vermiculite. After watering thoroughly, cover the seedling tray cover to keep moisture, and place it in an artificial climate chamber for culture. The culture conditions are: light time 16 h / d, temperature 23°C ± 1°C during the light period, temperature 18°C ± 1°C during the dark period, light intensity 20000-25000 Lux; After 7 days, uncover the seedling tray cover. When the roots of the seedlings grow out of the bottom of the seedling tray, transplant them together with the cultured vermiculite substrate into a large pot. The substrate in the pot is peat: nutrient soil: vermiculite = 1:1:1 (V:V:V); The seedlings after transplanting and potting are continued to be cultured in the artificial climate chamber in the early stage, and moved outdoors on November 20, and watered normally; (6) Bulb harvesting: Harvest the bulbs when 90-95% of the above-ground wilted leaves of the Iris hollandica seedlings reach the second year.

Images