CN114747480B - Method for inducing forest tetraploid in field in non-isolated manner - Google Patents

Abstract

The invention provides a method for inducing forest tetraploid in field in vitro, which comprises the step of carrying out heat shock treatment on the fruit sequence after pollination of the forest under the condition of non-vitro. The method of the invention induces the chromosome doubling of the synthons to obtain the tetraploid plants under the non-isolated condition on the infructescence in proper development state, and the chromosome doubling method of the invention has high efficiency of obtaining the tetraploid plants. The tetraploid induction method provided by the invention is suitable for field operation, breaks the restriction of test conditions, and opens up a new way for the in vitro culture of the long fruiting period forest induced zygote chromosome to double to generate tetraploid. The method has the advantages of simple operation, low cost, capability of obtaining a plurality of tetraploid new germplasm through one-time treatment, capability of avoiding generation of chimeras and mixed ploidy and the like, and has good prospect and great significance for promoting innovation and development of the germplasm of the forest in the south.

Description

A method for inducing tetraploidy of forest trees in vitro in the wild

Technical Field

The invention relates to a method for inducing tetraploid forest trees, in particular to an inducing method for inducing zygotic chromosome doubling to create eucalyptus tetraploid plants, and belongs to the field of plant genetic breeding.

Background Art

Eucalyptus (Eucalyptus spp.) is an important timber forest species in the world, providing more than 40% of the wood and pulp production for my country's industrial production every year. At present, the global research on eucalyptus genetic improvement, which is mainly based on hybrid breeding, has fallen into a bottleneck period, and no breakthrough varieties have been born for many years. In order to create high-yield and high-quality new germplasm of eucalyptus and further increase biological production, it is urgent to innovate the eucalyptus breeding technology system. In recent years, artificially induced tetraploids have been successfully applied to poplar genetic improvement, which has pointed out the direction for eucalyptus germplasm innovation. Tetraploid refers to an organism with four sets of chromosomes in its cells. Compared with ordinary diploids, tetraploid plants are usually sturdy plants, with significantly larger organs such as flowers, leaves and fruits, and stronger resistance to biological and abiotic adversities. At the same time, tetraploid germplasm can also be used as a parent to directly hybridize with diploids to obtain triploids with more obvious growth advantages. Therefore, artificially induced eucalyptus tetraploids are of great significance for breeding excellent polyploid germplasm of eucalyptus and significantly improving the productivity of eucalyptus plantations.

There are usually two ways to artificially induce plant tetraploidy. One is to use tissue culture methods in the laboratory, using cell division inhibitors such as colchicine to treat the meristem of diploid plants, etc., to induce plant somatic cell chromosome doubling to obtain tetraploid somatic cell tissue, thereby obtaining tetraploid regenerated plants. This method has been reported on eucalyptus (Han Chao et al. Study on polyploidy of asexual line Eg5 of Eucalyptus grandis induced by colchicine. Chinese Agricultural Science Bulletin, 2010, 26(24): 128-132; Han Chao et al. Study on polyploidy of Eucalyptus urophylla induced by colchicine. Chinese Agricultural Science Bulletin, 2010, 26(15): 149-153; Han Chao. Study on technical system of chromosome doubling induced by colchicine in eucalyptus. Chinese Academy of Forestry, 2010; Tan Deguan et al. Polyploidy induction of in vitro tissue of Eucalyptus Congo No. 12. Journal of Tropical Crops, 2005(02): 50-54; Hu Zhouhe et al. Preliminary study on polyploidy of eucalyptus induced by colchicine. Guangxi Forestry Science, 2004(04): 195-196+203; Tan Deguan. Eucalyptus Congo No. 12 (Eucalyptus Congo No. 12) 12ABL) Research on tissue culture and polyploid induction. South China University of Tropical Agriculture, 2004; KAPOOI et al. Experimental synthesis of eucalyptus allotetraploid. Hunan Forestry Science and Technology, 1986(04): 46-48), but there are also some problems. For example, applying mutagenesis treatment to meristem multicellularity often results in a large number of mixed ploids, and the ploidy of regenerated plants is complex and difficult to separate; mutagenesis treatment methods based on tissue culture regeneration systems are often only effective for individual genotypes, and an effective mutagenesis treatment can only obtain one polyploid germplasm, and the induction efficiency is not high.

Another way to artificially induce tetraploid plants is to directly induce plant zygotic chromosome doubling, that is, to use certain technical methods to apply mutagenesis at a specific time of zygotic cell development after fertilization, so as to induce zygotic cell chromosome doubling to obtain tetraploid plants. Compared with the somatic cell chromosome doubling that has been reported, induced zygotic chromosome doubling has the following three advantages: first, the induced tetraploid is a euploid tetraploid plant directly developed from a single zygotic cell, and will not produce mixed ploidy; second, the mutagenesis process does not rely on a specific laboratory environment and can be directly implemented on non-in vitro trees in the wild, with a wide range of applications; third, a single mutagenesis treatment can act on multiple infructescences and multiple fertilized zygotes in the ovary, and multiple new tetraploid germplasms can be obtained at the same time, with a high induction efficiency.

At present, artificial induction of plant zygotic chromosome doubling mainly adopts chemical agents such as colchicine solution, N2O gas, or physical means such as extreme temperature treatment to block the normal division process of zygotic cells, so as to promote the doubling of zygotic chromosomes to obtain tetraploidy. Among them, high temperature treatment is the best for inducing poplar zygotic chromosome doubling (Wang Jun et al. Physicochemical treatment induced zygotic chromosome doubling to breed hybrid tetraploids of Populus qinghaiensis. Journal of Beijing Forestry University, 2010, 32(05): 63-66; Shi Le et al. High temperature treatment induced chromosome doubling to obtain hybrid polyploids of poplar. Journal of Nuclear Agricultural Sciences, 2012, 26(08): 1118-1123; Lu Min. Research on the induction technology of triploid and tetraploid Populus sibiricum. Beijing Forestry University, 2013). However, the existing high-temperature-induced plant tetraploidy technology is mainly aimed at northern-adapted tree species such as poplars, which bloom at low temperatures in early spring, have a short flowering period and the development of flower buds is easy to identify and distinguish, and have a short fruit-setting period, which can be cut in vitro and harvested by indoor hydroponics. Southern-adapted tree species, represented by eucalyptus, usually bloom in the hot and humid summer, and the suitable environment requires the use of higher heat shock treatment intensity. The lush branches and leaves are easily damaged by heat and cause fruit drop; the flowering period is as long as several months, and the morphological changes of flower buds and infructescence are not obvious. It is difficult to determine the effective time to apply heat shock mutagenesis treatment during the several-month infructescence development period through experience or simple experiments; if the fruit-setting period exceeds half a year, it is impossible to harvest seeds by in vitro hydroponics, and non-in vitro mutagenesis operations can only be carried out in complex environmental conditions in the wild, which further increases the difficulty of choosing the timing of mutagenesis treatment and puts higher requirements on mutagenesis operation methods. Therefore, the methods that have been reported are not applicable to southern adaptive tree species represented by eucalyptus. The main defects are: it is unable to solve the problem of selecting the timing for in vitro induction of zygotic chromosome doubling in the wild, and there is no effective method to determine the zygotic development stage of the plant, making it difficult to accurately apply the mutagenesis treatment near the first mitotic period of the zygote, resulting in doubling failure; it is unable to solve the problem of operating methods for high-temperature mutagenesis treatment of trees under in vitro conditions in the wild, resulting in the branches and leaves of the trees being susceptible to heat shock damage; it is unable to solve the problem of preserving the activity of flower buds and infructescence after high-temperature treatment in the wild in summer, resulting in the withering and falling of tree branches and leaves and infructescence after mutagenesis.

Summary of the invention

The present invention aims at the technical problem that the induction treatment period and induction treatment conditions are difficult to determine in the process of inducing tetraploidization in the wild for trees that bloom in hot and humid summer, are insensitive to high temperature, have a long flowering period, unclear changes in the morphology of flower buds and infructescence, and have a long fruiting period, resulting in a poor tetraploid yield. The present invention provides a method for inducing tetraploidization of forest trees in the wild in vitro. The method of the present invention performs heat shock treatment on forest trees growing in the wild under in vitro conditions to induce zygotic chromosome doubling and obtain tetraploid plants. The method of the present invention overcomes the problem that the existing reported methods cannot be applied to the tetraploidization induction of southern trees such as eucalyptus.

The technical solution proposed in the present invention solves the following problems: it clarifies the effective treatment timing and treatment conditions for inducing zygotic chromosome doubling of Eucalyptus to obtain tetraploid by non-in vitro heat shock treatment in the wild; and establishes a method for inducing eucalyptus tetraploid by non-in vitro heat shock treatment in the wild, which can effectively preserve the vitality of eucalyptus flower buds and infructescence in summer.

To achieve the purpose of the present invention, the present invention provides a method for inducing tetraploidy of forest trees in vitro in the wild, comprising subjecting the infructescence of the forest trees after pollination to heat shock treatment in vitro.

Wherein, the trees are selected from eucalyptus, acacia or teak, preferably eucalyptus.

In particular, the eucalyptus tree is selected to be Eucalyptus urophylla.

The heat shock treatment is to heat the flower branches of the infructescence after pollination, that is, to heat the infructescence after pollination.

Particularly, the temperature of the heat shock treatment is 40-48°C; and the heat shock treatment time is 3-6h.

In particular, the heat shock treatment temperature is preferably 44±1° C.; the heat shock treatment time is preferably 6 hours.

Among them, after the trees are pollinated, heat shock treatment is carried out when the filaments and styles fall off on the infructescence or when the infructescence begins to swell.

In particular, it is preferred to perform heat shock treatment at the stage when filaments and styles fall off on the infructescence.

In another aspect, the present invention provides a method for inducing tetraploidy of forest trees in vitro in the wild, comprising the following steps in order:

1) Pollination is performed when the bud operculum on the tree branches changes from green to yellow or white and the bud operculum becomes loose along the abscission ring;

2) observing the infructescence on the flower branches after pollination, and heating the flower branches of forest trees at the stage when the filaments and styles fall off and the infructescence begins to expand, that is, performing heat shock mutagenesis treatment on the infructescence at the stage when the filaments and styles fall off and the infructescence begins to expand after pollination;

3) collecting mature seeds after heat shock mutagenesis treatment, and sowing and raising seedlings;

4) First, the ploidy level of the seedlings was preliminarily identified by flow cytometry; then, the tetraploid plants preliminarily identified by flow cytometry were secondary identified by somatic cell chromosome counting to determine the ploidy level of the plants and obtain tetraploid plants.

Wherein, the trees are selected from eucalyptus, acacia or teak, preferably eucalyptus.

In particular, the eucalyptus tree is selected to be Eucalyptus urophylla.

Wherein, the pollination in step 1) is to cut off the top of the bud capsule on the tree branch together with the top of the stigma with a blade, and then pollinate the style wound exposed by the incision.

A method of pollinating at the wound of the style exposed by the incision, completing pollination in one go without emasculating the flowers.

In particular, after pollination, the pollinated branches are covered with an isolation paper bag to isolate pollen contamination. The isolation paper bag is removed two weeks after pollination.

In particular, the pollen used in the pollination is other high-quality eucalyptus pollen collected in advance.

In particular, since the pollination was completed, the infructescence was collected continuously at intervals of 24 hours, the external morphological changes of the infructescence were observed and photographed, and the development process of the infructescence after pollination was divided into 5 stages according to the development time and external morphological changes of the infructescence after pollination;

The first stage is 1 to 6 days after pollination. The external morphology of the infructescence is that the top of the stigma is oxidized and blackened after being cut, and the yellow filaments stand upright around the style. At this stage, the zygote is not fertilized.

The second stage is 7 to 9 days after pollination, when the external appearance of the infructescence is that the filaments are withered and curled but have not fallen off. This stage is the double fertilization period;

The third stage is 10 to 22 days after pollination, when the external morphology of the infructescence is that the filaments gradually fall off and the styles gradually turn yellow and wither. This stage is the dormant zygote period;

The fourth stage is 23 to 26 days after pollination. The external morphology of the infructescence is that the filaments and styles have completely fallen off. This stage is from the end of zygotic dormancy to the 4-cell proembryo period.

The fifth stage is 27-28 days after pollination. Except that the infructescence begins to expand significantly, there are no other obvious changes in morphology. This stage is the period when the zygote further develops to the globular embryo stage.

The collecting of infructescence is collecting the infructescence in bags after pollination, and the bags are removed each time for collection and are put back immediately after collection.

Wherein, in step 2), the infructescence at the stage of filament and style shedding after pollination is subjected to heat shock mutagenesis treatment. At an appropriate time after pollination (i.e., the infructescence is at the stage of filament and style shedding), the entire flower branch where the infructescence after pollination is located is subjected to heat shock mutagenesis treatment (i.e., heating treatment).

Heat shock treatment is performed in the 4th and 5th stages of post-pollination development, i.e., 23-28 days after pollination; preferably, in the 4th stage, i.e., 23-26 days after pollination, when the filaments and styles have completely fallen off and the zygote development is in the period from the end of zygote dormancy to the 4-cell proembryo stage.

In particular, the temperature of the heat shock mutagenesis treatment in step 2) is 40-48°C, preferably 44±1°C; the heat shock mutagenesis treatment time is 3-6h, preferably 6h.

In particular, after the heat shock mutagenesis treatment, the method further includes: post-processing the flower branches, that is, spraying the leaves and infructescences on the flower branches with clean water or potassium dihydrogen phosphate aqueous solution precooled to 4-20°C until the leaf surface temperature drops below 10°C.

In particular, the post-treatment preferably uses spraying of an aqueous potassium dihydrogen phosphate solution precooled to 4-20°C until the leaf surface temperature drops below 10°C.

In particular, the temperature of the clean water or potassium dihydrogen phosphate aqueous solution is preferably 4° C.; the concentration of the potassium dihydrogen phosphate aqueous solution sprayed in the post-treatment is 0.05-0.2%, preferably 0.1%.

The purpose of spraying a large amount of pre-cooled 0.1% potassium dihydrogen phosphate aqueous solution is to quickly replenish water and cool down the leaves and infructescence, promote the repair of plant tissue damage after heat shock, thereby improving the vitality of the infructescence and increasing the tetraploid yield.

In particular, the heat shock mutagenesis treatment in step 2 is to heat the forest tree branches using the "Tree In-vitro Branch and Bud Heating Treatment Device" with patent number ZL200610113448.X.

Particularly, the temperature of the heat shock mutagenesis treatment is 40-48°C, preferably 44±1°C; the heat shock mutagenesis treatment time is 3-6h, preferably 6h.

In particular, the "in vitro branch and bud heating treatment device for trees" with patent number ZL200610113448.X is used to wrap the entire flower branch to be induced to undergo mutagenesis under in vitro conditions, and then heat it to increase the temperature inside the treatment device, heat the flower branch wrapped in the "in vitro branch and bud heating treatment device for trees", and perform heat shock mutagenesis treatment on the fruit clusters grown on the flower branch inside the treatment device.

The heat shock mutagenesis treatment of the present invention is directly carried out on the tree body, and the flower branch is still connected to the tree body, which is in a non-in vitro state. After the pre-treatment operation is implemented, a dedicated device (i.e., patent number ZL200610113448.X, patent name "Tree Non-In Vitro Branch Bud Heating Treatment Device") is used to perform temperature and time controlled heating treatment on the entire flower branch with pollinated fruit sequence.

In particular, before using the "tree in vitro branch bud heating treatment device" to heat the forest tree branches, it also includes: first spraying water to moisten the leaves and infructescence on the induced branches; then using wrapping materials to wrap the entire branch with infructescence.

The purpose of the pre-treatment is to keep the fruit clusters and leaves moisturized during the subsequent heat shock treatment and to prevent the fruit clusters from direct contact with the extremely high temperature electric heating plates of the "Tree In-vitro Branch and Bud Heating Treatment Device" with patent number ZL200610113448.X, thereby improving the vitality of the fruit clusters.

In particular, after spraying water until condensed water droplets flow down from the leaves, the entire flower branch with the infructescence is immediately wrapped with the silica aerogel material;

In particular, the wrapping material is selected from silica aerogel material, fireproof and heat-insulating cloth or ceramic fiber wool, preferably silica aerogel material.

In particular, after the flower branches are subjected to heat shock mutagenesis treatment, the "tree in vitro branch bud heating treatment device" with patent number ZL200610113448.X and the silica aerogel material wrapping the flower branches are removed, and then the clean water or potassium dihydrogen phosphate aqueous solution is sprayed.

The specific time for the seeds to mature in step 3) is 8 to 10 months after pollination, that is, around March to April of the following year.

Wherein, in step 4), when the seedlings grow to a height of about 10 cm, the ploidy level of the progeny seedlings is detected.

The method for inducing zygotic chromosome doubling to obtain eucalyptus tetraploid by non-in vitro heat shock treatment in the wild comprises: performing controlled pollination on eucalyptus planted in the wild (artificial pollination of eucalyptus by one-step pollination method), forming infructescence through bagging isolation, etc.; observing and recording the development state of infructescence after pollination, and applying non-in vitro heat shock treatment to the infructescence on the flower branch at a specific time when the infructescence further develops; sowing and raising seedlings after the seeds in the infructescence mature, and identifying and obtaining eucalyptus tetraploid plants therefrom; wherein the specific time for applying non-in vitro heat shock treatment to the infructescence on the flower branch is: when the infructescence develops to the fourth stage after pollination, that is, 23-26 days after pollination, when filaments and styles have completely fallen off; and the method for applying heat shock treatment is: wrapping the infructescence on the flower branch with silica aerogel material before treatment, and timely cooling the infructescence after heat shock treatment, wherein the efficiency of inducing zygotic chromosome doubling of eucalyptus to obtain tetraploid by heat shock treatment at 44±1°C for 6 hours is high.

Based on the above technical solutions, the advantages and positive effects of the present invention are as follows:

1. The method of the present invention clarifies for the first time the feasibility of obtaining tetraploidy by doubling the zygotic chromosomes of trees (preferably eucalyptus) that are induced to bloom in hot and humid summer, are insensitive to high temperatures, have a long flowering period, have unclear changes in the morphology of flower buds and infructescence, and have a long fruiting period, and obtains new tetraploid germplasm of eucalyptus, which effectively promotes the advancement of polyploid breeding technology of eucalyptus.

2. The method of the present invention determines the optimal treatment conditions for inducing zygotic chromosome doubling of Eucalyptus by non-in vitro heat shock treatment, that is, when the infructescence development is in the fourth stage (i.e., 23 to 26 days after pollination) and the filaments and styles are completely detached, the efficiency of inducing zygotic chromosome doubling of Eucalyptus to obtain tetraploids by heat shock treatment at 44±1°C for 6 hours is the highest; a complete operation method suitable for in vitro heat shock inducing zygotic chromosome doubling of Eucalyptus is provided, and a technical system for doubling zygotic chromosomes of Eucalyptus for breeding tetraploids is established, which adds a new way to forest tree breeding and promotes the innovative development of forest tree germplasm in the south. The present invention makes it more convenient to determine the timing of mutagenesis, and uses the developmental change characteristics of filaments and stigmas, combined with the length of time after pollination, to quickly determine the developmental state of the zygote, thereby determining the timing of implementing mutagenesis and improving the induction efficiency.

3. The method of the present invention aims to induce zygotic chromosome doubling after fertilization of Eucalyptus to obtain tetraploidy. No mixed ploidy or chimera is produced in the induction process, and the tetraploid induction efficiency is high. Heat shock mutagenesis treatment can be carried out after cross pollination of a specific parent combination to obtain a batch of new tetraploid germplasms of Eucalyptus with different genotypes at one time, effectively improving the induction efficiency of polyploidy of Eucalyptus, and the mutagenesis efficiency reaches more than 6%, which accelerates the breeding process of Eucalyptus improved varieties.

4. The method of the present invention has good scalability. Pre-treatment and post-treatment of the induced flower branches before and after heat shock treatment can effectively preserve the vitality of flower buds and fruit sequences and increase the tetraploid yield. It is particularly suitable for in vitro induction of tetraploids in southern broad-leaved tree species that bloom in summer and have a long fruit-setting period.

5. The tetraploid induction method provided by the present invention is suitable for field operation, breaking the constraints of experimental conditions, and opening up a new way to induce zygotic chromosome doubling to produce tetraploids for long-fruiting period trees that cannot be cultured in vitro.

6. The method of the present invention is simple to operate and has strong applicability in the mutagenesis operation method. It can be applied to field non-in vitro mutagenesis breeding of different tree species under various site conditions. Moreover, the method of the present invention has the advantages of low processing cost, and can obtain multiple tetraploid new germplasms through one treatment, avoiding the production of chimeras and mixed ploids, etc., which has good prospects and great significance for promoting the innovative development of southern forest germplasm.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of artificial pollination and the external morphology of flower buds and infructescence in an embodiment of the present invention, wherein a is a schematic diagram of artificial pollination of eucalyptus urophylla flower buds, and b to e are schematic diagrams of the external morphology of flower buds and infructescence at different developmental stages;

FIG2 is a schematic diagram of an in vitro heat shock treatment in the field according to an embodiment of the present invention;

FIG3 is a schematic diagram of sowing and raising seedlings in an embodiment of the present invention;

Fig. 4 is a diagram showing the results of plant flow cytometry ploidy detection and somatic cell chromosome counting in an embodiment of the present invention, wherein a and b are flow cytometry ploidy detection results, a peak at about 50 on the coordinate axis indicates diploid, and b peak at about 100 on the coordinate axis indicates tetraploid; c and d are somatic cell chromosome counting results, in c the cell has 22 chromosomes and is diploid, and in d the cell has 44 chromosomes and is tetraploid;

FIG5 is a comparison diagram of diploid and tetraploid plants of Eucalyptus urophylla in an embodiment of the present invention, wherein a is a diploid and b is a tetraploid.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific implementation examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. All other embodiments obtained by a person of ordinary skill in the art without creative work are within the scope of protection of the present invention.

Example 1

1. Artificial pollination:

Before the Eucalyptus urophylla sheds pollen, flower branches with large flower quantity are selected as candidate flower branches for mutagenesis treatment; artificial pollination is performed when it is observed that the caps of most flower buds change from green to yellow or white, and the caps of flower buds become loose along the abscission ring and are about to fall off;

The pollination method adopts a one-step pollination method, where the top of the bud capsule and the top of the stigma are cut off with a blade, and a cotton swab is used to apply pollen to the style wound exposed by the incision; wherein: the pollen used for artificial pollination comes from other excellent eucalyptus pollen. After pollination, the flower branches are covered with an isolation bag (acetic acid paper bag, as shown in Figure 1a).

In Figure 1a, the pollinated flower branches are covered with an acetic acid paper bag to isolate pollen contamination. The isolation bag is removed two weeks after pollination.

2. Observation of infructescence development stage:

Starting from the time when Eucalyptus urophylla completed pollination, the fruit sequences after pollination were continuously collected at a sampling interval of 24 hours, and the changes in the external morphology of the fruit sequences were observed and recorded by photographing.

Taking the time of artificial pollination of Eucalyptus urophylla as the starting point, the schematic diagram of the external morphological development changes of flower buds and infructescence after pollination is shown in Figure 1a, where a′ in Figure 1a is the flower bud after a single artificial pollination.

The results of external morphological observations of flower buds and infructescence after pollination are shown in Table 1. According to the duration of infructescence development and external morphological changes after pollination, infructescence development was divided into five stages (Table 1).

Stage 1: 1 to 6 days after pollination, the top of the stigma oxidizes and turns black after being cut, and yellow filaments stand upright around the style (Figure 1b); this stage is when the zygote has not yet been fertilized.

Stage 2: 7 to 9 days after pollination, the filaments on the infructescence can be observed to have withered and curled but not fallen off (Figure 1c); this stage is the double fertilization period.

Stage 3: 10 to 22 days after pollination, during which the filaments on the infructescence gradually fall off and the styles gradually turn yellow and wither (Figure 1d); during this stage, the zygote is in a dormant period.

In the fourth stage, 23 to 26 days after pollination, it can be observed that the filaments and styles on the infructescence have completely fallen off (Figure 1e); at this stage, the zygotes gradually end their dormancy and begin their first mitosis.

In the fifth stage, 27 to 28 days after pollination, it can be observed that except for the obvious swelling of the infructescence, there are no other obvious changes in appearance; at this stage, the zygote continues to divide and develops into a spherical embryo.

Table 1 Characteristics of the developmental stages of flower buds and infructescence after pollination

Example 2 Field non-in vitro heat shock mutagenesis treatment:

According to the duration of infructescence development and external morphological characteristics after pollination, infructescence at the 4th and 5th stages of development after pollination was subjected to in vitro heat shock mutagenesis in the field (Figure 2).

1. Pre-treatment

For the flower branches where the infructescence is located at the 4th and 5th stages, two groups of target flower branches are selected for pre-treatment at each stage according to the number of days after pollination, that is, two groups of flower branches with infructescence at different pollination days in each development stage of the 4th and 5th stages are selected for pre-treatment, that is, two groups of flower branches with infructescence from the 23rd to the 28th day after pollination are selected for pre-treatment, wherein the pre-treatment is to fully spray water on the leaves and infructescence on the target flower branches, and immediately use the silica aerogel material to wrap the entire flower branch with the infructescence after condensed water droplets flow down from the leaves;

The role of the silica aerogel material is to keep the fruit sequences and leaves moisturized during the subsequent heat shock treatment process and to prevent the fruit sequences from coming into direct contact with the extremely high temperature electric heating plates of the heat shock treatment device, thereby improving the vitality of the fruit sequences.

Taking the silica aerogel material wrapping the squid in the pre-treatment process as an example, other materials such as fireproof and heat-insulating cloth, ceramic fiber cotton, etc. are suitable for the present invention. Since it is used outdoors, in order to prevent the squid from being windproof, rainproof and light during the treatment process, the silica aerogel material is selected to be suitable for non-ex vivo use in the field.

2. Heat shock treatment

Each target flower branch is heat-shocked using the invention patent "Tree In-vitro Branch and Bud Heating Treatment Device" (patent number: ZL200610113448.X) that the applicant has obtained authorization for. The high-temperature heating bag and temperature controller of the "Tree In-vitro Branch and Bud Heating Treatment Device" are connected by a cable and connected to a power source.

The heating bag of the "tree in vitro branch bud heating treatment device" (as shown in Figure 2) is put on the flower branch to be treated with mutagenesis through the open end, and the open end of the bag is fastened to the flower branch to be treated with heat shock mutagenesis through a flexible rope and sealed and wrapped on the flower branch.

Place the temperature controller of the "device for heating the in vitro branches and buds of trees" in a safe place on the ground, and connect the bag body of the heating bag and the temperature controller via electric wires (Figure 2).

After the power is turned on, the heating bag raises the temperature to the preset temperature (44±1°C) according to the setting of the temperature controller and then maintains the constant temperature to ensure that the flower branches wrapped in the heating bag are at the preset temperature of 44±1°C. The timing starts after the treatment temperature is constant, and the heating treatment is terminated 3 hours and 6 hours after the start of the treatment.

3. Post-processing

After the heat shock treatment, the target flower branches were immediately post-treated, the heating treatment device and silica aerogel material were removed in time, and the leaves and infructescences on the flower branches were immediately sprayed with a large amount of 0.1% potassium dihydrogen phosphate aqueous solution precooled to 4°C until the leaf surface temperature dropped below 10°C.

The purpose of spraying a large amount of pre-cooled 0.1% potassium dihydrogen phosphate aqueous solution is to quickly replenish water and cool the leaves and infructescence, promote the repair of plant tissue damage after heat shock, thereby improving the vitality of the infructescence and increasing the tetraploid yield.

The post-treatment in the present invention is described by spraying a 0.1% potassium dihydrogen phosphate aqueous solution as an example, and other potassium dihydrogen phosphate aqueous solutions with a concentration of 0.05-0.2% are also applicable to the present invention; and a large amount of clean water can also be sprayed;

The temperature of the post-treatment spraying of clean water or potassium dihydrogen phosphate aqueous solution is described by taking 4° C. as an example, and other temperatures such as 4-20° C. are also applicable to the present invention.

The target flower branches were grouped and numbered and marked according to the temperature and duration of heat shock treatment. The temperature and duration of heat shock treatment are shown in Table 2.

Example 2A

Except for step 2), during the heat shock treatment, the temperature inside the heating bag was controlled to be maintained at 40° C., the rest was the same as in Example 2.

Example 2B

Except for step 2), during the heat shock treatment, the temperature inside the heating bag was controlled to be maintained at 48° C., the rest was the same as in Example 2.

When the temperature was set at 48°C, regardless of whether the treatment time was 3 hours or 6 hours, the branches dried up due to heat shock and no seeds could be obtained.

However, the branches treated with heat shock treatment at 40°C and 44°C showed no abnormality, and the infructescence could grow and bear fruit normally. When the seeds matured the following year, seeds were collected according to the treatment combination, and a total of 2197 treated Eucalyptus urophylla seeds were harvested.

Example 3 Ploidy detection:

1. Seedling cultivation

In March or April of the following year, after the seeds matured, the eucalyptus seeds of Example 2 and 2A were collected according to the labels, and a total of 2197 treated eucalyptus urophylla seeds were harvested.

The harvested seeds were sown and raised according to the marked groups (Figure 3). Under the treatment conditions of heat shock temperature of 40℃ and 44℃, the seeds harvested under the treatment time of 3 hours and 6 hours can all germinate (Table 2). The seedlings were transplanted when they grew to three pairs of leaves. After transplanting, 1074 seedlings of Eucalyptus urophylla survived (as shown in Table 2).

2. Preliminary identification of ploidy level of Eucalyptus urophylla progeny by flow cytometry

When the seedlings grew to 10 cm in height, some leaves were removed from the plants, chopped and filtered through a 50 μm nylon mesh. After filtration, 50 μg ml ^-1 of 4',6-diamidino-2-phenylindole (DAPI) was added to the cell suspension, and the ploidy level of the progeny of E. urophylla was analyzed using flow cytometry. The diploid leaves of E. urophylla were used as controls in the analysis.

Flow cytometry was used to detect the ploidy of 1074 Eucalyptus urophylla seedlings, and 4 tetraploid Eucalyptus urophylla seedlings were preliminarily identified (see Table 2). The results of seed ploidy detection by flow cytometry are shown in Figure 4. Figure 4a is the ploidy detection of diploid plants, and the peak of Figure a is around 50 on the coordinate axis for diploid plants; Figure 4b is the ploidy detection of tetraploid plants, and the peak of Figure b is around 100 on the coordinate axis for tetraploid plants.

3. Somatic cell chromosome count

Tetraploid plants initially identified by flow cytometry were ultimately secondarily identified by somatic cell chromosome counting to determine the ploidy level of the plants.

The young leaves of tetraploid plants identified by flow cytometry were fixed in Carnoy's fixative for 24 hours; the fixed samples were then washed with water until the Carnoy solution was completely removed, placed in 1N hydrochloric acid, and dissociated at room temperature for 15 minutes; the dissociated samples were then stained with Carbofuxin red stain and crushed to prepare slides; the sample slides were observed using an optical microscope, and the number of chromosomes in the sample was counted to determine the ploidy level.

Table 2 In vitro heat shock induced chromosome doubling in zygotic Eucalyptus urophylla

The results of the chromosome counting of diploid and tetraploid eucalyptus are shown in Figure 4, where the number of chromosomes of diploid Eucalyptus urophylla observed and counted under an optical microscope is 2n=2x=22, as shown in Figure 4c, 2n represents somatic cells, x represents the chromosome base number, and x=11 for eucalyptus in this embodiment, 2x represents two sets of chromosomes, diploid, with a total of 2×11=22 chromosomes; the number of chromosomes of tetraploid Eucalyptus urophylla plants preliminarily identified by the flow cytometer of the present invention under an optical microscope is 2n=4x=44, as shown in Figure 4d, the tetraploid 2n=4x=44, indicating that the somatic cells have 4 sets of chromosomes, are tetraploid, with a total of 4×11=44 chromosomes. Through the somatic cell chromosome counting, 4 tetraploid Eucalyptus urophylla plants were finally identified and confirmed

The diploid and tetraploid potted seedlings are shown in Figure 5, where 5a is a diploid and 5b is a tetraploid.

The test results showed that when the infructescence developed to the fourth stage, the heat shock treatment at 44±1℃ for 3-6 hours was used, and tetraploids were obtained in the groups 24-26 days after pollination. In particular, in the fourth stage, that is, 25 days after pollination, when the filaments and styles on the infructescence completely fell off, the efficiency of obtaining tetraploids by heat shock treatment at 44±1℃ for 6 hours was the highest, and the tetraploid yield reached 6.45%.

In many experiments of the present invention, tetraploids can be obtained at the stage when the filaments and styles of the fruit sequence completely fall off after pollination (usually 23-26 days after pollination).

Comparative Example 1

The infructescences that developed to the 4th and 5th stages after pollination obtained according to the method steps of Example 1 were not subjected to heat shock induction treatment, but were directly sown and raised as seedlings after the seeds matured in March or April of the following year; the progeny plants obtained by raising seedlings were tested for ploidy in the manner of Example 3, and the test results were as follows: no tetraploidy was found in both flow cytometry identification and somatic cell chromosome counting.

Comparative Example 2

After the heat shock induction treatment, the target flower branches and their attached flower buds and infructescences obtained by the method and steps of Example 1 that developed to the 4th and 5th stages after pollination were not post-treated, that is, the leaves and infructescences on the flower branches were not sprayed with a large amount of 0.1% potassium dihydrogen phosphate aqueous solution precooled to 4°C until the surface temperature of the leaves and infructescence reached below 10°C, and the rest was the same as Example 2. The flower branches and infructescences of each treatment group lost water, dried up, and became inactive due to the failure to cool down and replenish water in time after the heat shock treatment. Mature seeds could not be collected, and tetraploid eucalyptus plants could not be obtained.

Comparative Example 3

The present invention is the same as Example 2 except that a heating treatment device with a larger spatial volume is used, a bracket is used in the heating bag to keep the flower branch to be induced to undergo mutagenesis away from the bag body and away from the bag body of the heating bag, and the entire flower branch with infructescence is not wrapped with silica aerogel material.

After the heat shock induction treatment, the seeds matured in March or April of the following year, and then they were sown and seedlings were raised; the progeny plants obtained by seedling raising were tested for ploidy in the manner of Example 3, and the test results were as follows: when the infructescence developed to the fourth stage, a heat shock treatment at 44±1°C for 3-6 hours was applied, and tetraploids could be obtained. Among them, the efficiency of obtaining tetraploids by heat shock treatment at 44±1°C for 6 hours was the highest in the fourth stage, that is, 25 days after pollination, when the filaments and styles on the infructescence completely fell off.

Claims (1)

1. The method for inducing the forest tetraploid in the field in a non-isolated manner is characterized by comprising the following steps of:

1) Pollinating when the capsule cover of the flower bud on the eucalyptus urophylla flower branch turns from green to yellow or white and loosens along the shedding ring;

2) Observing the inflorescences on the pollinated flower branches, heating the forest flower branches in the inflorescences and the inflorescences falling-off stage and the inflorescences expanding stage, namely carrying out heat shock mutagenesis treatment on the inflorescences in the inflorescences and the inflorescences falling-off stage and the inflorescences expanding stage after pollination; the heat shock mutagenesis treatment is carried out on the pollinated inflorescences in the shedding stage of the filaments and the flower columns and the initial expanding stage of the inflorescences, the heat shock treatment is carried out on the 24 th to 26 th days after the pollination, the temperature of the heat shock mutagenesis treatment is 44 ℃ at the 24 th day after the pollination, and the treatment time is 3h; when the temperature of the heat shock mutagenesis treatment is 44 ℃ at the 25 th day after pollination, the treatment time is 6h; when the temperature of the heat shock mutagenesis treatment is 44 ℃ at the 26 th day after pollination, the treatment time is 6h;

3) Collecting mature seeds after heat shock mutagenesis treatment, sowing and raising seedlings;

4) Firstly, carrying out preliminary identification on the ploidy level of seedlings by adopting a flow cytometer; then, carrying out secondary identification on the tetraploid plant primarily identified by the flow cytometry through chromosome counting of somatic cells, and determining the ploidy level of the plant to obtain the tetraploid plant;

wherein after the heat shock mutagenesis treatment, further comprising: and (3) carrying out post-treatment on the flowering branches, namely spraying clear water or monopotassium phosphate water solution precooled to 4-20 ℃ on leaves and fruit sequences on the flowering branches until the temperature of leaf surfaces is reduced to below 10 ℃.

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