CN114747480B

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

Info

Publication number: CN114747480B
Application number: CN202210308666.8A
Authority: CN
Inventors: 杨珺; 刘钊; 康向阳; 邱炳发; 王建忠; 李晨赫
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2023-04-28
Anticipated expiration: 2042-03-25
Also published as: CN114747480A

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

Method for inducing forest tetraploid in field in non-isolated manner

Technical Field

The invention relates to an induction method of a forest tetraploid, in particular to an induction method for creating a eucalyptus tetraploid plant by doubling an induction zygote chromosome, and belongs to the field of plant genetic breeding.

Background

Eucalyptus (Eucalyptus spp.) is an important woody tree species worldwide, providing more than 40% of wood and wood pulp yield per year for industrial production in China. At present, the global eucalyptus genetic improvement research mainly based on cross breeding is in a bottleneck period, and breakthrough varieties are not produced for many years. In order to create a novel high-yield and high-quality germplasm of eucalyptus, the biological yield is promoted to be further improved, and a novel eucalyptus breeding technology system is urgently needed. In recent years, artificially induced tetraploids have been successfully applied to genetic improvement of poplar, which indicates direction for germplasm innovation of eucalyptus. Tetraploid refers to an organism that contains four sets of chromosomes within the cell. Compared with the common diploid, the tetraploid plant has the advantages of thick plants, obviously increased organs such as flowers, leaves, fruits and the like, and stronger resistance to biotic and abiotic stress. Meanwhile, the tetraploid germplasm can also be used as a parent and can be directly hybridized with diploid to obtain triploid with more obvious growth advantage. Therefore, the artificial induction of the eucalyptus tetraploid has great significance for breeding the eucalyptus polyploid fine germplasm and greatly improving the productivity of the eucalyptus artificial forest.

There are two general approaches for obtaining plant tetraploid by artificial induction, one is to use a tissue culture method in a laboratory, treat meristem of diploid plant with colchicine and other cell division inhibitor, induce chromosome doubling of plant cell to obtain tetraploid cell tissue, and obtain tetraploid regenerated plant. Such methods have been reported on Eucalyptus (Korean et al, colchicine-induced E.grandis clone, eg5 polyploid. Chinese agronomic report, 2010, 26 (24): 128-132; korean et al, colchicine-induced E.grandis polyploid. Chinese agronomic report, 2010, 26 (15): 149-153; han Chao. Colchicine-induced Eucalyptus chromosome doubling technical system. Chinese forestry science institute, 2010; tan Deguan et al. Eucalyptus 12 ex vivo polyploid induction. Tropical crop report, 2005 (02): 50-54; hu Zhouhe et al. Preliminary study of colchicine-induced Eucalyptus polyploid. Guangxi forestry science, 2004 (04): 195-196+203: tan Deguan. Eucalyptus 12 OI (Eucalyptus 12 ABL) tissue culture and research of Kapohlrabi, 2004 et al, experiments were synthesized on Eucalyptus tetraploid, forestry 6 (1986): 46). For example, subjecting multiple cells of a meristem to mutagenesis often results in a large number of mixed ploidy, the regenerated plant ploidy is complex and difficult to separate; the mutagenesis treatment method based on the tissue culture regeneration system is usually effective only aiming at individual genotypes, and only one polyploid germplasm can be obtained through one-time effective mutagenesis treatment, so that the induction efficiency is low and the like.

Another way to obtain plant tetraploid by artificial induction is to directly induce the chromosome doubling of plant zygote, i.e. to apply mutagenesis treatment at specific time of the development of the fertilized zygote cells to induce the chromosome doubling of the zygote cells to obtain tetraploid plants by adopting a certain technical method. Compared with the chromosome doubling of the somatic cells reported in the prior art, the induction of the chromosome doubling of the zygote has the following three advantages: firstly, the tetraploid obtained by induction is an euploid tetraploid plant directly developed from single zygote cells, and the euploid plant does not generate mixed ploidy; the mutagenesis process does not depend on a specific laboratory environment, can be directly implemented on a field non-isolated tree, and has wide application range; thirdly, one mutagenesis treatment can be applied to a plurality of infructescence and a plurality of fertilized synthons in ovary, a plurality of tetraploid new germplasm is obtained at the same time, and the induction efficiency is high.

At present, the artificial induction of plant zygote chromosome doubling mainly adopts chemical agents such as colchicine solution, N2O gas and the like, or physical means such as extreme temperature treatment and the like to block the normal division process of the zygote cell, so that the zygote cell chromosome doubling is promoted to obtain tetraploid. The method is characterized in that the chromosome doubling effect of the poplar zygote is optimized by high-temperature treatment (Wang Jun and the like; physical and chemical treatment is used for inducing the chromosome doubling to select and breed the populus hybrid tetraploid; beijing university of forestry, 2010, 32 (05): 63-66; dan Le and the like; high-temperature treatment is used for inducing the chromosome doubling to obtain the populus hybrid polyploid; nuclear agronomy, 2012, 26 (08): 1118-1123; lu Min; triclopyr and tetraploid induction technical research; beijing university of forestry, 2013). However, the existing high-temperature induced plant tetraploid technology is mainly aimed at northern adapted tree species which bloom in early spring such as poplar and have short flowering period, easily identified and distinguished flower bud development states, short fruiting period and capability of harvesting seeds through in-vitro indoor water culture. The southern suitable tree species represented by eucalyptus flowers in summer under damp and hot conditions, and the suitable environment requires higher heat shock treatment intensity, and the branches and leaves of the luxuriant tree species are easy to be damaged by heat to cause fruit drop; the flowering period is as long as several months, the morphological changes of the flower buds and the infructescens are not obvious, and the effective time for applying the heat-induced mutation treatment is difficult to determine through experience or simple experiments in the infructescens development period as long as several months; the seed can not be harvested by adopting in-vitro water culture in the fruit setting period exceeding half a year, and the non-in-vitro mutagenesis operation can only be implemented under the field complex environmental conditions, so that the difficulty in selecting mutagenesis treatment time is further increased, and the mutagenesis operation method has higher requirements. Therefore, the reported method is not applicable to the southern adapted tree species represented by eucalyptus, and has the main defects that: the problem of selection of the time for the outdoor non-in-vitro induced zygote chromosome doubling treatment cannot be solved, and no effective method is known for the plant zygote development stage, so that the mutagenesis treatment is difficult to accurately apply to the vicinity of the first mitosis stage of the zygote, and the doubling failure is caused; the problem of an operation method for carrying out high-temperature mutagenesis treatment on the tree body under the field non-in-vitro condition cannot be solved, so that branches and leaves of the tree body are easy to be damaged by heat shock; the problem of preservation of flower bud and fruit sequence activity after high temperature treatment in summer field cannot be solved, and the flower bud and fruit sequence of the tree body after mutagenesis is withered and falls off and the like.

Disclosure of Invention

Aiming at the technical problems that the existing forest with long flowering time, undefined bud and fruit sequence morphology and long fruit bearing time is difficult to determine in the field induced tetraploid process and the induced treatment conditions cause poor tetraploid yield, the invention provides a method for inducing the tetraploid of the forest in the field in a non-isolated manner. The method of the invention overcomes the difficulty that the prior reported method can not be applied to the induction of tetraploid of southern trees such as eucalyptus and the like.

The technical scheme provided by the invention solves the following problems: the effective treatment time and treatment conditions for obtaining tetraploid by inducing eucalyptus chromosome doubling through field non-isolated heat shock treatment are defined; the method for inducing eucalyptus tetraploid by field non-isolated heat shock treatment is established, wherein the activity of the flower buds and the infructescence of eucalyptus can be effectively preserved in summer.

In order to achieve the aim of the invention, the invention provides a method for inducing forest tetraploid in field in a non-isolated manner, which comprises the step of carrying out heat shock treatment on the fruit sequence after pollination of the forest in a non-isolated state.

Wherein the forest is selected from eucalyptus, acacia or teak, preferably eucalyptus.

In particular, the eucalyptus is selected as Eucalyptus urophylla.

Wherein, the heat shock treatment is to heat the pollinated fruit sequence with the flowering branch, namely to heat the pollinated fruit sequence.

In particular, the temperature of the heat shock treatment is 40-48 ℃; the heat shock treatment time is 3-6h.

In particular, the heat shock treatment temperature is preferably 44+/-1 ℃; the heat shock treatment time is preferably 6 hours.

After pollination, the heat shock treatment is carried out at the stage of falling off of filaments and flower columns on the fruit sequence or at the stage of expanding the fruit sequence.

In particular, it is preferable to perform the heat shock treatment at the stage of abscission of the filaments and the columns on the infructescence.

In another aspect of the present invention, a method for inducing a forest tetraploid in the wild non-in vitro is provided, comprising the steps of sequentially:

1) Pollinating when the capsule cover of the flower bud on the forest flower branch changes from green to yellow or white and looseness occurs 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;

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; and then carrying out secondary identification on the tetraploid plant which is 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.

In particular, the eucalyptus is selected as Eucalyptus urophylla.

Wherein, the pollination in the step 1) is to cut off the top of the flower bud capsule cap on the forest flower branch together with the top of the stigma by a blade, and then to shoot pollen at the wound of the pillar exposed by the cut.

And (3) applying pollen to the cut of the column exposed by the cut, and completing pollination once without emasculation.

In particular, after pollination, the pollination flower branches are sleeved with an isolation paper bag, so that the effect of isolating pollen pollution is achieved. The release paper bag was released after two weeks of pollination.

In particular, the pollens used in the pollination are other good eucalyptus pollens collected in advance.

In particular, after pollination, the fruit sequence after pollination is continuously collected at intervals of 24 hours, the external morphological change of the fruit sequence is observed, the record is photographed, and the development process of the fruit sequence after pollination is divided into 5 stages according to the development time of the fruit sequence after pollination and the external morphological change;

the 1 st stage is 1 to 6 days after pollination, the external morphology of the fruit sequence is that the top of the stigma is oxidized and blackened after cutting, yellow filaments stand upright around the columella, and the zygote is unfertilized at the stage;

the 2 nd stage is 7 to 9 days after pollination, the external morphology of the infructescence is that the filaments wither and curl but are not yet shed, and the stage is a double fertilization period;

the 3 rd stage is 10 to 22 days after pollination, the external morphology of the infructescence is that the filaments gradually drop off, the flower columns gradually become yellow and wither, and the stage is a dormant zygote period;

the 4 th stage is 23 to 26 days after pollination, the external morphology of the infructescence is that the filaments completely fall off, and the flower columns completely fall off, and the stage is the period from the end of the dormancy of the zygote to the 4-cell primordia;

the 5 th stage is 27-28 days after pollination, and the morphology is not changed obviously except that the infructescence begins to expand obviously; this stage is the stage of the zygote further development into the globular embryo.

The fruit sequence is collected and pollinated, and the fruit sequence in the bagging is removed from the bagging when the fruit sequence is collected each time and is sleeved back immediately after the fruit sequence is collected.

Wherein, in the step 2), the pollinated inflorescences in the stage of falling off the filars and the flower columns are subjected to heat shock mutagenesis treatment. And carrying out heat shock mutagenesis treatment (namely heating treatment) on the whole flowers where the pollinated fruit sequences are located at a proper time (namely, the fruit sequences are in a stage of shedding of filaments and flower columns) after pollination.

Stage 4 and stage 5 of the development of the pollinated fruit sequence, namely 23 to 28 days after pollination; preferably, the stage 4, namely 23-26 days after pollination, the filaments and the columns are completely detached, and the heat shock treatment is carried out during the period from the end of the synthon dormancy to the 4-cell primordial embryo.

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

In particular, the method further comprises the following steps of: 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 ℃.

In particular, the post-treatment is preferably carried out by spraying an aqueous solution of potassium dihydrogen phosphate pre-cooled to 4-20 ℃ until the temperature of the leaf surface falls below 10 ℃.

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

The aim of spraying a large amount of precooled 0.1% monopotassium phosphate aqueous solution is to rapidly supplement water and cool the leaves and the fruit sequences and promote plant tissue repair damage after heat shock, so that the viability of the fruit sequences is improved and the tetraploid yield is improved.

In particular, the heat shock mutagenesis treatment in the step 2 is to heat the flowers and branches of the forest by adopting a 'tree non-isolated branch bud heating treatment device' with the patent number of ZL200610113448. X.

In particular, the temperature of the heat shock mutagenesis treatment is 40-48 ℃, preferably 44+/-1 ℃; the heat shock mutagenesis treatment time is 3-6h, preferably 6h.

In particular, the whole flowers to be mutagenized are wrapped by adopting a 'tree non-isolated branch bud heating treatment device' with the patent number ZL200610113448.X under the non-isolated condition, then the internal temperature of the treatment device is increased, the flowers wrapped in the 'tree non-isolated branch bud heating treatment device' are heated, and the heat shock mutagenesis treatment is carried out on the inflorescences growing on the flowers in the treatment device.

The heat shock mutagenesis treatment is directly carried out on the tree body, and the flower branches are still connected with the tree body and are in a non-isolated state. After the pretreatment operation, the whole flower branches with pollinated fruit sequences are subjected to temperature control and time control heating treatment by using special equipment (namely a heating treatment device for non-isolated branches and buds of trees with a patent number of ZL200610113448. X).

In particular, before the heating treatment of the tree flowers by adopting the 'tree non-isolated branch bud heating treatment device', the device further comprises: firstly, spraying water to wet leaves and fruit sequences on the flowering branches to be induced; the entire flower branch of the fruit sequence is then wrapped with a wrapping material.

The pretreatment aims to preserve moisture of the infructescence and the leaves in the subsequent heat shock treatment, and avoid direct contact of the infructescence and an extremely high temperature electric heating plate of a tree non-isolated branch bud heating treatment device with the patent number of ZL200610113448.X, so that the viability of the infructescence is improved.

In particular, after water is sprayed until condensed water drops flow down from the leaves, the whole flower branches with the ordered fruit sequence are immediately wrapped by the silica aerogel material; the method comprises the steps of carrying out a first treatment on the surface of the

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

In particular, after the flowers are subjected to heat shock mutagenesis treatment, the 'tree non-isolated branch bud heating treatment device' with the patent number of ZL200610113448.X and the silicon dioxide aerogel material wrapping the flowers are removed, and then the clear water or the monopotassium phosphate aqueous solution is sprayed.

Wherein, the specific time for seed maturation in the step 3) is 8-10 months after pollination, namely 3-4 months before and after the next year.

And 4) detecting the plant ploidy level of the offspring seedlings when the seedlings grow to the height of about 10cm in the step 4).

The method for obtaining eucalyptus tetraploid by inducing zygotic chromosome doubling through field non-isolated heat shock treatment comprises the following steps: performing control pollination on eucalyptus planted in the field (performing artificial pollination on eucalyptus by a one-step pollination method), and forming an infructescence through bagging, isolation and the like; observing and recording the development state of the fruit sequence after pollination, and applying non-isolated heat shock treatment to the fruit sequence on the flowering branch at a specific time when the fruit sequence is further developed; sowing and raising seedlings after the seeds in the infructescence are mature, and identifying the seeds to obtain eucalyptus tetraploid plants; wherein, the specific time for applying the non-isolated heat treatment to the fruit sequence on the flower branch is as follows: the fruit sequence develops to the 4 th stage after pollination, namely, 23 th to 26 th days after pollination, and when the flower filaments and the flower columns are completely separated; the method for applying heat shock treatment comprises the following steps: the method has the advantages that the silicon dioxide aerogel material is adopted to wrap the infructescence on the flowering branches before treatment, the infructescence is timely cooled after heat shock treatment, and the efficiency of obtaining tetraploid is high after heat shock treatment for 6 hours at 44+/-1 ℃ to induce chromosome doubling of eucalyptus zygote.

In summary, the invention has the advantages and positive effects that:

1. the method provided by the invention has the advantages that the feasibility of obtaining tetraploid by doubling the chromosome of the forest (preferably eucalyptus) zygote with the characteristics of induction of damp-heat flowering in summer, insensitivity to high temperature, long flowering period, undefined bud and fruit sequence morphology change and long fruit setting period is clarified for the first time, the new germplasm of the eucalyptus tetraploid is obtained, and the technical progress of eucalyptus polyploid breeding is effectively promoted.

2. The method determines the optimal treatment condition of inducing the chromosome doubling of the eucalyptus zygote by non-isolated heat shock treatment, namely, when the development of the infructescence is in the 4 th stage (namely, 23 th to 26 th days after pollination) and the filars and the columella completely fall off, the efficiency of inducing the chromosome doubling of the eucalyptus zygote to obtain the tetraploid is highest by heat shock treatment for 6 hours at 44+/-1 ℃; provides a complete operation method suitable for non-isolated heat-induced chromosomal doubling of eucalyptus zygotes, establishes a technical system for chromosomal doubling breeding tetraploids of eucalyptus zygotes, adds a new way for forest breeding, and promotes innovation development of the germplasm of southern forests. The invention has the advantages that the mutagenesis time is more convenient to judge, the development state of the zygote is rapidly judged by utilizing the development change characteristics of the filaments and the stigmas and combining the time length after pollination, so that the time for implementing mutagenesis is determined, and the induction efficiency is improved.

3. The method aims at inducing the chromosome doubling of the fertilized zygote of eucalyptus to obtain tetraploid, the induction process does not generate mixed ploidy and chimera, and the tetraploid induction efficiency is high; the heat shock mutagenesis treatment can be carried out after hybridization pollination of specific parent combinations, a batch of eucalyptus tetraploid new germplasm with different genotypes is obtained at one time, the induction efficiency of eucalyptus polyploid is effectively improved, the mutagenesis efficiency reaches more than 6%, and the fine variety breeding process of eucalyptus is accelerated.

4. The method has good popularization, and can effectively preserve the activity of flower buds and fruit sequences and improve the yield of tetraploids by carrying out pretreatment and aftertreatment on the flowers to be induced before and after heat shock treatment, thus being particularly suitable for the non-in-vitro induced tetraploid of the southern broadleaf tree species which flowers in summer and has long fruit setting time.

5. 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.

6. The method is simple to operate, has strong applicability, and can be applied to field non-in-vitro mutation breeding of different tree species under various standing conditions; the method has the advantages of low treatment 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 southern forest germplasm.

Drawings

FIG. 1 is a schematic diagram of the external morphology of artificial pollination and flower buds and fruit sequences in the embodiment of the invention, wherein a is a schematic diagram of artificial pollination of the flower buds of Eucalyptus urophylla, and b to e are schematic diagrams of the external morphology of the flower buds and fruit sequences in different development stages;

FIG. 2 is a schematic diagram of a field non-ex-situ heat-shock treatment in accordance with an embodiment of the present invention;

FIG. 3 is a schematic diagram of seeding and seedling raising in an embodiment of the invention;

FIG. 4 is a graph showing the results of ploidy detection and chromosome counting of somatic cells of a plant in an embodiment of the present invention, wherein a and b are the results of ploidy detection of the flow cytometer, the peak value of the graph a is diploid around the coordinate axis 50, and the peak value of the graph b is tetraploid around the coordinate axis 100; c and d are chromosome count results of somatic cells, 22 chromosomes in the c diagram are diploid, and 44 chromosomes in the d diagram are tetraploid;

FIG. 5 is a comparative drawing of a Eucalyptus urophylla diploid, tetraploid plant in which a is diploid and b is tetraploid in the example of the present invention.

Detailed Description

The advantages and features of the present invention will become more apparent from the following description, taken in conjunction with the accompanying examples. These examples are merely exemplary and do not limit the scope of the invention in any way. All other embodiments obtained by those skilled in the art without making any inventive effort are within the scope of the present invention.

Example 1

1. Artificial pollination:

flowers with large flower quantity are selected as candidate flowers for mutagenesis treatment before the tail She Ansan powder; when most of the flower bud capsules are observed to be changed from green to yellow or white, and loosening occurs along the shedding ring, artificial pollination is carried out;

the pollination method adopts a one-step pollination method, the top of the flower bud capsule cover and the top of the stigma are cut off together by a blade, and pollen is applied to the wound of the pillar with the cut exposed by a cotton swab point; wherein: pollen used for artificial pollination is from other fine eucalyptus pollen. After pollination is completed, the flowers are sleeved with an isolation bag (acetate paper bag, as shown in figure 1 a).

In fig. 1a, the flower branches after pollination are sleeved with an isolating acetate paper bag, the isolating paper bag plays a role in isolating pollen pollution, and the sleeved isolating paper bag is released after pollination for two weeks.

2. And (3) observing the development stage of the infructescence:

and (3) continuously collecting the pollinated fruit sequence at sampling time intervals of 24 hours after the end eucalyptus urophylla finishes pollination, observing the external morphological change of the fruit sequence, and photographing and recording.

Taking the time of artificial pollination of Eucalyptus urophylla as a starting point, and the external morphological development change of the flower buds and the fruit sequences after pollination is shown in figure 1a, wherein a' in figure 1a is the flower buds after one-time artificial pollination.

The external morphology observations of the flower buds and the fruit sequences after pollination are shown in Table 1. The development of the infructescence was divided into 5 stages according to the duration of the development of the infructescence after pollination and the change of the external morphology (Table 1).

Stage 1: 1 to 6 days after pollination, the tops of the stigmas are cut and then oxidized to become black, and yellow filaments stand upright around the stigmas (figure 1 b); this stage is the period when the zygote has not yet fertilized.

Stage 2: from day 7 to day 9 after pollination, it was observed that the filaments on the fruit sequence withered and curled but had not yet fallen off (FIG. 1 c); this stage is the double fertilization period.

Stage 3: 10 to 22 days after pollination, during which the filaments on the inflorescences gradually drop off and the flower columns gradually yellow and wilt (fig. 1 d); the synthons are in sleep phase at this stage.

Stage 4, 23 to 26 days after pollination, it can be observed that the filaments and columns on the inflorescences have completely fallen off (fig. 1 e); at this stage the zygote successively ends dormancy, starting the first mitosis.

In the stage 5, the appearance is not changed obviously except that the fruit sequence begins to expand obviously after 27 to 28 days after pollination; at this stage the zygote continues to divide and develop into a globular embryo.

TABLE 1 bud and infructescence development stage characteristics after pollination

/>

Example 2 field non-ex-vivo heat shock mutagenesis treatment:

based on the length of time and external morphological characteristics of the postpollination infructescence development, field non-isolated heat shock mutagenesis treatment was performed on infructescence developed to stages 4 and 5 after pollination (fig. 2).

1. Pretreatment of

2 groups of target flowers are selected for pretreatment according to the number of days after pollination on the flowers where the fruit sequences growing to the 4 th and 5 th stages are located, namely 2 groups of fruit sequence-bearing flowers growing on different numbers of days after pollination are selected for pretreatment, namely, 2 groups of inflorescences growing on the fruit sequence from the 23 rd day to the 28 th day after pollination are respectively selected for pretreatment, wherein the pretreatment is to fully spray water to moisten the leaves and the fruit sequence on the target flower sequence until condensed water drops flow down from the leaves, and the whole flower sequence growing with the fruit sequence is immediately wrapped by using a silicon dioxide aerogel material;

the silica aerogel material has the function of moisturizing the infructescence and the leaves in the subsequent heat shock treatment process, and avoiding direct contact between the infructescence and an extremely high temperature electric heating plate of the heat shock treatment device, so that the viability of the infructescence is improved.

In the pretreatment process, the flower branches are wrapped by the silica aerogel material, and other materials such as fireproof heat-insulating cloth, ceramic fiber cotton and the like are suitable for the invention.

2. Heat shock treatment

The heat shock treatment is carried out on each target flower branch by adopting an invention patent (patent number: ZL200610113448. X) of the non-isolated branch bud heat treatment device of the tree, which is obtained by the applicant. The high-temperature heating bag of the tree non-isolated branch bud heating treatment device is connected with a temperature controller through a cable and is communicated with a power supply.

The heating bag (shown in figure 2) of the heating treatment device for the non-isolated branches and buds of the tree is sleeved on the flowers to be subjected to mutagenesis treatment through the opening end, the opening end of the bag is fastened on the flowers to be subjected to heat shock mutagenesis treatment through the flexible rope, and the bag is sealed and wrapped on the flowers.

The temperature controller of the "tree non-isolated branch bud heating treatment device" is placed at the ground safety place, and the bag body of the heating bag is connected with the temperature controller by electric wires (figure 2).

After the power is turned on, the heating bag is heated to the preset temperature (44+/-1 ℃) according to the setting of the temperature controller, and then the temperature is kept constant, so that the flowers wrapped by the heating bag are ensured to be at the preset temperature of 44+/-1 ℃. The timing was started after the treatment temperature was constant, and the heating treatment was released 3 hours and 6 hours after the start of the treatment.

3. Post-treatment

And immediately after the heat shock treatment is finished, carrying out post-treatment on the target flowering branch, timely removing the heating treatment device and the silicon dioxide aerogel material, and immediately spraying a large amount of 0.1% potassium dihydrogen phosphate aqueous solution precooled to 4 ℃ on the leaves and the fruit sequence on the flowering branch until the temperature of the leaf surface is reduced to below 10 ℃.

The post-treatment in the invention is illustrated by spraying 0.1% potassium dihydrogen phosphate aqueous solution, and other potassium dihydrogen phosphate aqueous solutions with the concentration of 0.05-0.2% are also suitable for the invention; but also can spray a large amount of clean water;

the temperature of the post-treatment spray clear water or the aqueous solution of potassium dihydrogen phosphate is illustrated by way of example at 4℃and other temperatures, such as 4-20℃are also suitable for the present invention.

And numbering and marking the target flower branches according to the heat shock treatment temperature and the time length. The heat shock treatment temperature and duration are shown in Table 2.

Example 2A

The procedure of example 2 was followed except that the temperature in the heated bag was kept at 40℃during the heat shock treatment of step 2).

Example 2B

The procedure of example 2 was followed except that the temperature in the heated bag was kept at 48℃during the heat shock treatment of step 2).

Under the treatment condition that the temperature is set to 48 ℃, no matter the treatment time is 3 hours or 6 hours, the branches are dried up under the influence of heat shock, and seeds cannot be received.

The branches under the treatment conditions of 40 ℃ and 44 ℃ are not abnormal, and the fruit sequence can grow normally and is firm. In the next year of seed maturation, the seeds are collected in a treatment combination and the treated eucalyptus urophylla seeds 2197 are harvested together.

Example 3 ploidy detection:

1. seedling raising

After 3-4 months of the next year, the eucalyptus seeds of example 2, 2A were collected according to the markers and the treated eucalyptus urophylla seeds 2197 were harvested together.

The harvested seeds were sown separately according to the marker group and grown in seedlings (fig. 3). Seeds harvested at treatment times of 3 hours and 6 hours at heat shock temperatures of 40℃and 44℃were able to emerge (Table 2). Transplanting when the seedlings grow to three pairs of leaves, and survival of 1074 eucalyptus urophylla seedlings after transplanting (as shown in Table 2).

2. Preliminary identification of ploidy level of eucalyptus urophylla offspring by flow cytometry

After the seedlings grow to 10cm in height, part of leaves are extracted from the plants, the leaves are cut up and filtered by a 50 mu m nylon net, and the filtered cell suspension is added with 50 mu g ml ^-1 In 4',6-diamidino-2-phenylindole (DAPI)) and the ploidy level of the progeny of Eucalyptus urophylla was analyzed using a flow cytometer, the diploid leaves of Eucalyptus urophylla were used as controls.

Ploidy detection was performed on 1074 eucalyptus urophylla seedlings using a flow cytometer to initially identify 4 tetraploid eucalyptus urophylla seedlings (see table 2). The result of the flow cytometry for identifying the ploidy of the seed seedling is shown in fig. 4, fig. 4a shows that the ploidy of the diploid plant is detected, and the peak value of the a diagram is diploid around a coordinate axis 50; FIG. 4b shows the ploidy test of tetraploid plants, with the peak of b being tetraploid around axis 100.

3. Somatic chromosome counting

The tetraploid plant which is primarily identified by the flow cytometry is finally subjected to secondary identification by somatic chromosome counting, so as to determine the ploidy level of the plant.

The tender leaves of the tetraploid plants identified by the flow cytometry are placed in a Carnot fixing solution for fixing treatment for 24 hours; then washing the fixed sample with water until the carnot solution is completely removed, placing the fixed sample in 1N hydrochloric acid, and dissociating the fixed sample at room temperature for 15 minutes; then the dissociated sample is dyed by using a carbofuchsin dye and crushed for tabletting; the sample preparation was observed using an optical microscope and the chromosome number of the sample was counted to determine the ploidy level.

TABLE 2 non-isolated heat-induced tail She Ange chromosome doubling

The chromosome count results of eucalyptus diploid and tetraploid are shown in fig. 4, wherein the chromosome number of the diploid eucalyptus robusta is 2n=2x=22, as shown in fig. 4c,2n represents the chromosome cell, x represents the chromosome cardinal number, and x=11, 2x of eucalyptus in the embodiment represents two sets of chromosome groups, diploid, and 2×11=22 total chromosomes; the number of chromosomes of the tetraploid eucalyptus urophylla plant preliminarily identified by the flow cytometer of the invention is all 2n=4x=44, as shown in fig. 4d, the tetraploid 2n=4x=44, which indicates: somatic cells have 4 sets of chromosomes, which are tetraploids, and total 4×11=44 chromosomes. By chromosome counting of somatic cells, 4 tetraploid eucalyptus urophylla are finally identified and confirmed

The diploid and tetraploid plant potted plant is shown in figure 5, wherein 5a is diploid; 5b is a tetraploid.

The detection result shows that: when the infructescence develops to stage 4, heat shock treatment is carried out for 3-6 hours at 44+/-1 ℃, and tetraploid is obtained from groups 24-26 days after pollination in the stage. Especially, the tetraploid is obtained in the 4 th stage, namely 25 days after pollination and when the flowers and the flower columns on the infructescence completely fall off, and the heat shock treatment is carried out for 6 hours at 44+/-1 ℃ so as to obtain the highest tetraploid with the tetraploid yield of 6.45%.

In the multiple experiments of the invention, tetraploid can be obtained at the stage of complete abscission of the inflorescence and the columella of the fruit sequence after pollination (usually 23-26 days after pollination).

Comparative example 1

The fruit sequences which are obtained according to the method step of the embodiment 1 and develop to the 4 th and 5 th stages after pollination are directly sown after the seeds are ripe in 3 to 4 months of the next year without heat shock induction treatment, and seedlings are grown; the progeny plants obtained from the seedlings were subjected to ploidy testing in the manner of example 3, with the following test results: no tetraploid was found by flow cytometry identification and somatic chromosome counting.

Comparative example 2

The fruit sequences obtained according to the method step of example 1 and developed to the 4 th and 5 th stages after pollination are not subjected to post-treatment on the target flower branches and the flower buds and fruit sequences growing thereon after heat shock induction treatment, that is, the leaves and fruit sequences on the flower branches are not sprayed with a large amount of 0.1% potassium dihydrogen phosphate aqueous solution precooled to 4 ℃ until the surface temperature of the leaves and fruit sequences reaches below 10 ℃, and the rest is the same as in example 2. The flowers and branches and the fruit sequence of each treatment group are dehydrated, dried and deactivated due to the failure of timely cooling and water supplementing after heat shock treatment. Mature seeds were not collected and tetraploid eucalyptus plants were not obtained.

Comparative example 3

The procedure of example 2 was repeated except that the heating apparatus having a large space volume was used, the flowers to be subjected to mutagenesis treatment were kept away from the bag body by using the holder in the heating bag, were not brought close to the bag body of the heating bag, and the whole flowers of the fruit sequence were not covered with the silica aerogel material.

After the heat shock induction treatment, the seeds are ripe in 3 to 4 months of the next year, sowing and seedling raising are carried out; the progeny plants obtained from the seedlings were subjected to ploidy testing in the manner of example 3, with the following test results: when the infructescence develops to the 4 th stage, the tetraploid can be obtained by heat shock treatment at 44+/-1 ℃ for 3-6 hours. Wherein the tetraploid is obtained by adopting the heat shock treatment at 44+/-1 ℃ for 6 hours in the 4 th stage, namely 25 days after pollination and when the flowers and the columns on the inflorescences completely fall off.

Claims

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;

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 ℃.