CN111937741B - Method for creating intergeneric distant hybrid of hibiscus and broad-leaved Maohua - Google Patents

Abstract

The invention discloses a method for creating intergeneric distant hybrids of hibiscus and broad-leaved Maohua. The method comprises the following steps: taking diploid hibiscus of Compositae as female parent and hexaploid Maohua of Compositae as male parent, and carrying out artificial hybridization. And (4) carrying out isolated culture on the immature embryo to obtain an F1 generation plant. And (3) carrying out hybrid morphological and cytological identification on the F1 generation material to finally obtain true hybrid progeny of the hibiscus and the broad-leaved Maohuaju. The invention overcomes the intergeneric distant hybridization obstacle of hibiscus and broad-leaved Maohua, obtains distant hybrid progeny, and creates a batch of new germplasm.

Description

Method for creating intergeneric distant hybrid of hibiscus and broad-leaved Maohua

Technical Field

The invention relates to the field of plant breeding, in particular to a method for creating a intergeneric distant hybrid of hibiscus and broad-leaved Maohuaju.

Background

Hibiscus mutabilis (Crossostephium chinensis), a single genus of Hibiscus of Compositae, is unique to China and is produced in coastal areas of southeast. Evergreen sub shrubs. The leaf is normally narrow and is in a shape of a reverse needle, the whole edge is always normal, and both sides are densely covered with grey soft hair, and the texture is thick; the flower diameter is about 0.5cm, the flower is light yellow, no tongue-shaped flower exists, and the late 11 months of the flowering season is 1 month in the next year. The hibiscus mutabilis is a rare natural heterochromatic leaf and aromatic plant, has the characteristics of salt and alkali resistance, wind resistance, few plant diseases and insect pests and the like, and is widely applied to gardens. Poplar petrel (2016) takes 8 parts of wild species of generalized chrysanthemum as experimental materials, and comprehensive analysis of physiological indexes under salt stress proves that hibiscus is an excellent salt-tolerant germplasm; after studies on the changes of anatomical structures and physiological indexes of hibiscus and other 4 chrysanthemum plants under the salt stress by forest double-wings (2017), the hibiscus has a unique salt tolerance mechanism. The domestic research on the hibiscus purpurea mainly focuses on the aspects of medicinal functions and salt tolerance mechanisms, and the research on breeding is very little.

Flos Chrysanthemi Indici (Chrysanthemum vestigium var. latifolia), Compositae family Chrysanthemum genus plant. Growing in scattered mode with less branches. Leaf-round shape, oval shape, thick villi, and large inflorescence diameter of about 4.5-5.0 cm. The flower is white, and blooms in the last 10 months, so that the ornamental value is better. After comprehensive evaluation of morphological and physiological indexes of broad-leaved Mao Huaju under salt stress (2018), the cloche and the like find that the broad-leaved Mao Huaju shows medium salt tolerance.

The chrysanthemum production is regionalized in China, and the saline-alkali soil in coastal and northern areas limits the cultivation and popularization of chrysanthemum. The method for cultivating the chrysanthemum variety with strong salt tolerance by utilizing the salt-tolerant wild germplasm (such as hibiscus purpurea) to carry out distant hybridization is an important direction for chrysanthemum breeding. Embryo rescue has been applied among chrysanthemum plants as an important means for overcoming distant hybridization obstacles, but the related technology is not applied to the hibiscus since hibiscus has no lingua, immature embryos are small and difficult to peel. Hybrid progeny of the hibiscus and the broad-leaved Maohua obtained by utilizing the embryo rescue technology inherits the salt tolerance of the hibiscus on one hand, can be used as genetic material for cytology and molecular research, and can obviously improve the hybridization affinity of hibiscus and chrysanthemum on the other hand when being used as a bridge.

Disclosure of Invention

The invention aims to provide a method for creating a intergeneric distant hybrid of hibiscus and broad-leaved Maohua.

In order to realize the purpose of the invention, the invention provides a method for creating a distant hybrid between hibiscus and broad-leaved Maohua, which takes hibiscus and chrysanthemum broad-leaved Maohua as parents to carry out hybridization and utilizes a young embryo rescue technology to obtain the distant hybrid.

Previous researches show that when the hibiscus mutabilis is used as a male parent and is hybridized with broad-leaved Maohua, the fructification rate is zero. Therefore, the invention uses diploid hibiscus as female parent and hexaploid broad-leaf Maohua as male parent to make hybridization and utilizes embryo rescue to create distant hybrid.

The method specifically comprises the following steps:

1) parent preparation: regulating and controlling the flowering phases of the amphiphilic plants to ensure that the flowering phases meet;

2) distant hybridization: when the male parent and the female parent bloom, castrating the female parent, taking fresh pollen of the male parent for pollination and bagging;

3) taking out ovules: taking the head-shaped inflorescence (taken once every other day) 8-24 days after the cross pollination (preferably 18 days after the pollination), and taking out ovules after disinfection and sterilization;

4) in-vitro culture of immature embryos: inoculating ovules into induction culture media with different hormone ratios for culture, and then transferring the ovules into a rooting culture medium for culture until seedlings emerge;

5) acquisition of hybrid generation F1: transplanting the seedlings into a matrix for hardening when the roots of the seedlings grow to be more than 1cm, and then putting the seedlings into a pot for conventional management;

6) screening true hybrids: true hybrids were screened from plants of generation F1 based on phenotypic observation and cytological identification of the parent and hybrid generation F1.

The method, step 1), comprises: the female parent hibiscus flower is cured by short sunshine for 8-10 hours from month 8, so that the blooming period is reached in the middle 10 th of month. Specifically, the short-day curing conditions are as follows: humidity is 70-75%, daytime temperature is 24-28 ℃, night is 20-22 ℃, illumination is 8-10 hours per day, and illumination intensity is 1600-.

The method, step 3), comprises: taking the head-shaped inflorescence after the cross pollination, sterilizing the head-shaped inflorescence, removing the ovary wall, and taking out the ovule.

Preferably, the method of sterilization comprises: sterilizing the surface of the capitula by 70-75% alcohol for 30-45 s, washing with sterile water, and adding 8-13% H₂O₂Sterilizing the aqueous solution for 10-15 min, and then sterilizing with sterile waterAnd (5) flushing.

The method as described above, wherein the induction medium in step 4) contains 6-BA and NAA; the rooting medium contains NAA.

Different hybrid combination embryo culture has different requirements on culture media. In other chrysanthemum allied plant hybridization, a document reports that MS +2.0mg/L KT +1.0mg/L IAA culture medium can be used for obtaining hybrid progeny for cultivating chrysanthemum morifolium citriodora and artemisia argyi (juneberry, 2011); it has also been reported in literature that the progeny of the hybridization between garland chrysanthemum and wormwood stalks can be obtained by using MS +0.2mg/L IAA medium, but the germination rate is only 12.9% (Hisao Ohtsuka, 2008). Related researches on obtaining callus and hybrid seedlings by culturing the embryos of the filial generation of hibiscus in intercross by using an MS culture medium containing 6-BA and NAA are rarely reported.

The induction culture medium is MS culture medium containing 1.0-2.0 mg/L6-BA and 0.2-1.0mg/LNAA, and pH5.6-5.8.

Preferably, the induction medium is MS medium containing 1.5mg/L of 6-BA and 0.5mg/L of NAA and having a pH value of 5.6; MS culture medium containing 2.0 mg/L6-BA and 0.2mg/L NAA, pH5.8; MS culture medium containing 2.0 mg/L6-BA and 0.5mg/L NAA, pH5.8; MS culture medium containing 2.0 mg/L6-BA and 1.0mg/L NAA, pH5.6; or MS culture medium containing 1.0 mg/L6-BA and 0.5mg/L NAA, and pH 5.8.

More preferably, the induction medium is MS medium containing 2.0 mg/L6-BA and 0.2mg/L NAA, pH 5.8.

The rooting culture medium comprises: 1/2MS +0.1mg/L NAA, pH 5.6.

In the method, the conditions for induction culture and rooting culture in the step 4) are as follows: the temperature is 20-25 ℃ (preferably 20 ℃), the daily illumination is 14-16h, and the illumination intensity is 1600-2000lx (preferably 2000 lx); after 4-6 weeks of induction culture, the culture is changed to rooting culture.

In the method, in the step 5), the matrix is formed by mixing vermiculite and perlite according to the volume ratio of 1: 1.

The seedling exercising conditions are as follows: culturing at 27-30 deg.C and relative humidity of 60-70% for 30-45 days.

The method as described above, step 6), wherein the phenotype is at least one selected from plant height, canopy width, leaf length, leaf width, leaf stalk length, etc. Wherein the plant height, crown width, leaf length, leaf width and leaf stalk length between the parent and the female parent are determined as true hybrids.

In the invention, the offspring material is observed by chromosome meiosis, and the true hybrid is determined when the chromosome number is expressed as tetraploid.

In the invention, the female parent is selected from diploid hibiscus (Cr. chinensis) of hibiscus, and the male parent is selected from hexaploid broad-leaf chrysanthemum (C. vestum) of chrysanthemum.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the salt-tolerant germplasm hibiscus purpurea is selected as a female parent, the medium salt-tolerant germplasm broad-leaved chrysanthemum is selected as a male parent, and salt-tolerant genes can be effectively introduced into the breeding process of chrysanthemum.

The invention compares the embryo development effect under the culture medium with different embryo development stages and different hormone proportions on the tissue culture technology. The result shows that the young embryo culture effect 18 days after pollination is the best, and the culture medium of MS +2.0 mg/L6-BA +0.2mg/L NAA and pH5.8 is most beneficial to embryo development and can reach the germination rate of 6.25 percent.

The authenticity of the intergeneric hybrid of hibiscus and broadleaf chrysanthemum is early identified by morphological observation and cytological identification.

The invention can realize the continuous development of the hybrid embryo of the hibiscus and the broad leaf Mao chrysanthemum and realize the intergeneric distant hybridization of the hibiscus and the broad leaf Mao chrysanthemum, thereby obtaining hybrid offspring.

The invention makes it possible to breed chrysanthemum by using new chrysanthemum germplasm and is favorable to the research of chrysanthemum genesis evolution and the kinship between hibiscus and chrysanthemum.

Drawings

FIG. 1 is a comparison graph of female parent hibiscus flower (left), male parent broad leaf flower (middle) and hybrid progeny (right).

FIG. 2 is a diagram showing the morphological comparison of the parental and hybrid progeny of hibiscus and latifolius florida in the preferred embodiment of the present invention. Wherein, A: comparing flowers and leaves of parents; b: asexual propagation of hybrid progeny; c: the hybrid progeny are morphologically contrasted with the parent.

FIG. 3 is a comparison of primary leaves of hibiscus, latifolius and their hybrid progeny in accordance with a preferred embodiment of the present invention. Wherein, A: flos Spilanthis Lanceolatae; b: flos Hibisci Mutabilis; C-G: hybrid progeny.

FIG. 4 is a diagram showing the cytological identification of hibiscus, broad-leaved Majorana and their hybrid progeny in a preferred embodiment of the present invention. Wherein, A: flos Hibisci Mutabilis; b: flos Spilanthis Lanceolatae; C-F: hybrid progeny.

Detailed Description

Aiming at the problems of low breeding utilization rate, narrow genetic basis, weak salt tolerance of cultivated chrysanthemum varieties and the like of the existing unique chrysanthemum resources in China, the salt-tolerant wild hibiscus is used as a female parent to be hybridized with broad leaf Mao Huaju, and the problems of hybrid embryo abortion, hybrid immature and the like of the two in distant hybridization are solved through a young embryo rescue technology, so that distant hybridization progeny of the hibiscus and the broad leaf Mao Huaju is finally obtained.

The invention provides a method for creating a intergeneric distant hybrid of hibiscus and broad-leaved Maohua, which comprises the following steps:

1) parent preparation: the female parent is hibiscus flower with 2n ═ 18, and the male parent is broad-leaved chrysanthemum flower with 2n ═ 54. The parents are adjusted in flowering phase to meet the flowering phase. A comparison graph of female parent flos Hibisci Mutabilis and male parent flos Spiraeae Fortunei is shown in FIG. 1.

2) Distant hybridization: when the male parent and the female parent both bloom, the female parent is castrated, and the male parent is pollinated with fresh pollen and bagged.

3) Taking out ovules: taking the head-shaped inflorescence 8-24 days after the cross pollination in the step 2), stripping the female florets on a superclean bench, carrying out surface disinfection and sterilization, then stripping the ovary wall, and taking out ovules.

4) In-vitro culture of immature embryos: under the aseptic condition, the ovule obtained in the step 3) is inoculated into induction culture media with different hormone ratios, the culture temperature is 20 ℃, the illumination is carried out for 16h every day, and the illumination intensity is about 2000 lx; a total of 5 media types were set, with 6-BA and NAA concentrations being varied, and approximately 9 ovules were inoculated per plate.

5) Rooting culture: after 4 weeks, the cells were transferred to rooting medium (1/2MS +0.1mg/L NAA, pH5.6) and cultured at 20 ℃ under light intensity of about 2000lx for 16h per day.

6) Obtaining hybrid F1 generation plants: transplanting the seedlings into a matrix (coarse vermiculite: perlite: 1 in volume ratio) for hardening seedlings when the roots of the seedlings grow to be more than 1 cm. After one month, the pot is filled with the fresh water and is managed conventionally.

7) And (3) morphological observation: morphological observation and statistics are carried out on the parent and the offspring materials, wherein the plant height, the crown width, the leaf length, the leaf width and the leaf stalk length between the parent and the offspring are determined as true hybrids.

8) And (3) cytological identification: and judging the authenticity of filial generations of the hibiscus and the broadleaf chrysanthemum by adopting chromosome counting, wherein the true hybrid is determined when the chromosome number is tetraploid.

Preferably, the flowering phase of the female parent material hibiscus flower used in the step 1) needs to be adjusted, and short-day curing is performed for 8 hours from 8 months, so that the full flowering phase is reached in 10 middle ten days.

Preferably, the pollination time in step 2) is 9-10 am or 3-4 pm, and the same inflorescence is pollinated repeatedly for 2 times, once a day.

Preferably, the bagging in the step 2) is a post-pollination sulfuric acid paper bag

Preferably, the head-shaped inflorescences which grow and develop for 8-24 days after the cross pollination are taken in the step 3).

Preferably, the method for sterilizing the female florets in the step 3) comprises the following steps: sterilizing with 70% ethanol for 30 s, washing with sterile water for 4 times, and adding 12% H₂O₂The aqueous solution was sterilized for 10 minutes and washed 5 times with sterile water.

Preferably, the culture medium for the in vitro culture of the immature embryo in the step 4) is MS culture medium containing 1.0-2.0mg/L of 6-BA and 0.2-1.0mg/L of NAA and having a pH value of 5.6-5.8. Specifically, culture medium a: 1.5 mg/L6-BA +0.5mg/L NAA, pH5.6; and (3) a culture medium B: 2.0 mg/L6-BA +0.2mg/L NAA, pH5.8; and (3) a culture medium C: 2.0 mg/L6-BA +0.5mg/L NAA, pH5.8; and (3) a culture medium D: 2.0 mg/L6-BA +1.0mg/L NAA, pH5.6; and (3) a culture medium E: 1.0 mg/L6-BA +0.5mg/L NAA, pH 5.8.

Preferably, the rooting medium in step 5) is 1/2MS +0.1mg/L NAA, pH5.6, i.e. 0.1mg/L NAA is added to 1/2MS minimal medium.

Preferably, the F1 generation plant transplanting method in step 6) is to open bottles and harden seedlings for 2-3 days, then transplant into a plug tray with coarse vermiculite and perlite in a ratio of 1:1, and after culturing for 30 days, put into a pot for conventional management.

Preferably, step 7) comprises measuring plant height, canopy width. Leaf length, leaf width, petiole length; three fully-unfolded leaves are randomly selected for each plant to measure the leaf length and the leaf width, and the leaf stalk length is measured respectively.

Preferably, the progeny with the progeny chromosome number of 36 in the cytological identification result of step 7) is determined to be true hybrid progeny.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. Example 1 obtaining intergeneric hybrid progeny of Hibiscus and Hibiscus latifolia by embryo rescue using young embryos of different embryo ages

Utilizing the young embryos with different embryo ages to perform embryo rescue to obtain the intergeneric hybrid progeny of the hibiscus and the broad-leaved Maohuaju. The method comprises the following steps:

1. parent preparation: the female parent is selected from special-produced hibiscus diploid salt-tolerant wild hibiscus, the male parent is selected from chrysanthemum hexaploid medium salt-tolerant wild hibiscus broad-leaved lonicera, and the male parent and the female parent are all planted in the small Tangshan base of the national flower engineering center. Adjusting the florescence of the hibiscus purpurea, putting the hibiscus purpurea in an artificial climate chamber in 8 months, starting short-day curing for 8 hours, and reaching the full florescence in 10 middle ten days; the short-day artificial climate chamber is set to have the humidity of 70 percent, the daytime temperature of 25 ℃, the night temperature of 22 ℃ and the illumination time of 6-14 points.

2. Distant hybridization: the cotton rose hibiscus and broad leaf Maohua are hybridized artificially in the greenhouse. Manually castrating the female parent and bagging the female parent 1-2 days before blooming, collecting fresh pollen of the male parent when the stigma stretches out and the fork presents a certain angle and secretes mucus, pollinating by using a brush pen, and then continuously bagging. Pollination is typically carried out at 10 am or 4 pm, and the same inflorescence is pollinated 2 times repeatedly, once a day.

3、Taking out ovules: and (3) taking the capitulum 8-24 days after the cross pollination in the step (2), and taking the capitulum once every other day. Stripping female florets from clean bench, sterilizing with 70% ethanol for 30 s, washing with sterile water for 4 times, and adding 12% H₂O₂The aqueous solution was sterilized for 10 minutes and washed 5 times with sterile water. The female ovary was cut open on sterile paper with a knife, the ovary wall was peeled off, and the ovule was removed.

4. In-vitro culture of immature embryos: inoculating the ovule obtained in step 3 into MS induction medium containing 1.0-2.0 mg/L6-BA and 0.2-1.0mg/L NAA and having pH of 5.6-5.8 under aseptic condition, culturing at 20 deg.C under illumination for 16 hr per day with illumination intensity of about 2000 lx. The results in Table 1 show that the success rate of inoculation is 0 for very immature embryos 8-10 days after pollination; the success rate of inoculation is also 0 for embryos 24 days after pollination, because the embryos are aborted. The ovule with the worst healing effect is 14 days after pollination, and the healing rate is 27.03 percent; the ovule 16 days after pollination is the best, and the healing rate reaches 70.79%. But the highest rate of plantlet formation (10.53%) occurred in ovules 18 days after pollination, much higher than in other time periods. Therefore, the optimal embryo rescue period for distant hybridization between hibiscus and latifolius is 18 days after pollination, which combines the healing rate and the seedling rate. This example was co-inoculated with 269 ovules, resulting in 17 hybrid seedlings.

TABLE 1 immature embryos of different embryo ages and the Effect on in vitro culture of immature embryos

5. Rooting culture and obtaining of hybrid F1 generation plants: transferring the germinated immature embryo obtained after 4 weeks into a rooting culture medium, 1/2MS +0.1mg/LNAA, pH5.6, culturing at 20 ℃, illuminating for 16h every day, and illuminating intensity of about 2000 lx; observing the condition that the young embryo generates cluster buds in a rooting culture medium until the young embryo develops into a seedling; when the root of the young seedling grows to be more than 1cm, the obtained hybrid F1 generation plant is subjected to bottle opening and seedling hardening for 2-3 days, and the young seedling is adapted to the autotrophic growth and the bacteria-bearing environment of the field by gradually reducing the relative humidity and enhancing the illumination. The plants were removed and the medium attached to their surface was washed and transplanted into a plug with a matrix of coarse vermiculite perlite: 1. After one month, the pot is filled with the fresh water and is managed conventionally. Finally obtaining the hybrid big seedling of 11 cottonrose hibiscus and broad-leaved Maohua chrysanthemum.

Example 2 obtaining intergeneric hybrid progeny of Hibiscus and Hibiscus latifolia by embryo culture using culture media with different hormone ratios

Performing embryo culture by using culture media with different hormone ratios to obtain intergeneric hybrid progeny of hibiscus and broad-leaved Mao Huaju. The method comprises the following steps:

1. parent preparation: the female parent is special diploid salt-tolerant wild hibiscus flower, the male parent is hexaploid wild broad-leaf Maohua flower, and the male parent and the female parent are planted in the small Tangshan base of the national flower engineering center. Adjusting the florescence of the hibiscus purpurea, putting the hibiscus purpurea in an artificial climate chamber in 8 months, starting short-day curing for 8 hours, and enabling the middle ten days of the hibiscus purpurea to reach the full florescence; the short-day artificial climate chamber is set to have the humidity of 70 percent, the daytime temperature of 25 ℃, the night temperature of 22 ℃ and the illumination time of 6-14 points.

2. Distant hybridization: the hybridization of hibiscus and broad-leaved Maohuaju is carried out in the greenhouse. Manually castrating the female parent 1-2 days before blooming, bagging and isolating, collecting the fresh pollen of the male parent for pollination when the stigma stretches out and the fork presents a certain angle and secretes mucus, and continuously bagging and isolating after pollination. Pollinating at 10 am or 4 pm, and pollinating the same inflorescence for 2 times repeatedly, once a day.

3. Taking out ovules: and (3) taking the head-shaped inflorescences after the hybridization pollination in the step (2), and putting the head-shaped inflorescences into a refrigerator for short-term storage. Stripping female floret on clean bench, sterilizing with 70% ethanol for 30 s, washing with sterile water for 4 times, and adding 12% H₂O₂The aqueous solution was sterilized for 10 minutes and washed 5 times with sterile water. The female ovary was cut open on sterile paper with a knife, the ovary wall was peeled off, and the ovule was removed.

4) In-vitro culture of immature embryos: inoculating the ovule obtained in the step 3 into an MS induction culture medium under the aseptic condition, and culturing at the temperature of 20 ℃ under the illumination for 16h every day with the illumination intensity of about 2000 lx. Inoculated on MS culture media with different hormone (6-BA, NAA purchased from Beijing Bayer Diels Biotech Co., Ltd.) ratios, respectively as culture media A: 1.5 mg/L6-BA +0.5mg/L NAA, pH5.6; and (3) a culture medium B: 2.0 mg/L6-BA +0.2mg/L NAA, pH5.6; and (3) a culture medium C: 2.0 mg/L6-BA +0.5mg/L NAA, pH5.8; and (3) a culture medium D: 2.0 mg/L6-BA +1.0mg/L NAA, pH5.6; and (3) a culture medium E: 1.0 mg/L6-BA +0.5mg/L NAA, pH 5.8. About 9 ovules were inoculated per plate as shown in figure 2. The results in Table 2 show that the germination rates of the 5 media are the lowest E-type media and the highest B-type media, the germination rates are 6.25% and the recovery rate is 46.88%. In general, the B-type culture medium (MS +2.0 mg/L6-BA +0.2mg/L NAA, pH5.8) is more suitable for rescuing the embryos of the hibiscus and the broad-leaved Maohua. This example was inoculated with a total of 269 ovules, resulting in 17 embryo-rescued seedlings.

TABLE 2 Effect of different hormone types of culture Medium on in vitro culture of immature embryos

Note: culture medium A: 1.5 mg/L6-BA +0.5mg/L NAA, pH5.6; and (3) a culture medium B: 2.0 mg/L6-BA +0.2mg/L NAA, pH5.6; and (3) a culture medium C: 2.0 mg/L6-BA +0.5mg/L NAA, pH5.8; and (3) a culture medium D: 2.0 mg/L6-BA +1.0mg/L NAA, pH5.6; and (3) a culture medium E: 1.0 mg/L6-BA +0.5mg/L NAA, pH 5.8.

5. Rooting culture and obtaining of hybrid F1 generation plants: transferring the germinated immature embryos obtained after 4 weeks into a rooting culture medium, wherein the culture temperature is 20 ℃, the illumination is 16 hours every day, and the illumination intensity is about 2000lx, and 1/2MS +0.1mg/LNAA are added; observing the condition that the young embryo has cluster buds in a rooting culture medium until the young embryo develops into a hybrid seedling; when the root of the young seedling grows to more than 1em, the obtained hybrid F1 plant is subjected to bottle opening and seedling hardening for 2-3 days, and the young seedling is adapted to the autotrophic growth and the bacteria-bearing environment of the field by gradually reducing the relative humidity and enhancing the illumination. Taking out the hybrid plant, cleaning the culture medium attached to the surface of the hybrid plant, and transplanting the hybrid plant into a plug tray with the matrix of coarse-grained vermiculite and perlite being 1: 1. After one month, the pot is filled with the fresh water and is managed conventionally. Finally obtaining hybrid progeny of 11 hibiscus and broad-leaved Maohua.

Example 3 identification of intergeneric hybrid authenticity of Hibiscus and Hibiscus latifolia Using morphological Observation and cytology

Morphological observation and cytology are used for identifying the authenticity of the intergeneric hybrid of the hibiscus and the broad-leaved Maohua. The method comprises the following steps:

1. morphological identification of hybrid authenticity

1) And (5) field planting the male parent and the female parent of the cross in the open in 5 months, and performing conventional maintenance management. Morphological observations and statistics were performed on hybrids and parents including: and (5) measuring the characters of the plant height, the crown width and the leaf part.

2) Randomly selecting three fully-unfolded leaves for each plant, measuring the leaf length and the leaf width, and respectively measuring the length of a leaf stalk; wherein the plant height, crown width, leaf length, leaf width and leaf stalk length between the parent and the female parent are determined as true hybrids.

The results are shown in Table 3, FIG. 2 and FIG. 3, and the results show that 8 strains are preliminarily identified as true hybrids and the hybrid form is similar to that of hibiscus as false hybrids in embryo rescue offspring obtained by the hybridization combination of hibiscus and broad-leaved Mao chrysanthemum. The leaf shape of the progeny of the true hybrid is closer to the broad egg shape of the broad-leaved chrysanthemum, which is probably related to the male parent of the broad-leaved chrysanthemum which is hexaploid and provides more chromosomes. The hybrid has thick and hard leafiness, blunt sharp edge and sparse hair, and is different from two parents; the crown width and the plant height of the progeny plants are between the parent and the maternal plant.

TABLE 3 plant type and leaf morphology characteristics of Hibiscus mutabilis, Hibiscus latifolius and hybrid progeny

Note: f: flos Hibisci Mutabilis; KY: flos Spiraeae Fortunei.

2. Chromosome identification of hybrid progeny

1) Cutting, culturing and rooting the progeny of hibiscus and broad-leaved Maohua, wherein the rooting substrate is perlite and coarse-grained vermiculite is 1: 1. Cutting fresh young root (preferably 8-9 o' clock under outdoor condition) to 1cm long, cleaning, placing into centrifuge tube containing distilled water, and treating for 24 hr under ice water coexistence condition.

2) Transferring into Carnot fixing solution (glacial acetic acid: ethanol ratio is 1: 3, volume ratio), and fixing at 4 deg.C for 24 h. Washing with 95% ethanol for 2 times, transferring into 70% ethanol, and storing at 4 deg.C. Before dissociation staining, the cells were washed with sterile water for 3 times, transferred into 1mol/L HCl solution, and dissociated for 10min in a water bath at 60 ℃. Washing with distilled water for 3 times, and rinsing for 5 min. The white part of the root tip was cut out from the slide glass, and a modified phenol magenta staining solution (Lixin, 2013) was dropped thereon and stained for 10 min. And covering a cover glass, covering the cover glass with a piece of filter paper, fixing the cover glass with the left hand, holding the large head of the dissecting needle with the right hand, and slightly and uniformly exerting force on the position where the root tip is located to knock out the cells at the root tip.

3) When a cell with better dispersion is observed and found by an optical microscope, the oil dropping is changed into a 100-fold oil mirror. For each material, 3 root tips were observed, with at least 10 cells per root tip.

4) The cytological identification result of hybrid progeny is tetraploid, the number of chromosomes is about 36 basically, and true hybrids are obtained when the number of chromosomes is greater than 18 of female parents.

The results are shown in FIG. 4, wherein 8 hybrid progeny are identified as tetraploids, the number of chromosomes is basically 36, the number of chromosomes is larger than 18 of the female parent, the hybrid progeny conform to the law of genetics, and the hybrid progeny are true hybrid progeny consistent with the morphological identification results.

Phenotypic character observation and cytological identification result determine that 8 filings of hibiscus and broad-leaved Mao Huaju are true hybrids.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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[6]Hisao Ohtsuka，Zentaro Inaba.Intergeneric hybridization of marguerite(Argyranthemum frutescens)with annual chrysanthemum(Glebionis carinatum)and crown daisy(G.coronaria)using ovule culture[J].Plant Biotechnology，2008，(25)，535-539。

Claims (5)

1. A method for creating a distant hybrid between hibiscus and broad-leaved Maohua, which is characterized in that hibiscus and chrysanthemum broad-leaved Maohua are used as parents to be hybridized, and a distant hybrid is obtained by utilizing a young embryo rescue technology;

performing distant hybridization by using diploid hibiscus as a female parent and hexaploid broad-leaf Maohua as a male parent;

the method comprises the following steps:

1) parent preparation: culturing the two parents to meet the florescence;

2) distant hybridization: when the male parent and the female parent bloom, castrating the female parent, taking fresh pollen of the male parent for pollination and bagging;

3) taking out ovules: taking the head-shaped inflorescence 18 days after the hybridization pollination, and taking out ovules;

4) in-vitro culture of immature embryos: inoculating the ovule into an induction culture medium for culture, and then transferring the ovule into a rooting culture medium for culture until seedlings emerge;

5) acquisition of hybrid generation F1: transplanting the seedlings into a matrix for hardening when the roots of the seedlings grow to be more than 1cm, and then putting the seedlings into a pot for conventional management;

6) screening true hybrids: screening true hybrid progeny from F1 generation plants according to phenotype observation of parent and hybrid F1 generation and cytological ploidy identification;

the induction culture medium is as follows: MS +2.0 mg/L6-BA +0.2mg/L NAA, pH5.8;

the rooting culture medium comprises: 1/2MS +0.1mg/L NAA, pH5.6;

the conditions for induction culture and rooting culture in the step 4) are as follows: the temperature is 20-25 ℃, the daily illumination is 14-16h, and the illumination intensity is 1600-; after 4-6 weeks of induction culture, the culture is changed into rooting culture;

in the step 5), the matrix is formed by mixing vermiculite and perlite according to the volume ratio of 1: 1;

the seedling exercising conditions are as follows: culturing at 27-30 deg.C and relative humidity of 60-70% for 30-45 days.

2. The method of claim 1, wherein step 1) comprises: the female parent hibiscus flower is cured by short sunshine for 8-10 hours from month 8, so that the blooming period is reached in the middle 10 th of month.

3. The method of claim 1, wherein step 3) comprises: taking the head-shaped inflorescence after the cross pollination, sterilizing the head-shaped inflorescence, removing the ovary wall, and taking out the ovule.

4. The method of claim 3, wherein the method of sterilizing comprises: sterilizing the surface of the capitula by 70-75% alcohol for 30-45 s, washing with sterile water, and adding 8-13% H₂O₂Sterilizing the aqueous solution for 10-15 minutes, and then washing with sterile water.

5. The method according to any one of claims 1 to 4, wherein the phenotype in step 6) is selected from at least one of plant height, canopy width, leaf length, leaf width, and petiole length.

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