CN113557954A - Method for obtaining hybrid between lagerstroemia and lagerstroemia - Google Patents

Abstract

The invention relates to a method for obtaining a hybrid between lagerstroemia and lagerstroemia, which comprises the following specific steps: 1) carrying out intergeneric artificial hybridization by using the crape myrtle leaves of the crape myrtle as a female parent and the crape myrtle leaves of the crape myrtle as a male parent; 2) spraying hormone on the stigma of the female parent after hybridization to preserve the fruit, taking immature fruit for embryo rescue, and obtaining a filial generation material; 3) and carrying out morphological observation on the hybrid progeny, and carrying out hybrid identification by adopting an SSR molecular marker to obtain the hybrid between the lagerstroemia and the lagerstroemia. The method provided by the invention realizes gene exchange between the lagerstroemia and the lagerstroemia, obtains the new species of intergeneric hybridization, and makes it possible to cultivate the new lagerstroemia species by intergeneric hybridization.

Description

Method for obtaining hybrid between lagerstroemia and lagerstroemia

Technical Field

The invention relates to the field of plant breeding, in particular to a method for obtaining a hybrid between lagerstroemia and lagerstroemia.

Background

Lagerstroemia plants are deciduous leaves or evergreen shrubs or trees of Lythraceae (Lythraceae), and are important summer flowering plants. There are about 55 species of plants in the genus of Lagerstroemia around the world, and there are many distributions in the southern Asia, the eastern Asia, the southeast Asia, and the northern Australia. At present, related work of lagerstroemia plant breeding, including germplasm resources, propagation, cultivation, pest control, molecular biology research and the like, is being carried out at home and abroad, resistance breeding, scented flower breeding, dwarf breeding, large flower breeding and new flower breeding become hotspots of lagerstroemia indica breeding research, and lagerstroemia indica varieties, dwarf lagerstroemia indica varieties, large flower lagerstroemia indica varieties and red flower lagerstroemia indica varieties which are resistant to powdery mildew are obtained by means of cross breeding.

The flower color is one of important ornamental characters of the lagerstroemia indica, and the lagerstroemia indica flower color has four color systems of purple, red, powder and white in nature, and does not have yellow varieties. Since the lagerstroemia plant has no yellow flower variety, the lagerstroemia indica variety which blooms yellow flowers can not be cultivated by using the traditional interspecific hybridization.

Yellow-flowering crape myrtle (Heimia salicifolia) in the crape myrtle is a related plant of the crape myrtle, yellow flowers are bloomed, and the yellow-flowering crape myrtle variety is hopeful to be obtained by performing intergeneric hybridization on the crape myrtle and the crape myrtle. The distant hybridization between two genera also has important significance for discussing the distant hybridization affinity and the gene exchange mechanism of the lagerstroemia indica and the related genera.

Disclosure of Invention

The invention provides a method for obtaining a hybrid between lagerstroemia and lagerstroemia aiming at the current situation that lagerstroemia plants lack yellow flowers so as to improve the flower color characters of the lagerstroemia plants.

The distant hybridization can effectively transfer and recombine two or more excellent characters which are evolutionarily accumulated, and the excellent characters which are difficult to obtain by the distant hybridization are obtained, thereby realizing the targeted improvement of the plants. Due to the existence of reproductive isolation, the distant hybridization can not form viable offspring generally under natural conditions, and with the increase of the genetic distance between parent races, the problems of incapability of pollen germination, incapability of pollen tube elongation, abortion of hybrid embryos and the like can occur, so that the difficulty in obtaining hybrid offspring is increased.

The invention provides a method for obtaining a hybrid between lagerstroemia and lagerstroemia after a plurality of attempts in different directions, which is characterized in that after the artificial hybridization of parents, 2,4-D is sprayed on a maternal stigma; after artificial pollination, when the hybrid embryo is transferred from heart-shaped embryo to torpedo embryo, embryo rescue is carried out to obtain hybrid progeny, and the hybrid progeny is identified by morphology and molecular marker to obtain the hybrid between lagerstroemia and lagerstroemia.

In the method provided by the invention, 20mg/L2,4-D is sprayed on the female parent flower column 2h and 24h after artificial hybridization; after artificial pollination for 20-25 days, immature fruits are taken for embryo rescue.

For the purpose of obtaining a hybrid between lagerstroemia and lagerstroemia, lagerstroemia indica is hybridized with lagerstroemia indica as a female parent and lagerstroemia indica as a male parent. Because the two parents have very far relationship and serious obstacle before fertilization, fruits using the crape myrtle as the female parent do not drop after hybridization, but the hybridized fruits are completely browned and withered after 10-15 days along with the extension of development time. The invention discovers that after the hormone 2,4-D is sprayed, about 10% -12% of fruits can still keep green (survive) after 15 days, and the development process of distant hybrid fruits is greatly prolonged.

The invention discovers that the possibility of successful embryo rescue is increased when hybrid embryos are transferred from heart-shaped embryos to torpedo embryos. Therefore, the immature fruit is taken for embryo rescue 20-25 days after artificial pollination. As the 2,4-D concentration is too high, the growth and development of plants are influenced, the invention further researches to find that the concentration of the 2,4-D which is most suitable for promoting the female parent to produce hybrid fruits and protecting the fruit development is 20 mg/L.

In the method provided by the invention, the embryo rescue culture medium contains 30g/L of sucrose, 7g/L of agar, 0.5mg/L of 6-BA and 0.5-1.5mg/L of GA₃And 0-0.5mg/L NAA, MS culture medium of pH5.8-6.0;

preferably, the embryo rescue medium contains 30g/L sucrose, 7g/L agar, 0.5mg/L6-BA and 1mg/L GA₃And 0.5mg/L NAA, MS culture medium of pH5.8-6.0。

The embryo rescue culture medium provided by the invention can enable filial generations to smoothly take roots and bud without changing the culture medium.

In the methods provided herein, the embryo rescue comprises: placing the fruit under running water to wash for more than 12h, washing the surface of the fruit with alcohol for 25-30s, soaking in sodium hypochlorite solution for 8-10min, washing the fruit with sterile water for 3-4 times, dissecting ovary, taking out seed, and inoculating the seed on embryo rescue culture medium.

In the method provided by the invention, the culture conditions for embryo rescue are as follows: the temperature is 24-26 ℃, the illumination intensity is 1500-.

In the method provided by the invention, the parent takes the crape myrtle in the crape myrtle genus as a female parent and the crape myrtle in the crape myrtle genus as a male parent.

Specifically, as a specific embodiment mode of the present invention, the method provided by the present invention includes the following steps:

(1) emasculating willow leaf crape myrtle and bagging at 5-7 in the morning to collect fresh pollen of the crape myrtle;

(2) at 9-11 am, artificially pollinating collected crape myrtle pollen to stigma of yellow crape myrtle, bagging for 1 time every day, repeating for 2 times, bagging with sulfuric acid paper bag after pollination, and changing into mesh bag after flowering;

(3) spraying 20mg/L2,4-D to the crape myrtle flower column 2h and 24h after artificial pollination;

(4) carrying out embryo rescue on fruits 20-25d after artificial pollination, placing the fruits under running water for washing for more than 12h, washing the surfaces of the fruits for 25-30s by using 75% alcohol, soaking the fruits for 8-10min by using 2% sodium hypochlorite solution, washing the fruits for 3-4 times by using sterile water, and inoculating seeds on a culture medium to obtain hybrid progeny; the culture medium contains 30g/L sucrose, 7g/L agar, 0.5mg/L6-BA, 0.5-1.5mg/L GA₃And 0-0.5mg/L NAA, MS culture medium with pH 5.8-6.0;

preferably, the culture medium contains 30g/L sucrose, 7g/L agar, 0.5mg/L6-BA, 1mg/L GA₃And 0.5mg/L NAA, MS medium pH 5.8;

(5) and performing morphological observation on the hybrid according to the parent morphology, and performing hybrid identification by adopting a molecular marker to obtain the hybrid between the lagerstroemia and the lagerstroemia.

In the method provided by the invention, the primer pair used for identifying the molecular marker is any one of the following primers:

YYJ-281

an upstream primer: 5'-GTTAGAGCCCTTCCGCACTT-3' (SEQ ID No.1),

a downstream primer: 5'-GAAGATCCAGCACAGCCCAT-3' (SEQ ID No. 2);

YYJ-656

an upstream primer: 5'-AGGAGGAAGGAGACGAGCTT-3' (SEQ ID No.3),

a downstream primer: 5'-CTCCTTCCCCTGGTAGTGGA-3' (SEQ ID No. 4);

YYJ-951

an upstream primer: 5'-GTTCCTGGAGACTTGGGCTC-3' (SEQ ID No.5),

a downstream primer: 5'-TCCGAACTTCCCGACGAATG-3' (SEQ ID No. 6);

YYJ-998

an upstream primer: 5'-AATCCGTGGCCAAGAGGAAG-3' (SEQ ID No.7),

a downstream primer: 5'-GGGAACTCCAATGTGGGCTT-3' (SEQ ID No. 8).

According to the understanding of the technical personnel in the field, the invention also requests to protect the application of the method in breeding a new variety of the lagerstroemia indica and researching the genetic relationship of the plants.

The invention has the beneficial effects that:

(1) according to the invention, 2,4-D is sprayed on the stigma of the female parent, so that the hybrid fruit can keep green (survive) after 15 days, and the development process of distant hybrid fruit is greatly prolonged.

(2) The invention discovers that the possibility of successful embryo rescue is increased when the hybrid embryo is transferred from the heart-shaped embryo to the torpedo embryo; therefore, the immature fruit is taken for embryo rescue 20-25 days after artificial pollination.

(3) The invention promotes the filial generation to take root and sprout by a specific embryo rescue culture medium under the condition of not changing the culture medium.

(4) The invention utilizes the lagerstroemia and the lagerstroemia indica to carry out distant hybridization, realizes the gene exchange between the lagerstroemia and the lagerstroemia, and obtains a new species of intergeneric hybridization.

(5) The method provided by the invention can enrich the genetic information of the lagerstroemia plants, enable the breeding of new lagerstroemia species by utilizing intergeneric hybridization to become possible, and can carry out the genetic relationship research of relatives.

Drawings

FIG. 1 is a photograph showing a comparison of leaves of Banaba (left), hybrid (middle) and Banaba (right) according to example 1 of the present invention.

FIG. 2 is a graph showing the result of the segregation test of the primer YYYJ-281 in the parent and the offspring in example 1 of the present invention.

FIG. 3 is a graph showing the results of the segregation test of the primer YYYJ-656 in the parent and the progeny in example 1 of the present invention.

FIG. 4 is a graph showing the results of the segregation test of the primer YYYJ-951 in the parent and the offspring in example 1 of the present invention.

FIG. 5 is a graph showing the results of the isolation and detection of the primer YYYJ-998 in the parent and the progeny in example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

Example 1

This example provides a method for obtaining filial generation of lagerstroemia and lagerstroemia.

(1) Material selection

In this example, lagerstroemia indica 'Sarah Favorite' of lagerstroemia was selected as the male parent; selecting Lagerstroemia salicifolia of the Lagerstroemia as a female parent.

(2) Hybridization method and hybrid acquisition

In summer of 2019, 361 pieces of yellow crape myrtle leaves are subjected to artificial emasculation, bagging and isolation at 5-7 am in a small-soup mountain nursery garden of the national floriculture center, fresh crape myrtle pollen of Sarah Favorite is collected and pollinated at 9-11 am, and the pollination is carried out for 2 times continuously once a day.

Picking off the sulfuric acid paper bags 2h and 24h after artificial pollination, spraying 20mg/L2,4-D on the stigma, and changing the sulfuric acid paper bags used for bagging into mesh bags after the stigma wilts.

The hybrid fruits using the yellow osmunda salicifolia as the female parent are hybridized with flowers pollinated by 361 individuals 20-25 days later, 15 hybrid fruits are obtained in total, and the fruit setting rate is 4.16%.

Washing hybrid fruit with flowing water for more than 12h, washing fruit surface with 75% alcohol for 25-30s, soaking in 2% sodium hypochlorite solution for 8-10min, and washing with sterile water for 3-4 times.

Preparing culture medium containing sucrose 30g/L, agar 7g/L, 6-BA 0.5mg/L, and GA 0.5-1.5mg/L₃And 0-0.5mg/L NAA, MS culture medium with pH of 5.8-6.0. Splitting the fruit surface, carrying out artificial pollination for 2 times, inoculating 720 hybrid seeds obtained in total on a culture medium for embryo rescue, culturing at 24-26 ℃, with the illumination of 1500-.

After emergence, 11 seedlings have weak growth, withering and yellowing and other premature senility phenomena, and finally 1 intergeneric filial generation plant is obtained, and the survival rate of the hybridized seedlings is 9.09%.

(3) Identification of crape myrtle and crape myrtle hybrid

The phenotype of the parents compared with the progeny of the cross is shown in FIG. 1. From the phenotype, the leaf size of the filial generation is between the parent and the female parent, the phenotype is closer to that of the female parent crape myrtle, the leaf size of the female parent crape myrtle is 3.48cm multiplied by 0.61cm, the leaf size of the male parent crape myrtle 'Sarah Favorite' is 6.51cm multiplied by 4.42cm, and the leaf size of the filial generation is 5.83cm multiplied by 1.15 cm.

Identifying and designing 4 pairs of primer pairs by SSR molecular markers, extracting DNA of parent and child young leaves for amplification, and comparing parent and child specific bands:

YYJ-281

an upstream primer: 5'-GTTAGAGCCCTTCCGCACTT-3'

A downstream primer: 5'-GAAGATCCAGCACAGCCCAT-3'

YYJ-656

An upstream primer: 5'-AGGAGGAAGGAGACGAGCTT-3'

A downstream primer: 5'-CTCCTTCCCCTGGTAGTGGA-3'

YYJ-951

An upstream primer: 5'-GTTCCTGGAGACTTGGGCTC-3'

A downstream primer: 5'-TCCGAACTTCCCGACGAATG-3'

YYJ-998

An upstream primer: 5'-AATCCGTGGCCAAGAGGAAG-3'

A downstream primer: 5'-GGGAACTCCAATGTGGGCTT-3'

As a result, it was found that the SSR molecular marker bands of 1 filial generation obtained exhibited markers specific to the parent and the female parent, and bands not possessed by the parent and the female parent (see FIGS. 2 to 5).

Therefore, the method provided by the invention can change the cross incompatibility of intergeneric species, obtain intergeneric hybrid varieties and provide background information for obtaining other intergeneric hybrid offspring.

Comparative example 1

The fruits of the yellow osmund willow which normally develop can keep green all the time and gradually expand, but the fruits which are hybridized with the crape myrtle cannot fall off, but the fruits can be gradually browned and withered on branches along with the extension of the development time.

2,4-D is not sprayed, fruits taking the crape myrtle as the female parent do not fall off after hybridization, yellowing begins to occur from 3 rd to 5 th after hybridization, the fruits cannot expand along with time, the hybrid fruits are completely browned and dried after 10 days to 15 days along with the time of the fruits on branches, and the drying rate is 100%.

After the yellow crape myrtle leaves and the crape myrtle are hybridized, 20mg/L2,4-D is sprayed, fruits using the yellow crape myrtle leaves as female parents do not fall off and can be expanded after hybridization, 10% -12% of fruits can still keep green (survive) after 15D, the development process of distant hybridization fruits is greatly prolonged, the hybridization fruits can develop to the torpedo embryo period, and the possibility of successful embryo rescue is increased.

Comparative example 2 different embryo rescue Medium formulations

The comparative example provides embryo rescue culture media with different formulas, and the emergence rate of the obtained hybrid.

In this comparative example, all the media were supplemented with sucrose (30 g/L), agar (7 g/L) and 6-BA (0.5 mg/L), and the pH was adjusted to 5.8-6.0, except for GA added₃As opposed to NAA concentration. The culture conditions are 24-26 ℃, the illumination intensity is 1500-.

(1) At 1mg/L GA₃And 0.5mg/L NAA MS culture medium, inoculating 144 seeds, and germinating 6 plants with the emergence rate of 4.17%;

(2) at 1mg/L GA₃And 0.5mg/L NAA in 1/2MS culture medium, inoculating 144 seeds, and germinating 3 plants with emergence rate of 2.08%;

(3) at 1mg/LGA₃And 144 seeds are inoculated on the MS culture medium without NAA, 1 plant emerges, and the emergence rate is 0.69%;

(4) at 0.5mg/L GA₃And 144 seeds are inoculated on the MS culture medium without NAA, 1 plant emerges, and the emergence rate is 0.69%;

(5) at 1.5mg/L GA₃And 144 seeds were inoculated on the MS medium without NAA, 0 seedlings emerged, and the rate of emergence was 0.00%.

Comparative example 3

This comparative example provides results of intergeneric hybridization of different parents.

(1) The fruits cannot be obtained by the hybridization combination of taking the lagerstroemia indica and lagerstroemia indica varieties 'Dallas Red', 'Catawba', 'Centenial Spirit', 'Near East' or 'Pocomake' as female parents and taking the yellow osmunda japonica or yellow osmunda salicina as male parents;

(2) by using the lagerstroemia indica variety 'Sarah Favorit' as a female parent and using the yellow osmunda japonica or the yellow osmunda salicifolia as a male parent, a small amount of hybrid fruits can be obtained, but seedlings cannot be obtained by embryo rescue;

(3) the fruits cannot be obtained by hybridization combination of using the crape myrtle as a female parent and using the crape myrtle varieties 'Dallas Red', 'Catawba', 'Centenial Spirit' or 'Near East' as a male parent;

(4) by using the crape myrtle as a female parent and using the crape myrtle in the Jiujima island or the crape myrtle variety 'Sarah Favorit' as a male parent, a small amount of hybrid fruits can be obtained, but seedlings cannot be obtained by embryo rescue.

Comparative example 4 different embryo rescue periods

The comparative example provides embryo rescue operation performed at different development stages of embryos to obtain the emergence rate result of hybrid seeds.

(1) Selecting fruits between 10 and 20 days after pollination for embryo rescue, wherein the rate of emergence is 0.00 percent;

(2) selecting fruits between 20 and 25 days after pollination for embryo rescue, wherein the rate of emergence is 1.53 percent.

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.

Sequence listing

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Claims (10)

1. A method for obtaining a hybrid between lagerstroemia and xanthoceras is characterized in that after the parents are artificially hybridized, 2,4-D is sprayed on the stigma of a female parent; after artificial pollination, when the hybrid embryo is transferred from heart-shaped embryo to torpedo embryo, embryo rescue is carried out to obtain hybrid progeny, and the hybrid progeny is identified by morphology and molecular marker to obtain the hybrid between lagerstroemia and lagerstroemia.

2. The method as claimed in claim 1, wherein 20mg/L2,4-D is sprayed on the female parent style 2h and 24h after artificial hybridization; after artificial pollination for 20-25 days, immature fruits are taken for embryo rescue.

3. The method of claim 2, wherein the embryo rescue medium comprises sucrose 30g/L, agar 7g/L, 6-BA 0.5mg/L, and GA 0.5-1.5mg/L₃And 0-0.5mg/L NAA, MS culture medium with pH 5.8-6.0;

preferably, the culture medium contains 30g/L sucrose, 7g/L agar, 0.5mg/L6-BA, 1mg/L GA₃And 0.5mg/L NAA, MS medium of pH 5.8-6.0.

4. The method of claim 3, wherein the embryo rescue treatment comprises: washing fruit under running water for more than 12h, washing fruit surface with 75% alcohol for 25-30s, soaking in 2% sodium hypochlorite solution for 8-10min, washing fruit with sterile water for 3-4 times, dissecting ovary, taking out seed, and inoculating the seed onto embryo rescue culture medium.

5. The method of claim 4, wherein the embryo rescue is performed under culture conditions selected from the group consisting of: the temperature is 24-26 ℃, the illumination intensity is 1500-.

6. The method of claim 5, wherein said parents are chosen from the species Lagerstroemia indica of the genus Lagerstroemia as the female parent and Lagerstroemia indica of the genus Lagerstroemia as the male parent.

7. The method of claim 6, comprising the steps of:

(1) emasculating willow leaf crape myrtle and bagging at 5-7 in the morning to collect fresh pollen of the crape myrtle;

(2) at 9-11 am, artificially pollinating collected crape myrtle pollen to stigma of yellow crape myrtle, bagging for 1 time every day, repeating for 2 times, bagging with sulfuric acid paper bag after pollination, and changing into mesh bag after flowering;

(3) spraying 20mg/L2,4-D to the crape myrtle flower column 2h and 24h after artificial pollination;

(4) carrying out embryo rescue on fruits 20-25d after artificial pollination, placing the fruits under running water for washing for more than 12h, washing the surfaces of the fruits for 25-30s by using 75% alcohol, soaking the fruits for 8-10min by using 2% sodium hypochlorite solution, washing the fruits for 3-4 times by using sterile water, and inoculating seeds on a culture medium to obtain hybrid progeny; the culture medium contains 30g/L sucrose, 7g/L agar, 0.5mg/L6-BA, 1mg/L GA₃And 0.5mg/L NAA, MS culture medium of pH 5.8-6.0;

(5) and performing morphological observation on the hybrid according to the parent morphology, and performing hybrid identification by adopting a molecular marker to obtain the hybrid between the lagerstroemia and the lagerstroemia.

8. The method of claim 7, wherein the primer pair used for identifying the molecular marker in step (5) is any one of the following:

YYJ-281

an upstream primer: 5'-GTTAGAGCCCTTCCGCACTT-3' the flow of the air in the air conditioner,

a downstream primer: 5'-GAAGATCCAGCACAGCCCAT-3', respectively;

YYJ-656

an upstream primer: 5'-AGGAGGAAGGAGACGAGCTT-3' the flow of the air in the air conditioner,

a downstream primer: 5'-CTCCTTCCCCTGGTAGTGGA-3', respectively;

YYJ-951

an upstream primer: 5'-GTTCCTGGAGACTTGGGCTC-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TCCGAACTTCCCGACGAATG-3', respectively;

YYJ-998

an upstream primer: 5'-AATCCGTGGCCAAGAGGAAG-3' the flow of the air in the air conditioner,

a downstream primer: 5'-GGGAACTCCAATGTGGGCTT-3' are provided.

9. Use of the method according to any one of claims 1 to 8 for breeding a new variety of lagerstroemia indica.

10. Use of the method of any one of claims 1 to 8 for the study of plant relationships.

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