Method for distant sexual hybridization of plants

Abstract

The invention relates to a method for distant sexual hybridization of plants, which comprises the following steps: selecting a good variety of a sexual propagation plant type expected to be obtained as a hybridization female parent, castrating before male flowers are mature, treating female flowers before insemination and carrying out reproduction isolation, selecting sperms of any interspecific and farther organisms with characters of the variety expected to be obtained, and carrying out distant sexual hybridization on the selected sperms. And adding a distant hybridization agent, uniformly stirring to form a sperm, inseminating the female parent, taking back a fertilized embryo, carrying out embryo rescue, culturing into a seedling, carrying out chromosome doubling on the obtained seedling, and transplanting into soil. In the mature period, harvesting true hybrids according to individual plants, and cultivating F2 and later generations by using a pedigree method until homozygosis. The bottleneck of distant sexual hybridization is broken through, high-yield and high-quality varieties can be cultivated, and the method has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant improvement. Specifically, it relates to a method for distant sexual hybridization of plants. Background Technology

[0002] Scientific research has proven that every revolutionary evolution in plants is triggered by allopolyploids formed through distant hybridization. Therefore, only distant hybridization can effectively promote plant evolution and cultivate breakthrough plant varieties. However, due to reproductive isolation between species, interspecific and distant hybridization is difficult to succeed.

[0003] However, modern scientific research shows that although different species may look very different, they share the same gene sequences (Mao Li, Different plant species share the same gene sequences, Science and Technology Daily, April 11, 2012, p. 1). Therefore, as long as the parent chromosomes meet, regardless of their kinship, they can pair or partially pair. Even if they cannot pair, chromosomes can be doubled. Thus, the inability of parent chromosomes to coexist is the fundamental reason for hybridization infertility between distantly related species. The reason why the parent chromosomes of distant hybrids cannot coexist is due to the presence of recognition proteins, namely glycoproteins, on the stigma and pollen / sperm surface. These proteins act like a lock, preventing distant pollen / sperm from entering. Therefore, by degrading the glycoproteins on the stigma and sperm surface, the genetic material, including the chromosomes of distant parents, can meet, allowing the chromosomes / genetic material of distantly related parents to pair / partially pair / combine, thus obtaining distant hybrid seeds. The enzymatic hydrolysate from the glycoprotein complete glycosylation kit provided by Laiziyou Biotechnology Co., Ltd. can degrade the recognition protein—glycoprotein—making distant hybridization possible. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art, adopt effective measures to break through the bottleneck of distant sexual hybridization of plants, realize distant sexual hybridization between any organisms with plants as the maternal parent, and breed the desired plant varieties.

[0005] Therefore, the present invention provides a method for sexual hybridization of plants, characterized in that the method includes the following steps:

[0006] (1) Select superior varieties of the desired sexually reproducing plant type as the hybridization female parent, remove the male flowers before they mature, treat the female flowers before fertilization and remove reproductive isolation;

[0007] (2) Select animal sperm or plant flowers from interspecific or more distant organisms with the traits of the desired variety, add a hybridization agent and stir to obtain a fertilization solution, and use the fertilization solution to fertilize the female parent; or apply the hybridization agent and the sperm or pollen to the stigma of the female parent for fertilization, preferably sealed in a parchment paper bag, and unpacked and operated again the next day when pollination is repeated, and so on for 2-11 days;

[0008] (3) Use insemination solution to fertilize the female parent, and harvest the fertilized eggs after successful hybridization;

[0009] (4) After fertilization, fertilized eggs are harvested at 7-70 days, embryos are rescued and cultured into seedlings, and after chromosome doubling, they are transplanted into the soil.

[0010] Furthermore, it also includes:

[0011] (5) Identify and remove false hybrids.

[0012] The plant distant sexual hybridization described in this invention refers to sexual hybridization between kingdoms, phyla, classes, orders, families, genera, and species, using plants as the female parent.

[0013] In a specific embodiment, the pre-fertilization treatment of the female flower in step (1) specifically involves removing the outer covering layer of the ovary to expose it, such as removing the sepals, petals, ovary wall, and integument. The removal of reproductive isolation before fertilization specifically involves dripping 1 to several drops of enzymatic hydrolysate into the ovary to thoroughly wet it; the enzymatic hydrolysate is a complete glycosyl hydrolysate obtained by animal glycoprotein hydrolysis.

[0014] The distant hybridization agent is formulated as follows: 100-300 mg / L gibberellin, 10-100 mg / L auxin, 1500-5000 ml / L enzymatic hydrolysate, and 1500-5000 mg / L compound amino acids. Preferably, the formulation is 150-250 mg / L gibberellin, 40-70 mg / L auxin, 2500-4000 μl / L enzymatic hydrolysate, and 2500-4000 mg / L compound amino acids. Preferably, the enzymatic hydrolysate is a complete glycosylation hydrolysate obtained by animal glycoprotein enzymatic hydrolysis, which can be purchased commercially. The compound amino acids are conventionally defined and can be prepared using the method described in CN200610031424.X, or can be purchased commercially.

[0015] In a specific implementation, the method of inseminating the mother with the insemination solution in step (3) is as follows: apply the insemination solution to the ovary and seal it; if necessary, repeat the insemination the next day, once a day, until successful fertilization. Specifically, a transparent bag, such as a parchment bag, can be used to tightly seal and isolate the ovary. Insemination can be repeated for 2-12 days, once a day, until the stigma is successfully fertilized.

[0016] In a preferred embodiment, the embryo rescue and seedling cultivation of the fertilized egg in step (4) specifically involves rescuing the embryo using MS medium and transplanting it into the soil.

[0017] Preferably, the chromosome doubling method involves using colchicine to double the chromosomes before transplanting them into the soil. Preferably, the true hybrids are further harvested from individual plants at maturity, and the F2 generation and subsequent generations are cultured using the pedigree method until they are homozygous.

[0018] In a more specific embodiment, the method for identifying false hybrids in step (4) is selected from one or more of the following three:

[0019] 1) Remove false hybrids that are similar to the maternal parent by morphological identification; 2) Remove individuals that are similar to the maternal parent by component identification; 3) Remove individuals that are similar to the maternal parent by genetic identification.

[0020] Further steps include the following:

[0021] (5) Purification and rejuvenation are aided by detecting metabolites of the variety using broad-targeted metabolomics technology. The method for propagating the variety is as follows: based on preliminary field selection and indoor re-selection of individuals with typical traits of the variety, individuals with unique metabolites that are absent in the maternal parent but present in the paternal parent are selected using broad-targeted metabolomics technology for propagation of the variety.

[0022] This invention breaks through the bottleneck of distant sexual hybridization in plants, enabling distant sexual hybridization between any organisms with plants as the female parent, cultivating groundbreaking high-yield and high-quality varieties, and has broad application prospects. Attached Figure Description

[0023] Figure 1 The current generation of ears of dairy cows and corn through sexual hybridization.

[0024] Figure 2 Dairy cow-maize cross (middle) and maternal parent 1314S (left, right).

[0025] Figure 3 Transcript read coverage. The X-axis represents the coverage of the transcript by reads, the Y-axis on the left represents the proportion of transcripts, and the Y-axis on the right represents the density of transcripts.

[0026] Figure 4 The distribution of reads on transcripts. The X-axis represents the location of transcripts (with 200 sliding windows), and the Y-axis represents the number of reads (calculated per sliding window).

[0027] Figure 5Box plot of expression levels of dairy cow genes in dairy cow maize 2. The X-axis represents the sample name, and the Y-axis represents log10FPKM (FPKM is the number of reads that are aligned to each gene in units of 1,000 bases per million sequences, the same below). The box plot of each region corresponds to five statistics (from top to bottom: maximum, upper quartile, median, lower quartile, and minimum).

[0028] Figure 6 Expression density plot of dairy cow genes in dairy cow maize 2. The X-axis represents log10FPKM; the Y-axis represents gene density, i.e., the ratio of the number of genes at this expression level to the total number of expressed genes.

[0029] Figure 7 Corn-fed papaya 1 (left) and the mother plant, local Zhanjiang papaya (right).

[0030] Figure 8 Transcript read coverage. The X-axis represents the coverage of the transcript by reads, the Y-axis on the left represents the proportion of transcripts, and the Y-axis on the right represents the density of transcripts.

[0031] Figure 9 The distribution of reads on transcripts. The X-axis represents the location of transcripts (with 200 sliding windows), and the Y-axis represents the number of reads (calculated per sliding window).

[0032] Figure 10 Box plot of maize gene expression in maize-papaya 1. The X-axis represents the sample name, the Y-axis represents log10FPKM, and each region of the box plot corresponds to five statistics (from top to bottom: maximum, upper quartile, median, lower quartile, and minimum).

[0033] Figure 11 Maize gene expression density in maize-papaya 1. The X-axis represents log10FPKM; the Y-axis represents gene density, i.e., the ratio of the number of genes at this expression level to the total number of expressed genes.

[0034] Figure 12 The distribution of maize gene expression levels in maize-papaya 1. The X-axis represents the sample name, the Y-axis represents the number of genes, and the color intensity indicates different expression levels: genes with FPKM <= 1 have extremely low expression levels, genes with FPKM between 1 and 10 have relatively low expression levels, and genes with FPKM >= 10 have medium to high expression levels.

[0035] Figure 13 Transcript read coverage. The X-axis represents the coverage of the transcript by reads, the Y-axis on the left represents the proportion of transcripts, and the Y-axis on the right represents the density of transcripts.

[0036] Figure 14 The distribution of reads on transcripts. The X-axis represents the location of transcripts (with 200 sliding windows), and the Y-axis represents the number of reads (calculated per sliding window). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0038] Example 1: Breeding of sexual hybrids of dairy cow and corn—Dairy Cow Corn 1 and Dairy Cow Corn 2

[0039] This embodiment is carried out according to the following method.

[0040] The tested maternal parent was the temperature-sensitive male-sterile maize line 1314S (selected from the segregating generation of Demeya 1), and the tested paternal parent was a Holstein dairy cow, whose sperm was purchased from the Nenjiang sales point of Heilongjiang Borui Genetics Co., Ltd. During the flag-picking period, tassels were removed daily, including the leaves, until all tassels in the entire region had been picked.

[0041] Insemination was performed when the female tassels were exposed. First, add 200 mg / L pectinase, 500 mg / L cellulase, 150 mg / L gibberellin, 155 mg / L auxin, 2300 μl / L enzyme hydrolysate, and 2500 mg / L compound amino acids to a measuring cup according to the required volume of somatic cell hybridization reagent. Add distilled water to bring the volume to a final volume and stir well. Refrigerate for later use. (The compound amino acids were purchased from Shanghai Haling Biotechnology Co., Ltd., and the enzyme hydrolysate was a complete glycosyl digestion enzyme hydrolysate from animal glycoproteins. Specifically, the enzyme hydrolysate used in this example was purchased from the complete glycosyl digestion kit of Shanghai Haling Biotechnology Co., Ltd.)

[0042] Purchase four vials of frozen cow semen (approximately 40-48 million sperm) daily at 8:00 AM, and immediately add them to an equal volume of distant hybridization agent and mix well. Before insemination, cut off the bracts at the top of the female ear rachis with scissors to expose the filaments neatly. First, drip 3-6 drops of 3000 ml / L enzyme hydrolysate onto the filaments to thoroughly wet them. Then, use a brush to apply the mixture of distant hybridization agent and cow semen to each female ear after the top has been cut. Seal the ear in a clean, transparent plastic bag. The next day, when repeating insemination, unpack the plastic bag, cut off the extended filaments, apply the semen, and seal the bag. Repeat this insemination process seven times.

[0043] Ten ears were randomly selected from both the post-treatment and control groups after 10 days. From each ear, one row was randomly selected to count the total number of florets and the number of grains set per ear. The grain set rate was calculated using the following method:

[0044] Hybridization seed set rate (%) = Number of seeds per row / Total number of florets per row × 100

[0045] All remaining embryos were retrieved, rescued using MS medium, and cultured into seedlings. Chromosomes were doubled using 0.3% colchicine, and the embryos were isolated and transplanted to the seed selection nursery in groups of one. One row per ear was selected. At the tasseling stage, homozygous superior ear rows and individual plants were selected, bagged, and self-pollinated. Homozygous superior ear rows were harvested in combination, and superior individual plants were harvested separately. The selected superior rows and individual plants were analyzed for quality using a DA-7250 near-infrared spectroscopy system, and those with better quality were selected.

[0046] Hybrid F1 plants selected were mixed and hybridized into second-generation hybrids, which were then isolated and cultivated. During flowering, hybrids were removed, and at maturity, they were harvested together. Selected superior individual plants from F1 were isolated and cultivated in groups. During tasseling, superior rows and individual plants with homozygous ears were selected, bagged, and self-pollinated. Superior rows and individual plants were harvested separately. The selected superior rows and individual plants were analyzed for quality in the laboratory. Two lines with better quality were selected and numbered Dairy Cow Corn 1 and Dairy Cow Corn 2, respectively.

[0047] The results of this embodiment are as follows.

[0048] 1. Seed setting rate of sexually crossed dairy cows and corn

[0049] The seed setting rate of sexually hybridized dairy cows and corn ranged from 88.6% to 97.1%, with an average of 92.8%. Figure 1 (Table 1).

[0050] Table 1. Seed setting rate of sexual crosses between dairy cows and corn

[0051]

[0052]

[0053] 2. Morphology and protein content of maize plants in dairy cows

[0054] Typical photos of cow-corn hybrid plants can be found here. Figure 2 The dairy cow-corn hybrid not only grows better than the maternal parent 1314S, but also has restored fertility.

[0055] The protein content of dairy cow corn 1 was 4.2 and 3.0 percentage points higher than that of 1314S and Demeya 1, respectively, while the protein content of dairy cow corn 2 was 4.8 and 3.6 percentage points higher than that of Demeya 1 and Demeya 1, respectively (Table 2).

[0056] Table 2. Partial Quality Data of Corn for Dairy Cows

[0057]

[0058] 3. Results of Maize 1 gene testing in dairy cows

[0059] (I) The bovine maize 1 gene was tested by Shanghai Lingen Biotechnology Co., Ltd. The results are as follows: A total of 413 bovine genes were expressed in bovine maize 1, and their expression levels (FPKM values, the same below) ranged from 1.59E-170 to 19.1, with an average of 0.17 (Table 3).

[0060] Table 3. Overview of gene expression levels in dairy cows and maize 1.

[0061]

[0062] The criteria for identifying dairy cow genes were selected based on the fact that they were not expressed in the 1314S (maize) sample but were expressed in dairy cow maize 1 and fpkm>1. The number of reliable dairy cow genes in dairy cow maize 1 was 7. Some information about these dairy cow genes is shown in Table 4.

[0063] Table 4 Information on some bovine genes expressed in bovine corn 1.

[0064]

[0065] (II) BGI Genomics was commissioned to perform gene analysis on the dairy cow-maize 2 genome. The results are as follows: the alignment rate between the dairy cow-maize 2 genome and the maize reference genome was 57.51%. The alignment rate between the maize genome reads that did not align with the dairy cow reference genome was 1.46%, with 203,233 reads and 13,744,932 new reads. The transcript read coverage is shown in the appendix. Figure 3 The distribution of reads on transcripts is shown in the appendix. Figure 4 The average alignment rate of the gene set was 1.23%. A total of 18 dairy cow genes were detected, all of which were known genes. The expression levels of dairy cow and maize genes are shown in the box curves. Figure 5 The expression density of maize 2 in dairy cows is shown in the figure. Figure 6 The expression levels of some bovine genes in bovine-maize 2 are shown in Table 5. A total of 13 new transcripts were detected, of which 3 belonged to new alternative splicing subtypes of known bovine protein-coding genes and 10 belonged to long non-coding RNAs.

[0066] Table 5. Expression levels of some dairy cow genes in maize-coated dairy cows.

[0067]

[0068] Example 2: Breeding of Maize Papaya 1 and Maize Papaya 2

[0069] This embodiment is carried out according to the following method.

[0070] 1. Preparation of corn pollen: Collect pollen from the male parent between 9 and 10 am and put it into the pollination box.

[0071] 2. Treatment of female papaya flowers: Select female flowers of local papayas that are about to bloom, cut off the nearby male flowers, and then cut off the upper half of the petals of the female flower to expose the stigma.

[0072] 3. Pollination: First, drip 1 to several drops of enzymatic hydrolysate into the ovary to thoroughly wet it; the enzymatic hydrolysate is a complete glycosyl hydrolysate obtained by animal glycoprotein hydrolysis. Apply the self-prepared distant hybridization agent to the stigma with a brush, then use another brush to apply corn pollen. Seal the bag with a clean, transparent plastic bag. The next day, when repeating the pollination, unpack the plastic bag and pollinate using the same method as the first time. Repeat this process three times. After pollination, count the total number of hybrid flowers.

[0073] 4. Fertilized eggs are removed 60 days after pollination, embryos are rescued and cultured into seedlings, and after chromosome doubling, they are transplanted into soil.

[0074] 5. Fruit setting rate survey

[0075] Harvest and cut the hybrid melons when their skins turn yellow, remove the seedless melons, count the number of seeded melons and the number of seeds in each type of melon, and calculate the seed set rate using the following company's calculation:

[0076] Hybridization fruit set rate (%) = Number of seeded melons / Total number of hybrid flowers × 100%.

[0077] The fruit set rate of the maize-papaya hybrid was 91.1%, with an average of 33 seeds per fruit (Table 6).

[0078] Table 6. Seed setting rate of maize-papaya hybrids

[0079]

[0080] 6. Offspring selection and sweetness analysis

[0081] The first generation of hybrids were planted in isolation, with each melon in a separate zone of 30 plants. Male plants were eliminated during the flowering period, and at maturity, high-yielding individuals with resistance to ring spot virus and high sweetness were selected from the hermaphroditic plants. For the second and subsequent generations, only exceptionally outstanding individuals were selected, and inferior plants were eliminated. In the fourth generation, some lines were homozygous, from which two superior lines were selected and designated as Corn Papaya 1 and Corn Papaya 2.

[0082] Ten individual plants of both corn-papaya 1 and local papaya were randomly selected, and one fruit was randomly selected from each plant. The sugar content was measured using an FW-32T saccharimeter produced by Hangzhou Fanwo Technology Co., Ltd. The test was repeated 10 times, and the average value was taken.

[0083] Among them, photos of corn and papaya plants can be found Figure 7 As can be seen, after hybridization with maize, the local papaya changed from an oval shape to a long and narrow shape (Table 7). The results of plant morphology, yield traits, resistance, and sweetness of maize-papaya are shown in Table 7 below.

[0084] Table 7. Plant morphology, yield traits, resistance, and sweetness of corn and papaya plants.

[0085]

[0086] The yield per plant of the Corn-Papaya 1 variety is higher than that of the local papaya (i.e., the female parent CK), it is highly resistant to ring spot virus disease, and its sweetness is 28% higher than that of the local papaya (Table 7).

[0087] 7. Maize gene detection in maize and papaya

[0088] Five fresh papayas from each of the two maize-papaya genomes (1 and 2) were submitted to BGI Genomics for maize genome testing. BGI Genomics' results for maize genome testing in maize-papaya genome 1 were as follows: the average alignment rate of the sample with the papaya genome was 84.60%; the alignment rate of reads that did not align with the papaya genome to the maize reference genome was 0.09%, with 4514 reads and 5067106 new reads. Transcript read coverage is shown in [link to transcript]. Figure 8 The distribution of reads on transcripts is shown in the figure. Figure 9 A total of 204 maize genes were detected, all of which are known genes. The expression levels of some maize genes in *Carica papaya* are shown in Table 8. Box plots, density plots, and distribution plots of maize gene expression levels in *Carica papaya* are shown in [Table 8]. Figure 10 , 11 A total of 7 new transcripts were detected, one of which belonged to a new alternative splicing subtype of a known protein-coding gene, and the remaining 6 belonged to long non-coding RNAs.

[0089] The results of BGI's detection of maize genes in Maize-Papaya 2 were as follows: the average alignment rate of Maize-Papaya 2 to the papaya genome was 81.23%, and the average alignment rate to the papaya gene set was 70.50%; a total of 18,015 expressed genes were detected. The average alignment rate of the sample to the maize genome was 0.03%, and the average alignment rate to the maize gene set was 0.04%; a total of 142 expressed maize genes and 174 transcripts were detected (Table 9). The expression levels of some maize genes in Maize-Papaya 3 are shown in Table 10; the transcript read coverage is shown in... Figure 13 The distribution of reads on transcripts is shown in the figure. Figure 14 .

[0090] Table 8 shows the expression levels of some maize genes in Maize Papaya 1.

[0091]

[0092] Table 9. Papaya and maize genes and transcript numbers expressed in Maize-Papaya 2 and its parent.

[0093]

[0094] Table 10 shows the expression levels of some maize genes in Maize Papaya 2.

[0095]

[0096] The above embodiments are merely best examples and are not intended to limit the implementation of the present invention.

Claims

1. A method for sexual hybridization of plants, characterized in that: The method includes the following steps: (1) Select superior varieties of the desired sexually reproducing plant type as the hybridization female parent, remove the male flowers before they mature, treat the female flowers before fertilization and remove reproductive isolation; (2) Select sperm or pollen from interspecific or more distant organisms with the traits of the desired variety, add a distant hybridization agent and stir well to obtain a fertilization solution, and use the fertilization solution to fertilize the female parent; or fertilize the stigma of the female parent by applying the distant hybridization agent and the sperm or pollen separately, preferably by sealing in a parchment bag, more preferably by unpacking the bag the next day and pollinating again, repeating for 2-12 days; (3) Harvest fertilized eggs after successful hybridization, preferably 7-70 days after successful hybridization; (4) The fertilized eggs were rescued and cultured into seedlings. After the chromosomes were doubled, they were transplanted into the soil. Furthermore, it also includes: (5) Identify and remove false hybrids.

2. The method as described in claim 1, characterized in that, The sexual hybridization mentioned refers to sexual hybridization between kingdoms, phyla, classes, orders, families, genera, and species.

3. The method as described in claim 1, characterized in that, The pre-fertilization treatment of female flowers described in step (1) specifically involves removing the outer covering of the ovary to expose it, such as removing the sepals, petals, ovary wall and integument.

4. The method as described in claim 1, characterized in that, The reproductive isolation before insemination mentioned in step (1) specifically involves dripping 1 to several drops of enzymatic hydrolysate into the ovary to wet it thoroughly; the enzymatic hydrolysate is a complete glycosylation hydrolysate obtained by animal glycoprotein hydrolysis.

5. The method as described in claim 1, characterized in that, The formulation of the distant hybridization agent is: 100-300 mg / L gibberellin, 10-100 mg / L auxin, 1500-5000 ml / L enzymatic hydrolysate and 1500-5000 mg / L compound amino acids. More preferably, the formulation is 150-250 mg / L gibberellin, 40-70 mg / L auxin, 2500-4000 μl / L enzymatic hydrolysate and 2500-4000 mg / L compound amino acids. Preferably, the enzymatic hydrolysate is a complete glycosyl hydrolysate obtained by animal glycoprotein enzymatic hydrolysis.

6. The method as described in claim 1, characterized in that, The method of inseminating the mother with insemination fluid in step (3) is to apply the insemination fluid to the ovary and seal it in a bag; preferably, once a day, repeated for 2-12 days.

7. The method as described in claim 1, characterized in that, The step (4) described above involves rescuing the fertilized egg from its embryo and culturing it into a seedling. Specifically, this involves rescuing the embryo and culturing it into a seedling using MS medium.

8. The method as described in claim 1, characterized in that, The chromosome doubling method involves doubling chromosomes with colchicine and then transplanting them into the soil. At maturity, each true hybrid is harvested individually. The F2 generation and subsequent generations are cultured using the pedigree method until homozygous.

9. The method as described in claim 1, characterized in that, The method for identifying and removing false hybrids in step (5) is selected from one or more of the following three: 1) Identify and remove false hybrids that are similar to the maternal parent by morphological identification; 2) Identify and remove individuals that are similar to the maternal parent by component identification; 3) Identify and remove individuals that are similar to the maternal parent by genetic identification.

10. The method as described in claim 1, characterized in that, Further steps include the following: The purification and rejuvenation of the variety is aided by detecting metabolites of typical individuals in the variety using broad-targeted metabolomics technology. The method for propagating the variety is as follows: based on preliminary field selection and indoor re-selection of individuals with typical traits of the variety, individuals with unique metabolites that are absent in the maternal parent but present in the paternal parent are selected using broad-targeted metabolomics technology for propagation of the variety.

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