CN112790104A - Method for identifying affinity of lily distant hybridization - Google Patents
Method for identifying affinity of lily distant hybridization Download PDFInfo
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Abstract
The invention discloses a method for identifying lily distant hybridization affinity, belonging to the technical field of distant hybridization affinity identification. According to the method, the lily is subjected to distant hybridization, the distant hybridization affinity of the lily is identified by comparing the ovary expansion rate and the pseudo expansion rate at different development times, and the higher the ovary expansion rate is, the lower the pseudo expansion rate is, the later the abortion time is, the higher the affinity of the hybrid parents is. The method for identifying the lily distant hybridization affinity by jointly using the ovary expansion rate and the pseudo expansion rate overcomes the defect that the actual germination condition of the embryo is not considered by only considering the ovary expansion rate when only using the ovary expansion rate, and avoids the interference of the ovary tissue pseudo expansion caused by pollen stimulation on the actual hybridization affinity judgment.
Description
Technical Field
The invention relates to a method for identifying affinity of lily distant hybridization, belonging to the technical field of identification of affinity of distant hybridization.
Background
Lily is an important flower integrating appreciation, medicine and eating, and is one of four cut flowers in the world. The lily resource is rich in variety, more than one hundred varieties of stock seeds are provided, the stock seeds are divided into 7 groups, each group of lily has respective unique advantages, for example, Asian lily in the rolling petal group has virus resistance, oriental lily with petiole group has the advantages of fragrance, large flower and brilliant color, and the trumpet group of lily has the characteristics of fast growth, low dormancy requirement and the like. A new variety with different lily advantages can be obtained by intercrossing. In the plant science, the hybridization between species and more than one species is defined as distant hybridization, the distant hybridization between different groups of lilies has obvious hybridization obstacle, the lilies in different groups can not be naturally hybridized, and technical measures such as cutting flower columns, embryo rescue and the like need to be adopted to overcome the hybridization obstacle. The method for obtaining distant hybridization progeny through technical measures such as embryo rescue and the like usually consumes a large amount of manpower, material resources and time, and the identification of the distant hybridization affinity among different species of the lily can provide powerful support for the establishment of hybridization plans such as parent matching, the number of hybridization groups and the like when a new lily variety is cultivated through distant hybridization.
Disclosure of Invention
The invention aims to provide a method for identifying affinity of lily distant hybridization, which comprises the following specific steps: performing distant hybridization on lily, and identifying the affinity of the lily distant hybridization by comparing the ovary expansion rate/pseudo expansion rate at the same development time, wherein the higher the ovary expansion rate is, the lower the pseudo expansion rate is, and the later the abortion time is, the higher the affinity of the parents of the hybrid is.
Preferably, the specific hybridization process of the distant hybridization according to the present invention is:
(1) when the mucus secreted by the stigma of the female parent is suitable for pollination, after the stigma of the female parent is cut at a position 0.4-0.6 cm away from the ovary of the female parent, selecting the stigma of the female parent which overflows and carries the mucus, dipping pollen of the male parent and coating the pollen on the cut of the stigma of the female parent to finish pollination; the cut was covered in time with a tin foil paper cylinder (about 1cm in diameter) to prevent pollen from falling onto the cut.
(2) And (3) removing the tin foil paper sleeve after 3-5 d, observing the swelling condition of the ovary, and calculating the swelling rate of the ovary at different times according to whether the ovary is swelled and the swelling time.
(3) And (3) cutting ovaries which grow and expand after pollination respectively at 20d, 30d, 40d, 50d and 60d after pollination for embryo rescue.
(4) And observing the germination condition of the embryo within 5 months after inoculation, counting the germination rate and the ovary pseudo-expansion rate, and counting the ovary pseudo-expansion rate by combining the ovary expansion rate and the embryo germination rate.
(5) Judging the affinity rate of the hybrid parents according to the ovary expansion rate/pseudo expansion rate and the ovary abortion time; the higher the rate of ovary expansion and the lower the rate of pseudo-expansion, the later the abortion time, the higher the affinity of the hybrid parents.
Further, in steps (2) and (4) of the present invention, the rate of ovary expansion is expanded ovary number/pollinated ovary number × 100%.
Furthermore, the embryo rescue method of the invention is different according to different development time, and the formula of the artificial culture medium is as follows: 1/2MS and 0.01-0.1 mg/L NAA, and the specific process is as follows:
and for the ovary which develops for 20 days, adopting an ovary slice culture method, namely cutting the complete ovary into slices with the thickness of 0.2-0.4 cm, and inoculating the slices on an artificial culture medium for in vitro culture.
For the ovary which develops for 30d, a embryo sac culture method is adopted, namely, embryo sac and endosperm and embryo which are wrapped by the embryo sac are completely taken out and then inoculated on an artificial culture medium for isolated culture.
For ovary developing for 40 days, endosperm culture method is adopted, i.e. embryo sac of endosperm outer layer is stripped, and endosperm containing embryo is taken out and inoculated on artificial culture medium for isolated culture.
For ovaries which develop for 50d and 60d, an embryo culture method is adopted, namely, endosperm is cut open, and complete embryos are taken out to be inoculated on an artificial culture medium for in vitro culture.
Further, in the step (4) of the present invention, the germination rate of embryos is equal to germination number/inoculation number × 100%.
Further, in the step (4), the rate of ovary pseudo-enlargement is equal to the rate of ovary enlargement/embryo germination x 100%.
The invention has the beneficial effects that:
the affinity of lily distant hybridization is identified by comparing the ovary expansion rate/pseudo expansion rate of different development time, and the higher the ovary expansion rate is, the lower the pseudo expansion rate is, and the later the abortion time is, the higher the affinity of hybrid parents is. The false expansion rate makes up for the defect that only the ovary expansion is considered and the embryo germination condition is not considered when the expansion rate is used alone) or the germination rate only considers the germination rate of the inoculated embryo and the number of the expanded ovaries is not considered, and avoids the interference of the ovary tissue false expansion caused by pollen stimulation on the actual hybridization affinity judgment.
Drawings
FIG. 1 is a pseudo-expanding condition of a pod pollinated for 40d by a Siberia (Siberia) x purple spot whitening (Lilium nepalense) cut boll;
FIG. 2 function (Oriental lily cultivar) х false pod expansion for cut boll pollination 50 days of Lanzhou lily (Asian lily traditional cultivar);
FIG. 3 development of purple pedicel (Oriental lily cultivar) х Liriopsis floribunda (Asian lily wild species) by crossing, combining, cutting, and pollinating 40d pods.
Detailed Description
The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the above description.
The instrument device in the embodiment of the invention comprises: superclean bench, greenhouse, plastic greenhouse, tissue culture room, superclean bench, etc.
The method for identifying affinity of lily distant hybridization specifically comprises the following steps:
(1) when the mucus secreted by the stigma of the female parent is suitable for pollination, a sharp blade is used for cutting off the stigma of the female parent at a position 0.5cm away from the ovary of the female parent, the stigma of the female parent overflowing and carrying a large amount of mucus is selected, and fresh and viable pollen of the male parent is stained and smeared on the incision of the stigma of the female parent, so that the pollination is completed.
(2) The cut is covered by cylindrical tinfoil paper with the diameter about 1.5 times of the ovary diameter in time to prevent pollen of other lilies from falling onto the cut.
(3) And (3) removing the tin foil paper bags after 3-5 d, observing the swelling condition of the ovary, and calculating the swelling rate at different time according to whether the ovary is swelled and the swelling time, wherein the swelling rate of the ovary is the swelling ovary number/pollination ovary number multiplied by 100%.
(4) And (3) cutting ovaries which grow and expand after pollination respectively at 20d, 30d, 40d, 50d and 60d after pollination for embryo rescue.
(5) The embryo rescue method is different according to different development time, and comprises the following steps:
for an ovary which develops for 20 days, an ovary slice culture method is adopted, namely, a complete ovary is cut into slices with the thickness of 0.2-0.4 cm and then inoculated on an artificial culture medium for in vitro culture, and the formula of the culture medium is as follows: 1/2MS + NAA 0.01-0.1 mg/L, the same below.
For the ovary which develops for 30d, a embryo sac culture method is adopted, namely, embryo sac and endosperm and embryo which are wrapped by the embryo sac are completely taken out and then inoculated on an artificial culture medium for isolated culture.
For ovary developing for 40 days, endosperm culture method is adopted, i.e. embryo sac of endosperm outer layer is stripped, and endosperm containing embryo is taken out and inoculated on artificial culture medium for isolated culture.
For ovaries which develop for 50d and 60d, an embryo culture method is adopted, namely, endosperm is cut open, and complete embryos are taken out to be inoculated on an artificial culture medium for in vitro culture.
(6) And (3) observing the germination condition of the embryo within 5 months after inoculation, and counting the ovary number of the embryo germination and the embryo germination rate, wherein the embryo germination rate is the ovary number/expanded ovary number of the embryo germination.
(7) And (3) counting the ovary false expansion rate, wherein the ovary false expansion rate is (1-embryo germination rate) multiplied by 100% according to the embryo germination rate.
(8) Judging the affinity of the hybrid parents according to the ovary expansion rate/pseudo expansion rate and the ovary abortion time; the higher the rate of ovary expansion and the lower the rate of pseudo-expansion, the later the abortion time, the higher the affinity of the hybrid parents.
Example 1
According to the hybridization combination of х purple spot lily (Asian lily wild species) in Siberian (oriental lily cultivar), the ovary expansion rate of 1000 pollinated flowers 20d, 30d, 40d, 50d and 60d reaches over 90% in 2014 by using a cut style method, but the embryo germination rate of embryo rescue at different times is 0, and the ovary pseudo-expansion rate is 100%.
The false amplification rate of the ovary of the hybrid combination of the lilium brownii (Asian lily wild species) х Siberian (Oriental lily cultivar) is 100 percent, so that the lilium brownii as a female parent has no affinity with the hybrid combination of Siberian, and a new lily variety with two resource advantages can not be cultivated and polymerized by using the lilium brownii as the female parent and hybridizing with Siberian.
Example 2
Function (oriental lily cultivar) х hybrid combinations of lilium davidii (asian lily traditional cultivar), using cut style and embryo rescue in 2014, the rate of ovary expansion, number of embryo germinating ovaries, and false expansion of 600 pollinated flowers 20d, 30d, 40d, 50d, 60d are shown in tables 1 and 2.
Note: "-" indicates that the early stage of in vitro germination is difficult due to too small embryo age or the late stage of ovary abortion is not suitable for statistical false enlargement rate.
From the data, the function (oriental lily cultivar) х Lanzhou lily (Asia lily traditional cultivar) hybrid combination has a false expansion rate of 70% after pollination for 50 days (at the moment, the lily embryo has developed and matured to the extent of easy germination and growth on an in vitro culture medium), has certain hybrid affinity, and under the condition of more pollinated flowers, the excellent oriental lily cultivar-function is taken as a female parent to be hybridized with the excellent edible lily in Asia lily-Lanzhou lily, so that hybrid offspring with the advantages of both the excellent oriental lily and the Lanzhou lily can be obtained for new variety breeding.
Example 3
The hybrid combination of purple pedicel (oriental lily cultivar) х lilium floribundum (asian lily wild species), the ovary expansion rates of 541 pollinated flowers 20d, 30d, 40d, 50d and 60d are respectively 100%, 75%, 62% and 45% in 2019 by using a cut style and embryo rescue method, the embryo germination rates of embryo rescue at different times are respectively 2.5%, 5.5%, 10.6%, 40.7% and 42.9%, and the ovary false expansion rates of 30d, 40d, 50d and 60d after pollination are respectively 68.5%, 48.2%, 18.9% and 0%.
Note: "-" indicates the statistical false expansion rate of early stage of embryo germination difficulty due to too small embryo age or late stage of ovary abortion
From the data, the purple pedicel (oriental lily cultivar) х lilium floribundum (asian lily wild species) distant hybridization combination has 75-62% of ovary expansion rate and the ovary false expansion rate is as low as 59.3% after pollination and hybridization for 40-50 days, which shows that the distant hybridization combination has high affinity, and a large amount of filial generations can be obtained through hybridization of the two distant hybridization combinations for polymerizing the advantages of the two or breeding new varieties with excellent variation.
Claims (6)
1. A method for identifying affinity of lily distant hybridization is characterized in that: performing distant hybridization on lily, and identifying the affinity of the lily distant hybridization by comparing the ovary expansion rate/pseudo expansion rate at different development times, wherein the higher the ovary expansion rate is, the lower the pseudo expansion rate is, and the later the abortion time is, the higher the affinity of the parents of the hybrid is.
2. The method for affinity identification of lily distant hybridization according to claim 1, wherein: the specific hybridization process is as follows:
(1) when the mucus secreted by the stigma of the female parent is suitable for pollination, after the stigma of the female parent is cut at a position 0.4-0.6 cm away from the ovary of the female parent, selecting the stigma of the female parent which overflows and carries the mucus, dipping pollen of the male parent and coating the pollen on the cut of the stigma of the female parent to finish pollination; covering the cut with tinfoil paper tube in time to prevent pollen of other Bulbus Lilii from falling onto the cut;
(2) removing the tin foil paper sleeve after 3-5 days, observing the swelling condition of the ovary, and calculating the swelling rate of the ovary at different times according to whether the ovary is swelled and the swelling time;
(3) cutting ovaries which grow and expand after pollination respectively at 20d, 30d, 40d, 50d and 60d after pollination for embryo rescue;
(4) observing the germination condition of the embryos within 5 months after inoculation, counting the ovary expansion rate and the embryo germination rate, and counting the ovary pseudo expansion rate according to the embryo germination rate;
(5) judging the affinity of the hybrid parents according to the ovary expansion rate/pseudo expansion rate and the ovary abortion time; the higher the rate of ovary expansion and the lower the rate of pseudo-expansion, the later the abortion time, the higher the affinity of the hybrid parents.
3. The method for affinity identification of lily distant hybridization according to claim 2, wherein: and (3) in the steps (2) and (4), the ovary expansion rate is equal to the number of expanded ovaries/the number of pollinated ovaries multiplied by 100%.
4. The method for affinity identification of lily distant hybridization according to claim 2, wherein: the embryo rescue method in the step (3) is different according to different development time, and the formula of the artificial culture medium is as follows: 1/2MS and 0.01-0.1 mg/L NAA, and the specific process is as follows:
(1) for an ovary which develops for 20 days, adopting an ovary slice culture method, namely cutting the complete ovary into slices with the thickness of 0.2-0.4 cm, and then inoculating the slices on an artificial culture medium for in vitro culture;
(2) for an ovary which develops for 30 days, adopting a embryo sac culture method, namely completely taking out embryo sac and endosperm and embryo which are wrapped by the embryo sac, and then inoculating the embryo sac and the endosperm and embryo on an artificial culture medium for isolated culture;
(3) for ovary which develops for 40d, adopting an endosperm culture method, namely peeling embryo sac at the outer layer of endosperm, taking out endosperm containing embryos, and inoculating the endosperm on an artificial culture medium for isolated culture;
(4) for ovaries which develop for 50d and 60d, an embryo culture method is adopted, namely, endosperm is cut open, and complete embryos are taken out to be inoculated on an artificial culture medium for in vitro culture.
5. The method for affinity identification of lily distant hybridization according to claim 2, wherein: and (4) in the step (4), the germination rate of the embryo is equal to the number of germination ovaries of the embryo/the number of inoculation ovaries multiplied by 100%.
6. The method for affinity identification of lily distant hybridization according to claim 2, wherein: in the step (4), the rate of pseudo-enlargement of ovaries is (1-germination rate) × 100%.
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Patent Citations (6)
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| US20170298382A1 (en) * | 2012-03-13 | 2017-10-19 | Pioneer Hi-Bred International, Inc. | Genetic reduction of male fertility in plants |
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