CN102577937A - Method for breeding azalea clonal cultivar - Google Patents

Method for breeding azalea clonal cultivar Download PDF

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Publication number
CN102577937A
CN102577937A CN 201210004689 CN201210004689A CN102577937A CN 102577937 A CN102577937 A CN 102577937A CN 201210004689 CN201210004689 CN 201210004689 CN 201210004689 A CN201210004689 A CN 201210004689A CN 102577937 A CN102577937 A CN 102577937A
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azalea
plant
breeding
individual plant
seed
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吴月燕
王忠华
李波
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Zhejiang Wanli University
Zhejiang Wanli College
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Zhejiang Wanli College
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Abstract

The invention discloses a method for breeding an azalea clonal cultivar. The method is characterized in that: azalea eminent trees with high ornamental value and an early flowering period are obtained in a manual physical mutation mode. The method comprises the following steps of: determining a breeding objective; collecting plump and mature seeds; performing physical mutation by using 50 to 250Gy of 60Co gamma rays in the dosage rate of 10Gy/min; sowing the seeds; cultivating nursery-grown plants for 3 to 6 years; selecting single plants, namely selecting in the aspect of the observation of biology, botanical characters, ornamental features and the like; analyzing genes with mutated characters; performing regional test on the eminent trees; summarizing cultivating measures; and popularizing. The eminent trees obtained by the method have a flowering period which can be advanced by 5 to 15 days commonly, can be white, red and white, red and the like, have polyphyll and are high in stress resistance.

Description

A kind of breeding method of azalea clone kind
Technical field
The present invention relates to the forest genetics technical field, specifically referring to utilize modern biotechnology (physical mutagenesis) means to obtain azalea choiceness kind is a kind of breeding method of azalea clone kind.
Background technology
The West cuckoo (Rhododendron simsii Planch) is called for short western cuckoo, has another name called belgian azalea.The West cuckoo is the good decorative flower of China, and it is numerous in variety, and pattern is abundant, comes in every shape.In view of the biological property of cuckoo self, it has high viewing and admiring with Landscape Application and is worth.At present, the selection to the azalea breeding mainly is that wild resource is carried out introduction and acclimatization both at home and abroad, and hybridizes to come the seed selection new varieties with cultivar, but this method probability of success comparatively complicated, that obtain improved seeds is lower.Along with the development of modern biotechnology, novel breeding technique such as using-system culture technique, induced-mutation technique, molecular marking technique and transgenic technology is cultivated new varieties has become possibility, but the example that success is reported is very few.
Cuckoo is easy to generate bud mutation in natural world the West; The part improved seeds are selected to cultivate successfully by natural bud mutation in the cultivation at present; But natural bud mutation probability is lower; Utilizing induced mutations can improve the variation probability of Western cuckoo greatly, is the good approach of seed selection the West cuckoo improved seeds, but utilizes the method for induced mutations breeding the West cuckoo also to rarely have report.
Summary of the invention
Technical problem to be solved by this invention is the situation to prior art, and a kind of method of Western cuckoo clone breed breeding is provided.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of method of Western cuckoo clone breed breeding; The main mode that adopts artificial physical mutagenesis; Obtain ornamental value height, the fine individual plant of morning in flowering stage; This method comprises the choosing of the confirming of breeding objective, breeding material, the selection of physical mutagenesis dosage, the management of nursery stock, the screening of variation individual plant, the molecule discriminating of variation proterties etc., specifically comprises the steps:
(1) confirming of breeding objective: with disease-resistant, higher ornamental value (like different colored type, patterns, long flowering stage etc.) is main breeding objective;
(2) choosing of breeding material: the plant of selecting to have kind (being), the robust growth of higher ornamental value and having got into the Sheng phase of blooming be maternal plant (general autumn or spring maternal plant the fruit pericarp just can gather when becoming brown or yellowish-brown; Generally gather before the capsule cracking in early spring in Zhejiang Province), behind maternal plant collection seed, select full, well-developed seed, be divided into irradiation treatment group and control group; Be specially: 900～1000 seeds are one group, and being divided into is 6 groups, and wherein 1 group is contrast (not adopting irradiation treatment), and 5 groups are used for radiation treatment;
(3) selection of physical mutagenesis dosage: through preliminary experiment, employing 50,100,150,200,250Gy carry out the seed that five groups (one group of corresponding dosage) are used for radiation treatment respectively 60The gamma-ray acute radiation of Co is contrast with radiationless processed group, and dose rate is 10Gy/min;
(4) sowing of seed: planting seed is in the plastic containers of 25～30cm, height 20～25cm; Matrix is the mixture (volume ratio is 6: 4) of peat and husk; Matrix dress basin (plastic containers) is preceding to carry out disinfection with high-pressure sterilizing pot; Basin soil amount is 800～1500g, and matrix contains organic 35～45gkg -1, alkali-hydrolyzable nitrogen 100～120mgkg -1, rapid available phosphorus 80～95mgkg -1, available potassium 200～250mgkg -1, pH6.0～6.5; The liver moss that 0.8～1.2 cm thick that tiles above the matrix shreds is watered permeable then; The seed of irradiation treatment and contrast evenly is seeded on the liver moss 180～200 in every basin; Container seedling places in the beam-connected greenhouse, and the temperature in the booth remains on 20～25 ℃, and the relative air humidity during the seed germination in the booth keeps 90～95%, with short rudiment, reduces to 65～75% behind the seed germination, to reduce disease; Soil conservation after planting is moistening, and relative moisture of the soil keeps 65～75%;
(5) management of nursery stock: germinateed in after planting 14～15 days, and when seedling grows to 2～3 leaves, transplanted in small container, select the field planting of non-woven fabric net bag individual plant for use, container diameter is 4～5cm, height 8～10cm, the same step of matrix (4) mesostroma; Container seedling places in the beam-connected greenhouse equally, and the temperature in the booth remains on 20～25 ℃, and temperature is too high shades with the sunshade net, and relative moisture of the soil remains on 65～75%; Upgrowth situation according to nursery stock changed basin on the 2nd year later on, cultivated plant blossom after 3～6 years;
(6) screening of variation individual plant: during seedling growth, carefully observe biology, the botany proterties of different disposal, selection and contrast (control group) are at the individual plant that notable difference is arranged aspect resistance, plant growing gesture, the flower (pattern, flower type, florescence etc.);
(7) molecule of variation proterties is differentiated: select fine individual plant, gather the young leaflet tablet of this fine individual plant and control group individual plant, adopt improved method of CTAB to extract Western cuckoo plant genome DNA, utilize the SRAP labeling technique to carry out the mutant gene analysis;
(8) regional test of fine individual plant and popularization: through biology, botany proterties and the observation of viewing and admiring characteristic, binding molecule is differentiated, obtains fine individual plant; This individual plant is carried out vegetative propagation (like cuttage, grafting), select different zones to make an experiment, fit kind of a scope, and sum up a whole set of cultivation step that is fit to this individual plant with clear and definite its.
The kind of choosing breeding material in the step (2) is like " fifth lunar month ", " white beautiful woman ", " snow beauty ", " purple cloud " etc.
Be preferably described basin soil amount in the step (4) and be 1000g, matrix contains organic 38gkg -1, alkali-hydrolyzable nitrogen 110mgkg -1, rapid available phosphorus 87mgkg -1, available potassium 237mgkg -1, pH6.3.
SRAP labeling technique described in the step (7) is carried out its Western azalea SRAP-PCR reaction system of mutant gene analysis: in the 20 μ L PCR reaction volumes, and MgCl 21.75mmol/L, dNTPs0.175mmol/L, Taq polymerase 1.5U, primer 0.2 μ mol/L, 10 * Buffer, 2.0 μ L, the DNA genome is 1.5M; The pcr amplification reaction program is: react 94 ℃ of preparatory sex change 5min of preceding 5 circulations; 94 ℃ of sex change 1min, 37 ℃ of renaturation 1min, 72 ℃ are extended 1min, 5 circular responses; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.
Above-mentioned primer utilizes TGAGTCCAAACCGGATA; TGAGTCCAAACCGGAGC; TGAGTCCAAACCGGAAT; TGAGTCCAAACCGGACC; TGAGTCCAAACCGGAAG; TGAGTCCAAACCGGTAA; TGAGTCCAAACCGGTCC; TGAGTCCAAACCGGTGC; GACTGCGTACGAATTAAT; GACTGCGTACGAATTTGC; GACTGCGTACGAATTGAC; GACTGCGTACGAATTTGA; GACTGCGTACGAATTAAC; GACTGCGTACGAATTGCA; GACTGCGTACGAATTCAA; GACTGCGTACGAATTCTG; GACTGCGTACGAATTCGA; GACTGCGTACGAATTCAG; 19 primers such as GACTGCGTACGAATTCCA (the common use) amplify bands of a spectrum and better repeated clearly.
Advantage of the present invention and beneficial effect:
1. the present invention successfully adopts 60Co gamma-rays induced mutations obtains the Western cuckoo breeding that the variation probability is high, handling safety is stable.
2. Western cuckoo breeding method of the present invention; The molecule of the sowing environmental condition control through confirming seed after exposure dose, dose rate, the irradiation, the management of nursery stock, screening, variation proterties is differentiated, the regional test of fine individual plant and popularization etc.; Obtain that the complete ornamental value of a cover is high, flowering stage (5-15 days ahead of time), proterties, good (pattern was for white, red and white, redness etc.; Polyphyll; The resistance height) individual plant the West cuckoo induced mutations breeding method has successfully realized the breeding of Western cuckoo induced mutations.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail (is example with " fifth lunar month " cuckoo kind mutagenic and breeding new varieties), but the present invention not only is confined to following examples.
Embodiment
Embodiment is an example with " fifth lunar month " kind, and " fifth lunar month " cuckoo originates in Japan, and China widely cultivates; The maternal corolla cerise of " fifth lunar month " cuckoo of adopting; Wealthy infundibulate, the filigree pale red utilizes induced mutations can successfully select the individual plant that produces good variation at aspects such as pattern, florescences.
(1) confirming of breeding objective: with higher ornamental value (like different colored types, pattern, than prematurity etc.) is main breeding objective;
(2) choosing of breeding material: selecting to have kind (being), the robust growth of higher ornamental value and to have got into the plant of the Sheng phase of blooming is maternal plant, general autumn or spring maternal plant the fruit pericarp just can gather when becoming brown or yellowish-brown; Generally gather before the capsule cracking in early spring in Zhejiang Province, selects full well-developed seed behind the seed collection, and about 1000 is one group, is divided into 6 groups, and wherein 1 group is contrast, and 5 groups are used for radiation treatment;
(3) selection of physical mutagenesis dosage: through preliminary experiment, employing 50,100,150,200,250Gy's 60The Co gamma-rays carries out acute irradiation, is treated to contrast with radiationless, and dose rate is 10Gy/min;
(4) sowing of seed: planting seed is in the plastic containers of diameter 25cm, height 25cm, and matrix is the mixture (volume ratio is 6: 4) of peat and husk, carries out disinfection with high-pressure sterilizing pot before the matrix dress basin, and about basin soil amount 1000g, organic 38gkg -1, alkali-hydrolyzable nitrogen 110mgkg -1, rapid available phosphorus 87mgkg -1, available potassium 237mgkg -1, pH6.3, above the tiling thickness be about 1 centimetre the liver moss that shreds, matrix is watered permeable.The seed of mutagenic treatment group and control group evenly is seeded on the liver moss about 200 in every basin; Container seedling places in the beam-connected greenhouse, and the temperature in the booth is general to keep about 20 ℃, and the relative air humidity during the seed germination in the booth keeps about 90%, with short rudiment; Reduce to behind the seed germination about 70%, to reduce disease, soil conservation after planting is moistening, and relative moisture of the soil keeps about 70%;
(5) management of nursery stock: after planting germinate about 15 days, when seedling grows to 2～3 leaves, transplant in small container, select the field planting of non-woven fabric net bag individual plant for use, its diameter is 4.5cm, height 10cm, the same step of matrix (4); Container seedling places in the beam-connected greenhouse equally, and the temperature in the booth generally keeps 20-25 ℃, and temperature is too high shades with the sunshade net, and relative moisture of the soil keeps about 70%.Upgrowth situation according to nursery stock changed basin later on the 2nd year, generally cultivated plant blossom after 4 years;
(6) screening of variation individual plant: during seedling growth, carefully observe biology, the botany proterties of different disposal, select and the individual plant of notable difference is arranged to impinging upon resistance, plant growing gesture, flower aspects such as (pattern, flower type, florescences etc.);
(7) molecule of variation proterties is differentiated: select fine individual plant, gather the young leaflet tablet of this fine individual plant and control group individual plant, utilize the SRAP labeling technique to carry out the mutant gene analysis; The optimum SRAP-PCR reaction system of the West azalea is: in the 20 μ LPCR reaction volumes, and MgCl 21.75mmol/L, dNTPs0.175mmol/L, Taq polymerase 1.5U, primer 0.2 μ mol/L, 10 * Buffer, 2.0 μ L;
The pcr amplification reaction program is: react 94 ℃ of preparatory sex change 5min of preceding 5 circulations; 94 ℃ of sex change 1min, 37 ℃ of renaturation 1min, 72 ℃ are extended 1min, 5 circular responses; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.
Primer adopts TGAGTCCAAACCGGATA; TGAGTCCAAACCGGAGC; TGAGTCCAAACCGGAAT; TGAGTCCAAACCGGACC; TGAGTCCAAACCGGAAG; TGAGTCCAAACCGGTAA; TGAGTCCAAACCGGTCC; TGAGTCCAAACCGGTGC; GACTGCGTACGAATTAAT; GACTGCGTACGAATTTGC; GACTGCGTACGAATTGAC; GACTGCGTACGAATTTGA; GACTGCGTACGAATTAAC; GACTGCGTACGAATTGCA; GACTGCGTACGAATTCAA; GACTGCGTACGAATTCTG; GACTGCGTACGAATTCGA; GACTGCGTACGAATTCAG; 19 primer amplifications such as GACTGCGTACGAATTCCA go out bands of a spectrum and better repeated clearly.
(8) regional test of fine individual plant and popularization: through biology, botany proterties and the observation of viewing and admiring characteristic, binding molecule differentiates, obtains a flowering stage (about 15 days ahead of time), petal is white but the petal tip is cherry double-colored fine individual plant; This individual plant is carried out vegetative propagation (like cuttage, grafting), select different zones to make an experiment, fit kind of a scope, and sum up a whole set of cultivation step that is fit to this individual plant with clear and definite its.

Claims (8)

1. the breeding method of an azalea clone kind is characterized in that: through the mode of artificial physical mutagenesis, obtain ornamental value height, the azalea fine individual plant of morning in flowering stage.
2. the breeding method of a kind of azalea clone kind according to claim 1 is characterized in that: these breeding method concrete steps comprise:
(1) confirming of breeding objective: with disease-resistant, higher ornamental value is main breeding objective;
(2) choosing of breeding material: selecting to have kind, the robust growth of higher ornamental value and got into the plant of the Sheng phase of blooming is maternal plant; Behind maternal plant collection seed, select full, well-developed seed, be divided into irradiation treatment group and control group, be specially: 900～1000 seeds are one group, and being divided into is 6 groups, and wherein 1 group is contrast, and 5 groups are used for radiation treatment;
(3) selection of physical mutagenesis dosage: through preliminary experiment, employing 50,100,150,200,250Gy carry out five groups of seeds that are used for radiation treatment respectively 60The gamma-ray acute radiation of Co is contrast with radiationless processed group, and dose rate is 10Gy/min;
(4) sowing of seed: planting seed is in the plastic containers of 25～30cm, height 20～25cm; Matrix is that volume ratio is 6: 4 the peat and the mixture of husk; Matrix dress basin is to carry out disinfection with high-pressure sterilizing pot before the plastic containers; Basin soil amount is 800～1500g, and matrix contains organic 35～45gkg -1, alkali-hydrolyzable nitrogen 100～120mgkg -1, rapid available phosphorus 80～95mgkg -1, available potassium 200～250mgkg -1, pH6.0～6.5; The liver moss that 0.8～1.2 cm thick that tiles above the matrix shreds is watered permeable then; The seed of irradiation treatment and contrast evenly is seeded on the liver moss 180～200 in every basin; Container seedling places in the beam-connected greenhouse, and the temperature in the booth remains on 20～25 ℃, and the relative air humidity during the seed germination in the booth keeps 90～95%, with short rudiment, reduces to 65～75% behind the seed germination, to reduce disease; Soil conservation after planting is moistening, and relative moisture of the soil keeps 65～75%;
(5) management of nursery stock: germinateed in after planting 14～15 days, and when seedling grows to 2～3 leaves, transplanted in small container, select the field planting of non-woven fabric net bag individual plant for use, container diameter is 4～5cm, height 8～10cm, the same step of matrix (4) mesostroma; Container seedling places in the beam-connected greenhouse equally, and the temperature in the booth remains on 20～25 ℃, and relative moisture of the soil remains on 65～75%; Upgrowth situation according to nursery stock changed basin on the 2nd year later on, cultivated plant blossom after 3～6 years;
(6) screening of variation individual plant: during seedling growth, carefully observe biology, the botany proterties of different disposal, select and the individual plant of notable difference is arranged to impinging upon resistance, plant growing gesture, flower aspect;
(7) molecule of variation proterties is differentiated: select fine individual plant, gather the young leaflet tablet of this fine individual plant and control group individual plant, adopt improved method of CTAB to extract Western cuckoo plant genome DNA, utilize the SRAP labeling technique to carry out the mutant gene analysis;
(8) regional test of fine individual plant and popularization: through biology, botany proterties and the observation of viewing and admiring characteristic, binding molecule is differentiated, obtains fine individual plant; This individual plant is carried out vegetative propagation, select different zones to make an experiment, fit kind of a scope, and sum up a whole set of cultivation step that is fit to this individual plant with clear and definite its.
3. the breeding method of a kind of azalea clone kind according to claim 2 is characterized in that: a kind of in " fifth lunar month ", " white beautiful woman ", " snow beauty ", " the purple cloud " of the kind of choosing breeding material in the step (2).
4. the breeding method of a kind of azalea clone kind according to claim 2 is characterized in that: in the step (2) on the maternal plant collection of seed with autumn or spring maternal plant the fruit pericarp gather when becoming brown or yellowish-brown.
5. the breeding method of a kind of azalea clone kind according to claim 2 is characterized in that: the described basin soil of step (4) amount is 1000g, and matrix contains organic 38gkg -1, alkali-hydrolyzable nitrogen 110mgkg -1, rapid available phosphorus 87mgkg -1, available potassium 237mgkg -1, pH 6.3.
6. the breeding method of a kind of azalea clone kind according to claim 2; It is characterized in that: SRAP labeling technique described in the step (7) is carried out its Western azalea SRAP-PCR reaction system of mutant gene analysis and is: in the 20 μ L PCR reaction volumes, and MgCl 21.75mmol/L, dNTPs0.175mmol/L, Taq polymerase 1.5U, primer 0.2 μ mol/L, 10 * Buffer, 2.0 μ L, the DNA genome is 1.5M.
7. the breeding method of a kind of azalea clone kind according to claim 6 is characterized in that: described pcr amplification reaction program is: react 94 ℃ of preparatory sex change 5min of preceding 5 circulations; 94 ℃ of sex change 1min, 37 ℃ of renaturation 1min, 72 ℃ are extended 1min, 5 circular responses; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.
8. the breeding method of a kind of azalea clone kind according to claim 6 is characterized in that: described primer is TGAGTCCAAACCGGATA, TGAGTCCAAACCGGAGC, TGAGTCCAAACCGGAAT, TGAGTCCAAACCGGACC, TGAGTCCAAACCGGAAG, TGAGTCCAAACCGGTAA, TGAGTCCAAACCGGTCC, TGAGTCCAAACCGGTGC, GACTGCGTACGAATTAAT, GACTGCGTACGAATTTGC, GACTGCGTACGAATTGAC, GACTGCGTACGAATTTGA, GACTGCGTACGAATTAAC, GACTGCGTACGAATTGCA, GACTGCGTACGAATTCAA, GACTGCGTACGAATTCTG, GACTGCGTACGAATTCGA, GACTGCGTACGAATTCAG, GACTGCGTACGAATTCCA totally 19 primers.
CN 201210004689 2012-01-09 2012-01-09 Method for breeding azalea clonal cultivar Pending CN102577937A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105803093A (en) * 2016-05-06 2016-07-27 上海市农业科学院 Hippeastrum rutilum SRAP-PCR reaction system and application thereof
CN106171467A (en) * 2016-08-03 2016-12-07 江西省·中国科学院庐山植物园 Rhododendronsimiarum seed propagation method
CN115836644A (en) * 2022-12-31 2023-03-24 浙江万里学院 Screening method and application of azalea hybrid progeny flower phenotype excellent single plant

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105803093A (en) * 2016-05-06 2016-07-27 上海市农业科学院 Hippeastrum rutilum SRAP-PCR reaction system and application thereof
CN106171467A (en) * 2016-08-03 2016-12-07 江西省·中国科学院庐山植物园 Rhododendronsimiarum seed propagation method
CN106171467B (en) * 2016-08-03 2021-06-01 江西省·中国科学院庐山植物园 Rhododendron Ericatum seed propagation method
CN115836644A (en) * 2022-12-31 2023-03-24 浙江万里学院 Screening method and application of azalea hybrid progeny flower phenotype excellent single plant

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Application publication date: 20120718