CN106561457B - A kind of radix pseudostellariae hybrid seed manually cultivates selection - Google Patents
A kind of radix pseudostellariae hybrid seed manually cultivates selection Download PDFInfo
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- CN106561457B CN106561457B CN201610972933.6A CN201610972933A CN106561457B CN 106561457 B CN106561457 B CN 106561457B CN 201610972933 A CN201610972933 A CN 201610972933A CN 106561457 B CN106561457 B CN 106561457B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
The present invention provides a kind of radix pseudostellariae hybrid seed and manually cultivates selection, comprises the following specific steps that: seed selects, seed disinfection, embryo strip, Seed embryo culture, proliferation amplification, the screening of hybrid seed tissue-cultured seedling, the screening of hybrid elite plant strain, the genetic stability detection of hybrid elite plant strain, hybrid elite plant strain expand numerous and transplant.Method provided by the invention is with workload is small, speed is fast, the period is short, less cost of power；Solve the problems, such as radix pseudostellariae hybrid seed germination rate it is low, vulnerable to insect pest, growth cycle is long, breeding coefficient is low, reproductive speed is slow, breeding heavy workload, the breeding period is long, work consuming is time-consuming, push radix pseudostellariae crossbreeding work to carry out.
Description
Technical field
The invention belongs to medicinal plant breeding fields, and in particular to a kind of radix pseudostellariae hybrid seed manually cultivates breeding side
Method.
Background technique
Radix pseudostellariae is perennial medicinal plant, is used as medicine with root tuber, is traditional strengthening by means of tonics Chinese medicine, strong with QI invigorating
Spleen, it is promoting production of body fluid and nourishing the lung the effect of.Radix pseudostellariae is mainly root tuber breeding and seminal propagation, and it is asexual numerous that artificial cultivation is mostly used root tuber progress
It grows, the accumulation of radix pseudostellariae disease, germplasm is made to degenerate.Radix pseudostellariae seminal propagation can cultivate virus-free seedling, can effectively prevent germplasm and move back
Change.
Hybridization is to concentrate in together the merit of two or more kinds by mating, using selection and is cultivated,
The method for obtaining new varieties, is breeding important method, the new varieties of breeding usually embody hybrid vigour.After hybridization pollination, harvest
Hybrid seed, usually sowed in experimental plot, rudiment, transplanting, by the field breeding of many years, observe its growth characteristics and
Morphological feature screens excellent hybridization single plant, carries out expanding propagation, forms strain, examines or check its genetic stability and economical character,
Breeding forms hybrid new breed.The Breeding Process of Hybrid is and its important and very very long, it usually needs 10 years left sides
The right time.
Radix pseudostellariae hybridization pollination carries out in 3-4 month, and fruit maturation after usually pollination one month harvests seed.Seed into
Row conventional seed planting, it is necessary to which by the low temperature induction in winter, second year spring could sprout, and a seed is sprouted to form single plant
Strain, by the growth of spring and summer, July, above-ground plant parts, which just turns to be yellow, withers, and a small root tuber is usually formed in underground,
Small root tuber is planted in the fall as seed ginseng, further expand it is numerous, by many years cultivation expand it is numerous, certain amount list could be formed
The plant of one seed culture harvests a certain number of root tubers, forms strain, cultivates into control, carries out individual plant selection, detects it
Genetic stability and economical character determine if the hybrid performance for having excellent, carry out breeding of new variety.It deposits in the prior art
In following disadvantage: (1) radix pseudostellariae hybrid seed carries out conventional seed planting, and there are dormancies, to pass through the low temperature induction in winter, the
2 year spring could sprout, and the time is long, not manageability, and germination rate is low；(2) radix pseudostellariae hybrid seed is smaller, conventional seed planting, is easy
It loses, also vulnerable to insect pest, there is invalid sowing situation；(3) hybrid seed sprouting only grows into single plant, harvests single fritter
Root, breeding coefficient is low, numerous by the cultivation expansion of many years, could form the strain of the single seed culture of certain amount, expand numerous speed
Degree is slow, time-consuming；(4) radix pseudostellariae hybrid seed single plant offspring carries out conventional herd breeding, and heavy workload, speed are slow, the period is long, time-consuming.
Currently, having no the report in terms of radix pseudostellariae crossbreeding, hybrid seed breeding.
Summary of the invention
In order to solve existing hybrid seed germination rate during existing radix pseudostellariae good variety selection it is low, vulnerable to insect pest,
Growth cycle is long, breeding coefficient is low, reproductive speed is slow, breeding heavy workload, breeding period length, the problem of work consuming time-consuming, improves
Hybrid seed seedling breakneck acceleration and accuracy shorten the crossbreeding period, save Hybrids Breeding cost, improve radix pseudostellariae hybridization and educate
Kind efficiency.The present invention provides a kind of radix pseudostellariae hybrid seed and manually cultivates selection.
To achieve the goals above, the present invention adopts the following technical scheme:
(1) seed is selected: being removed shrivelled hybrid seed using wet concentration method, is manually chosen the small seed of particle；
(2) seed disinfection: the conventional disinfection method of appliable plant tissue culture technology carries out the disinfection treatment of hybrid seed, sterile
After water cleaning, in spare on clean work station；
(3) embryo strips: in gnotobasis, carefully stripping exosper, endotesta, the perisperm of hybrid seed, has left
Whole embryo；
(4) Seed embryo culture: the embryonic breeding of hybrid seed is entered in embryo germination induced medium dark culture 7-10 days, is then turned
Enter low light culture, induction embryo is sprouted rapidly, and differentiates Multiple Buds；
(5) proliferation amplification: after embryo differentiation culture 30-60 days, Multiple Buds are taken out, carry out plant division, access methyl α-naphthyl acetate is dense
Degree is to promote bud to grow in the MS growth medium of 0.2mg/L, forms plant；Growth 50-70 days, single stipes of clip plant
Material is connect, same medium is accessed, further expands numerous growth, forms strain；
(6) screening of hybrid seed tissue-cultured seedling:
(a) each hybrid seed strain tissue-cultured seedling and its male parent tissue-cultured seedling, maternal tissue-cultured seedling preliminary screening: are compared
Cauline leaf grows speed, the growth characteristics of robust plant degree and including blade shape, thickness, color, stalk thickness, internode length
The morphological features such as short tentatively judge hybrid performance possessed by hybrid seed tissue-cultured seedling, distinguish hybrid tissue-cultured seedling and non-hybrid group
Train seedling；
(b) molecule screens: apply DNA molecular marker technology, further contrasting detection analyze each hybrid seed tissue-cultured seedling,
The DNA molecular diversity of male parent tissue-cultured seedling, maternal tissue-cultured seedling, the hybrid performance of accurate judgement hybrid seed tissue-cultured seedling retain miscellaneous
Kind tissue-cultured seedling, goes unless hybrid tissue-cultured seedling；
(7) screening of hybrid elite plant strain；Select that the speed of growth is fast, robust plant, microlith root form speed block, quantity
The hybrid strain more, microlith root is big；
(8) the genetic stability detection of hybrid elite plant strain: dense in methyl α-naphthyl acetate by the hybrid elite plant strain tissue-cultured seedling of screening
Degree is that continuous 3 subcultures expand numerous culture in the MS growth medium of 0.2mg/L, and each incubation time 30-60 days, detection is every time
Subculture expands the tissue-cultured seedling growth consistency and genetic stability of numerous culture, selects inheritance stability and grows consistent hybrid elite plant
System；
(9) hybrid elite plant strain expand it is numerous and transplanting: the hybrid elite plant strain tissue-cultured seedling screened is further expanded it is numerous, then
Transplant field production, breeding new varieties.
The present invention has the advantage that (1) carries out artificial sterile in-vitro inducing by stripping the embryo of radix pseudostellariae hybrid seed
Culture releases dormancy characteristics of different, and the embryo of seed is sprouted immediately, germination rate nearly 100%；(2) embryo is in inductive differentiation medium
In, it sprouts rapidly, and differentiate Multiple Buds, the growth coefficient of hybridization embryo is greatly improved, further group expands numerous growth, single miscellaneous
It hands over the embryo of seed that can expand numerous a large amount of plant out rapidly, quickly forms independent strain；(3) by comparing each cenospecies subgroup
The growth characteristics and morphological feature for training seedling and father, maternal tissue-cultured seedling, can tentatively judge hybrid performance possessed by hybrid seed；It answers
With DNA molecular marker technology, the DNA molecular diversity of comparative analysis hybrid seed tissue-cultured seedling, male parent tissue-cultured seedling, maternal tissue-cultured seedling
Analysis, further determines that the hybrid performance of hybrid seed tissue-cultured seedling, goes unless hybrid strain system tissue-cultured seedling, hybrid breakneck acceleration block,
Accuracy is high；(4) each hybrid strain tissue-cultured seedling, male parent tissue-cultured seedling, maternal tissue-cultured seedling are cultivated by control, observes more each tissue culture
Seedling plant strain growth speed, robustness, microlith root forms speed, quantity, size, and detects its genetic stability, breeding hybrid
Elite plant strain, superior hybrid breeding speed block, accuracy are high；(5) the hybrid elite plant strain screened is further expanded it is numerous, then
Transplant field production, breeding new varieties；Compared to conventional hybrid selection, this method workload is small, speed block, period are short, energy
Consumption is few.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1
A kind of radix pseudostellariae hybrid seed manually cultivates selection, the specific steps are that:
(1) seed is selected: shrivelled hybrid seed is removed using wet concentration method, it is artificial to reject the small seed of particle, and only protect
Stay granular size medium and the medium above hybrid seed；
(2) seed disinfection: the conventional disinfection method of appliable plant tissue culture technology carries out the disinfection treatment of hybrid seed, sterile
After water cleaning, in spare on clean work station；
(3) embryo strips: in gnotobasis, carefully stripping exosper, endotesta, the perisperm of hybrid seed, has left
Whole embryo；
(4) Seed embryo culture: the embryonic breeding of hybrid seed is entered into dark culture 7 days in embryo germination induced medium, is then transferred to
Low light culture, induction embryo is sprouted rapidly, and differentiates Multiple Buds.
(5) proliferation amplification: after embryo differentiation culture 50 days, Multiple Buds being taken out, and carries out plant division, and access concentration of NAA is
In the MS growth medium of 0.2mg/L, bud is promoted to grow, forms plant；Growth 50 days, single stipes of clip plant connects material, connects
Enter same medium, further expand numerous growth, forms strain.
(6) screening of hybrid seed tissue-cultured seedling:
A) preliminary screening: the stem of each hybrid seed strain tissue-cultured seedling and its male parent tissue-cultured seedling, maternal tissue-cultured seedling is compared
Leaf grows speed, the growth characteristics of robust plant degree and including blade shape, thickness, color, stalk thickness, internode length
Etc. morphological features, tentatively judge hybrid performance possessed by hybrid seed tissue-cultured seedling, differentiation hybrid tissue-cultured seedling and non-hybrid tissue culture
Seedling；
B) molecule screens: applying DNA molecular marker technology, further contrasting detection analyzes each hybrid seed tissue-cultured seedling, father
The DNA molecular diversity of this tissue-cultured seedling, maternal tissue-cultured seedling, the hybrid performance of accurate judgement hybrid seed tissue-cultured seedling retain hybrid
Tissue-cultured seedling is gone unless hybrid tissue-cultured seedling.
(7) screening of hybrid elite plant strain；Select that the speed of growth is fast, robust plant, microlith root form speed block, quantity
The hybrid strain more, microlith root is big；
(8) the genetic stability detection of hybrid elite plant strain: dense in methyl α-naphthyl acetate by the hybrid elite plant strain tissue-cultured seedling of screening
Degree is that continuous 3 subcultures expand numerous culture in the MS growth medium of 0.2mg/L, and each incubation time 30 days detects each subculture
The tissue-cultured seedling growth consistency and genetic stability for expanding numerous culture, select inheritance stability and grow consistent hybrid elite plant strain.
(9) hybrid elite plant strain expand it is numerous and transplanting: the hybrid elite plant strain tissue-cultured seedling screened is further expanded it is numerous, then
Transplant field production, breeding new varieties.
Embodiment 2
A kind of radix pseudostellariae hybrid seed manually cultivates selection, the specific steps are that:
(1) seed is selected: shrivelled hybrid seed is removed using wet concentration method, it is artificial to reject the small seed of particle, and only protect
Stay granular size medium and the medium above hybrid seed；
(2) seed disinfection: the conventional disinfection method of appliable plant tissue culture technology carries out the disinfection treatment of hybrid seed, sterile
After water cleaning, in spare on clean work station；
(3) embryo strips: in gnotobasis, carefully stripping exosper, endotesta, the perisperm of hybrid seed, has left
Whole embryo；
(4) Seed embryo culture: the embryonic breeding of hybrid seed is entered into dark culture 8 days in embryo germination induced medium, is then transferred to
Low light culture, induction embryo is sprouted rapidly, and differentiates Multiple Buds.
(5) proliferation amplification: after embryo differentiation culture 30 days, Multiple Buds being taken out, and carries out plant division, and access concentration of NAA is
In the MS growth medium of 0.2mg/L, bud is promoted to grow, forms plant；Growth 60 days, single stipes of clip plant connects material, connects
Enter same medium, further expand numerous growth, forms strain.
(6) screening of hybrid seed tissue-cultured seedling:
A) preliminary screening: the stem of each hybrid seed strain tissue-cultured seedling and its male parent tissue-cultured seedling, maternal tissue-cultured seedling is compared
Leaf grows speed, the growth characteristics of robust plant degree and including blade shape, thickness, color, stalk thickness, internode length
Etc. morphological features, tentatively judge hybrid performance possessed by hybrid seed tissue-cultured seedling, differentiation hybrid tissue-cultured seedling and non-hybrid tissue culture
Seedling；
B) molecule screens: applying DNA molecular marker technology, further contrasting detection analyzes each hybrid seed tissue-cultured seedling, father
The DNA molecular diversity of this tissue-cultured seedling, maternal tissue-cultured seedling, the hybrid performance of accurate judgement hybrid seed tissue-cultured seedling retain hybrid
Tissue-cultured seedling is gone unless hybrid tissue-cultured seedling.
(7) screening of hybrid elite plant strain；Select that the speed of growth is fast, robust plant, microlith root form speed block, quantity
The hybrid strain more, microlith root is big；
(8) the genetic stability detection of hybrid elite plant strain: dense in methyl α-naphthyl acetate by the hybrid elite plant strain tissue-cultured seedling of screening
Degree is that continuous 3 subcultures expand numerous culture in the MS growth medium of 0.2mg/L, and each incubation time 60 days detects each subculture
The tissue-cultured seedling growth consistency and genetic stability for expanding numerous culture, select inheritance stability and grow consistent hybrid elite plant strain.
(9) hybrid elite plant strain expand it is numerous and transplanting: the hybrid elite plant strain tissue-cultured seedling screened is further expanded it is numerous, then
Transplant field production, breeding new varieties.
Embodiment 3
A kind of radix pseudostellariae hybrid seed manually cultivates selection, the specific steps are that:
(1) seed is selected: shrivelled hybrid seed is removed using wet concentration method, it is artificial to reject the small seed of particle, and only protect
Stay granular size medium and the medium above hybrid seed；
(2) seed disinfection: the conventional disinfection method of appliable plant tissue culture technology carries out the disinfection treatment of hybrid seed, sterile
After water cleaning, in spare on clean work station；
(3) embryo strips: in gnotobasis, carefully stripping exosper, endotesta, the perisperm of hybrid seed, has left
Whole embryo；
(4) Seed embryo culture: the embryonic breeding of hybrid seed is entered into dark culture 10 days in embryo germination induced medium, is then transferred to
Low light culture, induction embryo is sprouted rapidly, and differentiates Multiple Buds.
(5) proliferation amplification: after embryo differentiation culture 60 days, Multiple Buds being taken out, and carries out plant division, and access concentration of NAA is
In the MS growth medium of 0.2mg/L, bud is promoted to grow, forms plant；Growth 70 days, single stipes of clip plant connects material, connects
Enter same medium, further expand numerous growth, forms strain.
(6) screening of hybrid seed tissue-cultured seedling:
A) preliminary screening: the stem of each hybrid seed strain tissue-cultured seedling and its male parent tissue-cultured seedling, maternal tissue-cultured seedling is compared
Leaf grows speed, the growth characteristics of robust plant degree and including blade shape, thickness, color, stalk thickness, internode length
Etc. morphological features, tentatively judge hybrid performance possessed by hybrid seed tissue-cultured seedling, differentiation hybrid tissue-cultured seedling and non-hybrid tissue culture
Seedling；
B) molecule screens: applying DNA molecular marker technology, further contrasting detection analyzes each hybrid seed tissue-cultured seedling, father
The DNA molecular diversity of this tissue-cultured seedling, maternal tissue-cultured seedling, the hybrid performance of accurate judgement hybrid seed tissue-cultured seedling retain hybrid
Tissue-cultured seedling is gone unless hybrid tissue-cultured seedling.
(7) screening of hybrid elite plant strain；Select that the speed of growth is fast, robust plant, microlith root form speed block, quantity
The hybrid strain more, microlith root is big；
(8) the genetic stability detection of hybrid elite plant strain: dense in methyl α-naphthyl acetate by the hybrid elite plant strain tissue-cultured seedling of screening
Degree is that continuous 3 subcultures expand numerous culture in the MS growth medium of 0.2mg/L, and each incubation time 50 days detects each subculture
The tissue-cultured seedling growth consistency and genetic stability for expanding numerous culture, select inheritance stability and grow consistent hybrid elite plant strain.
(9) hybrid elite plant strain expand it is numerous and transplanting: the hybrid elite plant strain tissue-cultured seedling screened is further expanded it is numerous, then
Transplant field production, breeding new varieties.
Claims (1)
1. a kind of radix pseudostellariae hybrid seed manually cultivates selection, which is characterized in that include following specific steps:
(1) seed is selected: being removed shrivelled hybrid seed using wet concentration method, is manually chosen the small seed of particle；
(2) seed disinfection: the conventional disinfection method of appliable plant tissue culture technology carries out the disinfection treatment of hybrid seed, and sterile water is clear
After washing, in spare on clean work station；
(3) embryo strips: in gnotobasis, carefully stripping exosper, endotesta, the perisperm of hybrid seed, leaves complete
Embryo；
(4) Seed embryo culture: the embryonic breeding of hybrid seed is entered in embryo germination induced medium dark culture 7-10 days, is then transferred to weak
Optical culture, induction embryo is sprouted rapidly, and differentiates Multiple Buds；
(5) proliferation amplification: after embryo differentiation culture 30-60 days, Multiple Buds being taken out, and carries out plant division, and access concentration of NAA is
In the MS growth medium of 0.2mg/L, bud is promoted to grow, forms plant；Growth 50-70 days, single stipes of clip plant connects material,
Same medium is accessed, numerous growth is further expanded, forms strain；
(6) screening of hybrid seed tissue-cultured seedling:
(a) preliminary screening: the cauline leaf for comparing each hybrid seed strain tissue-cultured seedling and its male parent tissue-cultured seedling, maternal tissue-cultured seedling is raw
Long speed, the growth characteristics of robust plant degree and including blade shape, thickness, color, stalk thickness, internode length form
Feature tentatively judges hybrid performance possessed by hybrid seed tissue-cultured seedling, distinguishes hybrid tissue-cultured seedling and non-hybrid tissue-cultured seedling；
(b) molecule screens: applying DNA molecular marker technology, further contrasting detection analyzes each hybrid seed tissue-cultured seedling, male parent group
The DNA molecular diversity of seedling, maternal tissue-cultured seedling is trained, the hybrid performance of accurate judgement hybrid seed tissue-cultured seedling retains hybrid tissue culture
Seedling is gone unless hybrid tissue-cultured seedling；
(7) screening of hybrid elite plant strain；Select the speed of growth is fast, robust plant, microlith root formed speed is fast, quantity is more,
The big hybrid strain of microlith root；
(8) the genetic stability detection of hybrid elite plant strain: by the hybrid elite plant strain tissue-cultured seedling of screening, it is in concentration of NAA
Continuous 3 subcultures expand numerous culture in the MS growth medium of 0.2mg/L, and each incubation time 30-60 days detects each subculture and expands
The tissue-cultured seedling growth consistency and genetic stability of numerous culture select inheritance stability and grow consistent hybrid elite plant strain；
(9) hybrid elite plant strain expands numerous and transplanting: further expanding the hybrid elite plant strain tissue-cultured seedling screened numerous, then transplants
Field production, breeding new varieties.
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| CN107027627B (en) * | 2017-04-20 | 2020-04-24 | 宁德师范学院 | Microtuber propagation method for young embryo culture of polygonatum cyrtonema |
| CN109618933A (en) * | 2019-01-23 | 2019-04-16 | 贵州大学 | A kind of radix pseudostellariae Seed embryo culture base and its cultural method |
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