CN104488694B - A method for rapidly cultivating transgenic maize inbred lines - Google Patents
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Abstract
The invention belongs to the field of crop breeding, and particularly relates to a method for quickly cultivating a transgenic corn inbred line. The method comprises the following steps: 1) and (3) assembling transgenic base materials: hybridizing the transgenic positive material with an excellent maize inbred line; 2) and (3) hybridization induction: hybridizing the induction line with the transgenic basic material; 3) and (3) haploid identification and selection: selecting haploid plants through seeds and field plants; 4) haploid doubling: doubling and selfing the haploid plant by a natural or artificial chemical doubling method; 5) identification of transgenic DH lines: PCR level detection is carried out on the obtained DH line, and a transgenic inbred line containing the target gene is confirmed. The method greatly shortens the cultivation process of the transgenic maize inbred line.
Description
Technical Field
The invention belongs to the field of crop breeding, and particularly relates to a method for quickly cultivating a transgenic corn inbred line.
Background
As a new method for crop genetic improvement, genetic engineering and transgenosis greatly improve the breeding quality and efficiency. The transgenic corn BT176 developed by the Sendai company as the first example of transgenic corn in the world is commercialized since 1996, the planting area of the transgenic crops is continuously increased, the number of 170 million hectares in 1996 is increased to 1.752 million hectares in 2013, the number of the transgenic crops is increased by more than 100 times in 18 years, and the global market value of only transgenic seeds in 2013 reaches $ 156 million, which accounts for 35% of the global $ 450 million commercial seed market. This growth has made transgenic technology the fastest growing crop technology available in modern agricultural history with considerable profit. At present, transgenic crops are mainly concentrated on seven major crops such as soybean, corn, cotton and the like. Corn is one of three grain crops in the world, important grain, channel and feed crops, and in 2013, the global transgenic corn planting area is 5740 ten thousand hectares, which accounts for 32 percent of the global corn total planting area. However, transgenic corn is not planted in China, and cotton and pawpaw are mainly used.
Aiming at the strategic requirements for guaranteeing food safety and developing the biological breeding industry, the 2008 national institute approves and implements a great special item for breeding a new transgenic organism variety, and the ultimate aim is to breed a large batch of a great new transgenic organism variety with disease and insect resistance, stress resistance, high quality, high yield and high efficiency, improve the overall level of agricultural transgenic organism research and industrialization, and provide powerful scientific and technological support for the sustainable development of agriculture in China. The method has great strategic significance for enhancing the independent innovation capability of agricultural science and technology, improving the biological breeding level of China, promoting agricultural efficiency improvement and income increase of farmers and improving the agricultural international competitiveness of China.
The cultivation of new transgenic corn varieties is a comprehensive system engineering, a good receptor system is established, the emphasis of corn genetic transformation is limited by a plant genetic transformation technology, the transformation efficiency of the existing genetic transformation method is low, for example, the most widely applied agrobacterium-mediated young embryo genetic transformation method in corn is limited by genotype, most of backbone inbred lines mainly developed in production cannot induce embryonic callus, and the sensitivity to agrobacterium infection is extremely low, so that the broad receptor system cannot be directly used for corn genetic transformation. At present, widely applied maize transgenic receptor genotypes at home and abroad are mainly concentrated on a few materials such as Hi-II, A188, H99 and the like, the materials contain recessive genes for controlling high induction rate of embryogenic callus and susceptibility to agrobacterium, and the maize transgenic receptor genotypes have higher genetic transformation efficiency as receptor materials. However, these several easily transformable recipient materials have poor agronomic performance. Therefore, the few transgenic maize receptor materials obtained by transformation need to be backcrossed with the backbone maize inbred line for backcross transformation, backcrossing is generally carried out for 6-7 generations, and then inbred for more than 3 generations, and finally a new transgenic maize inbred line with excellent agronomic characters and introduced with exogenous genes can be created, and the long breeding process greatly delays the research and development speed of transgenic maize breeding. Therefore, the method for establishing the rapid breeding of the transgenic maize inbred line becomes the combustion improver for accelerating breeding of new transgenic maize varieties.
In recent years, haploid breeding technology has gradually become a key technology for conventional breeding of corn in China. The Doubled Haploid (DH) technique is the fastest, simplest, economical, practical and direct method for breeding maize inbred lines. The basic process is to obtain homozygous diploid plant by artificially or naturally doubling the biologically induced haploid plant, and then to screen new lines from the homozygous diploid plant. The method is mainly characterized in that the line selection speed is high, and a stable pure line can be obtained only by 2 generations (more than 7 generations are needed for traditional inbred line breeding). At present, the method is gradually applied to the breeding of the conventional maize inbred line on a large scale. No attempt has been made to breed transgenic maize inbred lines. Based on the breeding requirement of the transgenic corn and the advantages of the haploid breeding technology, the haploid technology is applied to the cultivation of the transgenic corn inbred line, has important significance for accelerating the cultivation of the transgenic corn inbred line, and has good application prospect.
Disclosure of Invention
The invention aims to provide a method for quickly cultivating a transgenic maize inbred line.
The invention provides a method for rapidly cultivating a transgenic maize inbred line, which comprises the following steps:
(1) transgenic basal Material Assembly
The genetic basic material for breeding the transgenic maize inbred line by utilizing the cross-induced parthenogenesis method can be a cross combination containing target trait genes such as insect resistance and herbicide resistance, hybrid seeds (including single cross seeds, triple cross seeds and double cross seeds) or various groups (including comprehensive hybrid seeds) or F2 and F3 generation groups derived from the hybrid seeds, and the genetic basic material can be obtained by crossing the germplasm containing the target transgenic traits with materials such as excellent inbred lines and various groups under the isolation condition. The germplasm containing the target transgenic character can be obtained by an agrobacterium-mediated method, a gene gun method and the like. The basic material has the excellent characteristics of high yield, high quality, disease resistance, stress resistance, good comprehensive agronomic characters and the like.
(2) Induction of hybridization
Under the isolation condition, planting the matched transgenic basic material and induction line, wherein the transgenic basic material is 100 plants, sowing the induction line in a ratio of 4: 1, usually selecting induction lines of Stock6, Jigao lure No. 3, agriculture high lure series and the like, selecting the induction line with the flowering phase similar to the basic material as far as possible, or adopting measures of sowing in 2-3 stages and the like, ensuring that the two flowering phases meet, and smoothly completing the hybridization.
The induced cross pollination mode is the same as the conventional cross pollination mode. The induced transgenic base material is used as a female parent, a sulfuric acid paper bag is used for manual bagging isolation of the female ear immediately after the female ear is exposed from the transgenic base material, a haploid induction line is used as a male parent after the extracted filament grows to be 5cm (or 3 days for spinning), and fresh pollen (bagged in the previous day) is taken for pollination. Pollination time in extreme hot weather 11 am: before 00, the best result is obtained. Harvesting and properly keeping the hybrid ears after the hybrid ears are mature.
(3) Haploid identification and selection
Identification of haploid grains: harvesting corn ears with black or purple-red grain aleurone layers in autumn, and naturally drying for 20-30 days before threshing. The haploid is primarily identified according to the phenotypic characters (the top and the embryo color) of the kernel, and the pseudohaploid kernel is obtained by coloring (black or purple) the endosperm aleurone layer and colorless (the shield is in an inverted triangle shape and is obviously inwards sunken).
If artificial chemical doubling method is adopted for secondary identification, when the pseudomonoploid sprouts grow to 1-2cm, the sprouts with thick and strong embryonic roots are eliminated, and the sprouts with thin and weak embryonic roots are selected.
Haploid field confirmation: in the field, the haploid is identified according to the purple marker and morphological characteristics of the plant ABP1 before and after the pseudohaploid plant grows to 8-10 leaf stage. All the plants with purple sheaths and leaves and tall and strong plants of the seedlings are eliminated, and the haploid plants can be confirmed to be the haploid plants by slow growth, low plant height, few leaves, narrowness, short, uprush and light leaf color, and the doubling treatment is required.
(4) Haploid doubling
There are two methods of haploid doubling, natural and artificial.
The natural addition rate is low, generally between 1% and 5%, and different basic materials, ecological environments and planting time can influence the natural recovery rate. For haploid genotypes with higher pollen scattering rate, selfing and fructification can be realized by means of natural restoration of fertility. And (3) planting haploids in a plot with good isolation conditions, flat terrain and better fertility, watering in time and performing field fine management. When anther is exposed in the male ear of the haploid plant and obvious pollen is produced, the anther of the haploid plant is taken for fine selfing pollination, and the bag for sleeving the female ear is closed strictly after pollination, so that the pollution of the foreign pollen is avoided.
The haploid genotype with low natural doubling rate must be artificially doubled. The main doubling method comprises a seed soaking method, a root soaking method, a bud soaking method, an injection method, field herbicide spraying and the like. The shoot-macerating (cutting) method and the root-macerating method are described herein.
The method for soaking (cutting) buds is a method for soaking cut buds in colchicine solution. When the sprouts of the haploid seeds grow to 2-3 cm, manually cutting off the tips of coleoptiles by about 1-2mm, soaking the sprouts in a mixed solution of 0.06% of colchicine and 5% of dimethyl sulfoxide for 9 hours, cleaning the sprouts with clear water for 1.5 hours, putting the sprouts into a germination tray for seedling revival treatment, after 2 days of seedling revival, selecting a plot with good isolation conditions, flat terrain and good fertility for transplanting, transplanting according to the plant spacing of 20cm, watering in time and carrying out field fine management.
The root soaking method is to soak the root of haploid seedling with colchicine solution. The method can be carried out in a bud stage or a seedling stage, the root system of the haploid seedling in a 3-leaf stage is soaked in a 0.05 percent colchicine solution for 24 hours, after the soaking treatment is carried out by clear water, the seedling is transplanted in a land block with good isolation condition, flat terrain and good fertility, the transplanting is carried out according to the plant spacing of 20cm, watering is carried out in time, and field fine management is carried out.
During pollination, the female ears are covered with small bags in time, the male ears of each individual plant are carefully and repeatedly observed, pollen is taken out in time, pollen is scattered from the male ears, and pollination is carried out immediately (preferably 9-12 am). Generally, each single plant is pollinated for 2 times, and the pollen amount is large for 1 time.
(5) Identification of transgenic DH lines
And harvesting seeds after the selfed clusters are mature or pollinated for about 35 d. And (3) precisely sowing the obtained double haploid seed grains in the next season or in a greenhouse, and if the plant growth vigor is uniform, the seed grains are not separated, and accord with the character characteristics of the inbred line, so that the homozygous DH inbred line can be determined.
During growth, 100mg of fresh leaves of each DH line were cut into pieces and placed in 2ml EP tubes, ground using a high throughput tissue grinder, and then DNA of the samples was extracted using the TIANGEN plant genomic DNA extraction kit, solubilized with 100ul of ddH2O, and stored at-20 ℃ until use. The DNA concentration of the sample was measured by a Nanodrop2000 ultraviolet spectrophotometer, and the DNA solution was diluted to 25 ng/. mu.L to 50 ng/. mu.L depending on the measured concentration and stored at-20 ℃.
And carrying out PCR amplification on the diluted sample DNA, the positive sample, the negative control and the blank control. A PCR reaction system of 25. mu.L, comprising 2.5. mu.L of 10 XPCR buffer (containing Mg2+), 2. mu.L of 2.5 mmol. mu.L-1 dNTPs (each dNTP having a final concentration of 200. mu. Lmol. L-1), 0.5. mu.L of each 10. mu. mol. L-1 primer (having a final concentration of 200 nmol. L-1), 0.125. mu.L of 5U. mu.L-1 rTaq DNA polymerase, 2. mu.L of template DNA (100ng), and ultrapure water to a final volume of 25. mu.L.
PCR reaction procedure: denaturation at 94 deg.C for 5 min; performing 35 cycles of amplification reaction (94 ℃ denaturation 30s, 58 ℃ annealing 30s, 72 ℃ extension 30 s. the annealing temperature of PCR reaction can be changed according to Tm values of primer sequences of different target trait genes); extension at 72 ℃ for 10 min. And detecting the PCR amplification product by using 2% agarose gel electrophoresis and observing by gel imaging photography, and determining the PCR amplification product as a positive transgenic inbred line if a target PCR amplification product band is detected.
Currently, a great deal of special culture of new varieties of transgenic organisms is being implemented, and the aim is to identify and evaluate a batch of new transgenic germplasm with outstanding target characters. The homozygous transgenic inbred line obtained by the traditional backcross transformation and bicyclic line selection method generally needs more than 7 generations, and each generation needs a large amount of molecular detection, so that how to carry out quick backcross transformation so as to breed the homozygous transgenic inbred line is urgent. The invention can be applied to rapidly obtain the transgenic inbred line, and only needs 2 generations.
Drawings
FIG. 1 identified MON89034 gene transferred pseudohaploid grain
FIG. 2 PCR detection of MON89034 Gene
FIG. 3 protein level detection of MON89034 Gene
1：DH7；2：DH9；3：DH15；4：DH17
FIG. 4 shows the MON89034 gene transferred inbred line ears
DH7 on the left; right is DH9
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1:
1. test material
F1 is obtained by hybridizing an excellent maize inbred line Ji 201000 with a material containing MON89034 gene. In the second growing season, parthenogenesis induction line Nongda high-inducing No. 3 (provided by China university of agriculture) is taken as a male parent, a transgenic hybrid combination F1 plant is taken as a female parent for hybridization, monoploid is identified for the harvested F1 ear grains in autumn, and 226 pseudohaploid grains are identified (figure 1).
2. Haploid doubling
Adopting a chemical doubling bud soaking method, using Colchicine (COL) and Dimethyl sulfoxide (DMSO) as medicaments, obtaining 205 pseudohaploid seedlings, transplanting the pseudohaploid seedlings into an isolated field, identifying and determining 190 pseudohaploid plants through the field, carrying out self-crossing pollination in summer for 40 ears, harvesting 30 selfing ears in autumn, and threshing and storing the pseudohaploid seedlings, wherein the number of the seeds is 3-50.
3. PCR detection
And 4 parts of DH lines (DH7, DH9, DH15 and DH17) with more grains are selected to be planted in a greenhouse, and leaves are taken to carry out target gene detection in the five-leaf stage. The detection method is carried out according to the national standard of the people's republic of China (No. 1861 bulletin-4-2012 of the Ministry of agriculture) [ qualitative method for detecting insect-resistant corn 89034 and derived varieties thereof by using transgenic plants and product components thereof ].
The results show that: the target gene amplification product bands were detected by DH7 and DH9, whereas the target gene amplification product bands were not detected by DH15 and DH 17.
1: marker DL 2000; 2: blank control; 3: a positive control; 4: negative control; 5: DH 7; 6: DH 9; 7: DH 15; 8: DH17
4. Protein level expression assay
Taking the leaves to carry out PCR detection, and simultaneously taking another part of the leaves to carry out protein level expression detection. The protein level expression detection adopts a transgenic detection test strip, selects AS 003 BG, LS of Enviroogix, USA, and adopts a detection item Cry1 Ab.
(1) Method for extracting leaf tissue
a) Cutting off 2 layers of blades by using an off-line pipe cover, and placing the bottom of the pipe by using a cone; b) grinding with a cone for 20-30 seconds; c) sucking 0.5ml of extracting solution and pouring into a centrifuge tube; d) and (5) repeating the grinding.
(2) Test strip detection method
a) Placing the test strip in a grinding liquid; b) within 5 minutes, the presence of the control line indicates that the strip is functional, and if the sample contains the Cry1Ab gene, the 2 nd line (test line) will appear between the control line and the guard band within 5 minutes, and any clearly identifiable pink test line will be considered positive.
The results show (fig. 3): the detection result agrees with the molecular detection result, and detection lines appear in DH7 and DH9, while detection lines do not appear in DH15 and DH 17.
Thus, DH7 and DH9 were confirmed as transgenic inbred (DH) lines, whose ears were harvested after maturation (fig. 4).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. A method for rapidly breeding a transgenic maize inbred line comprises the following steps:
(1) and (3) assembling transgenic base materials: the genetic basic material for breeding the transgenic maize inbred line by utilizing a cross-induced parthenogenesis method is a cross combination, hybrid seeds, various populations or F2 and F3 generation populations derived from the hybrid combination, the hybrid seeds and the various populations containing the genes with the target traits of insect resistance and herbicide resistance, and the genetic basic material is obtained by hybridizing germplasm containing the target transgenic traits with excellent inbred lines and various population materials under the isolation condition;
(2) and (3) hybridization induction: under the isolation condition, planting the matched transgenic basic material and induction system, sowing 100 transgenic basic materials in a 4: 1 ratio to obtain the induction system, wherein the induction system is selected from Stock6, Jigao lure No. 3 and Jida lure series, and the induction system with the flowering phase similar to that of the basic material is selected, or sowing measures are taken in 2-3 stages to ensure that the flowering phases of the two materials meet and complete hybridization smoothly;
(3) and (3) haploid identification and selection:
identification of haploid grains: harvesting corn ears with black or purple red grain aleurone layers in autumn, and threshing after naturally airing for 20-30 days; the haploid is preliminarily identified according to the phenotypic character of the seed, the endosperm aleurone layer is colored into black or purple red, the embryo shield is colorless, the shield is in an inverted triangle shape, and the inward concave part is obvious to be pseudohaploid seed;
adopting an artificial chemical doubling method to carry out secondary identification, eliminating the plump and strong-growing buds with long embryonic roots when the pseudomonoploid buds grow to 1-2cm, and selecting the plump and weak-growing buds with thin embryonic roots;
haploid field confirmation: in the field, the haploid is identified according to the purple marker and morphological characteristics of the plant ABP1 before and after the pseudohaploid plant grows to 8-10 leaf stage; eliminating the plants with purple sheaths and leaves and tall and strong plants of the seedlings, and determining that the seedlings are haploid plants if the seedlings are slow in growth, low in plant height, few in leaves, narrow, short, more uprush and light in leaf color, and carrying out self-pollination treatment;
(4) doubling the haploid; natural doubling or artificial chemical doubling is adopted;
different basic materials, ecological environment and planting time can influence the natural recovery rate; the haploid genotype with higher pollen scattering rate depends on the natural restoration of fertility to realize self-pollination fructification; selecting a plot with good isolation conditions, flat topography and good fertility to plant haploids, watering in time and carrying out field fine management; when anther is exposed in the male ear of the haploid plant and obvious pollen is produced, the anther of the haploid plant is taken for fine selfing pollination, and the bag for sleeving the female ear is strictly closed after pollination, so that the pollution of the external pollen is avoided;
the artificial doubling treatment is needed to be carried out on the haploid genotype with very low natural doubling rate; the doubling method comprises a bud soaking method and a root soaking method;
the bud soaking method adopts a method of soaking cut buds by using a colchicine solution, when the buds of haploid particles grow to 2-3 cm, the tips of coleoptile are manually cut off by 1-2mm, the buds are soaked for 9 hours by using a mixed solution of 0.06% of colchicine and 5% of dimethyl sulfoxide, then cleaned for 1.5 hours by using clear water, put into a germination tray for seedling recovery treatment, after 2 days of seedling recovery, a plot with good isolation conditions, flat terrain and good fertility is selected for seedling transplantation, the seedlings are transplanted according to a plant spacing of 20cm, watering is carried out in time, and field fine management is carried out;
soaking the haploid seedling root by a colchicine solution; the method is carried out in a bud stage or a seedling stage, the root system of a haploid seedling in a 3-leaf stage is soaked in a 0.05 percent colchicine solution for 24 hours, after the soaking treatment is carried out by clear water, the seedling is transplanted in a land block with good isolation conditions, flat terrain and good fertility, the transplanting is carried out according to the plant spacing of 20cm, watering is carried out in time, and field fine management is carried out;
during pollination, covering a small bag on the haploid female ear in time, carefully and repeatedly observing the tassel of each single plant, taking pollen in time, finding pollen scattering of the tassel, and immediately pollinating; pollinating each single plant for 2 times, and obtaining the pollen with large amount for 1 time;
(5) transgenic DH line identification.
2. The method of claim 1, wherein: the hybrid seeds comprise single cross seeds, triple cross seeds and double cross seeds, and the various populations comprise comprehensive hybrid seeds; the germplasm containing the target transgenic character is obtained by an agrobacterium-mediated method and a gene gun method.
3. The method of claim 1, wherein: the hybridization induction in the step (2): the induced cross pollination mode is the same as that of the conventional cross pollination, the induced transgenic base material is used as a female parent, a sulfuric acid paper bag is used for manual bagging isolation of the female ear immediately after the female ear is exposed from the transgenic base material, a haploid induction line is used as a male parent after the female ear is extracted to have the filament length of 5cm or the silk is laid for 3 days, the pocket is laid one day before, and the fresh pollen is taken for pollination; pollination time in extreme hot weather 11 am: 00 before; harvesting and properly keeping the hybrid ears after the hybrid ears are mature.
4. The method of claim 1, wherein: harvesting seeds 35 days after the selfing ears are mature or pollinated; precisely sowing the obtained double haploid kernels in a greenhouse at the next season, and if the plants grow regularly and consistently, the kernels are not separated and accord with the character characteristics of an inbred line, and determining the plants to be a homozygous DH inbred line;
during the growth period, 100mg of fresh leaves of DH line were cut into pieces and put into 2ml of EP tubes, ground with a high throughput tissue grinder, and then DNA of the samples was extracted using the TIANGEN plant genomic DNA extraction kit, and 100ul of ddH was used2Dissolving O, and storing at-20 ℃ for later use; measuring the DNA concentration of the sample plate by using a Nanodrop2000 ultraviolet spectrophotometer, diluting the DNA solution to 25 ng/muL-50 ng/muL according to the measured concentration, and storing at-20 ℃;
and carrying out PCR amplification on the diluted sample DNA, the positive sample, the negative control and the blank control.
5. The method of claim 1, wherein: PCR reaction system 25. mu.L, including 10 XPCR buffer 2.5. mu.L, 2.5 mmol.L-1dNTPs 2μL，10μmol·L-1Primers were 0.5. mu.L, 5U. mu.L each-1rTaq DNA polymerase 0.125. mu.L, template DNA 2. mu.L, ultrapure water to a final volume of 25. mu.L.
6. The method of claim 1, wherein: PCR reaction procedure: denaturation at 94 deg.C for 5 min; 35 cycles of amplification reaction were performed: denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s; properly changing the annealing temperature of the PCR reaction according to the Tm values of the primer sequences of different target trait genes; extending for 10min at 72 ℃; and detecting the PCR amplification product by using 2% agarose gel electrophoresis and observing by gel imaging photography, and determining the PCR amplification product as a positive transgenic inbred line if a target PCR amplification product band is detected.
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