Experiments on oat transgenesis and its application in improving osmotic stress tolerance
Experiments on oat transgenesis and its application in improving osmotic stress tolerance
Oat, a temperate cereal crop widely grown worldwide, lacks tolerance to osmotic stress caused by drought and/or high salt. We have developed a genotype-independent system for efficient stem tip meristem regeneration, and four oat cultivars, Prairie, Porter, Ogle and Pacer, were able to efficiently differentiate regenerated seedlings from stem tip meristem in vitro. This experiment is based on "A Guide to Transgenic Technology and Field Identification of Wheat Crops" by H.D. Jones P.R. Hewley, ed. Hewley, eds.
Operation method
Oat transgenesis and its application in improving osmotic stress tolerance
Materials and Instruments
Oats Move 1. Nodule culture For more product details, please visit Aladdin Scientific website.
Ethanol Distilled water Clorox Bleach
Petri dishes MSI medium
( 1 ) Matured oats (Ogle, Pacer and Prairie ) seeds were used for shoot tip culture.
( 2 ) The lemma and palea of the seeds were removed by hand.
( 3 ) Seeds were surface sterilized by soaking in 70% ethanol (Sigma) for 5 min, then washed once with sterile distilled water, then soaked in 20% Clorox bleach and shaken continuously for 30 min at 0.5 g on a rotary oscillator (VWR International, Bristol, CT 06011) (see Note 1), and washed twice with sterile distilled water. twice.
( 4 ) After placing the seeds in petri dishes with moist filter paper under sterile conditions, cover with a layer of wet filter paper and leave the petri dishes at 3~5°C for 2~3 days to acclimatize the seeds to the treatment conditions. During this period, water should be added frequently to maintain humidity.
( 5 ) After placement, 10-12 sterilized seeds are placed in sterile Petri dishes (100 mm x 15 mm, diameter x height; VWR) filled with MSI medium (25 mL/per dish) for germination and incubated for one week in a growth chamber or 25°C growth chamber (see Note 2).
( 6 ) After seed germination, the shoot tip is cut into 3- to 5-mm segments with a sterile razor blade (Sigma) (see Note 3).
( 7 ) Place 5-7 cut buds horizontally in a Petri dish (100 mmx15 mm) inverted with MS2 medium and incubate for 4 weeks at 25°C under continuous light condition [ 60 μmol/( m2-s ) using 40W power cool white Econ-owatt fluorescent tubes, Philips Westinghouse, USA ] ( see Note 4 ).
( 8 ) Successional cultures were performed once a week by removing elongated leaves, embryonic sheaths, and stems from the cultured shoot tips, and then cutting them into 3~5 mm segments.
( 9 ) Succession cultures were carried out every 2 weeks with MS2 medium to maintain the growth of clumped shoots [ Fig. 10.2 (a) ]. 
( 10 ) After 8 weeks of culture, calculate the relative frequency of differentiation of clumped shoots for each species, i.e., the percentage of the number of shoot tips that produced clumped shoots compared with the total number of shoot tips in culture (see Note 5).
2. Microparticle bombardment
( 1 ) Take 30 mg of gold powder or tungsten powder and put it into a 1.5 ml centrifuge tube (VWR), add 1 ml of 100% ethanol to sterilize it by high speed vortexing for 30 min, and use it for bombardment with a gene gun.
( 2 ) During the shaking process, 50 μl of particulate ethanol suspension was taken into a new centrifuge tube and centrifuged at 10,000 g in a microcentrifuge (Brinkman, Westbury, NY 11590) for 30 s. Then 1 ml of distilled water was added, washed by vortex shaking and centrifuged. After two washes, the pellet was resuspended with 332 μl of sterile distilled water.
( 3 ) Add 15 μl of DNA sample containing 15 μg of pBY520 and pActl-D plasmids with 1:1 cationic molar ratio, 225 μl of 2.5 mol/L CaCl2 (Sigma), 50 μl of 0.1 mol/L spermidine (Sigma) to the resuspended pellet, and vortex and shake for 5 min at room temperature (see Note 6).
( 4 ) The particulate-DNA suspension was placed on ice for 10 min, and then centrifuged at 10000 g for 1 min.
( 5 ) Wash the particulate-DNA precipitate with 500 μl of 100% ethanol, vortex for 30 s, centrifuge for 1 min, and resuspend the particulate with 100 μl of 100% ethanol.
( 6 ) Depending on the size, place 2~3 clumps of shoots in an area of 1.5 cm in diameter in a Petri dish lined with MS3 medium (25 ml/dish), and then place the dish under the termination screen of the gene gun (see Note 7).
( 7 ) Aspirate 10 μl of microparticle-DNA suspension onto the center of the macrocarriers and use it for bombardment as soon as the ethanol evaporates (see Note 8).
( 8 ) Each shoot tip culture is bombarded again after 2 h (see Note 9).
( 9 ) The bombarded shoot tips are transferred to fresh MS2 medium (25 ml/dish) and grown for 4 weeks at 25°C under continuous light [60 μmol/( m2-s )], during which time a succession culture is performed [Fig. 10.2 (b)].
3. Selection of transgenic tissues
( 1 ) After 4 weeks, the clumped shoots bombarded by the gene gun were divided into small pieces of 5~10 mm in diameter (to avoid damaging the shoot meristematic tissue), and then 6~8 clumps of shoots were placed per dish on MS4 medium (25 ml/dish; 100x15 ), and the relative frequency of transgenic events was calculated (Table 10.1).
( 2 ) After 2 weeks, the transgenic buds (green ones) were succeeded on fresh MS4 medium and the non-transgenic buds (yellow or brown) were removed.
( 3 ) After 4 weeks of selective culture on MS4, the green clumped buds were divided into clusters of 5-10 mm in diameter and grown in succession on MS5 (25 mL/dish, 100x20 ) medium for 4-6 weeks.
( 4 ) After a total of 4 months of selection and proliferation, the fast-growing shoots were transferred to Magenta boxes (99 mmx 68 mm, LxD, Sigma; 2-3 clusters of shoots/Magenta) with MS6 medium (50-70 ml/Magenta) for nutrient growth, and then transferred to MS7 (50-70 ml/Magenta) medium for rooting. ) medium for rooting culture [ Fig. 10.2 ( c ) ].
( 5 ) When the transgenic plants grew to 5~10 cm tall with 2~3 leaves, they were transplanted into 8 cm plastic pots containing soil mixture, in which peat and perlite were mixed at 1 : 1, one plant per pot. They were grown until maturity in a greenhouse with a 16-h photoperiod and 70-80% humidity [Figure 10.2 (d); see Note 10].
( 6 ) The transgenic plants were allowed to self-pollinate and set fruit, the seeds were collected and stored independently by strain at 4°C and 70% humidity. 
4. Genetic analysis of transgenes
( 1 ) After low-temperature treatment, seeds (10-12 seeds per dish) were germinated on MS7 (25 ml/dish) medium for 1 week, which was used to analyze the genetic characteristics of the transgene, such as the genetic stability of the bar gene in the R, R1, and R2 generations (see Note 11).
( 2 ) Alternatively, seeds were germinated and grown for 2 weeks in a greenhouse using porous plastic trays (Griffin Green House and Nursery Supplies) in soil (Metro Mix 360 Soil). Spray the growing plants with herbicide (see Note 12).
( 3 ) After 1 week, count the number of seeds that germinated and did not germinate on the selection medium, or the number of plants that survived and died after herbicide treatment in the greenhouse.
( 4 ) The segregation of transgenes in the R., R1, and R2 generations was analyzed using the X2 test (53 ) (Table 10.2). 
5. Molecular detection of transgenic plants
(1) Polymerase chain reaction (PCR)
Detection of transgenes in transgenic plants (e.g., bar and hav1): DNA was extracted using leaves following the instructions of the REDExtract- N-Amp Plant PCR kit (Sigma-Aldrich, St. Louis, MO, Cat# XNA- P ).
② Forward (F ) and reverse (R ) PCR primers were used: e.g., bar-F: 5'-ATGAGCCCAGAACGACG-3'; bar-R: 5'-TCA GATCTCGGTGACGG-3'; hva1-R: 5'-TGGCCTCCAACCAGAACCAG-3'; hva1-R: 5'- ACGACTAAAGGA ACGACCAG; and hva1-R: 5'-TGGCCTCCAACCAG-3'. ACGACTAAAGGA ACGGAAAT-3'.
③ DNA amplification was performed on a PCR instrument (e.g., Per-kin Elmer/Applied Biosystem, Foster City, CA). Amplification reactions were initiated by denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min.
The PCR products were separated and analyzed on a 0.8% (m/V) agarose gel stained with EB and visualized under UV light. The transgene bands were 0.59 kb for bar and 0.70 kb for hva1 [Figure 10.3(a)]. 
(2) Southern hybridization analysis
Southern hybridization was performed using the sequences of the coding regions of the genes (e.g. hva1 or bar1) as probes [54].
② Genomic DNA was extracted from transgenic and non-transgenic plants using the Phytopure Plant DNA Extraction Kit (Amersham-Pharmacia Biotech).
(iii) Genomic DNA from 10 μg of R0, R1 and R2 generation plants was analyzed by Southern hybridization.
④ Genomic DNA was digested by HindIII and HindIII-BamH I restriction enzymes, and the fragments were separated by electrophoresis on 0.8% agarose gel.
⑤ The gel was denatured and neutralized and blotted onto Hybond-N+ membrane (Amersham-Pharmacia Biotech) following the method described by Sambrook et al [55].
(vi) The pBY520 plasmid was digested with the HindIII-BamH I enzyme, and the fragment with the hva1 coding region was isolated on 0.8% low-melting gel. A 1.0 kb fragment was excised and the fragment was purified using the QIAquick PCR purification kit.
(vii) The purified gene-specific DNA fragments were radiolabeled with the Rad Primer Labeling Kit (GIBC0 BRL) using the method described in the manual.
⑧ Hybridize the prepared membrane with the hav1 gene-specific probe according to the standard procedure [ 55 ].
⑨ Subsequently, the hybridized membranes were analyzed by radiographic autoradiography on X-ray film (Kodak) at -80°C [ Fig. 10.3 ( b ) ].
(3) Northern hybridization analysis
① Total RNA was extracted from the young leaves of transgenic and non-transgenic plants by TRIZOL (GIBCO BRL), as described in the instruction manual.
② 10 μg of total RNA was separated by electrophoresis in 1.2% agarose-formaldehyde denaturing gel according to the method of Sambrook et al.
(iii) The strands were blotted onto Hybond-N+ nylon membrane (Amersham-Pharmacia Biotech) according to the method of Sambrook et al [55].
(iv) The transcription of hva1 was analyzed with a 32P-labeled gene-specific probe according to the standard Northern hybridization procedure [55] [Fig. 10.3 ( c )].
(4) Western hybridization analysis
① Protein extraction, Western hybridization and immunodetection were performed according to the method of Xu et al [48].
② Grind about 0.1 g of fresh leaves of transgenic and non-transgenic plants in liquid nitrogen, and add 0.2 ml of protein extraction buffer to extract proteins.
(iii) The total protein concentration of each sample was determined by the method of Bradford using Bio-Rad protein analysis reagent (Bio-Rad) [56].
The total soluble proteins of 100 μg transgenic and non-transgenic samples were separated by electrophoresis on 12% SDS polyacrylamide gels.
⑤ After electrophoresis, the proteins were electrotransferred onto a nitrocellulose membrane (Amersham-Pharmacia Biotech) using a semi-dry transfer system (Bio- Rad ), as described in the instrument description.
(6) Incubate the Western membrane with primary antibody (e.g., rabbit antiserum) and secondary antibody (rabbit IgG with alkaline phosphatase, Sigma-Aldrich) immunized with the transgene-specific protein (e.g., HVA1).
(vii) Membrane coloration was performed in alkaline phosphatase buffer [ Fig. 10.3 ( d ) ].
6. Biochemical analysis of transgenes
( 1 ) Biochemical analysis of transgenes, such as GUS analysis, was performed on whole plants or different organs of transgenic and non-transgenic plants ( Fig. 10.2 (e )~(h ) ; [ 48 ] ).
( 2 ) To remove chlorophyll, green tissues were soaked in 70% ethanol for 2 h and then treated in 100% ethanol overnight (this step should be done after incubation at 37°C).
( 3 ) Vacuuming was performed to remove gas from the tissues.
( 4 ) The samples were immersed in GUS substrate [ 10 mmol/L EDTA (pH 7.0), 0.1 mol/L NaPO4 (pH 7.0), 1~5 mmol/L X-Gluc; X-Gluc can be dissolved in dimethylsulfoxide; Sigma] , and incubated at 37°C immediately after vacuum incubation ( 57 ) .
( 5 ) To observe the GUS expression site, freehand transverse sections were made and then observed under a Zeiss SV8 stereomicroscope or Zeiss Axioskop conventional microscope.
7. Evaluation of hva1 transgenic plants under stress conditions
(1) Salt stress under in vitro culture conditions
① Take seeds of 50 or 60 seeds per strain of transgenic and non-transgenic; see Note 12) for surface sterilization. Sow 10 to 12 seeds per dish on MS7 medium (25 ml/dish) and incubate in dark culture at 25°C for 4 days for germination (see Note 13). Use MS7 medium without glufosinate or dipropylphosphine for non-GM seeds.
② Split the germinated seeds into two groups, one without salt stress and the other grown under salt stress conditions.
(iii) From each independent transgenic and non-transgenic strain, 4-5 growing seedlings were transferred to MS8 medium (50-70 ml/Magenta) with or without NaCl and grown at 25°C and 60 μmol/( m2-s ) light. A minimum of four replicates was used.
After 6 days, the growth of transgenic and non-transgenic plants with and without salt was observed [ Fig. 10.2 (1) ].
⑤ Measurements such as plant fresh weight (see Note 14), plant length, root length, and dry weight (see Note 15; Table 10.3) were performed. 
(vi) Data were analyzed by ANOVA [58].
(vii) Tukey's difference test was used to compare means at the 95% confidence level.
⑧ Plants were transplanted into a peat: perlite (1 : 1, V/V ) mixture and further grown in the greenhouse.
(2) Salt stress under greenhouse (pot seeding) conditions
① Germinate seeds on MS7 medium according to the method in section 3.7.1.
② Transfer 1-week-old selected transgenic and non-transgenic control seedlings to small pots (8 cmx 4 cmx 6 cm) in a soil mixture of peat:perlite 1:1 (one seedling per pot).
(iii) These pots were placed in flat-bottomed trays with water and grown in the greenhouse for 1 week.
④ The growing seedlings were divided into 5 groups, with 8-10 seedlings from transgenic and non-transgenic strains in each group. The height and number of leaves of each plant were determined before the salt stress treatment.
⑤ Water the plants with different salt concentrations (0 mmol/L, 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L NaCl) once a day for 14 days (see Note 16).
(vi) Subsequently, water the plants for 1 week to allow them to recover.
(vii) After that, the salt stress treatment was repeated for 5 weeks without interruption.
The experiment was repeated at least 4 to 5 times.
⑨ Determine the plant height, root length, number of tillers, and fruiting rate of individual plants. Compare the growth differences between transgenic and non-transgenic plants under stress and non-stress conditions [see Note 1 7; Figs. 10.2 ( j) and 10.2 ( k)].
⑩ Data were analyzed statistically by ANOVA [58].
(11) Tukey's difference test was used to compare means at the 95% confidence level (Table 10.4 ). 
(3) Water deprivation or osmotic stress under in vitro culture conditions
① Seeds (R., R1, R2) were germinated on MS7 medium by referring to the method in section 3. 7.1 (see Note 18).
② Split the germinated seeds into two groups: one group grown under conditions without water deficiency or osmotic stress, and the other group grown under conditions of water deficiency or osmotic stress (see Note 19).
(iii) Transfer 4-5 seedlings from each independent transgenic and non-transgenic strain to MS9 medium with or without mannitol (50-70 ml/Magenta) in Magenta boxes and grow under light at 25°C. A minimum of 4-5 replicates should be grown. Minimum 4-5 replicates.
After 6 days, the growth of transgenic and non-transgenic plants was analyzed under osmotic and non-osmotic stress conditions.
⑤ Determine the growth condition of the plants as in the case of salt stress. The growth differences between transgenic and non-transgenic plants under stress and non-stress conditions were compared (Table 10.3).
⑥ Plants were transplanted into a soil mixture in which peat was mixed with perlite at 1 : 1 ( V /V ) for further growth in the greenhouse.
(4) Water deficit stress experiment under greenhouse (pot seeding) conditions
① Referring to the method in section 3. 7 .1, 30~40 seeds were germinated on MS7 medium (see Note 20).
② 1-week-old, screened transgenic seedlings and non-screened non-transgenic seedlings were used as controls and transferred to small pots (8 cmx4 cmx6 cm) containing a soil mix of peat and perlite 1 : 1. One seedling was planted in each pot.
(iii) The pots were placed in flat-bottomed trays with water and the plants were allowed to grow in the greenhouse for 2 weeks before water deficit treatment.
The plants were divided into two groups, one with water (group a) and the other without water (group b), with 8-10 seedlings of transgenic and non-transgenic strains in each group. The height and number of leaves of each plant were determined before the stress treatment.
⑤ Water was added to the flat-bottomed trays of group a plants for 1 week.
(6) Water was added to the flat-bottomed trays of group b plants for 2 days only.
(vii) After adding water for 2 days, completely remove the water from the pan of group b and leave it in a water-starved environment for the following week.
Repeat the process for 5 weeks.
At the end of the process, the number of tillers per plant was determined. Compare the growth differences between transgenic and non-transgenic plants under stress and non-stress conditions.
⑩ Data were statistically analyzed by ANOVA [58].
(11) Tukey's difference test was used to compare means at the 95% confidence level.