Plant Molecular Biology Reporter 19: 151–158, June 2001
`© 2001 International Society for Plant Molecular Biology. Printed in Canada.
`
`Protocols
`
`Serial Extraction of Endosperm Drillings (SEED) -
`A Method for Detecting Transgenes and Proteins
`in Single Viable Maize Kernels
`
`VARAPORN SANGTONG1, ERIK C. MOTTL1,2, MARY JANE LONG1,
`MICHAEL LEE1 and M. PAUL SCOTT1,2,*
`1Iowa State University, Department of Agronomy, Ames, IA 50011; 2USDA-ARS, Corn
`Insects and Crop Genetics Research Unit, Ames, IA 50011
`
`Serial extraction of
`
`endosperm drillings Sangtong et al.
`Abstract. We have developed a method for detecting a transgene and its protein product in
`maize endosperm that allows the kernel to be germinated after analysis. This technique
`could be highly useful for several monocots and dicots. Our method involves first sam-
`pling the endosperm with a hand-held rotary grinder so that the embryo is preserved and
`capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample
`buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the
`transgene can be detected in the first extract by SDS-PAGE with visualization by total pro-
`tein staining or immuno-blot detection. The second extract can be purified and used as
`template DNA in PCR reactions to detect the transgene. This method is particularly useful
`for screening transgenic kernels in breeding experiments and testing for gene silencing in
`kernels.
`
`Key words: ELISA, gene expression, PCR, transgene, Zea mays L.
`
`Introduction
`
`Because of recent advances in plant transformation research, transgenic crops are
`being incorporated into many breeding and research programs. A major difficulty
`with working with transgenic plants is gene silencing (Kumpatla et al., 1998).
`While transgenes are often inherited in a predicable manner, the expression of the
`transgene often varies in different lines due to position effects or epigenetic ef-
`fects. Thus, it is necessary to monitor inheritance and expression of the transgene
`each generation to verify that the transgene functions properly in the conditions
`tested. Inexpensive, high-throughput methods are needed to meet the demand of
`breeding programs for screening large numbers of plants.
`We have developed a method that allows detection of transgenes and their
`protein products in the endosperm of individual maize kernels. The embryo is not
`
`*Author for correspondence. Present address: 1407 Agronomy Hall, Iowa State
`University, Ames, IA 50011; e-mail: Pscott@iastate.edu; fax: +1 (515) 294-9359;
`ph: +1 (515) 294-7825.
`
`

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`152
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`Sangtong et al.
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`Figure 1. Schematic diagram of the SEED method.
`
`damaged so that the kernels can be analyzed, stored, and subsequently germi-
`nated. Nonlethal sampling methods that retain seed viability are frequently used
`to screen kernel traits. Our method couples nonlethal sampling with serial extrac-
`tion of the excised tissue to obtain extracts suitable for several types of analyses.
`Serial extraction was one of the first methods used to separate and classify seed
`components (Osborne, 1924); it is based on the differential solubility of the com-
`ponents being separated. Coupled with microsampling and analysis methods, se-
`rial extraction is a useful tool in molecular breeding.
`
`Materials and Methods
`
`The protocol is outlined schematically in Figure 1. All liquid handling steps are
`carried out
`in 96-well
`format, which lends
`itself
`to automation for
`high-throughput. All chemicals are molecular biology grade or equivalent. The
`protocol presented here is optimized for analysis of
`the wheat Glu1-Dx5
`transgene in maize. The parameters of SDS-PAGE, ELISA, PCR, and
`immuno-blotting will require optimization for detection of other transgenes and
`proteins.
`
`

`
`Serial extraction of endosperm drillings
`
`153
`
`Grinding apparatus
`
`Maize kernels are ground using a hand-held rotary grinder (Sears Craftsman vari-
`able speed rotary tool, model #61053, Sears Roebuck and Co., Chicago, Illinois,
`USA) with a flexible shaft attachment. A 3 mm2 patch of pericarp is removed
`with sand paper. The kernels are then placed on a small piece of weighing paper
`and kept in place by the operator’s thumbnail. The kernels are held embryo side
`down with the pedicel underneath the thumb. The rotary grinder is used at ap-
`proximately 50% full speed with a #105 Dremel grinding bit. The endosperm is
`carefully ground to avoid damaging the embryo or removing too much of the en-
`dosperm. Typical yields range from 10-20 mg of finely ground endosperm. This
`powder is transferred to a 1.5 mL round-bottom 96-well plate using a small glass
`funnel. Drill bits, funnels and fingers are thoroughly washed between samples to
`avoid contamination. A fresh sheet of weigh paper is used for each kernel. To in-
`crease the efficiency of the process, several funnels and drill bits are used so that
`they do not have to be washed between every kernel.
`
`Protein extraction for SDS-PAGE and immuno-blot analysis
`
`Protein extraction buffer (0.0625N Tris-HCl, [pH 6.8], 3.3% (W/V) SDS, 5%
`(v/v) 2-mercaptoethanol, 10% glycerol, 0.002% Bromphenol blue) is added to
`preweighed samples in a ratio of 10 to 1 µL/mg. One hundred to two hundred
`microliters of protein extraction buffer is added to 10-20 mg of ground endosperm
`in the ratio of 10 µL buffer per 1 mg ground endosperm. The 96-well plate is
`mixed for 10 min using a vortex mixer followed by 2 h of shaking at 1400 RPM
`at room temperature. The 96-well plate is then centrifuged for 5 min at 4000
`g-force; the supernatants are transferred to a new 96-well plate. These extracts are
`heated to 95°C for 5 min and stored at -20°C before analysis. The extracted endo-
`sperm pellet is used for DNA extraction.
`
`SDS-PAGE
`
`Extracted proteins are separated by electrophoresis on a 12% SDS-PAGE
`(Acrylamide/bis 37.5:1) gel (Laemmli, 1970) using a Mini-Protean II Electropho-
`resis Cell from BIO-RAD with a 20-well comb. Five microliters of protein extract
`is loaded in each well. Electrophoresis is carried out at 100 volts for 1.5 h. Gels
`are stained with 0.1% Coomassie Blue-R-250 in 1% acetic acid and 40% ethanol
`for 0.5 h and destained with 40% ethanol and 10% acetic acid for 1-3 h.
`
`Immuno-blot analysis
`
`Immuno-blot detection is carried out using a Bio-Rad Trans-Blot apparatus ac-
`cording to the manufacturer’s directions. Proteins are detected using a monoclonal
`antibody that specifically binds to the transgene product.
`
`DNA extraction
`
`DNA is extracted and purified from the endosperm pellet remaining in the 96-well
`plate after extraction of the proteins for SDS-PAGE. The DNA is extracted in
`300 µL of cell lysis buffer from the Puregene DNA purification kit (Gentra), and
`
`

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`154
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`Sangtong et al.
`
`purification is carried out using the Puregene protocol according to the manufac-
`turer’s directions. Since the purification is carried out
`in microplate format,
`centrifugation is conducted at 4000 g-force for 5 min in a swinging bucket
`microplate rotor. DNA resulting from this procedure is resuspended in 100 µL of
`1 mM Tris HCl, (pH 8.0), 0.1 mM EDTA. The DNA is allowed to resuspend by
`first heating to 65°C for 60 min, then shaking at 300 RPM and 50°C overnight in
`a shaking incubator.
`
`Polymerase chain reaction
`
`The transgene is detected by PCR using 5 µL of the purified DNA as template and
`primers specific to the transgene. A touchdown PCR protocol is used (Senior and
`Huen, 1993). Products were separated on 2% Metaphor (FMC Bioproducts)
`agarose gels and visualized by UV fluorescence of the ethidium bromide-stained
`DNA.
`
`Alternative protocol for ELISA analysis
`
`An alternative protocol can be used to detect the protein by ELISA rather than
`immuno-blot analysis. The protein extraction procedure for ELISA analysis is the
`same as that used for SDS-PAGE and immuno-blot analysis, except that our
`transgene product requires a different protein extraction buffer (70% ethanol,
`61 mM Sodium Acetate, and add 5% [v/v] 2-mercaptoethanol before use) and
`20-30 mg of endosperm is extracted in a ratio of 10 µL to 1 mg. The resulting ex-
`tract can be analyzed by SDS-PAGE and immuno-blot detection as well. The ex-
`tracted endosperm pellet was dried overnight at 4°C and then used for DNA
`extraction.
`ELISA analysis is conducted according to standard protocols (Harlow and
`Lane, 1999). Standards of purified transgene product were included in a range of
`5-120 ng/50 uL.
`
`Seed germination
`
`Because the sampling process damages the pericarp, we took precautions to pro-
`tect the seeds from fungal infection. Before planting in sterile soil, the seeds were
`treated with Chlorothalonil (tetrachloroisophthalonitrile 0.087%, Fung-Onil, Earl
`May). Seeds were germinated in 3" peat pots in the greenhouse and transplanted
`to the field.
`
`Results
`
`We have applied this method to analyze over 2000 kernels from populations of
`maize segregating for a transgene from wheat, the HMW-Glutenin 1Dx5 gene
`(Anderson et al., 1989). In wheat, expression of the glutenins is normally tissue
`specific (Shewry, 1995) with expression confined to the endosperm. We antici-
`pated a similar expression pattern in maize given the genetic similarity of these
`organisms (Bennetzen and Freeling, 1993).
`
`

`
`Serial extraction of endosperm drillings
`
`155
`
`Figure 2. (A) Characterization of proteins from endosperm drillings by SDS-PAGE with Coomassie
`Brilliant Blue staining. Lane 1: nontransgenic maize kernel. Lanes 2-11: transgenic maize kernels.
`Lane 12: wheat variety L88-6, which contains the 1Dx5 subunit. The same endosperm drillings were
`used to make Figures 2B and 2C. (B) Immuno-blot detection of SDS-PAGE-separated proteins. Lanes
`are labeled as in Figure 2A. (C) Detection of the transgene by PCR, agarose gel electrophoresis, and
`ethidium bromide staining. Lanes are labeled as in Figures 2A and 2B, except Lane 12 contains
`plasmid DNA of the construct used in transformation. DNA was purified from the same endosperm
`drillings used to make Figures 2A and 2B.
`
`Protein analysis
`
`In order to determine if the transgene was functional in maize endosperm, we
`tested for the accumulation of the protein it encoded using SDS-PAGE (Laemmli,
`1970). This method is sensitive enough that the drillings from a single mature ker-
`nel (about 20 mg) can be analyzed. The transgene product was sufficiently abun-
`dant to be visible by Coomassie Brilliant Blue staining (Figure 2A). In order to
`confirm that the bands in the Coomassie-stained gel were the product of the
`transgene, we ran duplicate gels and visualized one gel using immuno-blot detec-
`tion (Figure 2B) using a monoclonal antibody specific to wheat high molecular
`weight glutenin subunits. A band in the position of
`the prominent
`Coomassie-stained band was detected by this method, as well as a weaker band
`just below it. This lower band may be a product of proteolysis.
`The variation of the protocol utilizing ELISA analysis to detect the trans-
`gene product is valuable because it yields a quantitative measure of the target pro-
`tein (Table 1). Using ELISA, we were able to detect the transgene product in
`kernels that were scored negative by SDS-PAGE (e.g. Table 1, Sample 5).
`
`

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`156
`
`Sangtong et al.
`
`Table 1. Analysis of segregating F4 kernels using ELISA in the SEED
`protocola.
`
`Samples
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15 nontransgenic corn
`16 wheat
`
`17 plasmid
`
`1Dx5 concentration
`(ng/mg seed ± SD)b
`
`SDS-PAGE
`
`PCR
`
`-14.5 ± 3.6
`50.3 ± 7.3
`-9.3 ± 3.6
`55.5 ± 7.3
`6.2 ± 3.6
`42.5 ± 3.6
`55.5 ± 7.3
`-9.3 ± 3.6
`-11.95 ± 0.0
`-9.3 ± 3.6
`-9.3 ± 3.6
`-9.3 ± 3.6
`55.5 ± 7.3
`-6.7 ± 0.0
`-9.3 ± 3.6
`50.3 ± 7.3
`
`-
`+
`-
`+
`-
`+
`+
`-
`-
`-
`-
`-
`+
`-
`-
`+
`
`-
`+
`+
`+
`+
`+
`+
`-
`-
`-
`-
`-
`+
`-
`-
`+
`
`+
`
`aThese samples are different from those used in Figure 2.
`bWe interpret negative values to indicate no detectable expression.
`
`Genotype analysis
`
`A common goal is to develop transgenic lines that are homozygous for the trans-
`gene. As we advance plants in this breeding program, there are at least two reasons
`why a kernel may not express the transgene. First, it may not have inherited the
`gene and, second, the gene may have been silenced. To distinguish between these
`two possibilities, it was necessary to determine if the transgene was present in the
`kernels lacking expression of the transgene as measured by SDS-PAGE. After ex-
`traction for protein analysis, the kernel drillings contained enough DNA to extract
`and use for PCR analysis. We found that all kernels expressing the protein were
`positive in the PCR analysis, but some kernels that did not contain the protein con-
`tained the gene, while others did not. We concluded that kernels lacking both the
`gene and the protein did not inherit the transgene; while, in kernels containing the
`gene without detectable protein, the transgene was silenced. Caution is needed
`when interpreting PCR results, because the endosperm tissue will normally be con-
`taminated with small amounts of pericarp tissue, which could contain the transgene
`when the corresponding endosperm does not. Because pericarp tissue is present in
`very small quantities relative to endosperm, it should be possible to adjust the PCR
`conditions to distinguish between bands resulting from endosperm DNA and those
`resulting from pericarp DNA by comparing band intensities.
`
`Germination of analyzed seeds
`
`The sampled seeds were planted using sterile soil in a greenhouse before trans-
`planting to the field. Of 236 seeds planted, 191 (81%) germinated. Our normal
`
`

`
`Serial extraction of endosperm drillings
`
`157
`
`germination rate is about 90%, so we conclude that our sampling procedure has
`a minor impact, if any, on the ability of sampled seeds to germinate in carefully
`controlled conditions.
`
`Discussion
`
`In most high-throughput seed analysis strategies, grinding the seed requires a sub-
`stantial amount of labor. The serial extraction strategy employed by this method
`minimizes the grinding requirement by using the same ground material for several
`types of analysis. This method is particularly useful for the analysis of transgenes
`targeting cereal kernel quality traits, which often direct expression specifically in
`the endosperm. The method is applicable to any transgene containing a unique
`DNA sequence and encoding a product that is detectable in seeds by ELISA,
`immuno-blotting or total protein staining of SDS-PAGE. ELISA has the advan-
`tages of being quantitative and the throughput is higher than with immuno-blot
`analysis. Nontarget proteins that react with the antibody can interfere in this type
`of assay, so it is important to verify the specificity of the antibody. The throughput
`of the method is sufficiently high to be useful in a breeding program. One person
`can analyze 192 kernels in 4-5 d (two 96-well plates). Grinding the tissue takes
`the majority of the time.
`Nondestructive seed sampling methods have been in use for many years
`with several plant species because they are rapid and planting decisions can be
`based on the results of the analysis. This method includes these advantages and,
`in addition, the serial extraction allows it to give information both about the inher-
`itance and the expression of the transgene. This is particularly useful in situations
`where gene silencing is common.
`We have tested the method with corn; however, it also should be applicable
`to other cereals. It will be interesting to determine if this method could be applied
`to large-seeded dicots by sampling the cotyledons in place of the endosperm.
`
`Acknowledgments
`
`The authors wish to thank Dr. Roger Fido for the gift of the 1Dx5 antibody and
`Dr. Daniel Moran for his comments on the manuscript. This paper is a joint con-
`tribution from the Corn Insects and Crop Genetics Research Unit, USDA-ARS,
`and The Department of Agronomy, Iowa State University. Journal Paper No.
`J-19256 of the Iowa Agricultural and Home Economics Experiment Station,
`Ames, Iowa. Project no. 3495, and supported by Hatch Act and State of Iowa
`funds.
`
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