High-efficiency genetic transformation of maize by a mixture of

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 83, pp , February 1986 Genetics High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA (induced gene transfer/endosperm/aleurone color/anthocyanin pigmentation/gene expression) YASUO OHTA Institute of Agriculture and Forestry, University of Tsukuba, Ibaraki 305, Japan Communicated by Hitoshi Kihara, September 3, 1985 ABSTRACT High-efficiency genetic transformation was induced in a genetic stock of maize, Zea mays Linnaeus, by self-pollination of the recipient plants along with DNA of the donor. The highest frequency of transformed endosperm per ear was 9.29%. DNA was applied onto silks in a pollen/dna pasty mixture. The exogenous DNA transferred into endosperm expressed itself in endosperm formation. It is not known, however, whether the exogenous DNA segment in endosperm had been incorporated or whether it was present in the nucleus additionally, fragmentally, or in some other state. It was revealed that all of four possible cases had occurred as follows: exogenous DNA was transferred into (i) both embryo and endosperm of the same kernel, (it) embryo only, (ii) endosperm only, and (iv) neither embryo nor endosperm. It was also revealed that exogenous DNA that had entered into the embryo had been maintained through embryo formation, germination, vegetative growth and differentiation, and reproductive growth, and finally was manifested in embryo and/or endosperm of the following generation and acted in endosperm formation. The frequency of transformed endosperm in this generation, however, was rather low. It may be that exogenous DNA taken into the embryo was unstable. Genetic transformation in higher plants has rarely been reported, and obtaining high-efficiency transformation seems to be difficult. Genetic transformation induced by exogenous DNA given to germinating seeds was reported in Petunia hybrida Linnaeus (1) and Capsicum annuum Linnaeus (2). The same phenomenon in higher frequency after treatment by the same method was reported for Zea mays Linneaus (3), as was gene transfer by the use of sublethally irradiated pollen (4). Ohta and Sudoh have observed genetic transformation in C. annuum upon applying exogenous DNA to pollen grains and to stigmata at the time of pollination (5). The present study reports a similar experiment with maize. Here I describe a unique method to produce high-efficiency genetic transformation through pollen. The results presented also demonstrate that exogenous DNA has entered into both embryo and endosperm and its genes have been expressed. MATERIALS AND METHODS Materials. Maize genetic strains of the genetic stock familiar to most maize geneticists and cytogeneticists were provided by E. Amano (The Gamma Field, Japan); they had originally been obtained from H. H. Smith (Brookhaven National Laboratory, New York). The strains possessed in the homozygous state either all dominant alleles (the DNA donor) or all recessive alleles (the DNA recipient) of the following marker genes with the endosperm characters, on the full background for aleurone pigmentation such as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. 715 homozygous A1, A2, and R genes: C1 (abbreviated hereafter as C), a basic gene for anthocyanin pigmentation on aleurone tissue, located on chromosome 9 at position 26; Cl (I), an inhibitor of and dominant over C; sh1 (sh), shrunken endosperm, on chromosome 9 at 29; bzl (bz), modifier of aleurone color to bronzy, on chromosome 9 at 31; wxl (wx), waxy endosperm, the starch is amylopectin (very little amylose) and stains red with iodine, chromosome 9 at 59; pr, purple aleurone, on chromosome 5 at 46; Y1 (Y), yellow endosperm, presence of carotinoid pigments, with dose effect, on chromosome 6 at 17. Interactions of genes controlling anthocyanin and related pigments in aleurone tissue have been extensively studied and are well known (6). DNA Preparation. Seeds of the donor strain were sown in vermiculite in pots that were kept in a growth cabinet at 250C under 3000 lux of light (16 hr). One-month-old plants with three or four leaves were quickly frozen in liquid nitrogen and then crushed into a powder with a mortar and pestle. DNA was prepared by the method reported by Oono and Sugiura (7). The average size of DNA prepared was in the neighborhood of 14 x 106 daltons, which was estimated by agarose gel electrophoresis. The yield of DNA was 2.6 gg/g (fresh weight) of leaves. Application of DNA. DNA was suspended in 0.3 M sucrose (40 gg ofdna per ml) for adjusting the osmotic pressure: d of the solution was applied to each ear. A certain amount of fresh pollen of a recipient plant, taken at the time of full flowering, was immersed in the DNA solution, and the pasty pollen grain/dna mixture was placed, either immediately (less than 0.5 min) or about 5 or 10 min later, onto the silks of the same recipient plant, thus producing self-pollination (Fig. 1). Another method of DNA application was placing only DNA solution on the silks, and then pollinating as usual. Self-pollination without DNA was performed as control. Ear bags and tassel bags were applied as usual in all pollination, whether with or without DNA. RESULTS Hybridization Experiment as Control. Plants of the donor and the recipient strains were grown in the field in the year preceding the DNA application experiment. Selfing of both strains and ordinary crossing between them were carried out as controls. Ears of selfed donor and selfed recipient, as well as the recipient ears pollinated with pollen of the donor strain, served as the checks to the DNA-applied selling described below. The phenotype of the donor strain was colorless aleurone and nonshrunken (round) endosperm with yellow nonwaxy starch (Fig. 2a), whereas that of the recipient strain was bronzy red aleurone and shrunken endosperm with white waxy starch (Fig. 2b). However, the F1 hybrid between the recipient strain as the female parent and the donor strain as the pollen parent possessed a distinctly different phenotype: colorless aleurone and nonshrunken (round) endosperm with white or faintly yellow nonwaxy starch (Fig. 2c).

2 716 Genetics: Ohta Proc. Natl. Acad. Sci. USA 83 (1986) Ia - b - c- d - e - f- FIG. 1. Pollen/DNA paste applied to silks. FIG. 2. Ears showing endosperm phenotype on the cob. (a) Donor, selfed. Kernels have colorless aleurone, nonshrunken (round) endosperm, and yellow nonwaxy starch. (b) Recipient, selfed. Kernels have bronzy red aleurone, shrunken endosperm, and white waxy starch. (c) F1 hybrid between the recipient (b) as the female and the donor (a) as the pollen parent. Kernels have colorless aleurone, nonshrunken endosperm, and white or faintly yellow nonwaxy starch. (d) Ear 7, obtained by donor DNA-applied selfing of the recipient. Transformed kernels, near bottom, with colorless aleurone but shrunken endosperm and white waxy starch (see Fig. 4). All other kernels are normal, same as the recipient (b). (e) Ear, test-crossed, of plant 10, derived from a kernel with no phenotypic change on ear 5 (see Table 3). Three colorless but shrunken kernels (small pencil circles on the seed coat) can be seen among normal kernels on this side of the ear. (f) Ear, test-crossed, of plant 12, derived from another kernel with no phenotypic change on ear 5. Two unusually small kernels of nonbronzy color can be seen. FIG. 3. Examples of ears with only one or a few kernels set. Those kernels thus obtained have not been used as material for the DNA application experiments in following years, to avoid contamination. Selfing of the Recipient Plants with Donor DNA. Altogether, 40 ears were harvested from 43 pollinations; they are listed in Table 1 according to the mode of DNA application. Class 1,

3 Genetics: Ohta Proc. Natl. Acad. Sci. USA 83 (1986) 717 Table 1. DNA-applied pollination in Zea mays No. ears No. kernels Total no. Kernels with Polli- per ear kernels transformed Class nated Set Range Mean obtained endosperm 1. Pollen/DNA paste, immediate Pollen/DNA paste, -5 min later Pollen/DNA paste, "10 min later DNA only, then pollination Pollination without DNA (control) in which pollen grains were mixed with the DNA solution and the pollen/dna paste was immediately placed onto the silk, gave rise to the highest kernel set: the mean number of kernels per ear was 135.8, close to that of the control, In addition, only this class gave rise to transformed endosperm, which was found on four ears (cobs). Application of DNA/pollen paste about 5 or 10 min later (classes 2 and 3) gave rise to poorer kernel set than that with immediate application (class 1). Application of DNA solution to the silk followed by ordinary pollination with pollen of the same plant (class 4) gave rise to the poorest kernel set. Classes 2, 3, and 4 did not give rise to kernels with transformed endosperm at all. Several ears of the poorest kernel set are shown in Fig. 3. Analysis of the Transformed Endosperm. All kernels produced on class 1 ears with transformed endosperm were examined and classified into several categories according to the endosperm phenotype (Table 2). The ear (cob) obtained by self-pollination of the recipient line with donor DNA, when transformation was induced, gave rise to a quite different phenotype from either line or the F1 hybrid, as clearly seen in Fig. 2d. The ear was actually composed of kernels with several different phenotypes. The majority of the kernels was the same as those of the recipient strain, indicating no sign of transformation. The most distinct phenotype was colorless or nearly colorless aleurone but shrunken endosperm with white waxy starch, indicating that only a single gene (1) had been transferred. Fig. 4a shows kernels with colorless aleurone maintaining waxy starch, which were stained red by the iodine solution. A colorless and nonshrunken kernel revealed that this particular endosperm had received both I and Sh genes together. On the other hand, all other cases, such as colorless or bronzy purple, involved a single gene. The percentage of transformed endosperm obtained was 9.29 for ear 7, 7.40 for ear 5, 4.05 for ear 15, and 1.56 for ear 3. Analysis of Transformed Embryo. Since the transformed kernels were the results of double fertilization, there was a possibility that certain kernels, including those with no phenotypic change in endosperm, possessed embryos that Table 2. Classification of transformed endosperm No. kernels Genotype Ear Ear Ear Ear Endosperm phenotype inferred Colorless, nonshrunken I C C*, Sh sh sh Colorless ICC Nonbronzy Bz bz bz Nonwaxy Wx wx wx Bronzy purple Pr pr pr Yellow endosperm Yyy No phenotypic change Total no. of kernels *Other states such as I C C C, for example, might be possible, when I is additional, fragmental, or in some other state. had received a portion of donor DNA, although it was phenotypically unidentified. To examine such a possibility, all the transformed kernels classified by their phenotypes, of ears 5 and 7, as well as several randomly selected kernels of normal appearance from the same ears, were sown and grown into the next generation. Every ear of the plants thus grown was test-crossed-that is, pollinated with pollen of the recipient strain. The results are given in Table 3. The colorless and nonshrunken kernel, the only case of the recipient of two genes, did not germinate. Colorless kernels of ear 5 and bronzy purple kernels of ear 7 also did not germinate. On the other hand, the bronzy purple kernel of ear 5 did germinate, but the plant was dwarf; it flowered 3 weeks later than the others and gave small empty anthers, indicating that this individual must be haploid. Four plants were grown from the kernels with no phenotypic change in endosperm of ear 5. Individual 10 has set two ears; one bearing 8 kernels with colorless endosperm as well as 250 kernels of normal appearance, and another bearing 137 kernels of only normal appearance. Individual 12 gave rise to three nonbronzy and very small kernels (Fig. 2J) among 255 kernels. Individual 9 gave rise to one nonbronzy kernel. Regarding ear 7, seven plants were grown up from the 17 colorless kernels. Among them, three individuals, nos. 2, 9, and 13, gave rise to colorless kernels, while one individual, no. 5, gave rise to a nonbronzy kernel, revealing that the former three had received exogenous DNA for the gene I into the egg cell, thus into the embryo, and the latter had received exogenous DNA for Bz. None of the eight bronzy purple kernels germinated. On the other hand, 10 plants were grown up from kernels without phenotypic change in endosperm character. Among them, three individuals, nos. 15, 18, and 20, gave rise to colorless kernels, revealing that they had received exogenous DNA for I into the embryo, too. The other individual, no. 17, was dwarf, late maturing, and haploid, while another individual, no. 5, was also late maturing. DISCUSSION Precautions to Prevent Contamination. For this kind of experiment, it is most important to take precautions to prevent contamination and other mistakes. To prevent every possibility of receiving even a single pollen grain from the donor strain, the experiments were carried out in the following manner: (i) Hybridization between the recipient and the donor strains and selfing of both strains were carried out one year before the DNA application experiments. All the kernels thus obtained were never used as material for the DNA application experiments. (it) For the donor DNA-applied selfing of the recipient plants in the following year, the recipient strain was grown at a place isolated from any other experimental plants. (iii) Both ear bags and tassel bags were nevertheless applied as usual to secure controlled pollination. (iv) All the donor strain plants were, on the other hand, grown in a cabinet in a room, and 1-month-old plants, far before meiosis or reproductive cell division, were used for the DNA extraction.

4 718 Genetics: Ohta Proc. Natl. Acad. Sci. USA 83 (1986) FIG. 4. Starch type-i.e., waxy or nonwaxy-is examined by removing a small portion ofboth seed coat and aleurone tissue and then applying iodine solution. (a) Colorless kernels of ear 7 (Fig. 2d) were stained red and thus identified to be waxy. (b) Nonwaxy kernels, stained blue, of the donor. It can be said, therefore, that any donor trait found among recipient traits in the DNA-applied self-pollination in these experiments must be caused by the donor DNA. Were These Particular Kernels Really Transformed? Ifall of Table 3. Gene expression of exogenous DNA in the next generation (M2) Test-cross results Donor No. of No. of Individual Total no. No. kernels with Endosperm gene kernels plants identification kernels transformed Ear no. phenotype involved sown grown nos. obtained endosperm Remarks 5 Colorless and nonshrunken I, Sh 1 0 Colorless I 2 0 Bronzy purple Pr Dwarf, very late, haploid No phenotypic change colorless 137 All normal nonbronzy and very small nonbronzy 7 No analysis because of disease 7 Colorless I colorless colorless colorless nonbronzy 3, 7, All normal Bronzy purple Pr 8 0 No phenotypic change colorless colorless colorless 17 Dwarf, late, haploid 5 Late 1, 2, 4, 8, Al normal Recipient strain (control) All normal

5 Genetics: Ohta the genes concerned were transferred together by DNAapplied selfing-in other words, if a full set of genes was carried by donor DNA into an embryo sac and double fertilization took place as usual-the resulting embryo or endosperm should carry a full set of the genes. Then, the endosperm phenotype would be the same as that of the F1 hybrid. There was no such case. If, instead, only a small fragment of DNA carrying a single gene was transferred, the resulting endosperm phenotype would. differ from all three, the parents as well as the F1 hybrid and the phenotype must be determined case by case according to which gene was transferred. Such cases were found in the present experiments as shown in Figs. 2-4 and in Tables 2 and 3. Thus, this experimental system proved effective in identifying individual instances among the several different possible phenotypes according to the number of transferred genes. Gene Expression of the Exogenous DNA in Endosperm. The results given in Table 2 clearly demonstrated the following two facts: (i) exogenous DNA in the DNA-applied selfpollination was transferred through pollen into the embryo sac, and therefore into endosperm nuclei, and (it) exogenous DNA expressed its gene action during endosperm formation. It is not known, however, whether the transferred exogenous DNA was incorporated into the normal gene, as in the state I C C, for example, or whether it was present in the nucleus additionally, fragmentally, or in some other state-e.g., I C C C. The possibility of obtaining colorless aleurone as a result of losing C gene(s) instead gaining an I gene can be rejected, since the genotype of the pollen parent used for the test cross was C C homozygous. Gene Expression of the Exogenous DNA in Embryo. As maize kernels are the results of double fertilization, the endosperm phenotype can be observed directly on the cob. On the other hand, the gene expression of the exogenous DNA transferred into the embryo cannot be observed on kernels on the cob; the kernels must be sown, and the following generation of plants must be grown to identify the gene expression. There exist four possible cases: exogenous DNA is transferred into (i) both embryo and endosperm of the same kernel, (ii) embryo only, (iii) endosperm only, and (iv) neither embryo nor endosperm. Proc. Natl. Acad. Sci. USA 83 (1986) 719 With these considerations in mind, kernels with transformed trait(s) were classified and sown separately according to the phenotype. Kernels with no phenotypic change but on the same cob with transformed kernels were randomly selected and also sown. Gene expression of exogenous DNA on endosperm phenotype in the following generation was revealed by examination of the kernels on the cob obtained by test-cross. It was clearly demonstrated that all of the four possible cases occurred. It must be stressed that the fact that cases i and ii were confirmed to have taken place has special significance, since they demonstrate that exogenous DNA transferred into the embryo had passed through growing stages such as embryo formation, germination, vegetative growth and differentiation, and reproductive growth, and finally was manifested in embryo and/or endosperm of the following generation, and thus had acted as a gene in endosperm formation. The frequency of transformed endosperm in this generation, M2, however, was not so high as in ordinary inheritance through sexual reproduction. In other words, it may be said that exogenous DNA taken into the embryo can be inherited to the next generation but it will be unstable and often will be lost. Side Effects of Exogenous DNA. Mutagenic effects of exogenous DNA such as extremely tiny kernels, nongerminating kernels, high ratio of haploidy (2/22 = 9%, as compared to spontaneous haploidy in this material of 10-5) were observed in the course of this experiment. The DNA preparation was made by Dr. H. Uchimiya (Institute of Biological Science, University of Tsukuba), to whom I am thankful. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. 1. Hess, D. (1969) Z. Pflanzenphysiol. 60, Nawa, S., Yamada, M. A. & Ohta, Y. (1975) Jpn. J. Genet. 50, Korohoda, J. & Strzalka, K. (1979) Z. Pflanzenphysiol. 94, Pandey, K. K. (1983) Mol. Gen. Genet. 191, Ohta, Y. & Sudoh, M. (1980) Jpn. J. Breed. 30, Suppl. 2, Neuffer, M. G., Jones, L. & Zuber, M. S. (1968) The Mutants of Maize (Crop Science Society of America, Madison, WI). 7. Oono, K. & Sugiura, M. (1980) Chromosoma (Berlin) 76,