CA2004378C - Method for increasing the ability of a plant species to undergo androgenesis, and products produced therefrom - Google Patents

Abstract

ABSTRACT

This invention provides a method for producing germplasm of plant species exhibiting enhanced response to in vitro culture of male gametophytes. The method is used as a selection criterion for genes favoring in vitro androgenesis. After subjecting anthers to standard anther culture regeneration procedures, the regenerated plants are intermated and self-pollinated to generate valuable genetic variability for improved culture response. The transfer of increased male gametophyte culturability to other selected germplasm is also described.

37,427A-F

Description

20Q~3"~8 METHOD FOR INCREASING THE ABILITY OF A PLANT SPECIES TO
UNDERGO ANDROGENESIS, AND PRODUCTS PRODUCED THEREFROM
This invention relates to a method of producing novel germplasm of plants capable of increased levels of haploid and/or double haploid formation from cultured male gametophytes.
It is of great agricultural and economic interest to provide new plants which display an improvement in particular characteristics. Through proper breeding techniques, these characteristics can be introduced into new or existing genotypes of plant species which can then be marketed directly or used to produce superior hybrid plants.
The development of a hybrid variety conventionally involves three steps: (1) the selection of superior plants from various germplasm pools; (2) the selling of the superior plants for several generations to produce a series of inbred lines, which although different from each other, breed true and are highly uniform; and (3) the crossing of selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F1). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between any two inbreds will always be the 37~u27A-F -1-2QO~~~i'"~8 same. Once the inbreds that give the best hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parent is maintained.
Improvements by selective breeding have been relatively slow, since only a limited number of generations of plants may be propagated each year.
Therefore, relatively minor improvements in plants have been obtained only after years of rigorous work.
There has been much discussion about the potential utilization of haploids in plant breeding.
Since plant breeding is concerned with the development of genotypes to use directly or as parents of productive hybrids, the rapid advance to homozygosity which accompanies the doubling of haploids is an attractive feature. However, attempts at utilizing haploids in plant breeding have been frustrated by the lack of a reliable means of generating sufficient numbers of doubled haploid lines from a broad spectrum of commercially-important germplasm.
Numerous studies on the in vitro culture of ameto h tie cells with the aim of g p y producing haploid plants have been reported during the last two decades.
A large number of reviews, book chapters and symposia proceedings have been published as well [see generally Chu, "HaploidsinPlantlmprovement", In IK Uasil, WR Seoweroft, KJ Frey (eds.), Plant Improvement and Somatic Cell Genetics, New York: Academic Press, 1982, pp. 129-158; Heberle-hors, "In VitroHaploidFormationo~'Pollen:
A Critical ReUiew", Theor . Appl . Genet . ~, 361-374 ( 1985 ) ;
37,427A-F -2-~(~0<~~i'~8 and Hu and Yang, "HaploidsofHigherPlantsinVitro" Berlin, Heidelberg, Springer-Verlag, (1986)].
Early events during in vitro culture have been characterized at the cytological, ultrastructural and biochemical level [see Chen et al., "Segmentation Patterns and Mechanisms of Genome Multiplication in Cultured Microspores of Barley", Can. J. Genet. Cvtol. 26, 475-483 (1984);
Raghavan, "Protein Synthetic Activity during Normal Pollen Development and During Induced Pollen Embryogenesis in Hyoscyamus niger", J. Can Bot. 62, 2493-2513 (1984); and Huang, "Ultrastructural Aspects of Pollen Embryogenesis in Hordeum, Triticum and Paeonia" I n Hu H , Yang H ( eds ) : "Haploids of Higher Plants in Vitro", Berlin, Heidelberg: Springer-Uerlag, ( 1986)].
In vitro culture involves isolating immature anthers from plants and placing the anther or, male gametophytes isolated therefrom, onto a medium which induces the male gametophytes, which would normally be destined to become ollen p grains, to begin dividing and forming a cell culture from which plants can be regenerated. This method is known as androgenesis. The resulting cultures are haploid and contain only a single set of chromosomes from the original plants. The plants derived from these cultures are sterile unless chromosome doubling occurs, either spontaneously or by induction, to create doubled haploids which are fully fertile and completely inbred.
3 ~J
Anther culture provides a method for culturing male gametophytes (mierospores or young pollen gametophytes derived therefrom) directly in the anther.
A positive in vitro response will lead to the development of embryos and/or callus from which plants can be regenerated. For a general discussion of anther 37,427A-F -3-2Q0~~~'~8 _4-culture, see J. M. Dunwell, "Anther andDuaryCulture", In SWJ Bright and MGK Jones. (eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher, 1985, Dordreeht , pp . 1-44 ; and "Haploids from gametophytic cells -recentdeuelopmentsandfutureprospects", In CE Green, DA Somers, WP Hackett, DD Biesoer (eds.), Plant Tissue and Cell Culture, Laln R Liss, New York, pp 223-241).
Anther culture has been employed to obtain male gametophyte-derived callus, embryos and plants in well over 200 species [Maheshwari et al . , "Haploids from Pollen Grains-Retrospect a.nd Prospect", Amer . J . Bot . 6~, 865-879 (1982)]. However, the anther culture responsiveness varies considerably among species. A comparison of the overall responsiveness of anther culturability is made difficult, as the results reported in published studies are given in different bases. For example, anther culturability has been defined by the induction of male gametophytes that begin dividing, the number of embryos and/or callus per anther, the percentage of anthers producing at least one embryo and/or callus, the number of haploid plants regenerated, and the number of double haploid plants recovered.
The highest yield of responding anthers (anthers forming embryos and/or callus per 100 anthers plated) was found to be 87 percent in wheat [see A. M.
Wei , "Pollen Callus Culture in Triticum aertiuum", Theor. Appl .
Genet. 6~, 71-73 ( 1982)], 67 percent in rice [see S. L. Lin and H. S. Tsay, 1983, J. Air. Res., China, c i ted i n J . M. Dunwel l , "Anther and Ouary Culture", In SWJ Bright and MGK Jones, (eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher], 17 percent in maize [see Ting et al., "Improved Anther CultureofMaize" (Zea mays L.), Plant Seienee Lett. 2~, 139-145 (1981)] and 1 37,427A-F -4-~o0<~~~8 _5_ percent in barley (see Z. H. Xu and N. Sunderland, "Innoculation Density in the Culture of Barley Anthers", Sc i ent .
Sinic. 25, 961-968 (1982)]. In rye, 43 developing structures per 100 anthers were observed [see G. ~aenzel a t al . , "Increased Induction and Chromosome Doubling of AndrogeneticHaploidRye", Theor. Appl. Genet. 51, 81-86 (1977)]. Concerning plant regeneration, J. F. Petolino and A . M. Jones ["Anther Culture of Elite Genotypes of Maize", Crop Sci. 26, 1072-1074 (1986)] describe for maize that from 234 embr oils (of different y genotypes) transferred to regeneration medium, 43 developed into plants.
Frequencies of calli producing green plant per 100 cultured anthers are 72 percent in wheat [see J. W.
Ouyang et al . , "The Response of Anther Culture to Temperature in TriticumAestiuum", Theor. Appl. Genet. 66, 101-109 (1983)], in 12 percent rice [see L. J. Chen et al., "Medium Evaluation forRieeAntherCulture", in A. Fu jiwara (ed. ), "Plant Tissue Culture", pp. 551-551 . Jap. Assoe. Plant Tissue Culture Tokvo (1982)] and 10 percent in barley [see K. N. Kao, "Plant Formation from Barley Anther Cultures with FicollMedia", Z. Pflanzenzuchtg. 10 , 437-443 ( 1981 )].
Although relatively rapid progress has bean made in several species, many species of plants, unfortunately, have not shown detectable or significant anther culturability. The culture of intact anthers containing haploid male gametophytes has been used to obtain regenerable maize tissue cultures. However, production of positive results in maize anther culture has been particularly slow [see Nitsch et al., "Production o f Haploid Plants Zea mays and Pennisetum through Androgenesis", I n ED Earle, Y Demarley (eds.) Uariability in Plants Res~enerated from Tissue Culture, Prager Publishers, New York, 1982, pp. 69-91); Genovesi and Collins, Crop Sci.
37,427A-F -5-;goo<~~~~

22, 1137-1144 (1982); and Petolino and Jones, Crop Sci.
26, 1072-1074 (1986)].
However, response frequencies in cultured maize anthers have been very low in all but a few genotypes [see Ku a t al . , "Induction Factors and Morpho-cytological Characteristics of Pollen-derived Plants in Maize", ( Zea mays L . ) Proe Symp Plant Tissue Cult , (1978) Science Press, Peking, pp 35-42; Genovesi et al., "InuitroProductionof Haploid Plants o~'Corn via Anther Culture", Crop Se i . 22 , 1 137-1144 (1982), Dieu and Beckert, 1986; and Petolino and Jones, "AntherCultureofEliteGenotypeso~'Maize", Crop Sei. 26, 1072-1074 ( 1986 ) ] .
Maize genotypes differ with respect to their amenability to anther culture (Petolino and Jones, 1986, supra) suggesting that genetic factors are important in determining the level haploid production. Any attempt to use anther culture in commercial breeding will require a considerable improvement in the overall efficiency of doubled haploid seed recovery. Generally, the major problems in the use of anther culture have been the relatively low response frequencies and the difficulties associated with plant regeneration and chromosome doubling in all but a few genotypes.
However, the widespread use of anther culture of maize is inhibited, in part, by the low response frequency. It has been well documented that induction frequencies for anther culture systems are much greater than the frequency of appearance of macroscopic structures such as embryos or callus (i.e., survivability). This phenomenon of high induction and low survivability has been demonstrated in anther culture of maize [Peseitelli and Petolino, Plant Cell 37,427A-F -6-2Q0~~3'~8 _7_ Rep. 7, 441-444 (1988); and Pace et al., Theor Appl Genet ~, 863-869 ( 1986 ) ] .
Furthermore, it is generally recognized that abortion of induced male gametophytes represents a serious limitation to the achievement of high anther culture responses [see Sunderland and Dunwell, (1977) In: Street HE (ed) Plant Tissue and Cell Culture, University of California Press, Berkeley, pp 223-265;
Wenzel et al., Mol. Gen. Genet. 1~, 293-297 (1975); and Wernicke et al., Z. Pflanzenphysiol. 81, 330-340 ( 1978)].
Competition among male gametophytes for space and nutrients in the anther and the potential for inuitro selection and genetic transformation make the culture of isolated male gametophytes desirable. Isolated male gametophyte culture provides an alternate, readily available source of free haploid cells [see Sunderland and Dunwell, (1977), supra].
However, isolated male gametophyte culture requires a species demonstrating a relatively high response frequency of anther culturability.
Consequently, many species have not heretofore proven capable of being susceptible to isolated male gametophyte culture.
It was shown for Datura inoxia and Nicotiana tabacum that the androgenic response could be increased substantially by culturing isolated miorospores directly [see Sunderland and Dunwell, (1977), supra].
However, most reports of successful plant regeneration from in vitro cultures of maize nave involved relatively organized tissue cultures [see Hodges, TK et 37,427A-F -7-20U<~3'~8 al . , ( 1986 ) The Potential of Tissue Culture ~'or Maize Improvement, In: Shannon JC. Knieval DP, Boyer CD (eds) Regulation of Carbon and Nitrogen Reduction and Utilization in Maize, Waverly Press, Baltimore, pp 117-133]. Multicellular explants, such as immature embryos and inflorescences, have been used to establish embryogenic tissue cultures of many genotypes [see Duncan et al., Planta 165, 322-332 (1985)]. Plant regeneration from single somatic cells of maize following protoplast isolation has been reported [see Rhodes et al., Biotechnolo w 6, 56-60 (1988)], however, seed could not be obtained.
In 1977, :Vitsch did report an attempt to ' culture isolated maize microspores, but the results were preliminary and unpublished [see Nitsch ( 1977) Cultureo~' IsolatedMicrospores, In: J. Reinert and Y.P.5. Bajaj (eds) Applied and Fundamental Aspects of Plant Cell Tissue and Organ Culture, Spenger-Uelag]. Specifically, in Figure 3, Nitsch shows the development of a Few cell-like divisions, but never reports that embryo-like structures) (ELS) were formed. For lines demonstrating high androgenesis to be utilized in commercial maize breeding, plant regeneration and subsequent double haploid seed recovery is a prerequisite.
Thus, any attempt to use isolated male gametophyte culture in commercial breeding, particularly in maize, generally will require a considerable improvement in the overall efficiency of androgenesis in cultured plants.
As can be seen from the above discussion, male gametophyte culture techniques are still rather 37, ~+27A-F -8-_ g _ empirical, and as such it is difficult to draw generalizations from the prior art .
Surprisingly, the present invention provides a method that is capable of producing novel germplasm having increased ability to undergo androgenesis.
In one aspect, the present invention provides a method for preparing a plant having an improved ability to undergo androgenesis, the method comprising: (a) providing anthers from at least one heterozygous donor plant; (b) regenerating, from the anthers obtained from the donor plant, at least two male gametophyte-derived plants capable of being intermated; (c) intermating the regenerated plants to produce an F1 population; and (d) self-pollinating or cross-pollinating individuals of the F1 population to generate at least one F2 population, wherein the mean anther culture response frequency is at least 10 percent greater than the anther response frequency of the original donor plant.
In a second aspect, the present invention provides an F2 plant, plant cell or progeny thereof, produced by the method of the invention.
In a third aspect, the present invention is a method for producing an embryo-like structure (ELS) comprising the steps of: (a) providing at least one male gametophyte isolated from at least one anther from at least one donor plant or plant part, preferably maize, obtained from a plant cell of the invention, which is capable of being successfully anther cultured; and (b) incubating the male gametophyte in an ELS
induction medium to foster development of at least one ELS.

200<~~"~8 -~o-In a fourth aspect, the present invention is at least one maize seed which is generated from at least one male gametophyte.
Definitions As used herein the term "plant" includes seed capable of being germinated into a plant; plant cells;
plant protoplasts; plant cell or tissue cultures from '~~hieh a plant can be regenerated; plant calli; plant clumps; and plant cells that are intact in a plant or parts of a plant, such as flowers, kernels, ears, cobs, leaves, husks, stalks, and similar parts or tissues.
The present invention is applicable to any plant species which may be anther cultured. The term plant "species" is meant to include monocotyledons (e. g., the grasses, and the cereal crops such as maize, rye, barley, wheat, sorghum, oats, millet and rice); and the dicotyledons (e.g., broad-leafed plants such as tobacco, potato and alfalfa). The monocotyledons are preferred because plant regeneration is not as well documented as for many dicotyledons. Because of its commercial importance, maize is particularly preferred for use in the present invention due to its heretofore reticence to anther culture.
By "male gametophyte" is meant the haploid phase of the life cycle, including a mierospore of a higher plant. Generally, the male gametophyte develops in the following manner. Following mitosis and after release from the tetrad, each individual microspore is characterized by a large nucleus which occupies one-third to one-half of the cell volume. A germ pore appears prior to enlargement of the microspore while the 37,427A-F -10-~U~~-~3'~8 cytoplasm is dispersed throughout the cell. As the microspore increases in size, the nucleus remains near the pore and becomes smaller and more densely staining.
With the formation of a large central vacuole, the nucleus is pushed to the side opposite the germ pore and organizes a separate cell attached to the microspore wall. Within this cell, the nucleus enlarges and undergoes the first pollen grain mitosis. The daughter nuclei, which are initially similar in size, begin to differentiate while still within the original cytoplasm.
At this point, the vegetative nucleus is distinguishable by its larger size and more diffuse staining in contrast to the smaller and more densely staining generative nucleus.
The vegetative nucleus migrates to the opposite side of the pollen grain. The generative nucleus, along with a small amount of cytoplasm, is separated from the vegetative nucleus by the formation of an arch-shaped cell wall attached to the intine. Formation of a distinct cell wall around the vegetative nucleus appears to be delayed during migration. Upon reaching a position adjacent to the pore, a cell is formed which is similar to, but larger than, the generative cell.
Starch accumulation follows progrESSing from the tapetal or pore side until the entire grain is filled. The generative cell then detaches from the intine, becomes rounded, and migrates toward the vegetative cell where the second pohlen grain mitosis. In some species, pollen is shed before the generative cell division. In other species, such as maize, the generative cell division occurs whle the mierospore is still in the anther.
37,427A-F -11-200<~ i:~

For purposes of this invention, anther culture response is measured in terms of embryo-like structures per 100 anthers cultured (ELS/anthers). By "embryo-like structures" (ELS) is meant globular masses of cells resulting from repeated divisions of male gametophytes which are capable of continued growth and development.
Creation of Genotype Regardless of the previously attained ability of a species to undergo androgenesis, the present method will provide an enhancement in male gametophyte culture response frequency in the regenerated F1 progeny of at least 10 percent greater than the male gametophyte culture response frequency in either parent. The present invention will improve the ability to in vitro culture male gametophytes of a selected species, at perhaps significant levels. For example, in maize, a species for which androgenesis has been demonstrated at onl low levels, the male y gametophyte culture response of the F2 population will preferably be at least 18 ELS/anthers, and most preferably at least 30 ELS/anthers.
Plants containing heterozygous genotypes may be obtained from any convenient source known to the skilled artisan. For example, the plants may be naturally heterozygous, occurring from open pollination; wild relatives of inbred lines; mutations of inbred lines;
transformed inbred lines; and the progeny of crosses of inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. Exemplary crosses include single crosses (i.e., two inbred lines are crossed to produce F1 progeny), three-way crosses (i.e., three inbreds 37 , ~127A-F -12-2Q0~~ i'78 lines are crossed: [(A X B) X C]), and double crosses [i.e., Four inbred lines are crossed: (A X B) X (C X D)].
Plant breeding techniques suitable for production of such first generation hybrids are well--known to those skilled in the art. Such techniques are descr i bed in "Corn and Corn Improvement", Sprague ed . , American Society of ARronomy, Publication No. 18, Madison, Wisconsin (1977); Poelhman, J. H., "BreedingField Crop"s, Henry Holt and Company, New York, (1959); and Welsh, J . R. , "Fundamentals of Plant Genetics and Breeding", Wiley ( 1981 ) .
Preferably, at least one of the parents contains an agronomically-important trait. Although the phenotype of high androgenesis may be transferred to agronomically-important plants, as discussed below, it is more convenient to utilize agronomically-important plants directly in the present method. The presence of other traits should be selected so as not to affect the transfer of genetic factors which express the phenotype of high androgenesis.
In maize, a particularly preferred selection of plants is derived from a three-way cross of the following inbred lines: (H99 X FR16) X Pa9l. Seed for producing the inbred plants was obtained from Holden's Foundation Seeds, Williamsburg, IA, (H99 and Pa91) and Illinois Foundation Seed, Tolono, IL (FR16).
Anther Culture of the Donor Plant The first step of the method is to remove anthers from the selected donor plants. Anthers may be removed from the plant at any suitable stage of maturity. The stage of maturity will depend upon the 37,~27A-F -13-20th<l~~i'~8 -~~,-particular species. Generally, the anthers will be removed when they contain microspores at the early uninucleate-late binucleate stage of development.
Preferably, maize anthers are removed from the plant at between the late uninucleate-early binucleate stage of development.
Maturity of the anthers is determined microscopically by periodic sampling of plants.
Microscopic techniques are well-known in the art.
Generally, the stage of anther development is readily determined microscopically after treatment with nuclear staining, such as with acetocarmine or mithramyein.
After anthers from the donor plant have been isolated, the second step involves utilizing cell culture technology to isolate and characterize cell lines which express anther culturability. The anthers may be cultured by any standard technique which provides double haploid plants. The anther culture technique employed will of course depend upon the particular species used. For a general discussion of anther culture procedures see Dunwell (1985), supra; Keller et al . ( 1987 ) , supra; and Ba ja j , YPS, "In vitro Production off' Haploiu',s", In Evans DA, Sharp WR, Ammirato PV, Yamada Y
(eds.), Handbook of Plant Cell Culture, vol. 1, Macmillan, New York, 1983, pp 228-287.
Intermatinr~ of Regenerated Plants After producing a population of regenerated double haploid plants as discussed above, the regenerated plants should be randomly crossed to produce a series of F1 hybrids, and then self pollinate the F1 hybrids to produce an F2 population having genetic 37 , t+27A-F -14-~oo~~~~~

variability, i.e, segregating populations. Quite surprisingly, by intermating regenerated plants of anther culture to produce an F1 population and self-pollinating individuals of the Fl populations to generate an F2 population, the F2 progeny of those plants have an anther response frequency at least 10 percent greater than the anther culture response of the donor plant.
Exemplary techniques for intermating the regenerated plants include single, three-way and double crosses of the regenerated population; the techniques for performing the crosses has hereinbefore been described. Preferably, the individuals of the re enerated g population should be crossed in as many ways as possible in order to create several F2 populations.
Intermating of the regenerated plants is followed by creating a segregating population to produce maximum genetic variability in the F2 (SO) populations.
The F2 population is created by self-pollinating or cross-pollinating individuals of the F1 population. Any method of creating a segregating population to produoe maximum genetic variability may be employed to create the F2 population. Thus, the present invention contemplates pollinating individuals of the F1 population by self-pollination; or by being crossed with other plants suoh as with other members of the F1 population, or even nonresponsive plants; provided that the F2 progeny of those plants have an anther response frequency at least 10 percent greater than the anther culture response of the donor plant. Self-pollination of the Fl individuals is preferred, to maximize the release of genetic variability of the F1.
37,42TA-F -15-goo<~~ ~ s An exemplary method for maintaining a segregating population is as follows: at least 50 seeds are planted and the resulting individuals are intermated (using each plant as a male and female once). [See A.
R . Hal lauer , "Principles of Cultiuar Deueloprnent", in W. R . Fehr et al. (ed.), Crop Species, Macmillan Publishing Company, Vol. 2, 1987, Chapter 8 "Macize", pp.249-294.]
At least 25 ears from different plants should be harvested, the seed removed and mixed together in bulk.
From this bulked seed mix, a random sample of seeds can be saved.
Genetic Fixation of Se~re~atin~ Population Thereafter, the segregating populations are genetically fixed. Generally, genetic fixation may be accomplished by any standard technique.
Suitable techniques include (1) providing anthers from the progeny of intermated plants and subjecting the anthers to anther culture, or (2) inbreeding the progeny of the intermated plants.
Any suitable anther culture technique which provides double haploid plants can be used. Such techniques are described above (see Dunwell (1985), and Keller et al. (1987), supra).
Inbreeding involves the controlled self--pollination for several generations in order to develop true-breeding or homozygous inbred lines. Inbred lines are derived by a method of self-pollination and selection, usually over 5 or more generations (S1-S5), so that allelic pairs of genes on homologous chromosomes are homozygous or identical.
37,427A-F -16-goo<~~~~
-,7_ Plants which have been self-pollinated and selected for type over many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. The degree of inbreeding in a line is approached at the rate of about 50 percent per generation so that by the second generation the plants are about 75 percent homozygous and by the sixth generation the plants are about 98 percent homozygous.
Thereafter, all plants derived from self--pollination, sibling pollination, or random crossing with others in the inbred line theoretically should be essentially genetically identical and, therefor, should be essentially homozygous and uniform in appearance.
For maize, an exemplary embodiment of the present invention is the sibling pollination, or random crossing with others in the inbred line of a regenerated lant p produced by the anther culture of a hybrid plant of the (H99 X FR16) X Pa91 cross, which is described in greater detail in the experimental section below. As a result of that cross, a genetic factor that enhances the anther culturability of maize was first identified. As discussed therein, seeds containing the HAC genetic factor have been deposited with the American Type Culture Collection (ATCC), in Bethesda, Maryland.
However, as one aspect of the present invention is directed to intermating populations of regenerated plants, the invention is not limited to the exemplary populations deposited at the ATCC, i.e., those individuals which possess the HAC genetic factor.
Methods and plants are provided for producing male gametophyte cultures, callus cultures, plant tissue 37 , ~+27A-F -17-;goo<~~~~
-18_ culture, plant tissues, plants and seeds which express culturability and genetically transmit this trait to progeny.
Use of the HAC genetic factor, however, is preferred because it is stable and has been demonstrated to be capable of being transmitted to progeny over a number of generations. The HAC genetic factor is such that when a series of inbred lines are intermated, the average value of a line can be used to predict the response of a given cross. This is usually a function of additive gene effects and their interaction. This is typical of quantitatively inherited traits in maize (i.e., involving the interaction of more than one gene).
Individual hybrids can, however, deviate from the average performance of their parents. Thus, dominance or dominant types of epistasis may also play a role in the anther culture response.
The HAC
genetic factor, and effectively homologous derivatives thereof, can increase the ability to undergo androgenesis of both inbred and heterotic maize plants which possess other desirable characteristics. By "effectively homologous derivatives" is meant to include variants, modifications, and mutants that are functionally equivalent to the nucleotide or amino acid sequences of the HAC genetic factor. Thus, in this disclosure it will be understood that minor sequence variation can exist within homologous sequences and that any sequences exhibiting at least 80 percent homology are deemed functionally equivalent, provided that the homologous segment or homologous genes produce a unique phenotype in plants, namely F2 plants or their progeny, plants having a male gametophyte culture response frequency at 37, ~+27A-F -18-zoo<~3~~

least 10 percent greater than the male gametophyte culture response of the original donor plant. Homology is expressed at the fraction or percentage of matching bases (or amino acids) after two sequences (possibly of unequal length) have been aligned. The term alignment is used in the sense defined by Sankoff and Kruskal in Chapter One of their book, The Time Warps, String Edits, and Macromolecules: The Theory. and Practice of Seguence Comparison, Addison-Wesley, Reading, MA, (1983).
Roughly, two sequences are aligned by maximizing the number of matching bases (or amino acids) between the two sequences with the insertion of a minimal number of "blank" or "null" bases into either sequence to bring about the maximum overlap.
A skilled artisan will appreciate that as a result of the present invention, namely the HAC genetic factor, the source of HAC genetic factor is irrelevant.
Any source of DNA which provides the HAC genetic factor or an effectively homologous DNA sequence derived from the HAC genetic factor is now readily within the means of those of ordinary skill in the art.
A series of probes, made from genes of all or a part of the HAC genetic factor, could be used to find effectively homologous genes or gene segments in unknown plants, particularly maize plants. Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross-hybridization of nucleic acids under conditions of stringency as is well understood in the art [as described in Nucleic Aeid Hybridization, Hames and Higgens (eds.), IRL Press, Oxford, UK (1985)]. Given two sequences, algorithms are available for computing their homology: e.g. Needleham 37,427A-F -19-;~oo-~;~~~

and '~lunseh , J. Mol. Biol. 48 , 443-453 ( 1970 ) ; and Sankof f and Kruskal (cited above) pgs. 23-29.
Since seeds containing the HAC genetic factor have been deposited ~,~ith the ATCC, deposited under ATCC
designation numbers 40519 and 40520, a skilled artisan would appreciate that the HAC genetic factor may be modified by the addition, deletion, or nonconservative substitution of a limited number of various nucleotides or the conservative substitution of many nucleotides, provided that the proper reading frame is maintained.
Substitutions, deletions, insertions or any subcombination may be combined to arrive at a final construct. Exemplary techniques for additions, deletions and substitutions include oligonulceotide-mediated, site-directed mutagenesis and polymerase chain reaction.
Oligonueleotide site-directed mutagenesis in essence involves hybridizing an oligonueleotide coding for a desired mutation with a single strand of DNA
containing the region to be mutated and using the single strand as a template for extension of the oligonueleotide to produce a strand containing the mutation. This technique, in various forms, is described by Zoller, M.J. and Smith, M., Nuc.AcidsRes.
10, 6487-6500 (1982); Norris, K., Norris, F., Christiansen, L. and Fiii, N., Nuc.AcidsRes. 11, 5103-5112 (1983); Zoller, M.J. and Smith, M., DNA ~, 479-488 (1984); Kramer, W., Schughart, K. and Fritz, W. J., Nuc.
AcidsRes. 10, 6475-6485 ( 1982).
Polymerase chain reaction (PCR) in essence involves exponentially amplifying DNA in vitro using sequence specified oligonucleotides. The 37,427A-F -20-;~oo~.~~~a oligonucleotides can be incorporated sequence alterations, if desired. The polymerase chain reaction technique is described in Mullis and Faloona, Meth. Enz.
155, 335-350 (1987). Examples of mutagenesis using PCR
are described in Higuchi et al., lVucl. Acids Res. 16, 7351-7367 (1988), Ho et al., Gene 77, 51-59 (1989), and "Engineering Hybrid Restriction Genes Without the Use oj'Restriction Enzywes: Gene Splicing by Overlap Extension" , Horton et al . , Gene ~, 61 ( 1989 ) .
Transfer of Anther Culturability The method of the present invention now provides a means for producing plants, particularly maize plants, that have male gametophytes, either in or out of the anther, that are capable of being successfully cultured in vitro.
The present invention provides a genetically transmitted characteristic which can be selectively incorporated into progeny. Thus, traditional sexual techniques (e. g., breeding) or asexual techniques (e. g., in vitro procedures) or a combination of both, may be used to transfer the HAC genetic factor to other plants, such as commercially-important lines.
Sexual Techniques In plant breeding, after obtaining a genetically fixed variety having increased anther culturability, it may desirable to use the variety with enhanced anther culturability as a parent in a breeding program which will permit production of superior hybrids and inbred lines.
37,427A-F -21-~'(~0<~3'7~

Any plant containing the HAC genetic factor may be used as a breeding strain for developing other inbreds and hybrids. Specifically in maize, the HAC
genetic factor can easily be incorporated into proven maize inbreds and hybrids which possess other desirable characteristics, and all subspecies of maize, specifically including the dent corns, the flint corns, the soft or flower corns, the sweet corns, the pod corns, and the pop corns. Hybrids formed from a cross between a parent containing the HAC genetic factor and another parent will have an anther frequency response of approximately the average of the parents' anther frequency response.
Consequently, different strains possessing other genetic factors, or combinations of factors, may be crossed with strains of the same species having improved anther culturability to produce hybrids having increased anther culturability. Such crossing techniques are well-known in the art, and are described in detail above. Conveniently, the transfer of anther culturability is accomplished by pedigree breeding or backerossing (recurrent selection breeding).
Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complement the other. In the pedigree method, superior F2 plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self -pollination and selection (typically in five or more generations). The procedure can be 37 , ~+27A-F -22-goo<~~~s modified by anther culture of the F1, F2, F3, etc.
populations.
Backcrossing can be accomplished, for example, by first crossing a superior inbred (recurrent parent) to a donor inbred (non-recurrent parent), which carries the genetically transmitted characteristic of anther culturability. The F1 progeny~of this cross is then mated back to the superior recurrent parent followed by selection in the resultant progeny for the desired trait to be transferred from the nonrecurrent parent. After five or more backcross generations with selection for the desired trait, the progeny will be heterozygous far loci controlling the characteristics being transferred, but will be like the su erior p parent for most or almost all other genes. The procedure can be modified by anther culture of the F1, F2, F3, etc. populations.
After a number of homozygous lines displaying a range of desirable characteristics are produced, experimental hybrids which excel in many or all attributes identified may be produced. After such optimum combinations are determined, the parental lines are increased and large quantities of seedstock are produced by well-known means.
Asexual Techniques As an alternative or complementary strategy to traditional plant breeding, scientists have conducted research into the transformation of plants via genetic engineering techniques. Progress in the transformation of maize has been restricted by the limitations of presently available gene-transfer systems. Exemplary techniques for the delivery of rDNA (recombinant DNA) 37, ~+27A-F -23-zoo<~~~8 providing increased androgenesis (e. g., the HAC genetic factor) include microinjection (see Crossway et al. Mol.
Gen. Genet. 202, 179-185), or the gene gun technology (see Klein et al., Biotech 6, 559-563).
In Vitra Culture of Male Gametophyte The principle of in vitro culture of male gametophytes is to divert the normal development of the male gametophyte to an abnormal pathway, a sporophytic pathway, resulting in ELS and/or callus formation. ELS
and/or callus are expected to be haploid, carrying the gametic number of chromosomes in the sporophytic phase.
Thus, when the chromosomes of the haploid plant are doubled, the resulting plant will be homozygous.
The present invention thus provides male gametophytes that are well-suited for being in vitro cultured and regenerated into plants. Anthers may be obtained from any anther culturable genotype prepared by the method of the present invention. For genotypes which have demonstrated anther culturability, the present invention will provide increased efficiencies.
Once the plant is selected, its tassel may be harvested and pretreated. A preferred method of tassel pretreatment is set forth in Petolino and Jones (1986) supra. Generally, that reference teaches the following: tassels with anthers containing male gametophytes at the late uninucleate-early binucleate stage of development, as determined microscopically, may be removed from donor plants prior to emergence from the whorl. These microscopic techniques are well-known in the art. Genera_ly, the stage of male gametophyte development is readily determined microscopically after 37~~27A-F -24-~oo~~-~~~

treatment with nuclear staining; examples of suitable stains include acetocarmine and mithramycin.
Tassels are then wrapped in moist paper towels, covered with aluminum foil, and maintained at 8°C for 14 days. Before anther excision, tassels are surface sterilized for 15 minutes in a 0.5 percent sodium hypochlorite solution followed~by a sterile water rinse.
Preferably, only anthers from the central portion of the main tassel branch are used.
Either the tassels or the excised anthers may be pretreated prior to in vitro culture. Any particular physical or chemical pretreatment technique suitable to condition the male gametophytes for abnormal development may be employed. A preferred pretreatment method is to expose the tassel or excised anther to a temperature of between 4 to 12°C as set forth in Petolino and Jones, (1986), supra.
In one embodiment, male gametophytes may be removed from the anther from about the early uninueleate to about the late binucleate stage of development.
Preferably, the male gametophytes will be removed at between the late uninueleate and the early binueleate stage of development. Maturity of the male gametophytes is determined microscopically, as described above, by periodic sampling of plants.
The male gametophytes may be removed from the anthers by any suitable technique. Suitable techniques include mechanical removal or dehiscence of the male gametaphytes into an ELS induction medium.
Generally, mechanical removal of the male gametophytes may be accomplished by passing the anthers 37 , ~+27A-F _25_ 2Q0~'~3'7~

through a mesh screen as set forth in R. Lichter. _Z.
?flanzenphvsiol. 105, 427-434 (1982); or by microblending as set forth in Swanson et al., Plant Cell Reports 6,9~-97 (1987).
Generally, dehiscence comprises the shedding of male gametophytes into a liquid culture medium. Anthers floating in liquid medium presumably produce conditioning factors and release them into the medium.
[see Sunderland et al. Nature ~, 236-238 (1977)], ELS Induction The male gametophytes, whether contained in or removed from their anthers, may be cultured on the ELS
induction medium within a suitable range of temperatures and light intensities to foster the formation of ELS.
The male gametophytes are cultured preferably in the dark; and preferably at a temperature ranging from 20 to 32°C, most preferably between 26 to 29°C.
The ELS induction medium contains components which foster the growth of ELS from the male gametophytes. The ELS induction medium components useful herein are aqueous compositions (the water constituent is deionized and distilled), containing a basal salt mixture, and at least one carbon source.
The components of the ELS induction medium are preferably mixed with activated charcoal. Generally, the activated charcoal is added with the ELS induction medium components before autoclaving. However, if the activated charcoal is sterilized, it may be added after the other components have been autoclaved. Generally, the activated charcoal is added in an amount sufficient to absorb any deleterious by-products. Preferably, the 37 , ~+27A-F -26-goo<~~~~

activated charcoal is added with the other components in an amount of between 1 and 10 g/1, and most preferably between 3 and 7 g/1.
The activated charcoal is preferably filtered out prior to contacting the gametophytes with the ELS
induction medium. The activated charcoal may be Filtered by any process known to the skilled artisan.
An exemplary filtration technique includes passing medium through a sieve, e.g., a Nalgene'" filtration unit, or cheese cloth. Preferably, the sieve should have pores which are small enough to filter out the activated charcoal.
The basal salt mixture is a major component of the ELS induction medium. Suitable basal salt mixtures include YP, N6, MS, and B5, which are taught in Plant Tissue and Cell Culture (1977) University of California Press, Berkeley; and Chu et al., (1978) Proc. Symp.
plant Tissue Cult., Science Press, Peking, pp u3-56. A
preferable basal salt mixture which should be used is the YP basal salts as taught by Ku et al., (1978) Proe.
imp. Plant Tissue Cult., Science Press, Peking, pp 35-42.
Exemplary carbon sources include sucrose, glucose, and fructose, with sucrose being preferred.
Generally, the carbon source should be present in a concentration sufficient to support cellular growth.
Preferably, the carbon source will be present in an amount of between 10 and 200 g/1. Most preferably, when the carbon source is sucrose, the carbon source will be present in an amount of between 60 and 90 g/1.
37,~27A-F -27-I

Generally, the ELS induction medium may contain conventional organic components which are essential for Cell funCtlOn. Suitahlr? l~r~o~anin nnmnnncnt~ ,.~,.,1,...~., vitamins, amino acids, and hormones. Such organic components are taught in Street (1977), supra. An exemplary amino acid mixture includes casein hydrolysate. Preferably, the amino acid will be present in an amount of between 100 and 1000 mg/1. An exemplary hormone includes 2,4,5,-triiodobenzoic acid.
preferably, the hormone will be present in an amount of between 0.1 and 0.2 mg/1.
Except for filter sterilized components, the media in the ELS induction medium is normally sterilized b autoclavin Y g, e.g., utilizing 16 psi (110 kPa) steam.
The pH of the media prior to autoclaving normally ranges from 5.6 to 6Ø
The male gametophytes generally are cultured in the ELS induction medium until macroscopic ELS appear.
Preferably, the male gametophytes will be cultured in the ELS induction medium for a period of between 14 and 36 days, most preferably between 21 and 28 days. These ELS may be yellowish white with diverse shapes ranging from normal bipolar or globular embryos to multi-lobed or otherwise configured embryos. Preferable ELS will have distinct seutella and meristematie regions.
Suitable ELS for further culture are each C.5 to 1.5 mm in length.
Callus Induction Selected ELS are removed from the ELS induction medium and transferred to a callus induction medium. By "callus" is meant cells proliferating in a more or less 37,427A-F _2g_ 200<1~~~
_29_ disorganized manner without differentiating into organized structures (i.e., roots, leaves).
The ELS are cultured on the callus induction medium within a suitable range of temperatures and light intensities to foster the formation of callus. The ELS
are cultured in the callus induction medium preferably in the dark; and preferably at~a temperature ranging from 20 to 32°C, most preferably from 26 to 29°C.
The ELS induction medium components useful herein are aqueous compositions (the water constituent is deionized and distilled), containing a basal salt mixture, at least one carbon source, and at least one plant growth hormone.
The basal salt mixture is a major component of the callus induction medium. Exemplary basal salt mixtures include the YP, N6, MS, and B5 basal salts, which are taught in Street (1977), supra; and Chu et al., (1978), supra.
Exemplary carbon sources include sucrose, glucose, and fructose, with sucrose being preferred.
Generally, a carbon source should be present in a concentration sufficient to support cellular growth.
Preferably, the carbon source will be present in an amount of between 10 and 200 g/1. Most preferably, when the carbon source is sucrose, the carbon source will be present in an amount of between 10 and 40 g/1.
Generally, the plant growth hormone will be present in a concentration sufficient to induce callus proliferation as taught in Street (1977), supra.
preferably, the hormone will be present in an amount of 37,427A-F -29-X00<~ s'~8 _30_ between 0.1 and 10 mg/l. Exemplary hormones include dicamba, 2,~-D, and picloram.
Generally, the callus induction medium may contain other conventional organic components, such as vitamins and amino acids, as taught in Street, (1977), supra, An exemplary source of vitamins is the N6 formulations of vitamins [see Ku, (1978), supra]. Amino acid components preferably include enzymatic casein hydrolysate and L-proline.
The callus induction medium will beneficially contain a gelling agent. Suitable gelling agents include, for example, agar at a level of between 0.6 and 0.8 percent; Gelrite at a level of between 0.1 and 0.2 percent; and agarose (pure basis) at a level of between 0.2 and 0.4 percent.
Except for filter sterilized components, the media in the callus induction medium is normally sterilized by autoclaving, e.g., utilizing 16 psi (110 kPa) steam. The pH of the media prior to autoclaving normally ranges From 5.6 to 6Ø
Generally, the ELS will be cultured in the callus induction medium until displaying a compact, nodular appearance. Preferably, the ELS are cultured in the callus induction medium for a period of at least about 7 days, and most preferably between 10 and 1~1 days.
Once established, the compact nodular callus is preferably contacted with a chromosome doubling agent in an amount sufficient to induce chromosome doubling. By "chromosome doubling agent" is meant a mitotic poison, i.e., a chemical capable of interfering with mitosis.
37, ~+27A-F -30-l .31.
:or a detailed discussion of chromosome doubling agents see Jensen ( 1974 ) Chromosome Doubling Techniques in Haploids, In: K. J. Kasha, (ed.), Haploids in Higher Plants, University of Guelph, p 153-190. Exemplary chromosome doubling agents include nitrous oxide and colchicine.
Plant fteReneration Selected callus are individually transferred From the callus induction medium to a semi-solid or liquid regeneration medium. Incubation of the callus is carried out within a suitable range of temperatures and light intensities to foster the formation of plantlets.
The callus are preferably cultured in the plant regeneration medium at a temperature ranging from 26 to 29°C. The callus preferably will be exposed to a photoperiod ranging from 8 hours of light per 16 hours of dark to 18 hours of light per 6 hours of dark; more preferably, from about 16 hours of light per 8 hours of dark. The light may be diffused light as well as direct light and have an intensity of between 250 to 500 foot candles.
The plant regeneration medium may contain com onents which foster the p growth of plantlets from the callus. The plant regeneration medium components useful herein are aqueous compositions (the water constituent is deionized and distilled), containing a basal salt mixture and at least one carbon source.
The basal salt mixture is a major component of the plant regeneration medium. Exemplary basal salt mixtures include SH [described in Shenk, R. V., Can. J.
Bot. 50, 199 (1972)]; MS [described in Murashige, T., et al, Physiol. Plant 15, 473 (1962)]; B5 [described 37,427-F _31_ K:
_32_ in Gamborg, O.L.. Exp. Cell Res_. 50, 148 (1968)]; BL
[described in Blaydes, D. F., Physiol. Plant 19, 7~8-753 (1966)]; WH [described in White. P. R., The Cultivation of Animal and Plant Cells, 2d ed., Ronald Press, New York (1963)]; and N6 [described in Chih-Ching, C., et al.
(Sept.-Oct., 1975) Scientia Sinica, XVIII, No. 5, 659-668]. Preferably, the SH basal salts are employed.
Exemplary carbon sources include sucrose, glucose and fructose, with sucrose being preferred Generally, a carbon source should be present in a concentration sufficient to support cellular growth.
Preferably, the carbon source will be present in an amount of between 10 and 200 g/1. Most preferably, when the carbon source is sucrose, the carbon source will be present in an amount of between 5 and 50 g/1.
Except for filter sterilized components, the media in the plant regeneration medium is normally sterilized by autoclaving, e.g., utilizing 16 psi (110 kPa) steam. The pH of the media prior to autoclaving normally ranges from 5.6 to 6Ø
The callus are incubated in the plant re eneration medium until 8 producing root or shoot structures, e.g., between 2 to 5 nodes. Generally, incubation is conducted for a period of between 1 to 4 weeks.
It is preferable to subculture individual plantlets to fresh medium of the same composition.
From the plant regeneration medium, the plantlets are planted in soil or patting medium, e.g., in a greenhouse or in the field and under ambient 37,~+27A-F -32-zoo~~~~~

normal growth conditions the plantlets develop into whole fertile plants which flower and develop seeds.
Finally, the seeds are recovered to provide further generations of plants from which seeds can be recovered. The seeds are recovered from the plants propagated above or from descendants thereof by harvesting the seed pods and separating the seeds therefrom, for example, by hand or by using a thresher or combine.
The post plant-regeneration steps are readily carried out by conventional plant husbandry techniques.
1g Uses Tmprovements by selective breeding have been relatively slow, since only a limited number of generations of plants may be propagated each year.
Therefore, improvements in plants have been obtained only after years of rigorous work.
An important aim of traditional plant breeding is to engineer improved plants that are valuable as crop plants. In highly heterozygous, cross-pollinating crops, haploidy produced via anther culture creates a rapid method of producing pure breeding lines which can serve as parents in hybrid cultivar development.
. Production of haploids from F~ donors permits the breeder to effectively select desirable genetic recombinants. Because homozygous lines can be made rapidly available, a saving of time of up to 50 percent can be achieved in developing new cultivars.
The use of haploids has many potential applications in cellular biology research. One 37,427A-F -33-20o<~~~s _34_ application is cnuitro screening and selection using haploid cells, and to obtain mutants in a homozygous condition. Another application of haploidy is the production of desirable genetic translocations, substitution and addition lines through the culture of isolated male gametophytes of interspecific and intergeneric hybrids. The production of haploids and thereafter homozygous lines is a significant methodology for selecting rare genotypes, including those with recessive characters, and to include them in new combinations of crosses.
A still further benefit of anther culture is in the ability transform haploid maize via genetic engineering techniques. Exemplary techniques for the delivery of DNA into early-staged embryoids derived from mass-cultured male gametophytes include mieroinjection (see Crossway et al., Mol. Gen. Genet. 202, 179-185), or the gene gun technology (see Klein et al., Biotech, 6, 559-563). This technique would provide, upon chromosome doubling, a plant homozygous for the rDNA.
Examples The followin exam les are g p presented to further illustrate but not limit the scope of this invention.
All parts and percentages are by weight unless otherwise indicated.
Example 1 A. Formation of the Donor Plant A three-way cross was performed to create the donor plant. The inbred plants used in the three-way cross were H99, FR16, and Pa9l. Seed for producing the 37, ~+27A-F _34_ _35_ inbred lines was obtained from Holden's Foundation Seeds, Williamsburg, IA (H99 and Pa91) and Illinois Foundation Seeds, Tolono, IL (FR16). All lines were maintained by controlled self-pollination for two years prior to being used for crossing. The three-way cross, (H99 X FR16) X Pa91 was made by controlled pollination essentially following the procedures set forth in Hallauer (1987), described above.
Donor plants were field grown during April to August, 1985 in Champaign, IL. Tassels with anthers containing microspores at the late uninucleate-early binueleate state of development, as determined microscopically after treatment with acetocarmine, were removed from donor lams p prior to emergence from the whorl. Tassels were then wrapped in moist paper towels, covered with aluminum foil, and maintained at 8°C for 14 days. Before anther excision, tassels were surface sterilized for 15 minutes in a 0.5 percent sodium hypoehlorite solution followed by a sterile water rinse.
Only anthers from the central portion of the main tassel branch were used.
B. Anther Culture Sixty anthers were placed in a 20 x 60 mm Petri dish containing 20 ml of medium. The medium consisted of YP basal salts (see Ku et al., 1978, supra) with the addition of 5.0 g/1 activated charcoal, 500 mg/1 casein hydrolysate, 0.1 mg 2,3,4,5-triiodobenzoic acid, 120 g/1 sucrose, and 8.0 g/1 agar (Gibco) adjusted to pH 5.8.
Typically 3-6 dishes were obtained from each tassel harvested. Dishes containing freshly plated anthers 37,42TA-F -35-~oo-~-~~~

were sealed with Parafilm and placed in plastic boxes covered with aluminum foil.
After one week in the dark at 28°C, dishes were transferred to clear boxes and grown under cool white florescent lights (60 umol/m/sec) with a 16 hour photo period. Between 4 and 6 weeks later, anthers with recognizable embryo-like structures were apparent.
Embryo-like structures were yellowish-white and globular in appearance and resembled zygotic embryos displaying varying degrees of abnormal tissue proliferation.
Embryo-like structures were lifted from the anthers and placed onto a regeneration medium (YP with 1.0 mg/1 indole-3-acetic acid), 1.0 mg/1 kinetin, 146 mg/1 glutamine, and 30 g/1 sucrose). After 2-3 weeks, plantlets were placed on a hormone-free medium (YP salts only) and, after root formation, transferred to soil and grown to maturity in the greenhouse.
Two anther culture-derived embryo-like structures, obtained from two separate tassels, regenerated plants. The regenerated plants were grown to maturity during October to December, 1985. One plant (4139) produced an ear shoot and a tassel with no anther extrusion. A second plant (~~39) produced viable pollen but the ear shoot was late in development. The pollen from plant ~~39 was applied to plant 4139 resulting in the formation of a single F1 hybrid (139/39) seed.
The F1 hybrid seed was germinated and the resulting plant was self-pollinated, i.e., pollen from 37,427A-F -36-l one plant is used to fertilize itself to produce and F2 (SO) population.
C. Genetic Fixation of Se~re~atin~ Population 1. Self-Pollination of F2 Plants The F2 population was grown in the field in Champaign, IL during the summer of 1986 and fourteen plants were self-pollinated and grown ear-to-row.
The resulting fourteen S1 families were evaluated for their anther culturability. Evaluation was conducted by selecting tassels with anthers containing late uninueleate-early bi~ueleate mierospores, as determined by mithramycin/flourescent staining as set forth in Pace et al. , "Anther Culture of maize and the visualization o~'embryogenic microspores by ~lourescent microscopy", Theor. Appl. Genet. ,~, 863-869 ( 1987) .
Selected tassels were removed from the donor plants prior to emergence from the whorl. The results are set forth in Table 1.

37 , ~+27A-F -37-200-~~"78 Mean Anther Culture Response from Cultured Anthers of i4 S1 Families of Maize Anthers ELS+ Produced Genotype Cultured (Per 100 Anthers cultured) 139/39-O1 900 275.1 139/39-02 900 269.2 139/39-03 900 134.2 139/39-o4 9o0 126.0 139/39-05 900 238.4 139/39-06 900 64.2 139/39-07 900 79.8 139/39-08 900 30.4 139/39-09 900 248.0 139/39-10 900 57.6 139/39-11 900 130.0 139/39-12 900 87.5 139/39-13 900 70.9 139/39-14 900 119.8 +Embryo-Like Structures Means for the 14 S1 isolates ranged from 9.2~ and 41.3.
A population of S1 seed (ATCC No. 40519) containing an approximate equal distribution of 139/39-01 to 139/39-14 (hereinafter "139/39-bulk") was deposited at the ATCC on December 1, 1988.
37,427A-F -38-2004~~~

A distribution of response frequencies from individual plants from the original three-way cross, (H99 X FR16) X Pa9l, is presented in Fig 1A.

37,427A-F -39-2~~)~~~'~~

As can be seen in Figure 1A, the response frequencies were skewed toward the lower values and the overall mean response is 3.5 percent. Only 4 of the 53 (7.5 percent) plants displayed anther-culture response frequennies greater than 10 percent.
Individual anther culture response frequencies of the S1 plants are presented-in Fig. 1B.

37 , ~+27A-F -4 0-~oo:~~~~

As can be seen in Fig. 1B, a dramatic shift toward increased anther response was observed. The overall mean response frequency for the S1 plants was 23.u percent. A total of 54 of the 70 (77.1 percent) S1 plants evaluated had response frequencies greater than percent.
A single cycle of selection resulted in greater than a six-fold increase over the original three-way 10 cross in anther culture responsiveness as measured by the percentage of anthers producing embryo-like structures.
The two original plants from which the tassels were harvested (Fig. 1A), that ultimately lead to the regeneration of plants 439 and 41139, were not among the most productive based on their individual anther response (0.8 percent and 4.4 percent, respectively).
However, the intermating of male gametophyte-derived plants appears to be an effective means of shifting allelic frequencies toward increased responsiveness.
2. Anther Culture of S1 Plants Individuals of 139/39-05 were subjected to the anther culture technique set forth in Examples section "H. Anther Culture". Seed from the resulting regenerated plants, 139/39-DH (ATCC number 40520) was deposited at the ATCC on December 1, 1988.
3. Self-Pollination of S2 Plants Self-pollinations were made with four of the most responsive families (139/39-01, -02, -05, and -09) and S2 seed from the resulting ears were grown during April to October, 1988. Self-pollinations were made 37, ~127A-F -41-zoo<~~~~

within each of the families to produce novel S3 germplasm of this invention.
Since this material has undergone forced inbreeding for 4 generations [F1, F2 (SO), S1, S2]
approximately 95 percent of those loci which were heterozygous in the original F1 hybrid are now homozygous.
T-ransfer of Culturabilitv to Other Inbred Genotypes Crosses were made between the four selected S2 families and four other agronomically-important inbred genotypes (UAS 1, UAS 2, UAS 3, AND UAS 4) each of which is relatively non-responsive to anther culture.
The resulting F1 hybrids were evaluated for their anther culturability as measured by ELS formation.
The results are set forth in Table 2.

3o 37,427A-F -42-~oo~~~~

Mean Anther Culture Response of 16 F1 hybrids of Maize Anthers ELS Produced Cross er 100 A
th Cultured n ers P

cultured) 139/39-01 xUAS 1 6,840 5.7 139/39-02 xUAS 1 6,900 29.2 139/39-05 xUAS 1 5,880 17.7 139/39-09 xUAS 1 7,440 5,3 139/39-01 xUAS 2 6,240 30.7 139/39-02 xUAS 2 6,360 67.4 139/39-o5 xUAS 2 6,420 18.6 139/39-09 xUAS 2 7,320 47.3 139/39-01 xUAS 3 6,300 20.6 139/39-02 xUAS 3 6,600 29.1 139/39-05 xUAS 3 6,600 6,2 139/39-09 xUAS 3 6,720 32.5 LH139/39-01 x UAS 4 6,660 48.8 139/39-02 x UAS 4 7,680 171.1 139/39-05 x UAS 4 7,560 107.6 139/39-o9 x UAS 4 7,800 104.2 As can be seen from the Table 2, anther culture response frequencies ranged from 5.7 to 171.1 ELS/100 anthers. This demonstrates that the HAC genetic factor is effective in improving the anther culture response in otherwise unresponsive germplasm.
37,427A-F -43-20043~~

Example 2 The maize genotype used for donor plants (139/39-02), is a highly androgenic S2 line developed by intermating anther-derived doubled haploids [see Petolino et al., Theor. Appl. Genet. ~, 157-159, (1988)]. Donor plants were field-grown during April-August 1988 in Champaign, Illinois. Tassels from the donor plants were harvested and pretreated, following previously published procedures (see Petolino and Jones (1986), supra).
Anthers from the main tassel branch were inoculated (30 anthers per 60 x 20 mm plastic Petri dish) into about 10 ml of ELS induction medium.
Typically 3-6 dishes were obtained from each tassel harvested.
The ELS induction medium consisted of YP basal salts (see Ku (1978), supra) with the addition of about 60 g/1 sucrose, about 5 g/1 activated charcoal about 500 mg/1 casein hydrolysate, and about 0.1 mg/1 2,4,5 -triiodobenzoic acid adjusted to pH 5.8. Prior to use, the medium was filtered to remove the activated charcoal. Specifically, the activated charcoal was removed by filtration with a commercially available Filter sterilization unit having a pore size of about 5 microns.
Dishes containing freshly plated anthers were sealed with Parafilm'" brand film (commercially available from the American Can Company) and placed in plastic boxes covered with aluminum foil at 28°C.
Within 2 days, anthers cultured in a liquid medium dehisced, resulting in a stationary suspension of male 37,427A-F -44-goo<~~~s gametophytes. Maximum dehiscence occurred after 7 days with approximately 40 percent of the male gametophytes being liberated. Within 7 days, isolated male gametophytes exhibiting cell divisions were apparent.
By 12-15 days, multicellular masses were observed to break out of the exine resulting in the formation of ELS.
After 21 days, macroscopic ELS appeared. Most of the ELS which formed were submerged at the bottom of the plates and were not directly associated with the anthers floating on the surface of the medium. These structures were yellowish-white with diverse shapes ranging from normal bipolar of globular embryos to multi-lobed or otherwise abnormally configured embryos.
At 21 days, and weekly thereafter for three weeks, all ELS larger than 0.5 mm were counted and transferred to a callus induction medium. The total number of ELS
produced after 6 weeks was expressed per 100 anthers cultured, as set forth in Table 1.
The ELS was then transferred to a callus induction medium. The callus induction medium consisted of N6 major salts and vitamins (see Chu (1978), supra), B5 minor salts (see Gamborg et al. (1968) supra), 30 g/1 sucrose, 2.9 g/1 L-proline, 100 mg/1 myo-inositol, 100 mg/1 casein hydrolysate, 2.5 mg/1 dicamba, 0.1 mg/1 2,~f -D, and 8 g/1 TC-agar (obtained from Hazleton Research Products, Inc., Lenexa, Kansas).
After 2-3 weeks, callus, displaying a compact, nodular appearance, was transferred to a plant regeneration medium and maintained in a 16/8 hr photoperiod (60 ~mol m-2 sec-1) from cool-white light fluorescent lamps.. The regeneration medium consisted 37. ~127A-F -45-2U0<~~'~8 of SH basal salts (Schenk and Hildebrandt (1972), supra) and 10 g/1 sucrose.
After 4 weeks, individual plantlets were transferred from the plant regeneration medium to 10 x 25 mm culture tubes containing fresh regeneration medium for further development before transplanting to soil.
After root formation, the plantlets were transferred to soil and grown to maturity in the greenhouse at ambient temperature. The plantlets develop into whole fertile plants which flower and set seed and the seeds mature into viable seeds. The total number of cultures from which plants could be recovered (referred to as regenerable cultures) was determined and expressed per 100 ELS transferred, as set forth in Table 3.

37,427A-F -46-2U04~'~8 TABLE 3.
Formation of ELS and plant regeneration from cultured male gametophytes of maize.
._ Anthers Total Total Cultured ELS Produced Regenerable Cultures (per 100 Anthers) (per 100 ELS) ( 600 ~ 2,510 (418.3) 690 (27.5) As seen in Table 3, isolated male gametophytes produced ELS from which plants could be recovered.
Examale 3 The procedures of Example 2 were repeated with the exception that anthers cultured in the induction medium had male gametophytes mechanically isolated at 0, 3 and 7 days after culture initiation. Isolated.male gametophytes were isolated by gently pressing intact anthers against a 113 micron stainless steel sieve using a glass rod (see Lichter, (1982), supra). The contents of 30 anthers in a volume of 10 ml of induction medium resulted in a density of 6,000 +/- 500 male gametophytes ml-1 as determined by hemocytometer counts (see Street, (1977), supra).
The results Far ELS Produced/per 100 Anthers and Total Regenerable Cultures/per 100 ELS are set forth in Table 4.
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TABLE 4.
ELS formation and plant regeneration from male gametophytes mechanically isolated from maize anthers at various times after culture initiation.
Total Total PrecultureAnthers ELS ProducedRegenerable Time (Days)Cultured (per 100 Cultures Anthers) (per 100 ELS) 0 240 36 (15.0) 2 (5.6) 3 180 165 (91.1 23 ( 13.9) ) 300 I 568 (189.0)68 (12.0) ~

As seen in Table 4, isolated male gametophytes produced ELS from which plants could be recovered.
The present invention is not to be limited in scope by the cell line or seeds deposited, since the deposited embodiments are intended as 'single illustrations of one aspect of the invention and any cell lines or seeds which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will beoome apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.
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Claims (27)

1. A method for preparing a plant having an improved ability to undergo androgenesis, the method comprising:
(a) providing anthers from at least one heterozygous donor plant;
(b) regenerating, from the anthers obtained from the donor plant, at least two male gametophyte-derived plants capable of being intermated;
(c) intermating the regenerated plants to produce an F1 population; and (d) self-pollinating or cross-pollinating individuals of the F1 population to generate at least one F2 population, wherein the mean anther culture response frequency is at least percent greater than the anther culture response frequency of the donor plant.

2. The method of claim 1, wherein the donor plant is a monocotyledon or a dicotyledon.

3. The method of claim 2, wherein the monocotyledon is maize.

4. The method of claim 3, wherein the donor plant is obtained from a three-way cross of (H99 X FR16) X Pa91.

5. The method of claim 1, wherein step (c) comprises randomly intermating the regenerated plants by single, three-way or double crosses.

6. The method of claim 1, wherein step (d) comprises self-pollinating individuals of the F1 population.

7. The method of claim 1, wherein the F2 population produced in step (d) has a segregating population which is fixed genetically by a method of selfing or anther culture techniques.

8. A plant cell found in a plant produced by the method according to any one of claims 1 to 7.

9. A plant cell produced sexually, the plant cell having as an ancestor at least one plant cell as defined in claim 8.

10. A plant cell produced asexually, the plant cell being regenerated from the plant cell as defined in claim 8 or 9.

11. The plant cell according to claim 9 or 10, wherein the sexually or asexually produced plant cell contains a DNA
sequence effectively homologous to the HAC genetic factor.

12. A plant cell having the characteristics of a plant cell grown from 139/39-bulk.

13. The plant cell according to claim 12 having the characteristics of seed deposited under ATCC designation number 40519.

14. A maize plant cell having the characteristics of a plant cell grown from 139/39-DH.

15. The maize plant cell according to claim 14, having the characteristics of seed deposited under ATCC designation number 40520.

16. A method for producing an embryo-like structure (ELS) comprising:
(a) providing at least one male gametophyte from at least one plant or plant part obtained from a plant cell as claimed in any one of claims 8 through 15; and (b) incubating the male gametophytes in an embryo-like structure (ELS) induction medium to foster development of at least one ELS.

17. The method of claim 16, wherein the male gametophytes are isolated from anthers by a technique of mechanical excision or dehiscence.

18. The method of claim 16 comprising the further step of incubating the ELS in a callus induction medium to foster development of callus capable of being regenerated into at least one maize plant.

19. The method of claim 18 comprising the further step of incubating the callus on a plant regeneration medium to foster the germination of plantlets.

20. The method of claim 19 comprising the further step of cultivating at least one plantlet into at least one whole fertile plant.

21. The method of claim 20 comprising the further step of cultivating at least one whole fertile plant or a descendant thereof whereby it bears seeds.

22. An embryo-like structure (ELS) produced by the method according to any one of claims 16 to 21.

23. The embryo-like structure (ELS) according to claim 22, wherein the ELS is a maize ELS.

24. The embryo-like structure (ELS) according to claim 23, wherein the maize ELS comprises double haploid cells.

25. A method for the selection of maize mutants in a homozygous condition, comprising the step of in vitro screening and selection using haploid cells of maize developed by male gametophyte culture from a plant prepared by the method of claim 1.

26. A method for the production of genetic translocations, substitution and addition lines in maize, the method comprising the culture of male gametophytes of interspecific and intergeneric hybrids of maize prepared by the method of claim 1.

27. A method far the transformation of maize, the method comprising providing haploid cells of maize developed by male gametophyte culture from a plant prepared by the method of claim 1 and inserting genetic material into the haploid cells by a technique of microinjection apparatus or using a gene gun.