AU637602B2 - Method for increasing the ability of maize to undergo androgenesis, and products produced therefrom - Google Patents

Description

METHOD FOR INCREASING THE ABILITY OF MAIZE TO UNDERGO ANDROGENESIS, AND PRODUCTS PRODUCED THEREFROM

This invention relates to isolated male gametophyte culture, and the subsequent genetic transformation of the cultured male gametophytes. ^x*

c- 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

10 species which can then be marketed directly or used to produce superior hybrid plants.

.' The development of maize hybrids conventionally

_ involves three steps: (1) the selection of superior

15 plants from various germplasm pools; (2) the selfing of the superior plants for several generations to produce a series of inbred lines, which although different ^ffrom each other, breed true and are highly uniform; and (3) the crossing of selected inbred lines with unrelated .

20 inbred lines to produce the hybrid progeny (F-_j). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between any two inbreds will always be the same. Once

25 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 parents 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, 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. By definition, haploids are individuals which contain the gametic chromosome complement. Since maize 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 the chromosome complement of haploids is an attractive feature. However, attempts at utilizing haploids in maize 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 m vitro culture of gametophytic cells with the aim of 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 , "Haploids in Plant Improvement" , In IK Vasil , R Scowcroft, KJ Frey (eds.), Plant Improvement and

Somatic Cell Genetics, New York: Academic Press, 1982, pp . 129- 158 ; Heberle-Bors , ^"In Vitro Haploid Formation of Pollen: A Critical Review" , Theor . Appl . Genet . 71 : 361 -374 , 1985 ; and Hu and Yang, "Haploids of Higher Plants in Vitro" Springer- Verlag, Berlin, (1986).

Early events during invitro 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. Cytol.- 26:475-483 (1984); Raghavan , "Protein Synthetic Activity during Normal Pollen Development and During Induced Pollen Embryogenesis in Hyoscyamus niger", J. Can Bot. , 1984, 62:2493-2513; and Huang, "Ultrastructural Aspects of Pollen Embryogenesis in Hordeum, Triticim andPaeonia" In Hu H, Yang H (eds): "Haploids of Higher Plants in Vitro", Berlin, Heidelberg: Springer-Verlag, 1986).

The invitro culture involves isolating immature anthers from plants and placing the anther or, male gametophytes isolated therefrom, onto a medium which 0 induces the male gametophytes, which would normally- be destined to become pollen grains, to begin dividing and form a cell culture from which plants can be regenerated. This phenomenon is known as androgenesis. _c 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 0 fertile and completely inbred.

Anther culture provides a method for culturing male gametophytes directly in the anther. A positive in vitro response will lead to the development of embryos 5 and/or callus from which plants can be regenerated. For a general discussion of anther culture, see J. M. Dunwell, "Anther and Ovary Culture", In SWJ Bright and MGK Jones, (eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher, 1985, Dordrecht, pp. 1-44; and Keller et al. "Haploids from gametophytic cells - recent developments and future prospects", In CE Green, DA Somers, P Hackett, DD Biesoer (eds.), Plant Tissue and Cell Culture, Lain 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 and Prospect", A e . J. Bot. , 1982, 69:865- 879). However, the anther culture responsiveness varies considerably among species as well as among genotypes of the same species. A comparison of the overall responsiveness to anther culture is made difficult, as the results reported in published studies are given in different units. For example, anther culturability has been defined by the induction of male gametophytes that begin dividing, the number of embryos and/or callus produced 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.

Although relatively rapid progress has been made in several species, maize, unfortunately, has not shown detectable or significant anther culturability.

Although there has been much interest in developing anther culturable maize, there has been no description to date of significant anther culturability in maize. 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.

Although reports of haploid plant production

,- from maize gametophytes date back to 1975 (Ku et al.,

1978), response frequencies have been exceedingly low

(typically less than 1-2 embryos/100 anthers cultured).

(see Nitsch et al. , "Production of Haploid Plants Zea mays and

Pennisetum through Andro genesis"^', In ED Earle, Y Demarley

10 (eds.) Variability in Plants Regenerated from Tissue

Culture, Prager Publishers, New York, 1982, pp. 69-91);

Genovesi and Collins (1982) Crop Sci. 22: 1137-1144; and

Petolino and Jones, "Anther Culture o f Elite Genotypes of Maize",

(1986) Crop Sci. 26:1072-1074). 15

Even after a considerable amount of genotype screening , response frequencies have been undetectable in most genotypes and very low in those that do show any response , ( see Ku et al . , "Induction Factors and Morpho-

20 cytological Characteristics of Pollen-derived Plants in Maize" , ( Zea mays L . ) Proc Symp Plant Tissue Cult . , ( 1 978 ) Science

Press , Peking , pp 35-42 ; Genovesi et al . , "In vitro

Production of Haploid Plants of Corn via Anther Culture" , Crop

₂₅ Science , 22 , 1982 , pp . 1 137- 1 144 , Dieu and Beckert ,

1986 ; and Petolino and Jones , ( 1986 ) , supra .

Thus, none of the references provides a description of a procedure for producing maize ^•showing

30 detectable or significant anther culturability.

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. Unfortunately breeding for this trait has not been readily demonstrated, because anther culture is a difficult trait to assay.

It has been well documented that induction frequencies (departure from normal development) 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 (Pescitelli and Petolino, (1988) Plant Cell Rep. 7:441-444; and Pace et al., (1986) Theor Appl Genet 73:863-869).

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; enzel et al. (1975) Mol. Gen. Genet. 138, 293-297; and Wernicke et al. (1978) Z. Pflanzenphvsiol. 81, 330-340).

Competition among male gametophytes for space and nutrients in the anther and the potential for in vitro selection and genetic transformation make the culture of isolated male gametophytes desirable. Isolated male gametophyte culture involves removing the microspores from the anther and culturing them independently. Isolated male gametophyte culture provides a readily available source of free haploid cells (see Sunderland and Dunwell, (1977), supra). -7-

However, isolated male gametophyte culture requires a genotype demonstrating a relatively high anther culture response frequency . Consequently, many species have not heretofore proven capable of being amenable to isolated male gametophyte culture.

Most reports of successful plant regeneration from in vitrocultures of maize have involved relatively organized tissue cultures (see Hodges, TK et al., (1986) The Potential of Tissue Culture for Maize Improvement , In: Shannon JC, Knieval DP, Boyer CD (eds) Regulation of Carbon and Nitrogen Reduction and Utilization in Maize, averly 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., (1985) Planta 165:322-

332). Plant regeneration from single somatic cells of maize following protoplast isolation has been reported (see Rhodes et al., (1988) Biotechnology 6:56-60), however, seed could not be obtained. Only recently have fertile maize plants ben recovered from single, isolated protoplasts [see Shillito, et al., Biotechnology, vol. 7, page 581-587 (1989); Prioli and Sondahl, Biotechnology, vol. 7, 589-594 (1989)].

In 1977» Nitsch did report an attempt to culture isolated maize microspores, but the results were preliminary and unpublished (see Nitsch (1977) Culture of Isolated Microspores, In: J. Reinert and Y.P.S. Bajaj (eds) Applied and Fundamental Aspects of Plant Cell Tissue and Organ Culture, Springer-Velag) . Specifically, in Figure 3, Nitsch shows the development of a few cell-like divisions, but never reports that embryo-like structure(s) (ELS) or plants 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.

The combined difficulty of conventionally breeding genotypes for high anther culturability and the prerequisite for highly responding genotypes makes isolated gametoplyte culture in maize very difficult.

As can be seen from the above discussion, male gametophyte culture techniques are still rather 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.

One could not predict from the prior art at the time this invention was made, how to develop maize plants capable of increased levels of haploid and/or double haploid formation from cultured anthers and the use of such germplasm for anther culture, isolated male gametoplyte culture, and the like, and genetic transformation thereof.

In one aspect, the present invention provides a method for the production of a plant with an improved ability to undergo androgenesis, the steps of the method comprising: (1) providing anthers from at least one heterozygous donor plant; (2) regenerating, from the anthers obtained from the donor plant, at least two male gametophyte-derived plants capable of being intermated; (3) intermating the regenerated plants to produce an F-_] population; and (4) self-pollinating or cross- pollinating individuals of the F-_j population to generate at least one ?2 population.

In a second aspect, the present --invention provides an F2 plant, or progeny thereof, said plant containing a high anther culture (HAC) genetic factor, whereby the plant contains an anther culture response frequency at least 2 times greater than the anther response frequency of the original donor plant.

In a third aspect, the present invention is a method comprising the steps of: (a) providing at least one male gametophyte isolated from at least one anther from at least one donor maize plant containing the HAC genetic factor capable of being anther cultured; and (b) incubating the male gametophyte in an ELS induction medium to foster development of at least one ELS.

In a fourth aspect, the present invention is at least one callus which is generated from incubating an ELS; at least one plant which is generated from the callus; and at least one seed which is generated from at least one plant.

In a fifth aspect, the present invention provides at least one isolated male gametophyte containing a HAC genetic factor.

A sixth aspect, the present invention is a method for the transformation of maize, said method comprising providing haploid cells of maize developed by isolated male gametophyte culture and inserting genetic material into the haploid cells by a technique selected from the group consisting of particle bombardment, microinjection and infectious agents.

In a seventh aspect, the present invention is 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 isolated male gametophyte culture.

In a eighth aspect, the present invention is a method for the production of genetic translocations, substitution and addition lines in maize, said method comprising the culture of isolated male gametophytes of interspecific and intergeneric hybrids of maize.

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 which 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 the like.

By "male gametophyte" is meant the haploid phase of the life cycle, including a microspore of maize. 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 cytoplasm is dispersed throughout the cell. As the microspore increases in size, the nucleus remains near - l i ¬

the pore and becomes smaller. 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 graih 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 in contrast to the smaller 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 ^{* τ} 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 pollen grain mitosis occurs.

For purposes of this invention, anther culture response is measured in terms of embryo-like structures per 100 anthers cultured (ELS/ 100 anthers). By _*,

"embryo-like structures" (ELS) is meant globular^masses of cells resulting from repeated divisions ©f male gametophytes which are capable of continued growth and development and ultimately plant regeneration. Creation of Highly Anther Culturable Genotypes

Quite surprisingly, the present method will provide an enhancement in anther culture response frequency in the regenerated F2 progeny of 2 times greater, preferably 5 times greater and most preferably 10 times greater, than the anther culture response frequency in either parent.

This result is unexpected because the prior art would suggest that, even given a genetically determined trait susceptible to selection criteria, traditional breeding techniques would at best yield improved anther culturability of only up to about 10%. Conventional breeding techniques would suggest selecting genotypes based on their individual plant response to anther culture. Conventional breeding techniques are not a practical means of increasing androgenesis in subsequent generations, because of the need to sample within a genotype due to the rather significant non-genotypic, plant-to-plant variation in anther culture response [Petolino, et al., "Selection for Increased Anther Culture Response in Maize", Theor. Appl. Genet., Vol. 76, pp. 157-159 (1988)■. The fact that conventional breeding techniques are not suggestive of the present invention lies in the fact that artisans skilled in maize breeding and tissue culture have for many years attempted to develop highly anther culturable maize, with disappointing results previously reported. Jialin, "Breeding Haploid Corn by Anther Culture", H. Han, W. Hongyuan (eds), Haploids of Higher in Vitro, Springer- Verlag, Berlin, (1986).

Heterozygous plant 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 relativesrsof 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 F-_] progeny), three-way crosses (i.e., three inbreds 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)}.

Maize breeding techniques suitable for production of such first generation hybrids are well-known to those skilled in the art. Such techniques are described in "Corn and Corn Improvement", Sprague ed.» American Society of Agronomy, Publication No. 18, Madison, Wisconsin (1977); Poelhman, J. J3., "Breeding Field Crop"s , Henry Holt and Company, New York, (1959); and Welsh, J. R., "Fundamentals ofPlant Genetics and Breeding", , Wiley (1981). The disclosures of these volumes are herein incorporated by reference.

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 Pa91. 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 plant. Anthers may be removed from the plant at a suitable stage of maturity. 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 a nuclear stain. Acetocarmine and mithramycin are exemplary stains.

After anthers from the donor plant have been excised, the second step involves utilizing cell culture technology to isolate genotypes which express anther culturability.

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

(induction) may be employed. A preferred pretreatment method is to expose the tassel or excised anther to a temperature of between 4 and 12°C as set forth in Petolino and Jones, (1986), supra.

The anthers may then be cultured by any suitable technique for abnormal development. See, for example, Petolino and Jones, (1986), supra; and Dieu and Beckert (1986), supra. Generally, the anther culture techniques comprise culturing the anthers in a basal salt mixture. Suitable basal salt mixtures include YP,

10 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 43-56, the teachings of each being hereby incorporated by

15 reference. A preferably basal salt mixture is the YP basal salts should be used as taught by Ku et al., __; (1978) Proc. Symp. Plant Tissue Cult., Science Press, Peking, pp 35-42, the teachings of which are hereby

P₀ incorporated by reference.

Exemplary carbon sources include sucrose, glucose, and fructose, with sucrose being -preferred..

Generally, the carbon source should be present in a _. _pc- concentration sufficient to support cellular growth.

Preferably, the carbon source will be present in an amount of between 10 and 200 g/L. Most preferably, when the carbon source is sucrose, the carbon source will he present in an amount of between 60 and 90 g/L.

30

Generally, the anthers will be cultured in a medium that contains conventional organic components which are essential for cell function. Suitable organic components include vitamins, amino acids, and hormones.

35 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/L. An exemplary hormone includes 2,4,5,-triiodobenzoic acid. Preferably, the hormone will be present in an amount of 5 between 0.1 and 0.2 mg/L.

The components of the ELS induction medium are preferably mixed with activated charcoal. Generally, the activated charcoal is added with the ELS induction 10 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

15 activated charcoal is added with the other components in an amount of between 1 and 10 g/L, and most preferably between 3 and 7 g/L.

The activated charcoal is preferably filtered

20 out prior to contacting the gametophytes with the ELS induction medium. The activated charcoal may be filtered by any method known to the skilled artisan. An exemplary filtration technique includes passing medium _pt- 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.

30 The anthers are cultured for a period of time and within a suitable range of temperatures and light intensities to foster the formation of ELS. The anthers are cultured preferably in the dark; and preferably at a temperature ranging from 20 to 32°C, most preferably 35 between 26 to 29°C These ELS may be yellowish-white with diverse shapes ranging from normal bipolar or globular embryos to multi-lobed or otherwise abnormally configured embryos. Preferable ELS will have distinct scutella and meristematic regions. Suitable ELS for further culture are each 0.5 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

10 "callus" is meant cells proliferating in a more or less disorganized manner without differentiating into organized structures (roots, leaves, etc.).

The ELS are cultured on the callus induction

,j- 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.

20

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

25 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 basa **l salts,

30 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 35 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/L. Most preferably, when the carbon source is sucrose, the carbon source will be present in an amount of between 10 and 40 g/L.

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 0 between 0.1 and 10 mg/L. Exemplary hormones include dicamba, 2,4-D, and picloram.

Generally, the callus induction medium may contain other conventional organic components, such as

15 vitamins and amino acids, as taught in Street, 1977. 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. 0

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 5 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

30 sterilized by autoclaving, e.g., utilizing 16 psi (110 x 10^ kPa) steam. The pH of the media prior to autoclaving normally ranges from 5.6 to 6.0.

Generally, the ELS will be cultured in the ^•35 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 14 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 poisαra., i.e., a chemical capable of interfering with mitosis. For 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, the teachings of which are hereby incorporated by reference. Exemplary chromosome doubling agents include nitrous oxide and colchicine.

Plant Regeneration

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.

Intermating of Regenerated Plants

After producing a population of regenerated double haploid plants, the regenerated plants should be randomly crossed to produce a series of F-_j hybrid's, and then the F-_| hybrids are self-pollinated produce an F2 population having genetic variability, i.e, segregating populations. Quite surprisingly, by intermating regenerated plants from anther culture to produce an F-_j population and self-pollinating individuals of the F-_j populations to generate an F2 population, the F2 progeny of those plants have an anther response frequency at least 2 times greater than the anther culture response of the donor plant.

c Exemplary techniques for intermating the regenerated plants include single, three-way and double crosses; the techniques for performing the crosses has hereinbefore been described and incorporated by reference. Preferably, the individuals of the

10 regenerated 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

15 maximum genetic variability in the F2 (S ) populations. The F₂ population is created by self-pollinating or cross-pollinating individuals of the F-_j population. Any method of creating a segregating population to produce maximum genetic variability may be employed to create

20 the F2 population. Thus, the present invention contemplates pollinating individuals of the F-_| population by self pollination; or by being crossed with other plants such as with other members of the F-_{] p}c population, or even nonresponsive plants; provided that the F₂ progeny of those plants have an anther response frequency 2 times greater than the anther culture response of the donor plant. Self-pollination of the F-_j individuals is preferred, to maximize the release

30 genetic variability of the F-_j.

Genetic Fixation of Segregating Population

Thereafter, the segregating populations are

35 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 them 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 (S-_j-S^),^' so that allelic pairs of genes on homologous chromosomes are homozygous or identical.

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, therefore, should be essentially homozygous and uniform in appearance.

An exemplary embodiment of the present invention is the sibling pollination, or random crossing with others in the inbred line of a regenerated plant 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. As shown in the present examples, the seed deposited at ATCC is as follows: 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. Individuals of 139/39-05 were subjected to the anther culture and seed from the resulting regenerated plants, 139/39-DH (ATCC number 40520) was deposited at the ATCC on December 1, 1988.

The HAC genetic factor may be defined using restriction fragment length polymorphism (RFLP) mapping, as taught, for example in J. S. Beckmann and M. Soller, restriction fragment length polymorphism in plant genetic improvement. In plant Molecular and Cell

Biology, Vol 3, Oxford University Press, 1986, pp. 196- 250; and W0 84/04758, the teachings of which are hereby incorporated by reference

W0 84/04758 teaches a method of hybridization of restriction fragments with labeled probes until a genomic fingerprint of the tested variety is established. A comparison of the genomic fingerprints established with the genomic fingerprints of other individual plants or varieties, which have been established in the same way, determines the degree of -23-

relatedness or identity of individuals or varieties.- The differences in these genomic fingerprints which- * define the degree of genetic similarity are restricteon fragment polymorphisms. Comparison between the occurrences of a particular characteristic in a variety and the fingerprint of individual isolates by computer analysis will suggest which random clones used as probes are linked to the genes of interest.

Using RFLP mapping, the chromosomal location(s) of the HAC genetic factor in maize is determinable by one of ordinary skill in the art without undue experimentation. Thus, given the chromosomal location(s) of the HAC genetic factor, maize germplasm may be routinely RFLP-mapped to determine the presence,, or absence of the HAC genetic factor.

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 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

10 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,

15 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 ₀ 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

25 having a male gametophyte culture response frequency 2 times 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)

30 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 Sequence

-,.- Comparison, Addison-Wesley, Reading, MA, (1983).

Roughly, two sequences are aligned by maximizing the -25-

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 presently-disclosed 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 Acid Hybridization, Hames and Higgens (eds.), IRL Press, Oxford, UK (1985)]. Given two sequences, algorithms are available for computing their homology: e.g. Needleham and Wunsch, J. Mol. Biol , 48, 443-453 (1970); and Sankoff and Kruskal (cited above) pgs. 23-29.

Since seeds containing the HAC genetic factor have been deposited with the ATCC, 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 nuclqotides 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 oligonucleotide- mediated, site-directed mutagenesis and polymerase chain reaction.

Oligonucleotide site-directed mutagenesis in essence involves hybridizing an oligonucleotide 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 oligonucleotide to produce a strand containing the mutation. This technique, in various forms, is described by Zoller, M.J. and Smith, M. , Nuc. Acids Res. 10, 6487-6500 (1982); Norris, K., Norris, F., Christiansen, L. and Fiii, N., Nuc. AcidsRes. JJ_, 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 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., Nucl. Acids Res. 16, 7351 -7367 ( 1988 ) , Ho et al . , Gene 77 , 51 -59 ( 1989 ) , and "Engineering Hybrid Restriction Genes Without the Use of Restriction Enzymes: Gene Splicing by Overlap Extension" , Horton et al . , Gene 7_1_> 6 1 ( 1 989 ) . In Vitro Culture of Highly Anther Culturable Genotypes

Plants produced as above may in turn be used in subsequent anther cultures, by the- techniques taught above. However, the present invention also provides- isolated male gametophytes that are well-suited for being in vitro cultured and regenerated into plants.

Isolated male gametophytes may be obtained from any anther culturable genotype containing HAC. -

The principle of in vitro culture of isolated male gametophytes is to remove male gametophytes from the anther to divert the normal development 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.

Once the plant is selected, its tassel may be harvested and pretreated. A preferred method of tassel pretreatment as set forth in Petolino and Jones (193,6) supra.

In one embodiment, male gametophytes may be removed from the anther from about the early uninucleate to about the late binucleate stage of development. Preferably, the male gametophytes will be removed at between the late uninucleate and the early binucleate 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 gametophytes into an ELS induction medium.

Generally, mechanical removal of the male gametophytes may be accomplished by passing the anthers through a mesh screen as set forth in R. Lichter. (1982)

Z. Pflanzenphysiol. 105:427-434; or by icroblending as set forth in Swanson et al., (1987) Plant Cell Reports,

6:94-97.

A particularly preferred technique for the mechanical removal of male gametophytes comprises blending excised anthers using a commercial laboratory blender and a specialized blending attachment. Similar procedures have been described in Coumans et al., "Plant

Development from Isolated Microspores of Zea Mays L.",

Plant Cell Reports, 7, 1989, pp. 618-621. In this example, whole tassel segments were blended.

In this embodiment, the isolation procedure consists of blending excised anthers. A single isolation consisted of placing 150 to 300 anthers anthers into an appropriate blender attachment (preferably a 110 mL Waring MC-2) along with 40 to 60 mL. of the ELS induction medium. The container is placed on a commercial laboratory blender and blended for 5 to 15 seconds. This creates a slurry of gametophytes and anther wall material which is passed through an appropriately sized sieve, preferably 100 to 120 micrometer in size. The isolated gametophytes pass through the sieve and are then collected via centrifugation for 5 minutes at 1000 RPM and placed into ELS induction medium at an appropriate density, preferably 6000 to 15000 gametophytes per mL. The present invention also provides a method by which the viable male gametophytes can be separated either prior to or during first seven days of culture. A population of male gametophytes, whether within the anther or in isolated culture, consists of both viable and nonviable cells. Concentration of the viable cells is beneficial, particularly in isolated oultures .used for genetic manipulation. Transformation efforts" then can be focused on the viable male gametophytes, thereby increasing the chances of successful gene transfer and plant recovery. Removal of the dead microspores also improves the growing conditions for the developing ELS.

One example of a technique to collect viable male gametophytes is the use of a discontinuous -ϊd-ensity gradient. The gradient medium used here may consist of any suitable material having a density greater than the density of the ELS induction medium, such as sucrose, , Ficoll, or Percoll, preferably Percoll. The concentration of the gradient material should be 10 to

30 percent, preferably 20 percent in combination with 80 percent ELS induction medium. Three to five mL of this solution is placed in a centrifuge tube (preferably 10 mL size) and mixed with 1 mL of-a concentrated solution of 500,000 to 1,000,000 isolated male gametophytes. .^The pure ELS induction medium is layered on top of the*male gametophyte/Percoll solution and centrifuged at an rp'm and for a time effective to create a band of male gametophytes at the Percoll/ELS induction medium interface while the other male gametophytes are captured in a pellet at the bottom of the tube. Generally, the solution is centrifuged at 500 to 1500 rpm for 2 to 10 minutes, preferably at 1000 rpm for five minutes. The upper band retains approximately 30 to 70 percent of the male gametophyte of which 50 to 80 percent are viable. The lower pellet typically contains 0 to 2 percent viable male gametophyte.

_c 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. (1977) Nature 270:236-238.

10

Further, the isolated male gametophytes may be converted into protoplasts. By "protoplast" is meant a plant cell without a cell wall. Suitable techniques for producing maize protoplasts is set forth in EP 0 292

15 435. Generally, that reference teaches incubating the cells with an enzyme preparation which removes the cell wall. Suitable enzyme preparations are known in the art. For purposes of this invention, the term "male gametophytes" is intended to include protoplasts

20 thereof.

ELS Induction

The isolated male gametophytes may be cultured 25 on an ELS induction medium and under conditions which are essentially the same as used to culture anthers, as set forth above.

The present invention also provides a method by

30 which the developing ELS may be separated at a stage of development ranging from 3 to 28 days after culture initiation. The earlier stages of development are preferable for use in genetic transformation.

Separation of the ELS increases the efficiency of gene ^■55 transfer and plant recovery by isolating those ELS most likely to continue development. Removal of dead male gametophytes also improves the growing conditions for the developing ELS.

The induced male gametophytes an be separated _c from the non-induced or dead individuals by passing the liquid cultures through a sieve with a mesh size ranging from 74 to 150 micrometers. The induced male gametophytes are slightly larger in size than non- induced individuals and are retained by the sieve. They 10 are then transferred back into the ELS Induction medium. This separation can be done from 3 to 10 days, preferably at 7 days using an 88 micrometer mesh size. ELS beyond 10 days old can also be separated using mesh sizes ranging from 88 to 150 micrometers.

15

Callus Induction

Selected ELS are removed from the ELS induction medium and transferred to a callus induction medium, as 20 described above. „

Plant Regeneration

Selected callus are individually transferred

25 from the callus induction medium to a semi-solid or liquid regeneration medium, as described above.

From the plant regeneration medium, the plantlets are planted in soil or potting medium,

30 e.g., in a greenhouse or in the field and under ambient normal growth conditions the plantlets develop into whole fertile plants which flower and develop seeds.

Finally, the seeds are recovered to provide ,_c 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,

Uses

10 Improvements 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.

15

An important aim of traditional plant breeding is to engineer improved plants that are valuable as crop plants. In highly heterozygous, cross-pollinating 0 crops, such as maize, 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-_j donor plants permits the breeder to effectively select 25 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.

30 The use of haploids has many potential applications in cellular biology research. One application is in vitro screening and selection using haploid cells, and to obtain mutants in a homozygous condition. Another application of haploidy is the ^•3 - 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 methodolog for selecting rare genotypes, including those with recessive characters, and to include them in new combinations of crosses.

Transfer of Anther Culturability

The method of the present invention now provides a means for producing maize plants that have male gametophytes, either in or isolated from the anther, that are capable of undergoing embryogenesis in vitro .

The present invention provides a geneticall transmitted high anther culturability (HAC) characteristic which can be selectively incorporatedr into progeny. Thus, traditional sexual techniques (e.g., breeding) or asexual techniques (e.g., in vitrb 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 be 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.

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 culture response frequency.

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 backcrossing (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 modified by anther culture of the F-_j, 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 F-_| 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 for loci controlling the characteristics being transferred, but will be like the superior parent for most or almost all other genes. The procedure can be modified by^" anther culture of, for example, the F-_j, F2, F 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 _c research into the transformation of plants via genetic engineering techniques. A still further benefit of anther culture is in the ability transform haploid maize ^' via genetic engineering techniques. Progress in the transformation of maize has been restricted by the

10 limitations of presently available gene-transfer systems. Exemplary techniques for the delivery of rDNA (recombinant DNA) providing increased androgenesis (e.g., the HAC genetic factor) include microinjection (see Crossway et al. Mol. Gen. Genet., 202: 179-185), or

15 the gene gun technology (see Klein et al., Biotech, 6:559-563).

Conventional technologies for introducing biological material into living cells include particle

20 bombardment technology, microinjection mechanisms, infectious agents, and techniques for protoplast transformation (e.g., electroporation, uptake mechanisms, and fusion. These techniques provide, upon _2c chromosome doubling, a plant homozygous for the rDNA.

Relatively recently, technology has been developed to deliver substances into cells of intact tissues via particle bombardment.

30 One particle bombardment apparatus is set forth in the Examples section.

Another particle bombardment technology is set forth in High-velocity Microprojectiles for Delivering Nucleic Acids ^' into Living Cells ( 1987 ) , T . M. Klein , E . D . Wolf , R . Wu and J . C . Sanford , Nature , 337 ; and Delivery of Substances into Cells and Tissues using a Particle Bombardment Process ,

J. C. Sanford, T. M. Klein, E. D. Wolf, and N. Allen, Particular Sci. and Technol., 5:27-37. These references teach the acceleration of at small high-density, tungsten particles (microprojectiles) may be accelerated to high velocity via the following embodiments: (1) _, macroprojectile (plastic bullet) and stopping plate, (2) a transferred mechanical pulse, (3) a gas (e.g., air) discharge, and (4) a centripetal acceleration system.

A still third apparatus is set forth in published EP 0 270 356. Generally, the reference teaches an apparatus for injecting carrier particles carrying DNA into living cells character z^'ed in that it comprises: a spark discharge chamber; two electrodes extending into the spark discharge chamber and spaced apart by a spark gap, the electrodes being adapted for attachment to an external source of high voltage discharge; a carrier sheet held spaced above the spark discharge chamber, the carrier sheet receiving the carrier particles thereon; a retaining screen fixed in place above the carrier sheet; and a target surface held spaced above the retaining screen and carrying the cells so that a spark discharge generating a shock wave in the discharge chamber will accelerate the carrier sheet into the retaining screen so that the carrier particles are accelerated into the cells on the target surface.

Another technique is a direct method for the transfer of chromosomes by microinjection. For a general discussion of microinjection techniques, see Methods for Microinjection of Human Somatic Cells in Culture , ( 1973) , E.G. Diacumakos, In: Methods in Cell Biology, D.M. Prescott (ed.), Academic Press, NY, pp. 287-311; Microinjection of Tissue Culture Cells ( 1973) _> M. Graessman and A. Graessman, Methods in Enzymology, 101:482-492; Crossway A., Oakes J. V., Irvine J. M. , Ward B. , Knauf V. C, Shewmaker C. K. (1986), Integration of Foreign DNA following Microinjection of Tobacco Mesophyll Protoplasts, Mol. Gen. Genet. , 202:179-185; Crossway A., Hauptli H., Houck C. M., Irvine J. M. , Oakes J. V., Perani L. A. (1986), Micromanipulation Techniques in Plant Biotechnology , Biotechniques, 4:320-334; Reich T. J., Iyer V. N., Scobie B., Miki B. L. (1986), A Detailed Procedure for the Intranuclear Microinjection of Plant Protoplasts , Can. J. Bot. ; and Reich T. J., Iyer V. N. , Miki B. L. (1986), Efficient Transformation of Alfalfa Protoplasts by the Intranuclear Microinjection ofTiPlasmids, Bio/Technology, 4:1001-1004.

In addition to the systems mentioned above, there exist several infectious agents which can deliver nucleic acids into cells. Of primary importance are the Agrobacterium vectors for dicot plant cells (Genetic Transformation in Higher Plants (1986), R. T. Fraley, S. G. Rogers, and R. B. Horsch, CRC Crit. Rev. Plant Sci. , 4:1-46; and the retroviral vectors for animal cells (Prospects for Gene Therapy (1984), F. W. Anderson, Science, 226:401-409).

Retroviruses (RNA viruses) can be used to deliver genes into animal cells. When the virus enters the cell its RNA acts as a template for reverse transcription of complementary DNA which will integrate into the genome of the host cell. This DNA can be isolated and inserted into a plasmid. This plasmid, with additional genes added, can be used to transform cells with the aid of helper retroviruses. Electroporation is a method for introducing, a variety of molecules into cells and protoplast isolated therefrom by subjecting them to brief high-vol^*tage electric pulses. For a general discussion of electroporation, see Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells, (1988), K. Shigekawa and W. J. Dower, Biotechniques , 6. : 742 ; High-voltage Electroporation ofBabteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA , (1988), J. C. Miller, W. J. Dower, L. S. Tomkins, Proc. Natl. Acad. Sci. USA, 85.: 856-860; and A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation, (1988), I. G. Powell, M. G.^Achen, A. J. Hillier, B. E. Davidson, Appl. Environ. Microbiol. , 54:655-660. ^{' *}

One technique for uptake is the enhancement of membrane permeability by use of calcium (Ca) (Calcium Dependent Bacteriophage DNA Infection (1972), M. Mandel and A. Higa, J. Mol. Biol., 53:159-162); and temperature shock (Frozen-thawed Bacteria as Recipients of Isolated Coliphage DNA (1972), S. Y. Dityatkin, K. V. Lisovskaya, N. N. Panzhava, B. N. Liashenk, Biochimica et Biophysica Acta, 281:319-323). ^{Λ Λ}

A second technique for uptake is the use of surface-binding agents such as polyethylene glycol (PEG). For a general discussion of surface-binding agent, see High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA (1972), S. Chang, and S. N. Cohen, Mol. Gen. Genet., 168:111-115; Invitro Transformation of Plant Protoplasts With Ti-plasmid DNA ( 1982 ) , F. A. Krens, L. Molendijk, G. J. Wullems, and R. A. Schilperoort, Nature, 296:72); or such as calcium phosphate (A New Technique for the Assay oflnfectivity of Human Adenovirus 5 DNA (1973), F. L. Graham, and A. J. Van der Eb, Virology, 52:456; Transformation of Mammalian Cells with Genes from Procaryotes and Eucaryotes (1979), M. Wigler, R. Sweet, G. K. Sim, B. Wold, A. Pellicer, E. Lacy, T. Maniatis, S. Silverstein, and R. Axel, Cell, 16:777.

A third technique for uptake is the phagocytosis of particles into a protoplast. Suitable particles include liposomes (Liposome-mediated Transfer of 0 Plasmid DNA into Plant Protoplasts (1982), H. Uchimiya,

T. Ohgawara, and H. Harada, In: A. Fujiwara (ed.), Proc. 5th Intl. Cong. Plant Tissue and Cell Culture, Jap. Assoc. for Plant Tissue Culture, Tokyo, pp. 507-508); organelles ( Transplantation of Chloroplasts into Protoplasts of ^{c J} Petunia ( 1973 ) » I. Potrykus, 1. Pf lanzenphysiol. , 70:364

-366); or bacteria (Plant Cell Protoplasts Isolation and

Development (1972), E.C. Cocking, Ann. Rev. Plant

Physiol., 23:29-50). 0 Fusion can be induced with electric currents,

PEG, and Sendai virus particles. For a general discussion of cell fusion, see Methods Using HVJ (Sendai Virus) for Introducing Substances into Mammalian Cells (1980), ₅ T. Uchidaz, M. Yamaizumi, E. Mekada, Y. Okada, In: Introduction of Macromolecules Into Viable Mammalian Cells, C. Baserga, G. Crose, and G. Rovera (eds.) Wistar Symposium Series, Vol. 1, A. R. Liss Inc., NY, pp. 169-185; and H. Harris, Cell Fusion: The Dunham Lectures 0 (1970), Oxford University Press.

Examples

The following examples are presented to further 5 illustrate but not limit the scope of this invention. All parts and percentages are by weight unless otherwise indicated. ϊ Example 1

Development of High Anther Culturable (HAC) Genotypes

A three-way cross was performed to create the donor plant. The inbred plants used in the three-way cross were H99, FR16, and Pa91. Seed for producing the inbred lines was obtained from Holden's Foundation Seeds, Williamsburg, IA (H99 and Pa9D 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

__. binucleate state of development, as determined microscopically after treatment with acetocarmine, were removed from donor plants 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 hypochlorite solution followed by a sterile water rinse. Only anthers from the central portion of the main tassel branch were used.

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/L activated charcoal, 500 mg/L casein hydrolysate, 0.1 mg. 2,3,4,5-triiodobenzoic acid, 120 g/L sucrose, and 8.0 g/L agar (Gibco) adjusted to pH 5.8. Typically 3-6 dishes were obtained from each tassel harvested. Dishes containing freshly plated anthers 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 ELS were apparent.

ELS were yellowish-white and globular in appearance and resembled zygotic embryos displaying varying degrees of abnormal tissue proliferation.

ELS were lifted from the anthers and placed onto a regeneration medium (YP with 1.0 mg/L indole-3- acetic acid), 1.0 mg/L kinetin, 146 mg/L glutamine, and 30 g/L 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 ELS, obtained from two separate tassels, regenerated plants. The regenerated plants were grown to maturity during October to December, 1985. One plant (#139) 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 #139 resulting in the formation of a single F-_j hybrid (139/39) seed. The F-_| hybrid seed was germinated and the resulting plant was self-pollinated, i.e., pollen from one plant is used to fertilize itself to produce and F2 (S₀) population.

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 S-_j families were evaluated for their anther culturability. Evaluation was conducted by selecting tassels with anthers containing late uninucleate-early binucleate microspores, as determined by mithramycin/flourescent staining as set forth in Pace et al., "Anther Culture of maize and the visualization ofembryogenic microspores by flourescent microscopy", Theor. Appl. Genet., 73:863-869, 1987. the procedures of which are incorporated by reference. Selected tassels were removed from the donor plants prior to emergence from the whorl. The results are set forth in Table 1.

TABLE 1

Mean Anther Culture Response from Cultured Anthers of 14 S-_j

Families of Maize

^"Embryo-Like Structures

A population of S-_| 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. The two original plants from which the tasSels were harvested , that ultimately lead to the regeneration of plants #39 and #139, were not among the most productive based on their individual anther response . It was rather surprising, therefore, that the 14 Si lines responded as well as they did. Apparently, the intermating of male gametophyte-derived plants is an effective means of shifting allelic frequencies toward increased responsiveness.

Individuals of 139/39-05 were subjected to the anther culture technique described above. Seed from the resulting regenerated plants, 139/39-DH (ATCC number 40520) was deposited at the ATCC on December 1, 1988.'

Self-pollinations were made with four of the most responsive families (139/39-01, -02, -05, and -09) and ≤2 seed from the resulting ears were grown during April to October, 1988. Self-pollinations were made within each of the families to produce novel So germplasm of this invention.

Since this material has undergone forced inbreeding for 4 generations (F-_j, F₂ (S₀) , S-_j, S₂) approximately 95 percent of those loci which were heterozygous in the original F<_| hybrid are now homozygous.

Transfer of High Anther Culturability to Other Genotypes

Crosses were made between four selected S₂ families and four other agronomically-important inbred genotypes (UAS 1, UAS 2, UAS 3, and UAS 4) each of which is unresponsive to anther culture. The resulting F-_j hybrids were evaluated for their anther culturability as measured by ELS formation. The results are set forth in Table 2.

TABLE 2

Mean Anther Culture Response of 16 F-_j hybrids of Maize ^" .^"■

20.6

29.1

6.2

32.5

48.8 171.1 107.6 104.2

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. Crosses were made between an unresponsive maize variety and either 139/39-02 or any of the three parents from which 139/39-02 was derived.

The resulting F<_| hybrids were evaluated for their anther culturability as measured by ELS formation. The results are set forth in Table 3.

TABLE 3 Mean Anther Culture Response of Fi Hybrids of Maize

* Not an example of the present invention.

The data shows how an unresponsive line is made responsive with HAC but not with the parents of HAC. As mentioned previously, HAC appears to be a unique combination of genetic factors isolated in 139/39-Bulk after intermating anther-derived individuals. Table 3 shows a comparison of response data of attempts to transfer HAC to another unresponsive genotype.

Example 2

The maize genotype used for donor plants (139/39-02), is developed as set forth in Example 1. 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 c 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.

_ιn The ELS induction medium consisted of YP basal salts (see Ku (1978), supra) with the addition ^Jof abojαt ^ 60 g/L sucrose, about 5 g/L activated charcoal about 500 mg/L casein hydrolysate, and about 0.1 mg/L 2,4,5 -triiodobenzoic acid adjusted to pH 5.8. Prior to use,

15 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.

20

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.

25 Within 2 days, anthers cultured in a liquid medium ** dehisced, resulting in a stationary suspension of male gametophytes. Maximum dehiscence occurred after 7 days with approximately 40 percent of the male gametophytes

30 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.

35 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 4.

The ELS were 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/L sucrose, 2.9 g/L L-proline, 100 mg/L myo-inositol, 100 mg/L casein hydrolysate, 2.5 mg/L dicamba, 0.1 mg/L 2,4 -D, and 8 g/L 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 μmol m~² sec""^) from cool-white light fluorescent lamps. The regeneration medium consisted of SH basal salts (Schenk and Hildebrandt (1972), supra) and 10 g/L 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 4.

TABLE 4

Formation of ELS and plant regeneration from cultured male gametophytes of maize.

As seen in Table 4, isolated male gametophytes, dehisced from anther, produced ELS from which plants could be recovered.

Example 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~^ as determined by hemocytometer counts (see Street, (1977), supra). The hemacytometer used was a Speirs- Levy Eosinophil commercially available from C. A. Hausser & Son, Philadelphia, PA.

The results for ELS produced/per 100 Anthers and Total Regenerable Cultures/per 100 ELS are set forth in Table 5.

TABLE 5

ELS formation and plant regeneration from male

10 gametophytes mechanically isolated from maize anthers at various times after culture initiation.

15

0

As seen in Table 5, isolated male gametophytes, mechanically isolated from anthers, produced ELS from 25 which plants could be recovered.

Example 4

The procedures of Example 2 were repeated -_3Q except that the anthers had the male gametophytes mechanically isolated prior to the initiation of culture and the separation techniques previously described were employed and plating efficiencies were determined. All isolations were performed on anthers after excision from 35 the florets. The isolation procedure consisted of placing 60 anthers into a 110 mL stainless steel -53-

blending attachment (Waring MC-2) along with .0 mL of the ELS induction medium. In this experiment the Percoll gradient separation (at day 0) was used alone and in combination with the sieve separation (at day 7) and an evaluation of plating efficiencies was conducted for each treatment. The Percoll gradient consisted of a 3 mL lower layer of 20 Percoll/80 ELS induction medium and an upper layer of 1.5 mL pure ELS induction medium all contained within a in a 15 mL centrifuge tube. The isolated male gametophytes were added to the lower layer in a 1 L slurry which contained 500,000 to 1,000,000 male gametophytes and the tube was centrifuged at 1000 RPM for 5 minutes. The upper band of microspores was the saved.

The treatments in this study consisted of: the control, in which no separations were made; 0-day Percoll gradient separation only; 0-day Percoll gradient separation together with mesh size separation using 74 micrometer pore size at day 7; and 0-day Percoll separation together with 88 micrometer pore size performed at day 7. After separation, all gametophytes were returned to ELS induction medium at a density of between 7500 and 15,000 per mL. Both retained and released (those passing through the sieve) fractions were evaluated.

Microspore density was estimated using a hemocytometer with four counts for each plate. Density, total ELS, ELS per 100 anther equivalents (assuming 2500 gametophytes per anther) plating efficiency and RC data are given in Table 6 below. Table 6: ELS Formation and Plant Regeneration from Male

* AE = Anther Equivalent = 2500 gametophytes ** RC = Regenerable culture from which plants are obtained

Gametophytes Mechanically Isolated and Separated from Maize Anthers

Isolated male gametophytes in the control cultures yielded ELS response levels close to the range typically observed in intact anther cultures (see previous Examples 1 and 2) without the application of any separation procedures. Use of the percoll gradient alone resulted in a significant increase in plating efficiency. Further improvements in response level were observed with the combination of Percoll and sieve separation. The 88 micrometer mesh size was particularly effective providing a 16-fold efficiency improvement over the control. In terms of ELS production, the levels observed in this treatment were equivalent to 1200 ELS/100 anthers, exceeding intact anther culture response for this genotype. In addition, it was apparent that the separation procedures also enhanced the quality of the ELS as indicated by the recovery of RC. Studies have shown that the sieve technique, can also be used at later stages (21-28 days) of culture' to capture the larger more developed ELS. Using this . procedure, effective plating efficiencies should '^■ * approach a level equivalent to RC recovery (8 to 24 percent), as shown in Table 7 below. Table 7 ELS

* AE = Anther Equivalent 2500 gametophytes

Formation and Plant Regeneration from Male Gametophytes Mechanically Isolated and Separated from Maize Anthers

These techniques described above in Table 4 have been used to produce targets for particle bombardment as described in Example 5.

Example 5

Selected germplasm according to the present invention has also been transformed using particle '■

_ « bombardment technology.

Carrier particles coated with DNA-containing plasmids (pDAB199) are suspended in a carrier medium. More specifically, the plasmid is adsorbed to the surface of gold particles. The plasmid contains ,a gene which encodes for the enzyme beta-glucuronidase (GUS gene) and which is under the control of a 35s Cauliflower Mosaic Virus (CaMV) promoter. The gold particles are spherical powder of about 1.5-3.0 microns in diameter (commercially available from Alfa Products, Danvers, MA).

To accomplish adsorption, 50 μl of a plasmid solution (1.8 μg of DNA per μl of 0.01 M Tris buffer, pH 8.0, with 0.001 M ethylene diamine tetraacetic acid (EDTA)) is added to 400 μl of a suspension of gold carrier particles (300 mg of gold carrier particles per milliliter (mL) of distilled water). The DNA is precipitated by the addition of 74 μl of a 2.5 M calcium chloride solution and 30 μl of a 0.1 M spermidine solution. The coated carrier particles are allowed to settle to the bottom of an Eppendorf tube and the resultant clear liquid is completely drawn off. The carrier particles are resuspended in 500 μl ethanol (100 ) (carrier medium). One carrier particle is coated with approximately 10 copies of the plasmid.

The target matter preparation is as follows. Briefly, isolated male gametophytes are cultured in ELS induction medium. After 6 to 9 days in culture the induced MG (proembryos) are separated out by passing the cultures through an 88 micrometer mesh. A sample of approximately 100 mg of the proembryos is placed onto filter paper on top of agar solidified ELS induction medium in a 60 mm Petri plate. ELS in later stage of development (19 to 22 days after culture initiation) were also used. Preparation of the target matter from the older cultures is similar except that the separations are made using a 113 micrometer mesh. Male gametophytes cultured for 6 to 9 days (proembryos) and also for 19 to 22 days were separated by passing through an appropriately sized sieve (as previously described). These were then arranged into monolayer targets consisting of approximately 100 mg and were bombarded. The target area consists of a circle, 1 cm in diameter on a filter paper placed on top of agar- solidified ELS induction medium.

The apparatus used to accelerate the coated gold particles at the isolated male gametophytes, consisting of essentially the same apparatus described below. The apparatus 10 is described in greater detail in Figures 1, 2, and 3.

(1) As seen in Figure 2, source of gas 30. A 1A size gas cylinder (i.e., gas supply means 31a) filled with helium gas is in fluid communication with a stainless steel capillary tube having dimensions of 7' x 0.02" I.D. (i.e., gas supply line 33). The gas supply line is in operative combination with a standard two- stage pressure regulating valve, commercially available from Victor Equipment Co. (i.e., gas regulating valve 32a). The gas supply line is in turn in communication with a high pressure stainless steel gas chamber having dimensions of 1/8" O.D. x 3" length (i.e., gas r *_e__servoir

32b).

(2) As seen in Figure 3, source of propellable matter 20. The source of propellable matter comprises a 3 cubic centimeter (cc) Luer Lok sterile syringe 21a (i.e., propellable matter supply means) is in fluid communication with 1/10" Teflon (trademark of E. I. DuPont de Nemours Co.) FEP tubing (i.e., propellable matter supply line 23).

(3) As seen in Figure 1 ,* a recovery means 60. The recovery means is a clear 1/10" OD FEP Teflon overflow tube.

(4) As seen in Figure 1, a delivery means 50. The delivery means comprises a pyrex glass tube having dimensions of 10 cm x 1.2 mm O.D. x 0.8 I.D. mm.

(5) As seen in Figure 1, a dual three-way valve 70. The valve comprises two subvalves (subvalve 70a and 70b) commercially available from Rheodyne, Inc., Cotati, CA 94928 under the trade designation model 7030 ARV. The subvalves of the dual three-way valve 70 are in operative combination with a pnuematic actuator kit #41687 commercially available from Anspec Co., Ann Arbor, MI. The pnuematic actuator is driven by air supplied from a four-way solenoid valve, commercially available from the Automatic Switch Company (ASC0), Florham Park, NJ.

(6) As seen in Figure 1, a propellable matter reservoir 40. The propellable matter reservoir is an external loop 40 made of stainless steel, 1/16" O.D. is in operative combination with the first and second three-port valves.

The first three-port valve provides selective fluid communication between the propellable matter supply means or the gas reservoir, and the propellable matter reservoir. The second three-port valve provides selective fluid communication between the propellable matter„ reservoir and the recovery means or the delivery means.

The multipurpose valve is initially set in order to provide fluid communication between the 'the syringe and the propellable matter and to block pneumatic communication between the gas reservoir and the propellable matter reservoir.

The syringe which will deliver about 1 mL of the propellable matter (carrier medium suspension^*of coated particles), is placed in fluid communication with the propellable matter supply line. The propellable matter supply line is in fluid communication with the propellable matter reservoir.

The propellable matter regulating valve 22a is opened to provide fluid communication between the propellable matter supply means and the propellable matter reservoir. After a selected volume of propellable matter is emitted into the propellable matter reservoir, the propellable matter valve is closed.

Any volume of propellable matter in excess to the volume of the propellable matter reservoir is released through the recovery means.

The gas regulating valve 32a is opened to provide pneumatic communication between the gas supply means and the gas reservoir. A gas pressure of about

1000 psi is obtained in the gas reservoir. The gas regulating valve is then closed. The carrier particles coated with pDAB199 exit from the delivery means toward the proembyros and ELS in a vacuumed chamber. The pressure in the propulsion zone is about 0.1 atm absolute.

The operation is completed by selectively setting the valve in order to block the passage of gas from the gas reservoir through the valve into the delivery means, and to provide fluid communication between the syringe and the propellable matter overflow reservoir.

Following bombardment, the samples were returned to the liquid ELS induction medium and incubated in the dark for about 2 days at 27°C. Samples were taken from each of the individual targets and placed into a GUS assay. After an additional two days the expression of the GUS gene in the isolated male male gametophytes is observed by using the GUS histochemical assay (5-Br-4-Cl-3 indolyl-beta-D-glucuronic acid [χ~ -gluc] substrate samples were scored for GUS expression, which is indicated by the appearance of blue color. Results of this trial are given in Table 7 below.

-6 1 -

~

Table 7

GUS Expression in Transformed Isolated Male Gametophytes

Age of _Tnt-_{a . Hn} Mean No.

Sample sam_pled^' Expression

(days) ^Samp^le^d _Units*

6 5000 16.6

8 5000 9.8

9 3000 4.0

19 233 32.6

20 355 38.3 22 217 110.9

*Number of blue spots

Other variations will be evident to those skilled in the art. Therefore, the scope of the invention is intended to be defined by the claims.

>* _

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 become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims (1)

1. A method comprising the steps of: (a) providing at least one male gametophyte isolated from at least one anther from at least one donor maize plant which is capable of being successfully anther cultured; and (b) incubating the isolated male gametophytes in an embryo-like structure (ELS) induction medium to foster development of at least one ELS.

2. The method of Claim 1, wherein the donor anther culturable maize plant is prepared as follows:

(a) providing anthers from at least one heterozygous 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 F-_j population; and

(d) self-pollinating or cross-pollinating individuals of the F-_| population to generate at least one F2 population. 3. The method of Claim 2, wherein the donor plant is obtained from a three-way cross of (H99 X FR16) X Pa91.

4. The method of Claim 2, wherein step (d) comprises self pollinating individuals of the F-_| population.

5. The method of Claim 2, wherein the F£ population produced in step (d) has a segregating population which is fixed genetically by a method of selfing or anther culture techniques.

6. The method of Claim 1, wherein the donor plant is 139/39-DH or 139/39-Bulk.

7. The method of Claim 1, wherein the male gametophytes are isolated from anthers by a technique of mechanical excision or dehiscence.

8. The method of Claim 7, wherein the male gametophytes are isolated from anthers by density gradient comprising

(a) contacting the isolated male gametop'hytes in the ELS induction medium with a gradient medium having a density greater than the density of the ELS induction medium to form a discontinuous material;

(b) centrifuging the discontinuous material at an rpm and for a time effective to create a band of male gametophytes at the Percoll/ELS induction medium interface; and (c) collecting male gametophytes at the gradient medium/ELS induction medium interface.

9. The method of Claim 1-further comprising _j- the following step:

after incubating the the isolated male gametophytes in the ELS induction medium for a period of between 3 to 28 days, passing the ELS

10 induction medium containing the isolated male gametophyte through a sieve with a mesh size ranging from 74 to 88 micrometers.

10. The method of Claim 1 comprising the

_1C- further step of (a) autoclaving the components of the ELS induction medium, (b) mixing the ELS induction medium components, either before or after autoclaving, with activated charcoal, and (c) filtering out the activated charcoal prior to incubating the isolated male

20 gametophytes.

11. The method of Claim 1 comprising the further step of incubating the ELS in a callus induction medium to foster development of callus capable of being

25 regenerated into at least one maize plant.

12. The method of Claim 11 comprising the further step of incubating the callus on a plant regeneration medium to foster the germination of

30 plantlets.

13. The method of Claim 12 comprising the further step of cultivating at least one plantlets into at least one whole fertile plant.

35 14. The method of Claim 13 comprising the further step of cultivating at least one whole fertile plant or a descendant thereof whereby it bears seeds.

_c 15. At least one ELS produced by the method of

Claim 1.

16. At least one callus produced by the method of Claim 11.

17. At least one plant produced by the method of Claim 13.

18. At least one seed produced by the method of Claim 14. 15

19. The maize seed of Claim 18, wherein the seed is a double haploid.

20. A maize seed which is generated from at 20 least one isolated microspore.

21. The maize seed of Claim 20, wherein the seed is a double haploid.

25 22. 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 isolated male gametophyte culture.

30 23. A method for the production of genetic translocations, substitution and addition lines in maize, said method comprising the culture of isolated male gametophytes of interspecific and intergeneric hybrids of maize.

35 24. A method for the transformation of maize, said method comprising providing haploid cells of maize developed by isolated male gametophyte culture and inserting genetic material into the haploid cells by a technique selected from the group consisting of particle bombardment, and microinjection.