Methods for generating doubled haploid plants

Details Methods for generating doubled haploid plants Methods for generating doubled haploid plants

1999-08-26

2002-03-26

Campbell, Bruce R. (Department: 1661)

Multicellular living organisms and unmodified parts thereof and

Method of using a plant or plant part in a breeding process...

C800S268000, C800S271000, C800S275000, C800S278000, C800S299000, C800S320000, C800S320100, C435S430000, C435S430100, C435S468000

Reexamination Certificate

active

06362393

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods for generating doubled haploid plants from microspores, and to doubled haploid plants produced by the methods disclosed herein.
BACKGROUND OF THE INVENTION
Although plant breeding programs worldwide have made considerable progress developing new cultivars with improved disease resistances, yields and other, useful traits, breeding as a whole relies on screening numerous plants to identify novel, desirable characteristics. Very large numbers of progeny from crosses often must be grown and evaluated over several years in order to select one or a few plants with a desired combination of traits.
In a typical plant breeding experiment, two parent plants are crossed and the resulting progeny (the F1 generation) are screened and a plant (termed the F1 plant) identified that possesses a desirable combination of phenotypic traits. The F1 plant is then self-fertilized to yield a population of progeny plants (termed F2 plants) that must be individually analyzed to determine which F2 plants possess the desired combination of phenotypic traits originally introduced in the F1 plant. If, as is often the case, the desired phenotypic traits derive from the combined effect of several genes, then the number of F2 progeny plants that must be screened depends on the number of genetic differences between the parents of the F1 plant. Thus, the greater the number of genetically-controlled differences between parents of the F1 plant, the larger the number of F2 progeny that must be grown and evaluated, and the lower the probability of obtaining progeny with all the desired traits.
For example, if the two parents of the F1 plant differ by 25 gene alleles (not an unusually great number in breeding), more land than exists on the earth would be needed to grow all possible genotype combinations which can occur in the F2 generation derived from the self-fertilized F1 plant (Konzak, C. F. et al. In: Elliott, L. (ed.)
STEEP—Conservation Concepts and Accomplishment
, pp. 247-273, 1987. ). Further, once an F2 plant has been identified that exhibits the same, desirable, phenotypic trait(s) as the cross parents, the process of self-fertilization and analysis of the resulting progeny must be repeated several times until a homozygous population of plants is obtained which breed true for the desired phenotypic character, i.e., all progeny derived from the true-breeding population exhibit the desired, phenotypic trait (though the progeny may not be true-breeding for unselected traits).
One possible solution to the problem of screening large numbers of progeny is to produce them from the gametic cells as haploid plants, the chromosomes of which can be doubled using colchicine or other means to achieve instantly homozygous, doubled-haploid plants. In particular, doubled haploids can be produced from the microspores which normally give rise to pollen grains.
The life cycle of flowering plants exhibits an alteration of generations between a sporophytic (diploid) phase and a gametophytic (haploid) phase. Meiosis produces the first cells of the haploid generation which are either microspores (male) or megaspores (female). Microspores divide and develop within anthers to become mature male gametophytes (pollen). In normal development, microspores are genetically programmed for terminal differentiation to form mature pollen through two cell divisions. However, under certain conditions, microspores can be induced to initiate sporophytic development which leads to the formation of haploid or doubled haploid “embryoids”. These embryoids can give rise to mature plants, that are either haploids or doubled haploids, through subsequent sporophytic development. The process by which plants are produced from microspores is termed pollen-embryogenesis or androgenesis, i.e., from the male gametophyte. Androgenesis is of significant interest for developmental genetic research as well as plant breeding and biotechnology, since it is a means to produce genetically true-breeding, doubled haploid plants.
As shown in Table 1, by producing doubled-haploid (also termed polyhaploid) progeny, the number of possible gene combinations for any number of inherited traits is more manageable.
TABLE 1
Minimum size of F2 population needed to obtain all possible gene
combinations with various numbers of independently assorting gene pairs
Minimum Population Number Required
Number of Independently
Conventional Breeding
Doubled haploid
Assorting Gene Pairs
System
System
1
4
2
2
16
4
3
64
8
4
256
16
5
1024
32
10
1,048,576
1024
20
1,099,511,627,776
1,048,576
Thus, marked improvements in the economics of breeding can be achieved via doubled haploid production, since selection and other procedural efficiencies can be markedly improved by using true-breeding (homozygous) progenies. With doubled haploid production systems, homozygosity is achieved in one generation. Thus, the breeder can eliminate the numerous cycles of inbreeding necessary by conventional methods to achieve practical levels of homozygosity. Indeed, true homozygosity for all traits is not even achievable by conventional breeding methods. Consequently, an efficient doubled haploid technology would enable breeders to reduce the time and the cost of cultivar development relative to conventional breeding practices.
Thus, there is a need for a method of efficiently producing doubled haploid plants that is applicable to a wide variety of plant species.
SUMMARY OF THE INVENTION
In accordance with the foregoing, in one aspect the present invention provides methods of generating doubled haploid and/or haploid plants from microspores.
The methods of the present invention for producing plants from microspores include the steps of: selecting plant material including microspores at a developmental stage amenable to androgenic induction; subjecting the microspores to temperature stress to obtain stressed microspores; contacting the microspores with an amount of a sporophytic development inducer effective to induce sporophytic development and chromosome doubling, the contacting step occurring before, during, after, or overlapping with any portion of the temperature stress step; isolating the stressed microspores; and coculturing the isolated microspores with either ovary-conditioned medium or at least one live plant ovary. Preferably, microspores are subjected to nutrient stress at the same time that they are subjected to temperature stress. Preferably, microspores are contacted with an amount of an auxin and/or a cell spindle inhibiting agent before, during, after, or overlapping with any portion of the temperature stress step.
In the practice of the methods of the present invention, plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction. Preferably the selected plant material is tillers or branches bearing spikes or flowers that contain microspores in the mid uninucleate to early binucleate stages of development. The microspores are treated by contacting the selected plant material with an aqueous medium, such as water, and subjecting the selected plant material to temperature stress, and optionally to nutrient stress. Temperature stress is effected by incubating the selected plant material, in contact with aqueous medium, at a preferred temperature of from about 4° C. to about 40° C., more preferably from about 28° C. to about 35° C., most preferably at about 33° C., for a period of from about half an hour to about 72 hours. Nutrient stress is effected by utilizing, in the aqueous medium, an amount of at least one nutrient (such as nitrogen, calcium, phosphorus or sulfur) that is less than the amount of that nutrient necessary for the optimal growth and development of the microspores. Preferably nutrient stress is effected by utilizing water as the aqueous medium. Most preferably nutrient stress is effected by utilizing diluted NPB 98 as the aqueous medium, preferably NPB 98 medium diluted with an amount of water sufficient to dilute NPB 98 to less than or equa

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