Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2000-10-11
2002-12-10
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S420000, C435S410000
Reexamination Certificate
active
06492174
ABSTRACT:
BACKGROUND OF THE INVENTION
Present invention provides methods for initiating embryogenic cultures of plants. These methods provide somatic embryos needed for reproducing large numbers of plants by somatic embryogenesis. More specifically, the invention encompasses various methods and media compositions that can improve the frequency of initiation of embryogenic cultures.
THE NEED FOR HIGH QUALITY TREES
The forest products industry is a major economic entity. In the United States the industry had sales of $400 billion dollars in 1994. Coniferous softwood species make up the majority of the trees harvested. In the Southeastern United States, Loblolly pine (
Pinus taeda
L.) and its close relatives are the most important species. In the Pacific Northwest, Douglas-fir (
Pseudotsuga menziesii
(Mirb.) Franco) is probably the most important commercial species. Likewise, in Europe, Norway spruce (
Picea abies
(L.) Karst.) is probably the most important conifer species.
Forest productivity can be increased by planting tree farms with large numbers of elite, high-quality trees. Unfortunately, trying to produce such trees by sexual reproduction yields seeds of unpredictable quality. Further, asexual reproduction by rooting vegetative cuttings, as is easily practiced with angiosperm species such as fruit trees, is not practicable for most coniferous species. What is needed is a method of clonally propagating large numbers of genetically superior conifer trees.
SOMATIC EMBRYOGENESIS
Plant tissue culture is the broad science of growing plant tissues on or in a nutrient medium containing minerals, sugars, vitamins and bioactive small molecules such as hormones. By adjusting the composition of the media, cultured tissues can be induced to grow or differentiate into specific cell types or organs. Somatic embryogenesis is a type of plant tissue culture where a piece of a donor plant is cultured such that an embryogenic culture is initiated. A proliferating embryogenic culture forms multiple embryos. An embryo is a discreet mass of cells with a well-defined structure that is capable of growing into a whole plant.
Somatic embryogenesis is widely used in a variety of species. In some species, somatic embryogenesis is used to propagate desirable plant genotypes. In many crop species, somatic embryogenesis is used to propagate tissues that have been genetically transformed, in order to regenerate whole transformed plants. Although somatic embryogenesis protocols are widely used and have been adapted to numerous species, most species include recalcitrant genotypes that are not readily regenerated. Further, some transformation methods, such as agrobacterium infection, electroporation and particle bombardment can damage plant cells and such damaged cells are not easily regenerated into whole plants. Thus, there is a need in the art for methods and compositions that can improve the efficiency of somatic embryogenesis in a wide variety of plant families.
Presently, somatic embryogenesis is seen as the most promising technology for the efficient multiplication of valuable coniferous germplasm. Since the late 1970's, researchers have been working to develop methods of reproducing conifers by somatic embryogenesis. U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, 5,236,841, 5,413,930 5,491,090, and 5,506,136, herein incorporated by reference, describe various methods and media for conifer embryogenesis.
INITIATION OF EMBRYOGENIC CULTURES
Culture initiation begins with the selection of a suitable explant, that is any plant cell, tissue or organ capable of forming an embryogenic culture. A typical explant in conifer somatic embryogenesis is the megagametophyte, also called the ovule or the female gametophyte, which is extracted from a pollinated female cone and which may contain single or multiple zygotic seed embryos. Next, an embryogenic culture is initiated from this explant by inducing cells within the explant to proliferate into a tissue mass containing at least one somatic early stage embryo. The successful establishment of such a culture is known as initiation.
Explants
Some conifer protocols known in the art use a megagametophyte that has been split open to expose the zygotic embryo or embryos. In one method, the embryo, while remaining attached to the megagametophyte by the long suspensor, is removed from the megagametophyte so that it is no longer surrounded by megagametophytic tissue. The embryo and attached megagametophyte are then both placed on a culture initiation media. In Douglas fir, for example, the embryo lies in a groove or channel within the megagametophyte. With careful manipulation, the embryo may be “flipped out” or displaced from the groove, yet ideally, remain attached to the megametophyte by the suspensor. Alternatively, the embryo or embryos may be completely separated from the megagametophye and placed on the media, either alone or next to the megagemetophytic tissue. The megagemetophytic tissue is often placed near the embryo, as it may produce growth regulators that promote initiation.
Extrusion
When intact conifer megagametophytes are used as the explant, the somatic embryogenesis process comprises a distinct step called extrusion. Prior to this distinct step in development of such megagametophytes, explants can be considered as pre-extrusion megagametophytes. Pre-extrusion therefore denotes the time before extrusion in such megagametophytes. Extrusion is the process in which a mass of embryogenic tissue is extruded from the micropylar end of the megagametophyte when it is placed on a suitable culture media. Becwar et al. (1990) observed in loblolly pine (
Pinus taeda
) that the extruded embryogenic tissue comes from cell division and proliferation in the suspensor region of the zygotic embryo or embryos. Extrusion can be observed using low power microscopy. The proliferation and growth of zygotic tissues pushes the embryogenic tissue out of the megagametophyte and it appears as a mass of filamentous suspensor-like cells containing single or multiple zygotic embryos. The mass sometimes contains dense globules that may be somatic proembryos or very early stage somatic embryos. Successful extrusion is scored whenever some tissue that has come outside of the intact megagametophytes via the micropyle is visible. Successful extrusion does not necessarily indicate that initiation will follow. Typically, many of the extruded masses of tissue fail to proliferate and initiate an embryogenic culture.
Many conifer somatic embryogenesis protocols known in the art use intact megagametophytes as a preferred explant. However, prior to the discovery of the present invention, the entire initiation process including both extrusion and initiation, were carried out in a single media.
Initiation
Successfully initiating a high percentage of embryogenic cultures requires the proper medium and culturing conditions. In conifers, an embryogenic culture is successfully initiated when the zygotic embryo or zygotic embryogenic tissue mass, which has been either extruded or physically removed from a megagametophyte, undergoes division and proliferation. A successfully initiated culture will consist of a whitish translucent mucilaginous tissue mass that contains pre-embryonal masses of cells, filamentous suspensor-like cells and early stage somatic embryos. In a successfully initiated culture, new somatic embryos can often be seen growing directly from the somatic embryos. Visualization of initiation is aided by the fact that zygotic embryos, as well as extruded tissues, often become brown while initiated tissues are whiter and more translucent. Initiated cultures contain from one to dozens of somatic embryos. Initiation is considered successful when at least one somatic embryo is visible. The appearance of at least two somatic embryos provides a useful confirmation of successful initiation.
SUBSEQUENT STEPS TO REGENERATE WHOLE PLANTS
After embryogenic cultures are initiated, a number of subsequent steps are required to regenerate plantlets. First, the initiated somatic embryos are transferred to a
Peter Gary
Pullman Gerald S.
Finnegan Henderson Farabow Garrett & Dunner LLP
Hwu June
Institute of Paper Science & Technology
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