Multicellular living organisms and unmodified parts thereof and – Method of using a plant or plant part in a breeding process... – Breeding for pathogen or pest resistance or tolerance
Reexamination Certificate
1997-07-21
2001-09-04
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of using a plant or plant part in a breeding process...
Breeding for pathogen or pest resistance or tolerance
C800S268000, C800S275000, C800S320100, C800S300100, C435S412000, C435S424000, C435S430000, C435S430100
Reexamination Certificate
active
06284945
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a genotype of
Zea mays
(L.) which is auxin-autotrophic and from which protoplasts, which reproducibly and stably regenerate into normal fertile plants, can be produced, as well as the regenerated plants and parts and progeny of those plants. Additionally, the invention relates to a novel maize genotype starting from immature embryos on hormone-free media from which an auxin-autotrophic embryogenic callus is formed on the shoot basis of the seedlings which develop.
BACKGROUND OF THE INVENTION
Despite the fact that several alternative approaches such as microinjection into the cells, macroinjection or bombardment with DNA coated particles of the intact or cultured tissues have been claimed as methods for genetic transformation of crop plants, according to the available experimental data protoplasts are ideal and the most widely used objects for parasexual genetic manipulation methods including DNA transformation and somatic hybridization. These genetic manipulations require a methodology for protoplast isolation and culture. For practical applications obtaining fertile regenerants from the manipulated protoplasts is a basic prerequisite. Among the monocotyledonous crop species maize is one of the most studied tissue cultures. Several publications have shown that the potential for plant regeneration from 20 various cultured organs including anthers and immature embryoids is highly genotype-dependent (Green and Phillips, Crop Sci. 15, 417 (1975), Morocz, Tag-Ber., Acad. Landwirtsch.-Wiss. DDR, Berlin (1983), Duncan et al., Planta 165, 322 (1985); each of which is incorporated herein by reference). Improvements of culture conditions can only partially overcome these limitations (Duncan et al., Planta 165, 322 (1985)).
Dicotyledon plants can essentially be transformed via Tiplasmid vector systems with the aid of Agrobacterium tumefaciens. However, this system cannot be applied readily to monocotyledon plants. Potrykus et al. (Mol. Gen. Genet, 199, 183 (1985); incorporated herein by reference) and Lörz et al. (Mol. Gen. Genet, 199, 178 (1985); incorporated herein by reference) showed that plant protoplasts of monocotyledon plants can stably integrate foreign DNA into their genome. However, progress was first inhibited by the fact that the protoplasts could not be regenerated into fertile plants.
Over recent years there has been extensive research into the development of genotypes and processes to get the problem of plant regeneration under control. EP 29 24 435, incorporated herein by reference, relates to a process in which fertile plants can be obtained starting from a relatively non-mucilaginous, friable and granular maize callus. Shillito et al. (Bio/Technology 7, 581 (1989); incorporated herein by reference) have observed in this context that it is furthermore necessary for the regenerability into fertile plants to start from callus suspension cultures from which a dividing protoplast culture can be prepared which has the capability of regenerating plants. However, in this case, regenerable protoplasts could be isolated only after a long in vitro culture period of 7 to 8 months. Furthermore, the regenerated plants show abnormalities in morphology and fertility.
Prioli and Sbndahl (Bio/Technology 7, 589 (1989); incorporated herein by reference) relates to the regeneration and production of fertile plants from maize protoplasts of the Cateto maize inbred line Cat 100-1. The authors assume that protoplast regeneration into fertile plants is a function of a number of various factors, for example genotype, physiological state of the donor cells and the culture conditions. However, in this case, regenerable protoplasts could be isolated only after a long in vitro culture period of 20 to 40 months. Furthermore, the regenerated plants also show abnormalities with regard to morphology and reproductivity.
Thus, it would be advantageous to develop a new
Zea mays
(L.) genotype which can produce tissue and cell suspension cultures with the reproducible capacity to regenerate stably plants which are normal and predominantly fully fertile.
OBJECTS AND SUMMARY OF THE INVENTION
It is thus a primary object of the invention to provide a new
Zea mays
(L.) genotype capable of producing tissue and cell suspension cultures with the reproducible capacity to regenerate stably plants which are normal and predominantly fully fertile.
It is a further object of this invention to provide a novel maize genotype starting from immature embryos on hormone-free media from which an auxin-autotrophic embryogenic callus can be formed on the shoot basis of the seedlings which develop.
It has now been surprisingly found that a new
Zea mays
(L.) genotype can produce tissue and cell suspension cultures with the reproducible capacity to regenerate stably plants which are normal and predominantly fully fertile.
The inventive novel maize genotype is distinguished from the previously described regenerable genotypes by the fact that, starting from immature embryos on hormone-free media, an auxin-autotrophic embryogenic callus was formed on the shoot basis of the seedlings which develop. This callus can be subcultured on hormone-free media over a prolonged period (>12 months) while maintaining its embryogenetic potential. Under these culture conditions, fully-developed embryos were formed which occasionally differentiate spontaneously into plants. Moreover, a large number of adventive embryos was formed, in particular when the sucrose content in the medium was increased (6-9%). The formation of soft, granular callus which was composed of embryogenic cell aggregates (type I callus), can be induced reproducibly by changing the medium (reduction of the sucrose content to 2-3% and addition of 1-3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or dicamba). This type II callus was sub-cultured in liquid media in the form of a cell suspension culture and was suitable for isolating totipotent protoplasts. The protoplasts can be used, inter alia, for genetic transformation and can be regenerated under the conditions described in the invention into fertile transgenic maize plants.
Thus, the invention relates to a transgenic, protoplast-derived maize cell line obtained from an auxin-autotrophic maize genotype, and transgenic plants and parts of plants, and their progeny, which have been regenerated from this maize cell line. Additionally, the invention relates to a method of producing the transgenic maize cell line which comprises producing protoplasts from cell suspensions of the maize genotype by means of enzymes, incubating the protoplasts with DNA and selecting and regenerating the transgenic maize cell line from the DNA-containing protoplasts.
The invention relates to a genotype of
Zea mays
(L.) which is auxin-autotrophic and from which protoplasts, which reproducibly and stably regenerate into normal fertile plants, can be produced, as well as the regenerated plants and parts and progeny of those plants; a protoplast produced from the genotype, as well as cells regenerated from this protoplast, said cells having the capability to regenerate a fertile plant; and a method for producing a callus culture of
Zea mays
(L.) from which protoplasts can be isolated, which protoplasts are capable of being regenerated into fertile plants, which method comprises the steps of a) obtaining callus which is auxin-autotrophic, relatively non-mucilaginous, granular and friable on a callus maintenance medium and b) growing the callus of step a) on a callus maintenance medium.
The invention further relates to a transgenic, protoplast-derived maize cell line which has been obtained from an auxin-autotrophic maize genotype, and transgenic plants and parts of plants, and their progeny, which have been regenerated from this maize cell line. Additionally, the invention relates to a method of producing the transgenic maize cell line which comprises producing protoplasts from cell suspensions of the maize genotype by means of enzymes, incubating the protoplasts with DNA and selecting and regenerati
Donn Gunter
Dudits Denes
Morocz Sandor
Nemeth Janos
Benzion Gary
Frommer & Lawrence & Haug LLP
Hoechst AG
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