Plant Cell Reports
Plant Cell Reports (1991) 10:243-246
9 Springer-Verlag 1991
Callus formation and plant regeneration from somatic embryos of oat (Arena sativa L.) P. Bregitzer 1, W.R. Bushnell 2, H.W. Rines
2,
and D . A . Somers 3
1 USDA-ARS, Pacific West Area, P.O. Box 307, Aberdeen, Idaho 83210, USA z USDA-ARS, St. Paul, Minnesota, USA 3 University of Minnesota, St. Paul, Minnesota, USA Received February 8, 1991/Revised version received May 24, 1991 - Communicated by G. C. Phillips
Ai~act Globular-stage somatic embryos were isolated by vortexing friable, embryogenic callus of cat (Avena sativa L) followed by fractionation based on size. Somatic embryos were most frequently found in the 300-380/an size fraction. Friable, embryogenic callus was reinitiated from 55% of isolated somatic embryos Fertile plants were regenerated from 22% of isolated somatic embryos. Reinitiation of callus from somatic embryos and growth of friable, embryogenic callus was inhibited by the selective agents G418 and methotrexate These results suggest that somatic embryos isolated from friable, embryogenic callus of oat may be useful totipotent targets for particle acceleration-mediated transformation.
Introduction
Direct introduction of DNA into totipotent cells via particle a c c e l e r a t i o n (Klein et al., 1988) or microinjection (Neuhaus et al., 1987) bypasses the requirement of plant regeneration from protoplasts for the production of transgenic plants. These methods may be applicable to cereal species from which regenerable tissue cultures can be established. DNA delivery via particle acceleration and subsequent transient expression of the DNA has been demonstrated in cereal species (Klein et al., 1988~ Furthermore, transformed maize plants have been obtained following particle acceleration-mediated introduction of DNA into maize suspension culture cells (Fromm et al, 1990; Gordon-Kamm et al., 1990). Cells derived from embryogenic ~ o n cultures have been the preferred target an'~g Wansformation researchers (Vasil et al., 1990). However, establishment of regenerable suspension cultures of cereals is difficult and genotype dependent. In many cases, totipotency is lost before suitable suspensions are developed, presumably via the accumulation of unfavorable somatic mutations Because we Offprint requests to: P. Bregitzer
anticipated difficulties in developing embryogenic suspensions of oat (Avena sativa L.), we investigated a system in which suitable target cells for transformation were isolated directly from friable, embryogenic (FE) oat callus. Recently, we r e p o r t e d e s t a b l i s h m e n t a n d characterization of FIE callus from immature embryos of oat (Bregitzer et al, 1989). The FE oat callus is composed of loosely associated suspensor-borne somatic embryos that develop from undifferentiated, friable tissue. Under appropriate culture conditions, isolated oat somatic embryos developed directly into plantlets These results suggestedthat isolated sormtic embryos may be good targets for DNA introduction experiments However, only a few cells in a somatic embryo would be stably transformed subsequent to DNA introduction via the techniques of particle acceleration or microinjection, and it is unlikely that plants regenerated directly from a treated embryo would be uniformly Wansformed. A potential solution is to reinitiate callus from isolated somatic embryos prior to initiation of plant: regeneration, thus allowing the proliferation and selection of transformed cells to produce a uniformly transformed callus from which non-chimeric plants could be regenerated. The objectives of this study were 1) to develop a procedure to isolate oat somatic embryos from FE callus, 2) to determine if regenerable FE callus could be establ~ed from the son-atic embryos and 3) to de~anine if the growth of cat scamtic embryo
Plant Cell Reports (1991) 10:243-246
9 Springer-Verlag 1991
Callus formation and plant regeneration from somatic embryos of oat (Arena sativa L.) P. Bregitzer 1, W.R. Bushnell 2, H.W. Rines
2,
and D . A . Somers 3
1 USDA-ARS, Pacific West Area, P.O. Box 307, Aberdeen, Idaho 83210, USA z USDA-ARS, St. Paul, Minnesota, USA 3 University of Minnesota, St. Paul, Minnesota, USA Received February 8, 1991/Revised version received May 24, 1991 - Communicated by G. C. Phillips
Ai~act Globular-stage somatic embryos were isolated by vortexing friable, embryogenic callus of cat (Avena sativa L) followed by fractionation based on size. Somatic embryos were most frequently found in the 300-380/an size fraction. Friable, embryogenic callus was reinitiated from 55% of isolated somatic embryos Fertile plants were regenerated from 22% of isolated somatic embryos. Reinitiation of callus from somatic embryos and growth of friable, embryogenic callus was inhibited by the selective agents G418 and methotrexate These results suggest that somatic embryos isolated from friable, embryogenic callus of oat may be useful totipotent targets for particle acceleration-mediated transformation.
Introduction
Direct introduction of DNA into totipotent cells via particle a c c e l e r a t i o n (Klein et al., 1988) or microinjection (Neuhaus et al., 1987) bypasses the requirement of plant regeneration from protoplasts for the production of transgenic plants. These methods may be applicable to cereal species from which regenerable tissue cultures can be established. DNA delivery via particle acceleration and subsequent transient expression of the DNA has been demonstrated in cereal species (Klein et al., 1988~ Furthermore, transformed maize plants have been obtained following particle acceleration-mediated introduction of DNA into maize suspension culture cells (Fromm et al, 1990; Gordon-Kamm et al., 1990). Cells derived from embryogenic ~ o n cultures have been the preferred target an'~g Wansformation researchers (Vasil et al., 1990). However, establishment of regenerable suspension cultures of cereals is difficult and genotype dependent. In many cases, totipotency is lost before suitable suspensions are developed, presumably via the accumulation of unfavorable somatic mutations Because we Offprint requests to: P. Bregitzer
anticipated difficulties in developing embryogenic suspensions of oat (Avena sativa L.), we investigated a system in which suitable target cells for transformation were isolated directly from friable, embryogenic (FE) oat callus. Recently, we r e p o r t e d e s t a b l i s h m e n t a n d characterization of FIE callus from immature embryos of oat (Bregitzer et al, 1989). The FE oat callus is composed of loosely associated suspensor-borne somatic embryos that develop from undifferentiated, friable tissue. Under appropriate culture conditions, isolated oat somatic embryos developed directly into plantlets These results suggestedthat isolated sormtic embryos may be good targets for DNA introduction experiments However, only a few cells in a somatic embryo would be stably transformed subsequent to DNA introduction via the techniques of particle acceleration or microinjection, and it is unlikely that plants regenerated directly from a treated embryo would be uniformly Wansformed. A potential solution is to reinitiate callus from isolated somatic embryos prior to initiation of plant: regeneration, thus allowing the proliferation and selection of transformed cells to produce a uniformly transformed callus from which non-chimeric plants could be regenerated. The objectives of this study were 1) to develop a procedure to isolate oat somatic embryos from FE callus, 2) to determine if regenerable FE callus could be establ~ed from the son-atic embryos and 3) to de~anine if the growth of cat scamtic embryo
