ST. LOUIS, MISSOURI

Monsanto Agricultural Company

WOODLAND, CALIFORNIA

PetoSeed, Inc.

ST. PAUL, MINNESOTA

University of Minnesota

Development and availability of germplasm with high Type II culture formation response

--C. L. Armstrong, C. E. Green and R. L. Phillips

Type II (friable, embryogenic) cultures of maize were first developed and described in the early 1980's (Green, Plant Tissue Culture, ed. A. Fujiwara, pp. 107-108, 1982). These cultures have been used successfully in a variety of applications, including the recent production of fertile, transgenic maize plants following microprojectile bombardment (Fromm et al., Bio/Technology 8:833-839, 1990; Gordon-Kamm et al., Plant Cell 2:603-608, 1990). In anticipation of increased interest from the maize genetics community in utilizing this tissue culture technology, we describe here the development and availability of "user-friendly" germplasm for the establishment of Type-II cultures.

Most published reports on Type-II cultures have utilized the inbred lines A188, B73, or the F1 hybrids between these two lines. Using published procedures, initiation frequencies for B73 are extremely low (typically less than 1 in 100 immature embryos). Initiation frequencies are much better for A188 and the F1 hybrid, but are variable and dependent on environmental conditions. For the inexperienced researcher, high quality Type-II cultures from A188 and the F1 can be difficult to maintain. Establishment of embryogenic suspension cultures from these genotypes also requires a reasonable amount of tissue culture experience.

We selected two partially inbred lines (Hi-II Parent A and B) out of the cross between A188 and B73 which have a greatly improved Type-II culture response (Fig. 1). Each parent was derived from a different F2 embryo. The Type-II culture initiation frequency from immature embryos from each line is nearly 100% when incubated on a modified N6 medium contain 1 mg/L 2,4-D and 25mM L-proline (Armstrong and Green, Plant 164:207-214, 1985). The cross between the two selected lines has been designated "Hi-II", and has much better plant vigor than the two parental lines while maintaining the excellent culture response. While selection was based on immature embryo response, immature "Hi-II" tassels also form Type-II cultures at a high frequency (William Petersen, Monsanto, unpublished results). Embryogenic suspension cultures can be established relatively easily from "Hi-II" callus, and such suspensions have been used to generate fertile transgenic plants (Fromm et al., Bio/Technology 8:833-839, 1990).

The "Hi-II" germplasm should be useful in any experiments requiring a high-frequency Type-II initiation response from immature embryos or tassels. Many F1 combinations with the "Hi-II" germplasm have formed excellent Type-II cultures, and therefore it should be useful for researchers desiring to produce Type-II cultures of specific dominant or cytoplasmic genetic stocks which are in a recalcitrant background. We feel this material should be particularly useful for researchers with little or no experience with corn tissue culture. It is more "forgiving" than most genotypes, and will respond reasonably well under a wide variety of in vitro culture conditions.

The "Hi-II" germplasm has some significant limitations which must also be considered. The parents are only partially inbred. One consequence of this is that regenerated plant vigor is somewhat variable from line-to-line. While generally quite acceptable, regenerated plants are not as vigorous as from A188xB73 F1 cultures. A second limitation is that the parental lines are somewhat difficult to propagate.

Samples of seed of "Hi-II" and the "Parent A" and "Parent B" lines can be obtained by contacting Chuck ArmstrongCL (Monsanto, Mail Zone GG4H, St. Louis, MO, 63198). Recommended culture media and growth conditions are as described by Armstrong and Green (Planta 164:207-214, 1985), but modified to contain 0.2% PhytagelTM (Sigma) in place of 0.7% Difco-Bacto agar, and including 10µM AgNO3 (Dr. Dave Songstad, Monsanto, unpublished results).

Figure 1.  Development of "Hi-II" germplasm.

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