TRANSPLANTATION OF NUCLEI IN NEUROSPORA CRASSA

WILSON, JAMES F. (Hartnell Coll., Salinas, Calif.) Transplantation of nuclei in Neurospora crassa. Amer. Jour. Bot. 50(8): 780-786. Illus. 1963.-Heterocaryons of N. crassa were produced artificially by cell-to-cell transfer of protoplasm of genetically marked strains. The injected hyphal sections were excised and cultured separately. The criteria of prototrophic growth and recovery of conidia of the donor genotype were used to prove that functional nuclei had been transplanted. Reciprocal injections were made with 2 pairs of macroconidial and 3 pairs of microconidial strains. Growth curves and conidial ratios of these artificial heterocaryons were compared to the corresponding natural heterocaryons. In each pair of heterocaryons produced by reciprocal injection, the growth curve of only one was equivalent to the naturally formed heterocaryon of that pair. The growth curve of the other artificial heterocaryon differed either in rate or in the lag period before growth occurred. There was no apparent relation between conidial ratios of the natural heterocaryons and the artificial heterocaryons with comparable growth curves. Injection of normal nuclei into a morphological temperature-sensitive mutant produced an apparent heterocaryon which grew with normal morphology at 34 C. The apparent stability of Neurospora nuclei mav make in vitro studies possible. SUCCESSFUL nuclear transplantation has been achieved in remarkably few species. Among the animal cells used for nuclear transplantation studies are protozoa (Comandon and De Fonbrune, 1939; Tartar, 1953) and amphibian eggs (Briggs and King, 1952). In plants, Burgeff (1914) transplanted nuclei artificially in Phycomyces nitens by the simple expedient of squeezing the contents of a sporangiophore of one mating type into a sporangiophore of the opposite mating type. Recently, Zetsche (1962) has reported transplantationi of functional nuclei in Acetabularia. None of the above organisms has been particularly well suited for genetic analysis, thus limiting the investigator's ability to exploit the nuclear transfer technique. This paper describes heterocaryons produced by nuclear transplantation in an organism plentifully supplied with genetic markers-Neurospora crassa. MATERIALS AND METHODS-Except for an improved type of microforge, the equipment used was the same as described in an earlier publication (Wilson, 1961). The new microforge consists of 2 Emerson micromanipulators and a stageless compound microscope mounted horizontally. By using a lOX objective and a 15X ocular in the final stages of pipette fabrication, it is possible to manufacture pipettes with sharp tips and orifices of less than 0.5g. However, most of the pipettes used for nuclear transplantation had terminal openings of 3.0-4.0,u. 1 Received for publication January 20, 1963. This work has been supported by Grants G 10711 and G 18880 from the National Science Foundation. The author wishes to thank Dr. Laura Garnjobst of The Rockefeller Institute for furnishing the cultures used in these experiments and for helpful advice. The ANeurospora strains used are given in Table 1. The general methods of preparation of microcultures for micro-inijection, the making of special pipettes, and the injection techniques employed have already beeii described (Wilson, 1961). The following summary of the injection techniquie contains some minor variations of the original description. Microcultures of the strainis listed were prepared by inoculating 22 X 30-mm coverslips covered with a thin layer of minimal agar medium (Beadle and Tatum, 1945) containing the necessary supplement if a nutritionally deficient mutant were used. The donor microculture was inverted over a de Fonbrune chamber filled with silicone oil. The micropipette was filled with cytoplasm and nuclei by inserting the pipette into several cells and removing the contents. The tip of the pipette was filled with silicone oil, and the donor microculture was interchanged for the host microculture chamber filled with a glycerol solution concentrated enough to produce incipient plasmolysis in the recipient hyphae. Injections of donor cytoplasm aind nuclei were made indirectly, by piercing a cell next to a septum and making the injection through the septal pore leading into the next cell. Within a few seconds, the septal pore usually became plugged, so that the injected material was retained. A hyphal section consisting of 2 or 3 cells, including the inijected cell, was cut out, dropped to the bottom of the chamber, and transferred to Petri dishes of minimal medium with a Pasteur pipette. After overnight incubation at 30 C, those sections growing on the minimal medium were transferred on agar blocks to minimal agar slants or to growth tubes containing minimal agar mediuim. If the injected cell was to