For several years it has been known that regenerable tissue cultures (cultures capable of plant regeneration) can be initiated from immature embryos of several maize genotypes. Recently, explants of other tissues such as immature tassels, ears, and nodal stem sections also have been used successfully (S. J. Molnar et al., MGNL 54:52, 1980; T. Rice et al., Propagation of Higher Plants Through Tissue Culture, Univ. of Tenn. Symp. Proc., p. 262, 1979). This study was undertaken to determine optimal conditions for culturing young tassels, and to identify genotypes which will produce regenerable cultures under those conditions.
Immature tassels were removed under sterile conditions from 6- to 7-week-old field grown plants and then cut into 1-2 mm long cross-sections. The pieces were placed on modified MS medium (1.0 mg/l 2,4-D) in 100 x 25 mm plastic Petri dishes and incubated at 26 C under a 16/8 hr photoperiod. All tissues were examined after 3 and 5 weeks for the presence of scutellar-like bodies usually indicative of regenerability for embryo-derived cultures. A positive culture response was scored if one or more pieces of a tassel possessed these bodies. Immature embryos from the same 13 genotypes (4-5 ears, 50 embryos per ear) also were used to initiate cultures; their response was scored similarly. When tassel-derived cultures were transferred to MS medium without 2,4-D, not all tissues with scutellar-like bodies produced plants capable of growing to maturity. Some genotypes (W22, W23, Mangelsdorf Multiple Tester, Coe's Stock 6) produced plants that grew up to 2" tall, but then died.
The frequency of positive culture response from immature tassels ranged from 0% (A632 and ND 203) up to 100% (A188 x A619 (Table 1). Other types of growth responses were observed, including root formation, secretion of mucilage, enlargement of florets, and callus growth. Each genotype had a characteristic growth pattern, which was genotype specific for all tassel sizes. The average number of positive-responding pieces per tassel is another measure of genotypic response and revealed further differences among genotypes (Table 1).
The tassel response often, but not always, corresponded to the positive response rate of immature embryos. Vigor of cultures derived from either explant source was similar for each genotype.
Tassels from progeny of a self-pollinated A188 plant that had been regenerated from a tassel-derived tissue culture gave about the same positive response (85.7%) as did tassels from progeny of normally grown A188 plants (82.0%).
Further experiments with A188 tassels were performed to determine additional important factors for culture initiation. When tassels were cut into sections of varying lengths (25 tassels per treatment), the response of 3 mm sections (60.0%) was slightly lower than with 1 mm sections (72.0%). The developmental stage as measured by tassel size was an important factor (Table 2). Although explant pieces from any position in the tassel could give a positive response, pieces from the basal half of branches of 2-3 cm long tassels had the highest response rate.
Initiating cultures from immature embryos is mechanically easier than from tassels, but it may be useful to initiate cultures from tissues taken later in the sporophytic life cycle than embryonic stages. Plants are usually 6-7 weeks old when the tassels are 2-3 cm long; this allows time for the expression of certain genetic markers. Cytogenetically altered plants, such as haploids, monosomics, or those with deletions, can be identified via genetic markers and then cultured by the immature tassel technique. We have used this technique to establish regenerable tissue cultures from haploid and monosomic plants. (Supported in part by USDA/SEA Competitive Research Grant 5901-0410-8-0149-0 and NSF Graduate Fellowship).
C. A. Rhodes, C. E. Green and R. L. Phillips
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