The primary factors influencing establishment of regenerable tissue cultures in maize are genotype, source and stage of donor tissue, and culture media composition (C. E. Green, Hort. Sci. 12:131-134, 1977). During the summer of 1980, 48 inbred lines from The People's Republic of China were planted at 2 dates (9 May and 24 May) and grown in the field at St. Paul, MN. Plants were selfed or sibbed and 12 to 16 days later 2 ears of each line were used as a source of immature embryos for tissue culture initiation. Embryos were isolated aseptically according to the standard procedure (C. E. Green and R. L. Phillips, Crop Sci. 15:645-649, 1975) and placed scutellar-side-up on Murashige and Skoog media containing 0.75 mg 2,4-D per liter. For most lines 30 embryos from each of 4 ears were tested (2 plants from the early and late planting dates); however, only 2 ears (1 date) were used for 4 genotypes.
The resultant tissue culture growth from each embryo was evaluated 4-5 weeks after initiation according to the following:
1. Mixed callus - lobular tissue proliferation from the scutellum, mixed
with areas of leaf morphogenesis.
2. Callus - lobular tissue proliferation only, no areas of leaf morphogenesis.
3. Enlarged scutellum - smooth enlarged embryo, little to no tissue
proliferation, no areas of leaf morphogenesis.
4. Developing embryo - highly organized, root and/or shoot emergence,
no tissue proliferation.
5. Senescent embryo - no germination, no visible change in embryo morphology.
Two weeks after the first classification the cultures were examined again to confirm the initial observations. Data from the final classification are reported here as the distribution of 48 inbred lines according to their frequency of response within each of the 5 classes.
The mixed callus response is the most appropriate for the establishment of tissue cultures capable of organogenesis and plant regeneration. The data in Table 1 first were broken out according to the frequency of mixed callus formation and then according to the frequency of the other 4 response classes. Five lines formed mixed callus cultures more frequently than any other class; however, the average frequency for all lines was only 12% and embryos of 18 lines did not form any mixed callus cultures. The most frequent response (class 4) was that which led only to shoot and/or root emergence from the embryo axis. This result suggested that the interaction of 2,4-D concentration (.75 mg/l) and embryo size might not have been appropriate for mixed callus formation from some lines. An examination of average embryo sizes recorded at the time of isolation for individual ears revealed that the 18 poorest lines (0% class 1) and the 9 best lines (21-80% class 1) had similar ranges (.9-3.1 mm vs. 1.3-3.7 mm) and identical overall mean embryo length (2.0 mm). Moreover, a range of embryo sizes had been obtained for most inbred lines by sampling embryos from different ears. These observations indicate that there was a pronounced genotypic effect, which for most of the 48 inbred lines did not favor mixed callus formation.
Table 1. Tissue Culture Response of
Immature Embryos from 48 Inbred Lines from China.
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| Distribution of 48 inbred lines according to frequency of mixed callus | (1) Mixed callus | (2) Callus | (3) Enlarged scutellum | (4) Developing embryo | (5) Senescent embryo | |||||||
| % Range | No. Emb | No. Lines | % Emba | No. Linesb | % Emba | No. Linesb | % Emba | No. Linesb | % Emba | No. Linesb | % Emba | No. Linesb |
| 0 | 1924 | 18 | 0 | 0 | 17 | 3 | 34 | 5 | 44 | 9 | 5 | 1 |
| 1-20 | 2273 | 21 | 8 | 0 | 13 | 2 | 26 | 4 | 51 | 15 | 1 | 0 |
| 21-40 | 432 | 4 | 28 | 0 | 4 | 0 | 13 | 0 | 55 | 4 | 0 | 0 |
| 41-60 | 348 | 3 | 47 | 3 | 9 | 0 | 18 | 0 | 25 | 0 | 2 | 0 |
| 61-80 | 180 | 2 | 77 | 2 | 2 | 0 | 10 | 0 | 8 | 0 | 2 | 0 |
| Total | 5157 | 48 | 12c | 5 | 13c | 5 | 27c | 9 | 45c | 28 | 3c | 1 |
aPercent of embryos from all inbred lines within the range indicated in left-most column.
bNumber of inbred lines for which that class was the most frequent response.
cAverage of all embryos.
Inocula from 15 mixed callus cultures were transferred to media lacking 2,4-D to determine the frequency of plantlet regeneration (Table 2). White Fung Kur 1 and Chi 31 were the best lines both for the formation of mixed callus cultures and for the frequency of recovery of plantlets from cultures. Plants were not recovered from 2 lines, but the frequency of plantlet recovery from the other 11 lines did not seem related to the frequency with which mixed callus cultures had been established.
These results suggest that the 2 lines, White Fung Kur 1 and Chi 31, would be appropriate genotypes to use in maize tissue culture work. Other lines among the 48 tested also could be used but the conditions employed for the culture procedure may need to be altered for satisfactory performance. The 18 lines that were not cultured successfully seem to have a strong genotypic effect against culturability, although suitable combinations of embryo size and developmental stage and media composition might be found upon further experimentation. These 18 lines apparently are not closely related as they were obtained from different locations in China. All 48 lines have been maintained and small amounts of seed are available by contacting BGG.
You-ju Xie and Burle G. Gengenbach
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