Cucurbit Genetics Cooperative Report 5:10-11 (article 5) 1982
Robert D. Locy and Todd C. Wehner
North Carolina State University, Raleigh, NC 27650
Previous research (2) indicated that cotyledon explants formed adventitious shoots in culture whereas hypocotyl explants did not. A possible explanation for that is that hypocotyl explants must rely primarily on nitrogen supplied from the medium, but cotyledon explants have additional sources of nitrogen in their tissues. The form of nitrogen in the Murashige-Skoog (MS) medium is ammonium plus nitrate (1). The objective of this study was to determine whether the form of nitrogen available in the tissue culture medium affects shoot growth.
The experiment design was a factorial in a randomized complete block with 6 lines, 9 nitrogen sources, and 2 blocks. Each treatment unit consisted of 5 Petri plates of 5 shoot tips (1 to 2 mm long) each. The 6 lines were ‘Chipper’, ‘Wisconsin SMR18’, ‘Sumter’, M41, ‘Pacer’, and ‘Poinmarket’. The 9 nitrogen sources were nitrate (NO3-), ammonium (NH4+) nitrate plus ammonium (standard Murashige-Skoog medium), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu) glutamine (Gln), alanine (Ala) and serine (Ser). The nitrogen sources were used in the standard Murashige-Skoog medium in place of nitrate plus ammonium in concentrations of 20 mM for the amino acids, 20.2 mM ammonium succinate, or 40.4 mM potassium nitrate. Shoot tips were grown on the medium for 60 days at 25°C before shoots and callus were counted and weighed. Percent shoots was calculated as (weight of shoots) x 100/(weight of callus + weight of shoots).
The shoot tips placed in culture all formed callus with the exception of those on the medium containing serine. New shoots originated from axillary buds on the original shoot tips. Serine was a poor nitrogen source because there was only 1 shoot tip per Petri plate and no growth of either shoots or callus (Table 1). The best nitrogen source for the growth of shoots in culture was asparagine. It had the greatest shoot number per petri plate, the greatest percent shoots, and the second greatest shoot weight. The nitrate plus ammonium nitrogen source produced the greatest shoot weight, but it promoted tremendous callus growth. Thus, the Murashige-Skoog nitrogen source (nitrate plus ammonium) was best for callus, not shoot growth.
Table 1. Shoot and callus growth on 9 nitrogen sources in in vitro culture.z
Nitrogen Source |
No. Shoots per Petri Plate |
Shoot Weight (g) |
Callus Weight (g) |
Percent of Total that is Shoots (by Wt.) |
| Asn | 7.4 | 1.0 | 0.6 | 59 |
| Gln | 3.8 | 0.6 | 1.2 | 40 |
| NO3- | 4.8 | 0.7 | 1.2 | 38 |
| NH4+ | 4.9 | 0.9 | 1.7 | 33 |
| Asp | 4.1 | 0.4 | 0.8 | 33 |
| Glu | 4.4 | 0.4 | 1.0 | 32 |
| NO3- + NH4+ | 5.8 | 1.6 | 2.7 | 29 |
| Ala | 3.7 | 0.5 | 1.4 | 27 |
| Ser | 0.7 | 0.3 | 0.0 | 100 |
| LSD (5%) | 1.0 | 0.2 | 0.4 | 6 |
| CV (%) | 27. | 37. | 38. | 17. |
z Data are means per Petri plate over 6 lines, 2 blocks and 5 subsamples.
Of all nitrogen sources, callus weight was not correlated with either shoot weight or shoot number, but it ws negatively correlated with percent shoots (r = -0.68**). Shoot number was correlated with shoot weight (r = 0.21**). It was thought that the heavy callus growth on the Murashige-Skoog nitrogen source may have promoted the heavy shoot growth. This is because shoots rely upon callus growth for most of the sources (Table 2). However, the Murashige-Skoog nitrogen source (NO3- + NH4+) showed a negative correlation between shoot and callus growth. Thus it appears that shoots grew large in spite of the large amount of callus growth on that nitrogen source.
Table 2. Correlation (r) and coefficient of determination (r2) of shoot weight with callus weight for shoot tip cultures grown on 9 nitrogen sources.
Nitrogen Source |
r |
r2 (%) |
| Asn | .57** | 32 |
| Gln | .49** | 24 |
| NO3- | -.18 | 3 |
| NH4+ | .62** | 39 |
| Asp | 41* | 17 |
| Glu | .13 | 2 |
| NO3- + NH4+ | -.63** | 39 |
| Ala | .59** | 35 |
| Ser | .59** | – |
| Mean | .25 | 6 |
*, ** Significant at the 5 and 1% levels, respectively.
In conclusion, the Murashige-Skoog nitrogen source is not the best one to use for tissue culture experiments in which shoot growth, but not callus growth, is desired. In that situation (such as when the shoot tip is the unit of selection), asparagine is probably the best nitrogen source to use in the medium.
Literature Cited
- Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
- Wehner, T. C. and R. D. Locy. 1981. In vitro adventitious root and shoot formation of cultivars and lines of Cucumis sativus L. HortScience 16:759-760.