Biochemical Basis of Adventitious Root Formation on Etiolated Stem Segments

Segments (2.5-cm-long) of Populus nigra L. obtained from etiolated axillary branches did not root in water or auxin alone, but rooted in 0.5% ribose, glucose and sucrose and more profusely with 0.1 mg/1 in dole-acetic acid (IAA) or in dolebutyric acid (IBA) added to the medium. 5-fluorodeoxyuridine (FUDR), 5-fluorouracil (FU), actinomycin-D and cycloheximide inhibited rooting. The deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein contents of segments cultured in glucose plus IAA were higher than in water or in glucose/ IAA solutions containing cycloheximide or actinomycin-D. New isoenzymes of peroxidase and IAA-oxidase developed in solutions containing IAA and glucose, as did two new low-molecular-weight RNAs. New isoenzymes also developed in solutions containing actinomycin-D and cycloheximide. The physiological significance of these facts is discussed, and a biochemical explanation for the root initiation process is proposed. INTRODUCTION Nanda et al. (1968, 1970), Nanda and Anand (1970) and Nanda (1971) demonstrated that seasonal changes in auxin effects on rooting (which relate to> morphophysiological status) are determined by changes in the levels of endogenous regulatory substances and nutritional status. These in turn are caused by changes in temperature and light conditions prevailing during the annual cycle of plant growth. It has also been shown that a proper balance between auxin and nutritional levels is necessary for optimal production of adventitious roots (Nanda et al, 1971; Nanda and Jain, 1971, 1972) and also for differentiation of callus (Kumar, 1972; Nanda et al. 1974). This paper describes the results of some experiments that were undertaken to study the mechanism of action of auxin at the subcellular level, and the role of nutrition on the biochemical basis of adventitious root formation in Populus nigra L. MATERIAL AND METHODS Fifteen-cm stem cuttings of Populus nigra L. taken from trees growing on the University campus were planted in sand in earthenware pots in the dark. The etiolated branches that developed from the axillary buds were cut into* 2.5-cm segments and were planted vertically in holes on polythene sheets stretched over 10-cm Petri dishes containing the test solutions. Experiments were carried out in an air-conditioned room N.Z. J. For. Sci. 4 (2): 347-53 348 New Zealand Journal of Forestry Science Vol. 4 (28 ± 2°C) and all test solutions contained 30 fJiM chloramphenicol to prevent microbial growth. At periodic intervals observations were recorded of the number of rooted segments and roots per rooted segment. Determinations of DNA, RNA and protein contents were made by the modified methods of Burton (1956), Mejbaum (1939) and Lowry et al. (1951), respectively. In Experiment 4 the crude enzyme extract was obtained by homogenising the weighed segments at 4°C in 0.067 M phosphate buffer (pH 7.0). After repeated centrifuging at 15,000 rpm the supernatant was collected and stored till used. The total activity was assayed with a Bausch and Lomb Spectronic 20 photocolorimeter for peroxidase by the method of Mitra et al. (1970), for IAA-oxidase by that of Gordon and Weber (1951) and for NAD-dependent glutamate dehydrogenase (GDH) by that of Thurman et al. (1965). Isoenzymes were separated by disc electrophoresis on 10% polyacrylamide gels at 4°C following the method of Ornstein (1964) and Davis (1964). The gels were stained by the methods cited above, except for IAA-oxidase where the technique of Endo (1968) was utilised. Experimental details are given separately in each experiment. EXPERIMENTATION AND RESULTS Experiment 1 This experiment was designed to study the relative effectiveness of some soluble sugars and starch in the rooting process. Three hundred and sixty stem segments were divided into 18 groups of 20 segments each. Half the segments in each group were kept in continuous light (3200 lux) and the other half in continuous darkness. The treatments and results are presented in Table 1. They show that no roots were initiated on segments in water or auxin solution alone, but roots developed on segments in sugar solutions. No segments rooted in starch in the light but a few rooted in darkness. Both the number of rooted segments and the number of roots per segment were similar in glucose and sucrose but were lower in ribose. Auxins added to sugar or starch increased both the number of rooted segments and the number of roots; IB A was more effective than IAA in both light and dark. The increase due to auxins was most pronounced with 1.0% starch and 0.5% sucrose, less so with glucose and least with ribose. With 0.5% starch the effect was markedly higher in light than in darkness. Experiment 2 This experiment was conducted to study the effect on rooting of some inhibitors of protein and nucleic acid synthesis in relation to nutritional and regulatory factors. The inhibitors were 5-fluorodeoxyuridine (FUDR), 5-fluorouracil (FU), actinomycin-D and cycloheximide; the full range of treatments is shown in Table 2. The results (also presented in Table 2) show that roots were not initiated on segments . cultured in water or IAA alone but were produced in glucose and more profusely in IAA plus glucose. FUDR, actinomycin-D and cycloheximide inhibited rooting completely in glucose alone and decreased it appreciably in glucose plus IAA. The inhibitory effect increased with concentration until 5.0mg/l where none of the segments rooted. FU at 1.0mg/l did not affect the number of segments that rooted, but it did decrease the number of roots per segment slightly; at 5.0mg/l it decreased both parameters. No. 2 Nanda et al. — Adventitious Root Formation 349 TABLE 1—Effect of different sugars and starch alone, or together with IAA and IBA, on the number of etiolated stem segments of Populus nigra (2.5 cm) that rooted, the number of roots per rooted segment and per segment (figures within parentheses) in continuous light or darkness. Treatment No.of segments No. of roots per rooted rooted segment Light Dark (per segment) Light Dark Control IAA 0.1 mg/1