Tissue culture is used to mass propagate plants of commercial interest (Trueman et al. 2007; Pant 2014; Trueman et al. 2018; Suprasanna and Jain 2022), including some Corymbia species and their hybrids that include attractive ornamentals (Smith et al. 2007). The present authors attempted to propagate tip cuttings (45 mm long) of spotted iron gum, C. maculata (Hook) K.D. Hill & L.A.S. Johnson (syn. Eucalyptus maculata Hook: Myrtales, Myrtaceae) using the eucalyptus medium of Gribble (1999). This medium had been modified to propagate an E. urophylla × E. grandis hybrid principally by increasing the concentration of ferric EDTA from 0.1 to 2.0 mM. This study’s spotted iron gum plantlets remained relatively healthy for 2 to 3 wk before some leaflets began to scorch.

Trial-and-error has been used to resolve mineral imbalance in tissue culture (Kanashiro et al. 2009). This is relatively inefficient because of the number of minerals potentially involved. In contrast, mineral analysis of plantlets has systematically guided modification of media composition to accommodate the particular needs of certain species, such as passionfruit (Passiflora edulis) (Monteiro et al. 2000), baby’s breath (Gypsophila paniculata) (Gribble et al. 2002), Persian walnut (Juglans regia L.) (Ashrafi et al. 2010), and white gum (E. dunnii Maiden) (Oberschelp and Gonçales 2016). Nonetheless, this approach appears to not have gained wide acceptance, which is surprising given the widespread adoption of foliar analysis for field-grown plants and the consequent wide botanical scope of databases of foliar sufficiency levels, for example, Bryson and Mills (2014).

Mineral analysis was tested to diagnose the cause of the malnutrition of the spotted iron gum plantlets in a simple experiment. Spotted iron gum shoots about 45 mm long were aseptically excised and transferred to the eucalyptus medium of Gribble (1999) modified as follows: (1) iron (Fe3+) EDTA at 0.1 and 2.0 mM; (2) target pH values of 6 and 7 (Chris Newell, personal communication) where the achieved pH levels were 5.2 and 6.8; and (3) plus and minus 0.5 g of activated charcoal per kg (Pan and Van Staden 1998; Thomas 2008). Bromocresol purple was added at 3.0 mg L−1 as a pH indicator. Each plantlet was cultured in 25.0 mL of medium contained in a 120 mL capacity, sterile, screw-capped, polycarbonate jar. The jars were randomized under cool-white, fluorescent lights with 16 h light/ 8 h dark period. The light intensity was approximately 50 micromoles m−2 s−1, and the temperature was 25 ± 2°C. The three variables were in factorial combination (2 × 2 × 2 = 8 treatments), and there were initially 19 plantlets per treatment.

After 6 wk, the majority of plantlets in the higher iron treatments had foliar scorching, and 6 plantlets died, that is, 13 to 16 replicate plantlets remained per treatment. Living plantlets were cut just above the callus, and any shed leaves were discarded to minimize contamination of the harvested material by the medium. The presence of roots was observed qualitatively, but no data were recorded. Harvested plantlets were dried at 65 ± 2°C and weighed. The small dry mass per plantlet necessitated compositing all the replicates of a treatment for mineral analysis. The resulting 8 samples were analyzed for nitrogen (N) using a dedicated Dumas combustion system (Leco, Germany) and for phosphorus (P), sulfur (S), boron (B), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), and molybdenum (Mo) by inductively coupled plasma-optical emission spectroscopy following digestion of 300.0 mg of sample in nitric acid and hydrogen peroxide (Wheal et al. 2001). For future studies, the greater sensitivity of inductively coupled plasma-mass spectroscopy would allow the analysis to be completed on less than 100.0 mg of sample, which would make mineral analysis of plantlets even more attractive.

The initial medium pH had no effect on plantlet growth (P > 0.05), which is unsurprising since the pH near the plantlet bases—inferred from the color of the bromocresol purple in the medium—declined from the initial values to <5.2 within a week. Analysis of the combined pH data for the remaining four treatments (2 Fe × with or without charcoal) showed that the plantlet dry weights were depressed in the high Fe medium. This weight reduction was even greater when high Fe was in combination with activated charcoal, no doubt because the charcoal increased the plantlet Fe concentration (R2 = 0.938, Fig. 1). The deleterious effect of charcoal was possibly due to the increased root development that it caused (observed but not measured). A similar stimulation of root growth was reported in previous studies where it was attributed variously to binding of plant growth regulators or the removal of organic toxins antagonistic to root production (Pan and Van Staden 1998; Ashrafi et al. 2010). However, the effect of charcoal on root development may also be related to the obstruction of light as demonstrated for olive (Olea europaea L.) (Mencuccini 2003).

Figure 1.
figure 1

Relation between dry weight and the iron concentration of the plantlets of spotted iron gum, C. maculata (Hook) K.D. Hill & L.A.S. Johnson (syn. Eucalyptus maculata Hook: Myrtales, Myrtaceae) grown in media with two different concentrations of Fe and of activated charcoal. Values are means (n = 26 to 32) and bars represent standard error values.

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The onset of yield depression between 220.0 and 440.0 mg Fe kg−1 in the plantlets (Fig. 1) may be interpreted as either a toxicity or an imbalance. Certainly, it is far in excess of the 39 to 50 mg Fe kg−1 in juvenile leaves of spotted iron gum required to satisfy the growth requirement of greenhouse plants (Dell and Robinson 1993), which are expected to also have minimal surface contamination by Fe from the soil. In contrast, the leaves of the E. urophylla × E. grandis cross grown in tissue culture at the present study’s high Fe concentration were just 91 mg Fe kg−1 (Gribble et al. 2002), demonstrating a radical difference between native Australian tree genotypes in the Fe concentration in the medium required for healthy growth in vitro.

The mineral analysis had other benefits. For example, for the low Fe treatment, the plantlet concentrations of three elements remained outside the sufficiency ranges suggested for spotted iron gum (Dell and Robinson 1993). That is, the Cu concentration was 2 to 3 mg kg−1 when the adequate range is 6 to 12 mg kg−1; and the Mn concentration was 420 to 490 mg kg−1 when the adequate range is 22 to 32 mg kg−1. In addition, the concentration of Mo was 4.5 to 6.3 mg kg−1; and, although no authoritative data were found for the Mo requirement of spotted iron gum, it is unlikely to exceed 0.5 mg kg−1 (Reuter and Robinson 1997; McGrath et al. 2010). That is, the mineral analysis detected multiple mineral mismatches between the requirements of spotted iron gum plantlets and the media composition. Taken in context with similar successes in other studies, the present authors suggest that mineral analysis of plantlets, together with the published data on foliar nutrient sufficiency ranges, provides a systematic, efficient approach to optimizing the mineral composition of media for the in vitro production of plants of commercial interest.