Screening, selection and clonal propagation of Amaranthus dubius genotypes with different calcium and iron content.
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Decontamination of Amaranthus dubius field-derived nodal explants was achieved in a 10 min soak with 1% (v/v) NaOCl and 2 drops of Tween 20® followed by three rinses in sterile distilled water, immersion in an antibiotic solution (¼ strength Murashige and Skoog basal salt medium, 50 μg l-1 rifampicin, 100 μg l-1 streptomycin/penicillin), at 1500 rpm for 5 h. Of the tested plant growth regulators, MS media supplemented with 2 mg l-1 benzylaminopurine (BAP) + 0.5 mg l-1 indole-3-acetic acid (IAA), and 0.1 mg l-1 IAA, respectively, gave the best in vitro responses of 4 shoots/nodal explant and 100% rooting. Plantlets were acclimatised over 21 days (d) in soil (S) and 1soil:1vermiculite (v/v) (1S:1V) substrates; a significant increase in the number of leaves occurred up to 21 d (6.8 to 16.2 in S and 6.3 to 13.1 in (v/v) 1S:1V). At 21 d in (v/v) 1S:1V, there were more leaves than in S, in contrast longer plant height and root length were observed in S than in (v/v) 1S:1V. The post-acclimatisation yield was 2 plants/nodal explant. The variation in calcium (Ca) and iron (Fe) content within a population of greenhouse-germinated A. dubius seedlings was then evaluated, and specific genotypes were selected to investigate the effects of micropropagation, acclimatisation in S and (v/v) 1S:1V and physiological age (time) on their growth and Ca and Fe accumulation. After 60 d, using inductively coupled plasma-optical emission spectrometry, the content of leaf Ca ranged from 246.3 to 765.3 mg 100 g-1 dry mass (DM) and the Fe from 5.3 to 26.7 mg 100 g-1 DM. Based on the significant differences of these levels amongst the parent genotypes seven were selected and were ‘ranked’ as G47 > G45 > G11 > G41 = G8 > G39 > G15 for Ca and as G47 = G45 > G39 = G41 > G8 > G15 > G11 for Fe. Nodal explants of the selected parent genotypes were subjected to the established micropropagation protocol (using S and (v/v) 1S:1V during acclimatisation). The post– acclimatisation yield was 2 to 4 plants/nodal explant. Over the 21 d of acclimatisation in the two substrates, there were clear genotypic effects on all the tested growth parameters in S. There were significant increases in S–grown plants in the number of leaves of G39 (7.0 to 10.6) and G47 (7.0 to 13.3), the plant height of G11 (5.6 to 12.3 cm) and the root lengths of G8 (6.6 to 17.3 cm) and G41 (10.0 to 16.6 cm). When grown in (v/v) 1S:1V, the plant height significantly increased from d 0 to 21 for G8 (7.0 to 12.3 cm) and G47 (6.3 to 10.6 cm). With regards to the effect of substrate, only the clones of G8 preferred nutrient-poor soil to produce more leaves (8) than when grown in S at d 21 (5). After transferring the clones to the greenhouse for 90 d, no significant differences in the root:shoot dry masses amongst the clones were observed on each substrate, and the substrate had no effect on the root:shoot dry mass for each genotype. After acclimatisation and transfer of the clones into the greenhouse it was observed that both physiological age (time, i.e. 15, 30, 60, 80 and 90 d) and substrate influenced their accumulation of Ca and Fe. Over time, in both substrates, the Ca content increased while Fe content decreased. Significant interactions were found between the genotype and substrate for both Ca and Fe, and between physiological age (time) and genotype for Fe only. In S, the clones of all the parent genotypes matched the Ca content of their parents at 15 d while for Fe that of five of the seven selected genotype clones were similar in Fe content to their parents at 60 d. Clones of four of the seven selected parent genotypes accumulated higher Ca and Fe levels when grown in (v/v) 1S:1V than in S at certain time intervals. In S, the Ca ‘rankings’ of all the clones did not match their respective parent genotypes at any time interval while in the case of Fe, the clones of two genotypes in S (G47 and G11) and one genotype in (v/v) 1S:1V (G47) matched their respective parents between 60 to 90 d of growth in the greenhouse. In conclusion, nodal explants of the selected A. dubius genotypes with varying Ca and Fe contents, were clonally propagated in vitro using BAP and IAA and the yield after acclimatisation was 2 to 4 plants/nodal explant. The physiological age (time) and substrate affected the number of leaves of the cloned genotypes, whilst in the case of Ca and Fe, these levels were influenced by micropropagation, physiological age and substrate type. Phenotypic plasticity can be further evaluated by exposing the clones of the selected genotypes to varying water, salinity and heat stresses. Additionally, investigations to understand the clones’ ability to accumulate Ca and Fe would be valuable, in this regard, quantifying inhibitory factors and exploring the effects of substrate properties such as pH and porosity are suggested.