Inhibition of strigolactones promotes adventitious root formation

Abstract

Roots that form from non-root tissues (adventitious roots) are crucial for cutting propagation in the forestry and horticulture industries. Strigolactone has been demonstrated to be an important regulator of these roots in both Arabidopsis and pea using strigolactone deficient mutants and exogenous hormone applications. Strigolactones are produced from a carotenoid precursor which can be blocked using the widely available but broad terpenoid biosynthesis blocker, fluridone. We demonstrate here that fluridone can be used to promote adventitious rooting in the model species Pisum sativum (pea). In addition, in the garden species Plumbago auriculata and Jasminium polyanthum fluridone was equally as successful at promoting roots as a commercial rooting compound containing NAA and IBA. Our findings demonstrate that inhibition of strigolactone signaling has the potential to be used to improve adventitious rooting in commercially relevant species.

Adventitious root formation is the process of root initiation from a non-root tissue and the ability to induce these roots is central to plant industries worldwide. However many plant species do not readily produce adventitious roots and considerably constraining forestry and horticulture industries and limiting the use of this approach in plant conservation. Despite a long history of clonal propagation, we are only recently beginning to understand adventitious root formation at the genetic, molecular, and biochemical levels.^1-3

Several hormones have been shown to control adventitious root formation including auxin^1,3-6 and cytokinin,^7-11 however their mechanisms of action have yet to be fully elucidated and it can be expected that other signaling pathways play important roles. One signaling pathway that we showed recently to be a potent regulator of adventitious rooting is that of strigolactone.¹² Strigolactones are a group of carotenoid-derived hormones originally discovered for their promotion of mycorrhyzal association¹³ and parasitic weed seed germination¹⁴ and more recently have been found to negatively regulate axillary bud outgrowth in shoots.^15,16 In our previous work,¹² we showed that mutants which are defective in strigolactone production or signaling in Arabidopsis and pea have increased numbers of adventitious roots formed on hypocotyls or cut stems respectively. These results prompted us to test if chemical inhibition of strigolactones could improve adventitious rooting in stem cuttings.

The carotenoid inhibitor fluridone has been demonstrated to reduce strigolactone production^14,17 and is a widely available compound found in herbicides. This existing availability of fluridone means it could immediately be used in field applications and as such, we wanted to know if fluridone could be used to promote adventitious rooting in stem cuttings from a diversity of species.

Inhibiting strigolactone promotes adventitious rooting in Pea

Previously we have used pea stem cuttings to demonstrate that strigolactone inhibits adventitious root formation.¹² To examine the effect of fluridone on adventitious root formation we applied either 500 nM (Fig. 1, experiment A) or 2.5 µM fluridone (Fig. 1, experiment B) to the bases of pea cuttings. The cuttings were taken above the second scale leaf from 2-week old seedlings (five leaves expanded including scale leaves) and the bases were maintained in the dark.¹² The seedlings were etiolated for 5 d (experiment A) or 7 d (experiment B) to elongate the lower internodes of this variety of pea. For both concentrations of fluridone the number of adventitious roots was increased (Fig. 1) but not to the same level as in the strigolactone deficient rms1. This discrepancy could be due to fluridone inhibiting all carotenoid-derived pigments which are important for stress tolerance.¹⁸ Trials using lower doses or different application lengths may further enhance the root inducing effect of fluridone. In experiment B (Fig. 1) the plants were etiolated longer than in experiment a (Fig. 1), which may explain the difference in number of adventitious roots between the two experiments. ABA is also inhibited by fluridone, however, it is unlikely that the increase in rooting is due to reduced ABA because ABA has been shown to promote adventitious rooting in Vigna radiate and Hedera helix^19,20 and has no effect in Arabidopsis (data not shown). These results demonstrate that fluridone could be used to induce adventitious rooting in stem cuttings of pea.

Figure 1. Fluridone (inhibitor of carotenoids) promotes adventitious root formation. Number of adventitious roots formed from two experiments (A and B). Both experiments used pea cuttings from Térèse background. In Experiment A, 0 or 500 nM of fluridone was applied to bases of wild type while in Experiment B, 0 or 2.5 µM of fluridone was added to wild type. The strigolactone deficient mutant rms1 was included as an additional control. Means are presented with standard error bars. Different letters represent means that are significantly different (p < 0.05, student t-test).

Fluridone promotes adventitious rooting in diverse species

Next we tested if fluridone could also be used to improve adventitious rooting in other species in a way that could be used in industry. To do this we took cuttings (10–15 cm long) from parent plants of Tradescantia fluminensis, Trachelospermum jasminoides, Jasminium polyanthum, Plumbago auriculata and Pongamia pinnata which had been maintained in the University of Queensland campus gardens and watered automatically every second day. The bases of the cuttings were dipped for 10 min in 0, 100 or 500 nM fluridone or in standard rooting hormone (Take Root, Multicrop Australia Pty) which contained 0.5% IBA and 0.5% NAA. After treatment cuttings were placed in potting mix (peat:cocopeat:perlite:sand 4:4:1:1) with 42 g of micromax, 5 g K2SO4, 46 g gypsum and 36 g of superphosphates in every 50 L. A total of 51, 40, 45, 40 and 20 cuttings were set in each treatment for Tradescantia, Trachelospermum, Jasminium, Plumbago and Pongamia respectively. Environmental conditions were: 28°C +/− 2°C; 70% of full sunlight; 80% humidity provided by two minutes of mist (misting system by Neta, 1.5 L per minute) every two hours.

Fluridone improved the rooting percentage and number of adventitious roots formed on cuttings of Plumbago and Jasminium (Fig. 2C‑F). Pongamia cuttings treated with fluridone also tended to have improved adventitious rooting (Fig. 2A and B) although this was not statistically significant in this experiment. In contrast, Fluridone had no effect on the rooting percent or the number of adventitious roots formed in Tradescantia or Trachelospermum (Fig. 2G‑J). These two species are easy plants on which to induce adventitious roots and further improvement of rooting may be difficult.

Figure 2. Rooting percentage and number of adventitious roots is sometimes improved with fluridone treatments. Cuttings of Pongamia (A and B) n = 3(5) [3 biological replicates (5 cuttings in each biological repeat)], Plumbago (C and D) n = 5(8), Jasminum (E and F) n = 3(15), Trachelospermum (G and H) n = 5(8) and Tradescantia (I and J) n = 3(17), treated with: 0, 100 or 500 nM fluridone; a commercial rooting hormone (RH) containing 0.5% IBA and 0.5% NAA; or a combination of RH with 100 nM fluridone (RH⁺100F). Values for rooting percentage (A, C, E, G and I) and number of adventitious roots (B, D, F, H and J) were calculated relative to the average for the control treatment and means are presented ± standard error. Different letters represent means that are significantly different (p < 0.05, student t-test)

The commercially available rooting hormone, which contains 0.5% Indole-3-Butyric Acid and 0.5% 1-NaphthaleneAcetic Acid (w/w) also improved adventitious rooting in most species with the exception of Trachelospermum (Fig. 2B‑J). Furthermore, fluridone, compared with the commercially available product, was equally as effective for improving adventitious rooting. We then tested if fluridone applied together with the commercial product could additively improve adventitious rooting. In Plumbago, Jasminium, Trachelospermum and Tradescantia rooting hormone together with fluridone had the same effect on rooting percentage as either compound applied alone (Fig. 2C,E,G and I). In Plumbago and Tradescantia the combined treatment reduced the number of adventitious roots compared with the rooting hormone alone (Fig. 2D and J) while in Trachelospermum this was the only treatment to significantly improve the number of adventitious roots (Fig. 2H).

These results demonstrate that fluridone (a known inhibitor of strigolactone) can improve adventitious rooting in a range of species to a similar level as commercially available rooting hormone mixes.

Rasmussen A, Mason MG, De Cuyper C, Brewer PB, Herold S, Agusti J, et al. Strigolactones suppress adventitious rooting in Arabidopsis and pea. Plant Physiol. 2012;158 doi: 10.1104/pp.111.187104. In press.