Plant Cell Tiss Organ Cult DOI 10.1007/s11240-017-1244-1 ORIGINAL ARTICLE Etiolation and flooding of donor plants enhance the capability of Arabidopsis explants to root Mehdi Massoumi1,2 · Frans A. Krens1 · Richard G. F. Visser1 · Geert‑Jan M. De Klerk1 Received: 26 February 2017 / Accepted: 28 May 2017 © The Author(s) 2017. This article is an open access publication Abstract Rooting of cuttings depends not only on the sucrose level. As low sucrose favors the juvenile state and rooting treatment and the genotype, but also on the condi- juvenile tissues have a higher capability to root, the low tion of the cuttings at the time of excision. The physiologi- sucrose levels may also play a role. cal and developmental conditions of the donor plant may be decisive. We have examined in Arabidopsis the effect Keywords Adventitious root formation · Arabidopsis of two donor plant pre-treatments, etiolation and flooding, thaliana · Donor plant pre-treatment · Etiolation · Flooding on the capability of flower stem and hypocotyl segments to root. For etiolation, plantlets were kept in the dark, hypoco- tyls up to 12 days and plantlets for 12 weeks. Flooding was Introduction applied as a layer of liquid medium on top of the semi- solid medium. This procedure is also referred to as “double Vegetative propagation is the predominant propagation layer”. Both pre-treatments strongly promoted rooting and method in horticulture and forestry and various major agri- we examined possible mechanisms. Expression of strigol- cultural crops (Hartmann et al. 2011). Since cuttings that actone biosynthesis and signaling related genes indicated are excised from the donor plants obviously have no roots, that promotion by etiolation may be related to enhanced adventitious root (AR) formation is indispensable in veg- polar auxin transport. Increased rooting after flooding may etative propagation (De Klerk et al. 1999b). Treatment have been brought about by accumulation of ethylene in the with auxin is the common way to induce ARs (De Klerk cutting (ethylene has been reported to increase sensitivity et al. 1999a), but a significant number of crops is recal- to auxin) and by massive formation of secondary phloem citrant to this treatment. Despite much research, no other (the tissue close to which adventitious roots are induced). general applicable rooting treatments have been developed. Both pre-treatments also strongly lowered the endogenous An alternative way to increase rooting is donor plant pre- treatment aiming at cuttings with an increased capability to respond to auxin. In this context, we have previously exam- Communicated by Sergio J. Ochatt. ined rejuvenation of donor plants (Massoumi et al. 2017). In the present paper, we deal with etiolation and with flood- Electronic supplementary material The online version of this ing, the other two rooting promoting pre-treatments (De article (doi:10.1007/s11240-017-1244-1) contains supplementary Klerk 2002). material, which is available to authorized users. When applied during the rooting treatment, light (qual- * Mehdi Massoumi ity, intensity and duration) often influences the rooting of [email protected]; [email protected] cuttings (e.g., Daud et al. 2013). Studies on the effect of light on rooting have provided evidence both for possible 1 Wageningen U&R Plant Breeding, Wageningen University and Research, P. O. Box 386, 6700 AJ Wageningen, synergistic and antagonistic interactions of light with plant The Netherlands growth regulators e.g., auxin and cytokinins (CKs) (Fett- 2 Euro-Tiss Company, Laageinde 6, Neto et al. 2001; Wynne and McDonald 2002). 4016 CV Kapel Avezaath Buren, The Netherlands Vol.:(0123456789)1 3 Plant Cell Tiss Organ Cult Light changes also rootability of cuttings when the Apart from rejuvenation and etiolation, flooding also donor plant has been treated. Keeping donor plants for has been reported to enhance rootability (Voesenek and some period (weeks) in the dark, a pre-treatment usually Sasidharan 2013). This is mediated by an accumulation of referred to as etiolation, often improves the rootability endogenous ethylene brought about by a reduction in gas of cuttings (Hammerschlag et al. 1987; Klopotek et al. release from submerged tissue (Vidoz et al. 2010; Vis- 2010; Koukourikou-Petridou 1998; Shi and Brewbaker ser et al. 1996). It should be noted that the diffusion rate 2006). Researchers have attempted to relate the stimu- of gases in water is 10,000 times slower than in air (Jack- lation by etiolation with anatomical, physiological and son 1985). Roots are most prone to flooding and the first molecular changes (Maynard and Bassuk 1988; Haissig to suffer from oxygen shortage. Several mechanisms have and Davis 1994; Hartmann et al. 2011; Sorin et al. 2005) been described that help maintain root function through an but the mechanism is still not understood. A complicat- improved oxygen supply during flooding. Establishment of ing factor is the plural role of sucrose, the product of a lateral diffusion barrier (Bramley et al. 2010), formation photosynthesis: energy source, building block and signal of internal gas spaces (aerenchyma) (Colmer and Voesenek molecule. With respect to plant hormones, it was initially 2009) as well as initiating organogenesis to replace the believed that brassinosteroids play a key role in etiola- original root system with ARs roots are those which have tion but this has been refuted. It has been suggested that been thoroughly addressed (Maurenza et al. 2012; Zhou gibberellin (GA1) plays such role: after exposure of de- et al. 2012). etiolated seedlings to light, there is an inhibition of stem In addition to the positive influence of flooding on AR growth caused in part by a rapid drop in GA1 (reviewed formation potential while the root system is still connected, in Symons and Reid 2003). There are various reports on flooding has also been reported to pose similar effects on the effect of etiolation on changing endogenous indole- cuttings. Shibuya et al. (2013, 2014) reported that soaking 3-acetic acid (IAA) in cuttings. An increased IAA level the basal cuttings of Carolina poplar (Populus canadensis has been reported in etiolated stem parts of eucalyptus Moench.) and Japanese cedar (Cryptomeria japonica D. (Fett-Neto et al. 2001), carnation (Agulló-Antón et al. Don) in warmed water at controlled low-air-temperature 2011) and pea (Koukourikou-Petridou 1998). Addition- improves early initiation and development of ARs. How- ally, light would affect the level of endogenous auxin ever, it seems that the better rooting response of cuttings either by influencing its transport or its metabolism into treated with warm water is because of temperature gradient conjugates or via photo-oxidation (Ding et al. 2011; created by warm water at their basal portion while cooling Normanly et al. 2004; Sassi et al. 2012). There are few their apical ends. reports about the influence of light on auxin signaling. It Double layer (a layer of liquid medium on top of the has been reported that light has a contrasting effect on the semi-solid medium) is the tissue culture equivalent of expression of AUXIN RESPONSE FACTOR (ARF) genes. flooding. The effect of double layer on rooting has been While it positively affects expression of ARF6 and ARF8 examined occasionally and a strong increase was observed (both positive controllers of AR initiation), it negatively (De Klerk 2002; Maene and Debergh 1985). In the present regulates expression of ARF17 (negative controller of AR study, we investigated the effect of flooding/double layer initiation) (Gutierrez et al. 2009). Auxin signaling hap- culture as another donor plant pre-treatment on rootability pens through the SCFTIR1-Aux/IAA-ARF pathway. In of Arabidopsis explants cultured in vitro. Understanding its Arabidopsis TIR1 (TRANSPORT INHIBITOR RESIST- underlying mechanisms may help application of this tech- ANT 1) and AFBs [AUXIN SIGNALING F-BOX PRO- nique to improve in vitro rooting of other plant species at TEIN 1 through 5 (AFB1–5)] are F-box components of commercial scale. a nuclear SCF-type E3 ubiquitin ligase, which target the Aux/IAA (AUXIN/INDOLE-3-ACETIC ACID INDUCI- BLE) proteins for degradation (Gray et al. 2001; Petroski Materials and methods and Deshaies 2005; dos Santos Maraschin et al. 2009). Whether light influences auxin signaling via change in Plant materials expression of TIR1 and AFBs is not clear. In the present research, we studied mechanisms underly- Arabidopsis thaliana (Col-0) seeds (Lehle Seeds, Round ing the effect of etiolation, a donor plant pre-treatment, on Rock, USA) were surface-sterilized with 70% (v/v) ethanol AR formation of Arabidopsis explants cultured in vitro. We for one minute followed by 2% (w/v) sodium hypochlo- mainly focused on the changes in endogenous sugar level rite for 10 min. Then the seeds were rinsed three times for as well as in expression of auxin signaling and strigolac- 10 min with sterile distilled water. They were germinated tone (SL) biosynthesis/signaling related genes as caused by in Petri dishes up to the seedling stage (for hypocotyl etiolation. explants) or containers up to flowering plants (for flower 1 3 Plant Cell Tiss Organ Cult stem explants) using half-strength MS (MS1/2) basal salt we applied IBA (10 µM) and IAA (30 µM) for rooting mixture including vitamins (Murashige and Skoog 1962), of hypocotyl and FS explants, respectively. The selected 3% (w/v) sucrose and 0.7% (w/v) Micro-agar (Duchefa, concentrations are also based on the findings of Mas- Netherlands). To synchronize germination, the seeds were soumi and De Klerk (2013). Considering that IAA and first stratified in the dark for 3 days at 4°C. Then they were IBA are sensitive to photo-oxidation (although IBA to a transferred to 20 °C under long day (16 h light/8 h dark) lesser extent) and auxins are only required during the first conditions (30 μmol m− 2 s− 1, Philips TL33).