Adventitious Root Formation in C hlora hlorant nthus hus s p. Stems via Waterlogging and Auxin Treatment using Hydroponically-grown Seedlings and Stem Cuttings Cumigad, I.D., Eduardo, A.J., and Gloria, P.C ., Institute of Biology, University of the Philippines Diliman, Quezon City
ABSTRACT Adventitious Adventitious roots are roots that develop from any organ of the plant except the embryonic embryonic root. Although part of the normal development, development, formation can be a response to stress conditions. In this experiment, we studied the formation of adventitious roots in Chloranthus sp. seeds and stem cuttings by hypoxia and phytohormone treatment. Chloranthus seeds were germinated in nutrient solution, acclimatized, before they were transferred to polystyrene rafts. Hypoxia was induced by deoxygenizing and flushing of nitrogen until oxygen concentration were 0.4-1.0 mg/L. For the auxin treatment set-up, the nutrient solution was flushed with NAA and ethylene. Induction of adventitious roots from cuttings was induced by dipping 0.5 cm of the cutting the into powder ontaining 8 g/kg IBA. Cross sections were viewed under the microscope at 72, 96, 144, 192, and 240 hours post-excision. The formation of the meristemoid cells were observed after 72 hours while globular meristem formation was observed 96 hours after. The dome-shaped meristem and primordia were already present after 144 hours and vascularization at 192 hours. The root emerged 240 hours post-excision. Both set ups yielded the same expected results and successfully induced formation of adventitious adventitious roots. Further research could could look into the effects of varying varying light intensities, moisture, moisture, and temperature in Chloranthus adventitious root formations.
INTRODUCTION Adventitious Adventitious roots r oots are plant roots that form from any non-root tissue and are produced both during normal development and in response to stress conditions, such as flooding, nutrient deprivation, and wounding. In horticulture, agriculture and forestry, adventitious root (AR) formation is a prerequisite for the vegetative propagation of many important crops and it is also a common practice to maintain genetic identity in progeny (Hartmann et al., 2011). The de novo formation novo formation of ARs is a complex developmental process during which the stem base of cuttings undergoes a series of anatomical and physiological transformations with the use of phytohormones. Phytohormones are chemicals that regulate plant growth, in which they affect the gene expression and transcription levels, cellular division, and growth of the plant (Srivastava, 2002). Plant hormones are not nutrients, but chemicals that in small amounts promote and influence the growth, development, and differentiation of cells and tissues. There are five major hormone classes which are based on their structural similarities and on their effects on plant physiology, namely: Abscisic Acid, Auxins, Cytokinins, Ethylene and Gibberellins. (Weier et al., 1979). To stimulate root growth from cuttings of plants for vegetative propagation, auxins in the
form of 1-Naphthaleneacetic acid (NAA) and Indole-3-butyric acid (IBA) are commonly used (Osborne and McManus, 2005). Traditionally, phases of AR formation are classified into (i) induction, (ii) initiation of ARs, and (iii) expression (Kevers et al., 1997; Li et al., 2009). Induction of founder cells occurs before any histological changes become apparent and is associated with the physical separation of the cutting from the stock plant. Disruption of polar auxin transport caused by severance of the cutting leads to the accumulation of indole-3acetic acid in its stem base, which is considered to be a prerequisite for induction of the founder cells (De Klerk et al., 1999; Garrido et al., 2002; Pop et al., 2011; Ahkami et al., 2013). Furthermore, disturbance of existing cell connections and transport of assimilates causes a depletion of assimilates that in turn leads to the establishment of a new carbohydrate sink in the stem base (Ahkami et al., 2009). Chloranthus is Chloranthus is a type genus of the family Chloranthaceae, which consists of fragrant shrubs or herbaceous plants that are commonly occurring in Southeast Asia, the Pacific, Madagascar, Central and South America, and the West Indies. The family consists of four extant genera (Hedyosmum ( Hedyosmum,, Ascarina, Ascarina, Sarcandra, Sarcandra, and Chloranthus), Chloranthus ), totalling about 77 known species (Christenhusz and Byng, 2016). It
is a plant of high importance and significance, since it is among the early-diverging lineages in the angiosperms with unknown closely-related families among other families of angiosperms (Angiosperm Phylogeny Group, 2009). In particular, these plants are neither monocot nor dicot and fossils assigned to the family Chloranthaceae are among the oldest angiosperms known (Angiosperm Phylogeny Group, 2009). Chloranthus plants are used in traditional medicine, in which aromatic oils may be extracted from the leaves. Given that the entire plant has anti-stress, antioxidant, antiinflammatory, detoxifying, blood activating, and antibacterial effects, it has not yet been cultivated by artificial introduction with the use of stem cuttings (He et al., 2009). In this study, Chloranthus sp. seeds and stem cuttings were used to induce adventitious root formation. Formation of adventitious roots were induced by hypoxia (waterlogging) and phytohormone (auxin and ethylene) treatment in Chloranthus sp. seeds. Meanwhile, IBA, a form of auxin was used to induce adventitious root formation in Chloranthus sp. stem cuttings. MATERIALS AND METHODS Adventitious root induction via hypoxia and ethylene treatment with growth starting from the seed (adapted from Visser et al., 1996)
unstirred agar solution (0.1% w/v with nutrient concentration same as before). The agar solution was deoxygenized by vigorous flushing of nitrogen gas. Oxygen concentration was estimated to be at 0.4-1.0 mg/L. Auxin Treatment Plants were transferred from containers to nutrient solution flushed with NAA and ethylene. 5% ethylene and NAA concentration was maintained all throughout the experiment. Adventitious root induction using Chloranthus sp. Cuttings subjected to Indole3-butyric acid (IBA) (adapted from Bryant & Trueman, 2015) Seedlings were grown in 2.8L pots and maintained with day-night cycle similar to the previous method. When considerable height was reached, cuttings were made from the plant. The cuttings were dipped 0.5cm into powder that contains 8 g/kg IBA for 3 sec, and placed 1 cm deep into a 70mL micropropagation tube. Cuttings were harvested 7, 14, 21, and 28 days. Microscopy Thin cross-sections were made from the cuttings and examined under high power objective (HPO) of a bright field microscope. RESULTS
Plant Growth Chloranthus sp. Seeds were collected and sown in trays filled with polyethylene grains and nutrient solution (Table IA). After germination, seeds were subjected to a 16-hour day cycle under PFFD light (27°C) and an 8-hour night cycle (10°C). After 1 week, seeds were exposed in a climate room with 16-hour day cycle (higher PFFD intensity) and 8-hour night cycle (22°C). Uniform seedlings were then transferred to polystyrene rafts that floated on nutrient solution in 20-L containers (containing about 6-8 plants per raft). Aeration was accomplished by flushing air through bubble stones. Hypoxia Treatment This treatment was simulated by transferring grown plants from nutrient solution to
In the figure, adventitious root (AR) formation can be seen in response to application of auxin in the form of indole-3-butyric acid on Chloranthus sp. stem bases. Five developmental stages can be seen from the figure (indicated in roman numerals), namely: meristemoid formation (I) which occurred 72 hours post-excision, globular meristem formation (II) which occurred 96 hours post-excision, formation of domeshaped meristems and AR primordia (III) which occurred 144 hours post-excision, AR primordia with developing vascularization and elongation of cells (IV) which occurred 192 hours postexcision, and emergence of AR (V) which occurred 240 hours post-excision.
Figure 1. Anatomy of adventitious root (AR) formation from the vascular ring of Chloranthus sp. stem bases (cross-section). co , cortex; pp, pith parenchyma; vr , vascular ring.
DISCUSSION In this study, adventitious roots were induced in response to two stresses, namely: (1) hypoxia (waterlogging of grown plants) and (2) in response to wounding of the stem (cuttings). Under aerated conditions, gaseous ethylene escapes from plant tissues. However, during hypoxic conditions, water acts as a physical barrier, which traps ethylene in the plant (Musgrave et al., 1972). Gibberellins enhance the ethylene-promoted adventitious root growth, while abscisic acid reduces the ethylene-promoted adventitious growth (Steffens et al. 2006). Ethylene triggers reactive oxygen species production, and together they trigger epidermal programmed cell death for root emergence and cortical programmed cell death for lysigenous aerenchyma formation. In dicots, auxin, in addition to ethylene signaling is the main requirement for de novo adventitious root initiation (Steffens et al., 2012). Wound-induced adventitious roots are central to the propagation of forestry and horticultural species. In intact plants, cytokinin and strigolactones are predominantly produced in the root, while auxin is predominantly produced in the shoot. On wounding, jasmonic acid peaks within 30 minutes and is required for successful root development (Ahkami et al., 2009). Reactive oxygen species, polyphenols, and hydrogen sulfide also increase and promote adventitious rooting via reducing auxin degradation. Auxin builds up in the base of the cutting, acting upstream of nitric oxide to promote adventitious root initiation (Zhang et al., 2009). Auxin, nitric
oxide, and hydrogen peroxide (H2O2) increase soluble sugars, which can be used for root development (Li et al., 2009). Furthermore, levels of root initiation inhibitors such as cytokinin and strigolactones are reduced with the removal of the original root system (Rasmussen et al., 2012). At later stages, auxin inhibits primordia elongation while ethylene promotes adventitious root emergence. As the new root system establishes, the production of cytokinin and strigolactones is restored (Gomez-Roldan et al., 2008). The formation of adventitious roots occurs naturally and is part of the development of most plants. Adventitious root formation has three major stages namely: induction, inititation and expression. Induction is when the cells are introduced to stimuli that promote adventitious root growth, this is usually characterized by the dedifferentiation of the cells. Initiation on the other hand occurs when the cell is determined to form an adventitious root, marked by the formation of root initials and root primordia, and expression refers to the maturation and further elongation of the primordia leading to root emergence (Beeckman, 2009). The cell’s competence will determine whether differentiation will occur. Competence refers to the ability of specific, differentiated cells to respond to stimuli which induces root-formation (Altamura,1996). The molecular basis for cell competence have not been determined yet; however, it is theorized that cell receptors to the root-inducing stimuli play a huge factor in the cell’s competence (Mohnen, 1994). When a competent cell is stimulated by root-inducing
factors, the cell becomes determined to initiate root formation. Complete determination is achieved when the inducer is removed and the cell continues to develop into a root. Once a competent cell is determined by inducers, it causes the differentiated cell to dedifferentiate, forming adventitious root initials (Altamura et al.,1991). There are no cells specified to be adventitious root initials, however, in some plants like willow (Salix ), adventitious root initials are present but remain dormant until the stem has been cut and placed in the water (Hartmann and Kester, 1993). The key stage in adventitious root formation is the formation of the root initials. The root initials are small meristematic cells with chromophil nuclei and nucleoli and no starch (Altamura, 1996). Root initials usually develop near the vascular tissues and their continuous divisions cause the formation of a root dome or the root primordia. Once the root primordia have been formed, the continuous divisions of the meristematic cells lead to the further elongation of the primordia leading to its emergence and maturation; emergence and maturation can be distinguished with the formed connection to the vascular ring (Beeckman, 2009). Hypoxia refers to a state of lack of oxygen which usually occurs in plants after floods since gas exchange is 10,000 times slower in water than in air (Armstrong et al., 1991). Well adapted plants such as those thriving in wetlands, develop adventitious roots with aerenchyma - air channels that allow gas diffusion to the shoot (Dawood et al., 2013). Flooding alone does not induce formation of adventitious roots, increased ethylene production caused by the flooding results to various responses depending on the plant species. Ethylene responses may vary from epinasty, hyponastic growth, shoot elongation, aerenchyma formation and adventitious root development, all of which aids the plant escape the hypoxic environment, or improve oxygen uptake (Jackson, 2002; Visser and Voesenek, 2004; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008). Ethylene and auxin signals are intertwined in producing adventitious roots. Hypoxia leads to an increased biosynthesis of ethylene which are then perceived by the NR receptor, triggering the transport of auxin towards the flooded part of the plant. The accumulation of auxin in the stem
stimulates further ethylene production, causing a flux of auxin in the base of the stem. This accumulation of auxin at the plant’s base lead to the formation of adventitious roots (Vidoz et al., 2013). Aside from auxin, other hormones are involved in adventitious root formation namely: cytokinins, gibberellins, abscisic acid, and ethylene. Cytokinin promotes cytokinesis, vascular cambium sensitivity, vascular differentiation, and root apical dominance (Aloni et al., 2006). Root tips are the major sites of cytokinin synthesis (Aloni et al., 2004). There are reports that exogenous cytokinin application inhibits adventitious root formation (Bollmark and Eliasson, 1986). Also, endogenous expression of zeatin (a type of cytokinin) in pea plants showed strong inhibition of adventitious roots in the hypocotyl (Kurohah & Satoh, 2007). Since cytokinins are transported in the root, they travel using the transpiration system or the xylem sap. In the context of zeatin and pea plant, zeatin is first converted into trans-zeatin riboside before translocation into the shoot. And the transconfiguration of zeatin is responsible for adventitious root inhibition (Kurohah & Satoh, 2007). Gibberellic acid (GA) is a tetracyclic diterpenoid compound that stimulates plant growth and development (Gupta & Chakrabarty, 2013). GA was shown to work synergistically with ethylene to induce adventitious roots in rice (Steffens et al., 2006). They claim that GA activity controls root emergence in rice and GA concentration controls root growth. They also claim that endogenous GA promotes growth induced by ethylene. In Arabidopsis, GA signalling which results to AR growth is allowed via the degradation of DELLA proteins (Fu et al., 2002). Hence, there is a close relationship between ethylene and GA. Ethylene, together with GA, positively regulates AR formation (Steffens et al., 2006). It plays a central role in regulating AR root growth because ethylene-insensitive transgenic plants showed no or reduced AR growth. This hormone accumulates in flooded plant parts because its gaseous form traps it in the intercellular spaces (Musgrave et al., 1972). Endogenous accumulation causes nodal adventitious root primordia to exert mechanical force on overlying epidermal cells which result in epidermal
apoptosis (Steffens et al., 2012). The close relationship between ethylene and GA is established by experiments showing that ethylene alone can induce root emergence but GA cannot however presence of both results in growth. This is explained via the GA signalling which involves DELLA proteins. DELLA proteins accumulate in the nucleus as a response to ethylene while the response of DELLA to GA is mentioned in the above paragraph. Hence, a crosstalk is formed using this protein which links their relationship (Steffens et al., 2006). While synergistic relationship exists between GA and ethylene, abscisic acid exerts an antagonistic effect on them (Steffens et al., 2006). Abscisic acid (ABA) is a competitive inhibitor of GA activity and of ethylene signaling (Steffens et al., 2006). It is an important signal molecule for abiotic stress adaptation but also acts as a developmental signal. It is inhibitory on seedling root growth (Beaudoin et al., 2000) and development of lateral roots (De Smet et al. 2003). ABA negatively controls root emergence. 10uM of ABA resulted to a 50% decrease in AR penetration in rice (Steffens et al., 2006). Similarly, it inhibits root growth and was shown to primarily act using the ethylene signaling pathway. This was established because ABA was not able to affect ethylene mutants. So far, only the hormones central to AR formation has been discussed. Equally important are the genes at play to induce ARs. Whole genome transcriptomic analysis reveals that genes associated with metabolism (GO Term: biological process) and catalytic activity (GO Term: molecular process) are highly upregulated in shoots subjected to AR stimulating factors (Park et al., 2017). In the context of hormone-associated genes, auxin genes are both up- and down-regulated while ethylene and GAassociated genes are down-regulated. Cytokinin related genes were found to be the same (Park et al., 2017). CYP79B3, a gene encoding cytochrome P450 enzyme of auxin biosynthesis, was highly upregulated (Park et al., 2017). This is involved in the indole-3-acetaldoxamine (IAOx) pathway among multiple pathways for auxin biosynthesis. Logically, auxin responsive factor genes are also significantly upregulated and as a consequence, transcriptional repressors of auxin elements are downregulated (Park et al., 2017).
Specific genes responsible for certain steps in AR were also identified in numerous studies. ROOT REDIFFERENTIATION (RRD) were identified in Arabidopsis (Sugiyama, 2003). RRD1 and RRD2 are for active cell proliferation while RRD4 is needed in hypocotyl generation from callus. SCARECROW-LIKE1 and SHORTROOT genes identified from forest species are found to be involved in the earliest stages of AR formation as they play a role in auxin-signaling pathways in rooting-competent cuttings (Sanchez et al., 2007; Sole et al., 2008; Ricci et al., 2008). PINHEAD/ZWILLE-like proteins were upregulated during root initiation phase where they play a role in cell replication and cell wall weakening (Brinker et al., 2004). Most importantly, Auxin Response Factors (ARFs) are transcription factors which regulate auxin response genes (Guilfoyle and Hagen, 2007). They play an important role in allowing auxin to produce its specific effects in inducing AR. Aside from endogenous factors which affect AR induction; external factors are of relevance in determining a response. When a stem cutting with axillary bud is used to induce AR, rooting is exhibited. However, when the buds are removed or the phloem beneath the node is removed, rooting is not induced (Kibbler et al., 2004). Presence of leaves in a cutting help induce rooting because carbohydrates and auxin from the leaf travel to the AR initiation sites (Koyuncu & Balta, 2004). Polyphenol oxidase and IAA oxidase are group of compounds known as rooting co-factors which act synergistically with auxin to induce rooting in selected cuttings to induce AR (Jackson & Harney, 2011). Environmental factors such as moisture and temperature are shown to be important in adventitious root induction (Briske & Wilson, 1978). Their experiment utilized Bouteloua grama (Blue Grama) which needed 96% moisture and 15°C to form AR. Light intensity, duration and oxygen concentration are equally important in AR induction (Lee, 2011). The physiological status of a plant should be also considered. This includes (1) stock plant health and etiolation, (2) carbohydrate content, (3) mineral nutrition, and (4) girdling. Factors for the selection of wood to be used should also be considered and include the following: (1) lateral versus terminal shoots (2) proximal versus distal selections (3) flowering versus vegetative shoots and (4) ‘heel’ versus ‘non-heel’ cuttings (Lee, 2011) .
Knowledge of AR induction mechanisms and techniques are valuable because it is usually used in asexual reproduction of important crops. Hence, knowledge derived from basic and applied research regarding AR technologies are indispensable in agriculture and horticulture (Bagherabadi, 2016). SUMMARY AND CONCLUSION In this experiment, we studied the feasibility of the formation of adventitious roots by hypoxia and phytohormone treatments in Chloranthus sp. seeds and cuttings. Both set-ups yielded the same expected results and successfully induced formation of adventitious roots. The mechanisms of actions of the two treatments were also studied. Waterlogging leads to a hypoxic environment due to the slow diffusion of gas in water. The hypoxic environment leads to
an increased biosynthesis of ethylene which then triggers the transport of auxin to the flooded part of the plant. The increased concentration of auxin leads to the production of more ethylene, forming a positive feedback loop, further increasing the auxin concentration which initiates the adventitious root formation. In line with this, cuttings with leaves showed more production of adventitious roots since carbohydrates and auxin from the leaf travel to the AR initiation sites. The effects of other hormones such as gibberellic acid and abscisic acid were also explored and the roles of genes such as CYP79B3 and the RRD in adventitious root formation were identified. Environmental factors affecting adventitious root formation in Chloranthus were not fully explored. Further research could look into the effects of varying light intensities, moisture, and temperature in Chloranthus adventitious root formations.
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APPENDIX Table IA. List of micronutrients and macronutrients included in the nutrient medium Reagent
Concentration
Ca(NO3)2
2mM
K2SO4
1.25mM
MgSo4
0.5mM
K2PO4
0.5mM
Fe-EDTA
90 uM
NaCl
50uM
H3BO3
25uM
MnSO2
2uM
CuSO4
0.5uM
H2MoO4
0.5uM
