Identification of the HAK gene family reveals their critical response to potassium regulation during adventitious root formation in apple rootstock

Adventitious root formation is a bottleneck during vegetative proliferation.Potassium(K^(+))is an essential macronutrient for plants.K^(+)accumulation from the soil and its distribution to the different plant organs is mediated by K^(+)transporters named K^(+)transporter(KT),K^(+)uptake(KUP),or high-affinity K^(+)(HAK).This study aimed to identify members of the HAK gene family in apples and to characterize the effects of K^(+)supply on adventitious root formation and on the expression of HAK genes and the genes that putatively control auxin transport,signaling,and cell fate during adventitious root formation.In this study,34 HAK genes(MdHAKs)were identified in the apple(Malus×domestica‘Golden Delicious’)genome.A phylogenetic analysis divided MdHAKs into four clusters(Ⅰ,Ⅱ,Ⅲ,andⅣ),comprising 16,1,4,and 13 genes,respectively.The syntenic relationships revealed that 62.5%of the total MdHAK genes arise from genomic duplication events.Chromosome location,domain structure,motif analysis,and physico-chemical characteristics were subsequently investigated.Furthermore,the application of K^(+)indicated the emergence of adventitious roots at 8 d and produced more adventitious roots at 16 d than the K^(+)-free control(CK)treatment.In addition,various MdHAKs showed root-specific expression in B9 apple rootstock stem cuttings and enhanced expression during the initiation and emergence stages of adventitious root formation in response to K^(+)treatment.Additionally,K^(+)treatment enhanced the expression levels of MdPIN1,MdPIN2,and MdAUX1.Further data indicated that a higher expression of MdWOX11,MdLBD16,and MdLBD29 and of cell cycle-related genes contributed to the auxin-stimulated adventitious root formation in response to K^(+).

Identification of WRKY Transcription Factors Related to Saikosaponin Biosynthesis in Adventitious Roots of Bupleurum chinense

Objective To identify the genes of WRKY transcription factors（TFs） from roots of Bupleurum chinense and genes that potentially regulate saikosaponin（SS） biosynthesis.Methods Firstly,the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data.Secondly,qPCR was used to screen such genes of WRKY TFs that could be induced by NaCI and PEG6000 in adventitious roots of B.chinense.Meanwhile,saikosaponins（SSs） in treated adventitious roots were determined by HPLC.The roots were collected at 0,2,4,8,12,24,48,and 72 h after treatments,and 120 h only for PEG.Finally,the tissue-specific expression was analyzed on screened genes by qPCR.Results The 27 genes were grouped into three categories：There were nine in Group Ⅰ,15 in Group Ⅱ,and two in Group Ⅲ.Four genes of WRKYTFs,BCWRKY6,BCWRKY16,BCWRKY32,and BCWRKY35 were obviously induced by NaCI in adventitious roots of B.chinense,while only BCWRKY32 was induced by PEG.The content of SSs increased at different levels in NaCI and PEG6000 treatment.Three genes including BCWRKY6,BCWRKY32,and BCWRKY35,expressed most in roots,were similar to the accumulation pattern of SS.Conclusion The three WRKY genes,BCWRKY6,BCWRKY32,and BCWRKY35,may be involved in the biosynthesis of SS.

Co-cultured adventitious roots of Echinacea pallida and Echinacea purpurea inhibit lipopolysaccharide-induced inflammation via MAPK pathway in mouse peritoneal macrophages

Objective:In order to elucidate the biological activity of the Co-cultured adventitious roots(ARs) of Echinacea pallida and Echinacea purpurea and provide theoretical basis for its application,and the antiinflammatory activities and potential mechanisms of Co-cultured ARs were studied.Methods:The experimental materials were obtained by bioreactor co-culture technology and used in the activity research.In this study,mouse macrophages induced by lipopolysaccharide(LPS) were used as in vitro model.Different concentrations of AR extract(50-400 g/mL) were used to treat cells.The expression of pro-inflammatory cytokines was determined using enzyme linked immunosorbent assay.The inducible nitric oxide synthase and cyclooxygenase-2 expression,mitogen-activated protein kinase(MAPK) phosphorylation,and the inhibitor of nuclear factor-kappa B-a levels were determined by the Western blot analysis.Results:In the co-cultured ARs,total flavonoids and total caffeic acid were determined,and the contents of both bioactive compounds were significantly higher than those ARs from the single-species culture.Compared with the control group,the large amount of pro-inflammatory mediators was released after LPS stimulation.However,in the extract groups with different concentrations(25,50,and 100 g/mL),the production of these pro-inflammatory mediators was inhibited in a dose-dependent manner.Furthermore,the levels of phosphorylation of MAPK proteins,including p-p38, p-c-Jun N-terminal kinase,and p-extracellular regulated protein kinases were significantly(P <0.05) decreased in the extract groups,revealing that the AR extract probably involved in regulating the MAPK signaling pathway.Conclusion:Collectively,our findings suggested that the co-cultured ARs of E.pallida and E.purpurea can inhibit production of pro-inflammatory mediators in mouse peritoneal macrophages and possess the anti-inflammatory effect by regulating MAPK signaling pathways.

Adventitious Root Regeneration:Molecular Basis and Influencing Factors

Plant regeneration is a self-repair of the plant body in response to adverse conditions or damaged structures,and root regeneration allows the plant body to better adapt to its environment by supplementing the roots’structure.Previous research has shown that adventitious roots can be made to occur from scratch in two ways.Studies that simulate adventitious root regeneration through natural conditions allow the regeneration process to be broadly divided into three stages:the perception of early signals,the massive accumulation of auxin,and the transformation of cell fate.The strength of regeneration,in turn,is influenced by wounding,stress,hormones,etc.This study mainly reviews the molecular mechanisms of de novo adventitious roots and discusses how the environment,hormones,and its own development in Arabidopsis thaliana.

Changes in endogenous hormone levels and redox status during enhanced adventitious rooting by rare earth element neodymium of Dendrobium densiflorum shoot cuttings

The effects of neodymium nitrate （Nd^3＋） on the adventitious rooting of Dendrobium densiflorum shoot cuttings were studied. The addition of Nd^3＋ （5 μmol/L） to culture medium significantly increased rooting frequency. Histological investigation showed that Nd^3＋ did not change the process of root initiation. Nd^3＋ did not influence total endogenous cytokinin levels, but significantly increased the level of endogenous indole-3-acetic acid （IAA） in the base of shoot cuttings. Compared to the control, the ratio of IAA/cytokinins was very high in the Nd^3＋ treatment. These results suggested that the enhanced rooting frequency may be related to the increase in endogenous IAA level in Nd^3＋ treatment. Analysis of enzyme activities showed that the enhanced accumulation of the endogenous IAA by Nd^3＋ should not be attributed to inhibition of IAA decomposition by IAA oxidase or promotion of cytokinin decomposition by cytokinin oxidase. Besides, Nd^3＋ increased the ratio of reduced glutathione （GSH） and oxidized glutathione （GSSG） in the process of adventitious rooting while the ratio of ascorbate （ASC） to dehydroascorbate （DHA） was not affected.

Regulation of phytohormones on root primordium initiation and adventitious root formation in the etiolated shoots of Paeonia suffruticosa'Yinfen Jinlin'

Tree peony is well known and sought after for its large, colorful flowers. Its propagation is via vegetative methods. Mech- nisms of the adventitious rooting and the regulation of rooting processes are the principles and techniques of plant propagation and improvement. Microstructures and fluctuations of phytohormones in the adventitious rooting were studied with the etiolated soft- wood shoots of Paeonia suffkuticosa ＇Yinfen Jinlin＇. There are no pre-primordia in the shoots of the cultivar. Adventitious roots are produced in five stages： shoot selection, primordium initiation, primordium growth, conducting tissue differentiation and root protru- sion. Primordia initiated in the cortex. The contents of the endogenous hormones, IAA, ABA and GA, were 5.842, 0.873 and 1.043 nmol·g^-1 FW on the bases of shoots, respectively. CTKs which included isopentenyl adenine （iPA）, zeatin riboside （ZR） and dihy- drozeatin riboside （DHZR） were 0.949, 0.695 and 2.034 nmol·g^-1 FW, respectively. DHZR is active among CTKs. The ratio of IAA to GA, CTK and ABA clearly increased at the stage of primordium initiation, while they showed low levels at the stages of primor- dium growth. The ratios were restored at the shoot levels at the stage of root protrusion. IBA provoked primordia initiation in the cortex, the vascular cambium, the pith and even in the callus induced on the base of shoots. ]AA levels in the treated shoots increased gradually to its highest level （three times of control） at the stage of conducting tissue differentiation. The ratios of IAA to GA, CTK and ABA clearly decreased at the stage of primordium initiation. The ratio of IAA to ABA is regulated at 10：1.

Genome-wide identification of WOX gene family in apple and a functional analysis of MdWOX4b during adventitious root formation

The plant-specific WUSCHEL-related homeobox(WOX)genes are crucial for plant growth and development.Here,we systematically identified the MdWOX gene family in apple at the genome-wide level,and analyzed the phylogenetic relationships,conserved motifs,gene structure,and syntenic relationships of the MdWOX genes.A total of 18 MdWOX genes were identified and phylogenetic analysis placed them into three clades.The phylogenetic relationships among the WOXs were further supported by the analyses of gene structure and conserved motifs.Chromosomal distribution and synteny analysis revealed that whole-genome and segmental duplications have played key roles in MdWOX gene family expansion.Moreover,the MdWOX genes exhibit tissue-specific expression patterns and MdWOX4a,MdWOX4b,MdWOX5b,MdWOX11/12a,and MdWOX11/12b may play essential roles in adventitious root development.The adventitious rooting ability was enhanced in MdWOX4b transgenic tobacco lines.The results of this study provide useful information for future functional studies on MdWOXs in the development of apple rootstocks.

Optimization of Factors Influencing Adventitious Rooting in Hybrid Larch

Optimization of in vitro adventitious root induction contributes to the development of a large-scale production system of hybrid clone seedlings of larch(Larix spp.).We used orthogonal testing to investigate the factors that affect the rooting of hybrid larch shoots-activated carbon,vitamin B1,sucrose,glycine,glutamic acid(Glu),cysteine,pH,and induction time in the dark and indole-3-butyric acid(IBA).Variance analysis showed that the effects of sucrose concentration on rooting rate,survival rate,and average root number,pH on the average number of roots;and Glu on survival rate were all significant(p<0.05)when compared to the mock treatments.Among all of the 45 treatments,the highest rooting rate was 90%,the highest survival rate was 100%,and the highest average root number was 11.8.The theoretically optimal procedure of in vitro root induction of hybrid larch shoots is shoot induction on L9 modified medium(pH 6.7)containing 2 mg.L^(-1)naphthalene acetic acid,0.25 mg.L^(-1)IBA,1/3 macroelements,7.5 g.L^(-1)sucrose,and 5 mg.L^(-1)Glu for 8 days,and then root induction on L9 modified medium without auxin.

Proteomic Analysis through Adventitious Rooting of Pinus radiata Stem Cuttings with Different Rooting Capabilities

In forest production systems, vegetative propagation of elite clones through adventitious rooting is a common practice. In Chile, adventitious rooting is the main methodology for vegetative reproduction of Pinus radiata. However, the capability of produce adventitious roots in gymnosperms decreases with aging. While it is true that some efforts have been made to identify markers or/and regulators of the aging process and adventitious rooting, molecular mechanisms that regulate both processes are scarcely known, especially at protein level. This research evaluated qualitative and quantitative changes in protein accumulation during the adventitious rooting process of P. radiata stem cuttings, with different rooting capabilities. Beside, an analysis of morpho-anatomical changes was performed in stem cuttings with high and low rooting capabilities, during the adventitious rooting process. It was observed that juvenile 1-year-old stem cuttings rooted in a 100%, while aged stem cuttings (3-year-old) presented only a 20% of rooting. According to the results of differential protein accumulation, univariate and multivariate analysis indicated that in total, 114 and 89 proteins were differentially accumulated in juvenile and aged cuttings, respectively. Also, identification of such proteins showed the presence of proteins related to cell wall organization and the presence of a protein related with proper distribution of auxin PIN transporter, both key in the new meristem formation process during adventitious rooting.

Evaluation of total protein,peroxidase,and nutrients measured by pXRF for the determination of tissue rejuvenation/reinvigoration of Eucalyptus microcorys

Ontogenetic aging of tissues and the gradual decrease of adventitious rooting are known challenges for the clonal propagation of woody species,hampering clonal forestry programs.This study examined possible signatures of tissue rejuvenation/reinvigoration in different propagated materials of Eucalyptus microcorys by analyzing the total protein profile,peroxidase activity,macro-and micronutrient contents,and adventitious rooting of mini cuttings.The analyses were performed on E.microcorys shoots which were successfully obtained by seminal and grafting propagation,micropropagation with epicormic shoots,and indirect organogenesis.Among four mature trees used in the propagation,tissues from the one with the best propagation results were investigated for signs of tissue rejuvenation and/or reinvigoration.Five individuals from each technique were randomly selected and transferred to a semi-hydroponic"channel"system.After four weeks in the seedbed,the total protein,peroxidase activity,nutrient content and rooting of the mini cuttings were evaluated.SDS-PAGE enabled the differentiation of leaf samples obtained by grafting from the other propagation techniques,as revealed by two distinct bands.Materials obtained by micropropagation with epicormic shoots showed the highest peroxidase activity,while those obtained by seminal propagation and from the selected mature tree showed the lowest peroxidase activity.A portable X-ray fluorescence spectroscope(pXRF)identified adequate nutrient content in most of the nutrients tested in materials obtained by seminal and grafting propagation,and by indirect organogenesis.The analysis of adventitious rooting showed that the highest rooting percentage was observed in mini cuttings from seminal propagation(75%)followed by indirect organogenesis(35%).Based on principal component analysis,it was concluded that rooting of mini cuttings from both seminal propagation and indirect organogenesis was associated with phosphorous,sulphur,and potassium contents,which suggests a higher level of tissue rejuvenation/reinvigoration in these propagated plants.Further studies are recommended to search for other methods that present similarities with the responses to adventitious rooting in forest species and thus optimize the rescue and propagation of plants with distinct ontogenetic stages.

Factors involved in the success of Castanea henryi stem cuttings in different cutting mediums and cutting selection periods

Chinquapin(Castanea henryi) is a dual-purpose tree species in China valued for as a source of timber and starch.We investigated the effect of four cutting mediums(pure vermiculite;peat:river sand at 3:1 v/v;peat:krasnozem at 1:1 v/v;and pure krasnozem) and three stem cutting periods(March,May,and July) on rooting performance of C.henryi cuttings.Different cutting periods and cutting mediums greatly influenced the rooting rate of C.henryi,ranging from 3.35 to 77.31%.Principal component analysis indicated that the best combination of cutting period and cutting medium was semi-hardwood cuttings(May cuttings)+krasnozem.Histological evidence indicated that adventitious root initials were present by week 5-6,and that the site of root primordia initiation was observed in the vascular cambium.Stem anatomical structures observed at different periods indicated that a xylem/radius ratio of29.90-37.42% and a fractured phloem fiber ring are indicative of rooting success.The relational model between rooting index and medium properties indicated that nutrient content and porosity significantly influenced callus production.However,pH strongly affected C.henryi root formation,with the Pearson correlation coefficients for May and July cuttings of-0.856 and-0.947,respectively.Our protocol is helpful to achieve mass clone propagation of improved C.henryi genotypes,thus overcoming a common hurdle in chinquapin breeding programs.

Plantlet regeneration of adult Pinus massoniana Lamb. trees using explants collected in March and thidiazuron in culture medium

A protocol for micropropagation using nodal explants from mature Pinus massoniana trees has been developed. Time of explant collection is crucial for the initial success of aseptic culture. Explants collected in early March gave the highest percentage of explant survival (64.5%) and shoot-forming percentage (52.3%). Thidiazuron (TDZ) concentration significantly influenced shoot formation; 4 mu M TDZ was optimum, with 4.8 shoots produced per explant with a mean length of 7.1 cm after 120 days of culture. Regenerated shoots rooted for 60 days in basic medium with 1 mu M NAA were ready for growth in pots. This is the first report on plantlet regeneration in vitro from mature trees of P. massoniana that provides a reliable method for propagating selected elites.

The roles of microRNAs in regulating root formation and growth in plants

MicroRNAs(miRNAs) are small(ca. 20-24 nucleotides) non-coding RNAs that have recently been recognized as key post-transcriptional modulators of gene expression;and they are involved in many biological processes in plants, such as root growth and development. The miRNAs regulate root elongation, lateral root(LR) formation and adventitious root(AR) development in response to hormone signaling, nutrient uptake and biotic/abiotic stress. This review provides multiple perspectives on the involvement of miRNAs in regulating root growth and development in plants. We also discuss several crucial mechanisms of miRNAs, their relationships with transcription factors and the target gene-mediated hormone signaling interactions in the regulation of root growth and development.

Mini-cutting technique for Khaya anthotheca: selection of suitable IBA concentration and nutrient solution for its vegetative propagation

Khaya anthotheca is a hardwood species from Africa and recently introduced to Brazil. This species yields high-quality wood for diverse applications, but little has been done toward an effective propagation method for large-scale production in nurseries. We evaluated the effects of different concentrations of macro and micronutrients（i.e., 100, 50 and 25% of the concentration of a referenced solution） combined with indole-3-butyric acid（IBA） at 0 and 2 g L-1 on the survival rate of mini-stumps and the survival and adventitious rooting of K. anthotheca mini-cuttings. The mini-stumps were grown from a source of seeds imported from Ghana, West Africa. The ministumps survived at a high rate（97%）. Consistently high shoot yields were obtained from the mini-stumps（average of 1.8 per mini-stump）. High survival percentage of minicuttings and adventitious rooting were observed when solutions with 100 and 50% of the nutrients concentration were combined with 2 g L-1 IBA. Histological sections analyzed through optical microscopy indicated the tissues from mini-cutting stems were juvenile, which might have stimulated adventitious rooting. These results have important implications for further work aimed at establishing propagation strategies for K. anthotheca, which are of prime importance for assisting breeding programs of this species.

Vegetative propagation of Apuleia leiocarpa(Vogel)J.F.Macbr

Apuleia leiocarpa is an environmentally and economically significant Atlantic Forest species threat-ened by ongoing exploitation.The vegetative propagation of the species could be a step forward to enable implanta-tion of clonal seed orchards and multiplication of selected trees but remains unknown to date.This study assessed the mini-cutting technique for propagation of A.leiocarpa and determined the time required for rooting and viable mini-cutting type and sizes.The results show that it is possible to propagate A.leiocarpa with mini-cuttings derived from mini-stumps produced with seeds;the mini-stumps pre-sented regrowth capacity and remained constantly produc-tive for 1 year;10-cm A.leiocarpa mini-cuttings should remain under misting conditions for at least 40 days,and the vegetative propagation of A.leiocarpa with intermediate 7-to 10-cm mini-cuttings resulted in more vigorous clonal seedlings than smaller mini-cuttings.

A Molecular Framework for the Control of Adventitious Rooting by TIR1/AFB2-Aux/IAADependent Auxin Signaling in Arabidopsis

In Arabidopsis thaliana,canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR(ARF)family that interact with auxin/indole acetic acid repressors(Aux/IAAs),which themselves form co-receptor complexes with one of six TRANSPORT INHIBITOR*!/AUXIN-SIGNALLING F-BOX(TIR1/AFB)proteins.Different combinations of co-receptors drive specific sensing outputs,allowing auxin to control a myriad of processes.ARF6 and ARF8 are positive regulators of adventitious root initiation upstream of jasmonate,but the exact auxin co-receptor complexes controlling the transcriptional activity of these proteins has remained unknown.Here,using loss-of-function mutants we show that three Aux/IAA genes,IAA6,IAA9,and IAA17,act additively in the control of adventitious root(AR)initiation.These three IAA proteins interact with ARF6 and/or ARF8 and likely repress their activity in AR development.We show that TIR1 and AFB2 are positive regulators of AR formation and TIR1 plays a dual role in the control of jasmonic acid(JA)biosynthesis and conjugation,as several JA biosynthesis genes are up-regulated in the tir1-1 mutant.These results lead us to propose that in the presence of auxin,TIR1 and AFB2 form specific sensing complexes with IAA6,IAA9,and/or IAA17 to modulate JA homeostasis and control AR initiation.

Genome-wide Identification and Expression of ARF Gene Family during Adventitious Root Development in Hot Pepper(Capsicum annuum)

Auxin response factors(ARFs) are transcription factors that activate or repress the expression of primary/early auxin response genes by binding to auxin-responsive elements(Aux REs) in their promoter regions. The ARFs play important roles in diverse developmental processes.To explore the ARF gene family in hot pepper(Capsicum annuum L.), we performed a genome-wide identification and expression analysis. In this study, 19 pepper ARF genes(Ca ARFs) clustered into three phylogenetic groups(I, II, and III) were comprehensively analyzed. Conserved domain analysis showed that all Ca ARFs contained a B3 DNA-binding domain and a middle domain, but two members lacked the carboxyterminal dimerization(CTD) domain. The number of introns in Ca ARF genes ranged from 1 to 13 and the gene structure was similar among genes in the same phylogenetic group. Additionally, prediction of Ca ARFs promoter elements and putative targets for micro RNAs suggested that the regulation of Ca ARFs may occur at both transcriptional and posttranscriptional levels. Most Ca ARFs were expressed in more than one tested tissue, and most Ca ARFs were identified as being responsive to exogenous auxin. Moreover, time-course transcription profiles of Ca ARFs revealed their roles in adventitious rooting of hypocotyl cuttings from pepper seedlings. Therefore, our results will provide a foundation for better understanding the regulatory mechanisms and molecular functions of Ca ARFs in hot pepper.

Improvement of Growth and Periplocin Yield of Periploca sepium Adventitious Root Cultures by Altering Nitrogen Source Supply

Objective To increase the ultimate yield of periplocin in Periploca sepium adventitious root cultures by a two-stage culture based on nitrogen source.Methods Firstly,the effects of nitrogen source(NH4+-NO3-) at different ratios and different total initial nitrogen amounts on the accumulation of biomass and secondary metabolites in adventitious root cultures of P.sepium were investigated,and growth and production media for the two-stage culture based on the above results were established.Results The highest biomass and periplocin content were obtained in the culture medium of 15 mmol/L total nitrogen amount with NH4+-NO3-(1:2) and 30 mmol/L total nitrogen amount with nitrate as the sole nitrogen source.By adopting a fed-batch cultivation strategy,the dry weight adventitious root,periplocin content and yield were increased by 136%,108%,and 389%,respectively when compared with those of the control,reaching up to 8.13 g/L,157.15 μg/g,and 1277.63 μg/L,respectively.Furthermore,it was found that in the process of two-stage culture,the adventitious roots grew thicker significantly after they were transferred into production medium directly.Conclusion The ultimate yield of periplocin in P.sepium adventitious root cultures could be significantly increased by a two-stage culture based on nitrogen source.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.

A Fluorescent Alternative to the Synthetic Strigolactone GR24

Strigolactones have recently been implicated in both above- and below-ground developmental pathways in higher plants. To facilitate the molecular and chemical properties of strigolactones in vitro and in vivo, we have devel- oped a fluorescent strigolactone molecule, CISA-1, synthesized via a novel method which was robust, high-yielding, and used simple starting materials. We demonstrate that CISA-1 has a broad range of known strigolactone activities and further report on an adventitious rooting assay in Arabidopsis which is a highly sensitive and rapid method for testing biological activity of strigolactone analogs. In this rooting assay and the widely used Orobanche germination assay, CISA-1 showed stronger biological activity than the commonly tested GR24. CISA-1 and GR24 were equally effective at inhibiting branching in Arabidopsis inflorescence stems. In both the branching and adventitious rooting assay, we also demonstrated that CISA-1 activity is dependent on the max strigolactone signaling pathway. In water methanol solu- tions, CISA-1 was about threefold more stable than GR24, which may contribute to the increased activity observed in the various biological tests.