Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

Rhizosphere competent <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in the management of <Emphasis Type="Italic">Heterodera cajani</Emphasis> on sesame

Biological control of the cyst forming nematode Heterodera cajani was studied on sesame using plant growth promoting rhizobacteria (PGPR) Pseudomonas aeruginosa LPT3 and LPT5. Based on plant growth promoting attributes, two fluorescent pseudomonads, LPT3 and LPT5 were evaluated for their efficacy against cyst forming nematode Heterodera cajani that parasitize Sesamum indicum. Pseudomonas aeruginosa LPT5 produced IAA, HCN, chitinase, glucanase and siderophore, and also solubilized inorganic phosphate in vitro. Moreover, LPT5 resulted in mortality of second stage juveniles of H. cajani, which was 13% higher as compared to P. aeruginosa LPT3. Interestingly, when both strains were inoculated together for the management of H. cajani on Sesamum indicum the population of H. cajani was reduced significantly, in field trial. Approximately 60% reduction in cyst and juveniles population was recorded with LPT5 coated seeds, while LPT3 resulted in 49% reduction in cyst and juvenile population as compared to control. Plants grown with seeds bacterized with LPT5 and reduced doses of urea, diammonium phosphate (DAP), muriate of potash (K) and gypsum gave maximum increase in yield, in comparison to that of plants raised under the influence of recommended or full doses of the chemical fertilizers. Pseudomonas aeruginosa LPT5 also showed excellent root colonization.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of microshoots of <Emphasis Type="Italic">Cotinus coggygria</Emphasis> Mill,a woody ornamental plant

Studies on rooting of microshoots of smokebush (Cotinus coggygria Mill, var. Royal Purple), a woody ornamental, were carried out in vitro. Microshoots were rooted in a mixed-auxin regime (indole 3-acetic acid, indole butyric acid [IBA], and naphthalene acetic acid) or singly in the above auxins and the 2,4 dichlorophenoxyacetic acid (2,4-D) over a wide concentration range. Indole butyric acid at 10 μM proved to be the most suitable treatment, producing less basal callus, 100% rooting, and earlier root emergence than the other treatments. No roots were formed with 2,4-D. A 6-day root induction period was obtained with 10 μM of IBA. Histological studies revealed increased mitotic activity after 3 d in culture in the medullary ray cells, which led to root primordium formation, several of which were formed simultaneously around the base of the explant. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 10. Thus, direct rhizogenesis occurred with the IBA treatment, as opposed to the roots that were formed in the basal callus under the mixed-auxin regime.     

Identification and Characterization of an Autolysin Gene, <Emphasis Type="Italic">atlh</Emphasis>, from <Emphasis Type="Italic">Streptococcus downei</Emphasis>

An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in grain biopreservation

Cereal grain is a major component of food and feed in large parts of the world. The microbial flora on cereal grains may interfere with hygiene and storage stability, palatability and bioavailability of minerals and proteins may depend on the composition of the microbial population. Therefore, it is of primary interest to control the microbial species present on cereal grain. Inoculation of the biocontrol yeast Pichia anomala to cereal feed grain improved feed hygiene by reduction of moulds and Enterobacteriaceae, and enhanced the nutritional value by increasing the protein content and reducing the concentration of the antinutritional compound phytate. P. anomala strains showed a high phytase activity, for some strains also considerable extracellular phytase activity was observed. A certain maximum in biomass concentration was never exceeded indicating cell density induced growth inhibition of P. anomala.     

Hormonal control of the outgrowth of axillary buds in <Emphasis Type="Italic">Alstroemeria</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

We study apical dominance in Alstroemeria, a plant with an architecture very different from the model species used in research on apical dominance. The standard explant was a rhizome with a tip and two vertically growing shoots from which the larger part had been excised leaving ca. 1 cm stem. The axillary buds that resumed growth were located at this 1-cm stem just above the rhizome. They were released by removal of the rhizome tip and the shoot tips. Replacement of excised tips by lanolin with indole-3-butyric acid (IBA) restored apical dominance. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-napthylphthalamic acid (NPA) reduced apical dominance. 6-Benzylaminopurine (BAP) enhanced axillary bud outgrowth but the highest concentrations (> 9 μM) caused fasciation. Thidiazuron (TDZ) did not show improvement relative to BAP. Even though the architecture of Alstroemeria and the model species are very different, their hormonal mechanisms in apical dominance are for the greater part very similar.     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

Ultrastructure of cyanobacterium <Emphasis Type="Italic">Nostoc</Emphasis> sp. f. <Emphasis Type="Italic">Blasia</Emphasis> cell forms in persisting populations

Cell clusters formed in persistent populations of Nostoc sp. f. Blasia, a cyanobacterium capable of cell differentiation, under prolonged storage in the dark at low temperatures were studied for the first time. Cell reorganization was observed, including changes in the ultrastructure of thylakoids, the cell wall peptidoglycan layer, and carboxysomes. Subcellular structures involved in intercellular communication within the clusters were revealed (structures similar to microplasmodesms and contact pores, secretory vesicles, etc.) Persistence of cyanobacterial populations was concluded to result from formation not only of specialized dormant cells (akinetes), but also L-forms, as well as from the modification changes of the clustered vegetative cells. A cluster containing the vegetative cells and L-like forms within a common intercellular matrix is considered a structural unit at the supracellular level, which is responsible for survival of cyanobacterial populations when mass akinete formation does not occur.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

The presence of <Emphasis Type="Italic">Zygina</Emphasis> sp. and <Emphasis Type="Italic">Puccinia myrsiphylli</Emphasis> reduces survival and influences oviposition of <Emphasis Type="Italic">Crioceris</Emphasis> sp.

A leaf beetle, Crioceris sp. (Coleoptera: Chrysomelidae), was introduced into Australia as a biological control agent of bridal creeper (Asparagus asparagoides L. Druce) during October 2002. Rearing of Crioceris sp. is labour intensive therefore all releases of Crioceris sp. have been under 1000 individuals, which may be too low to ensure establishment if high mortality and high competition with other agents occurs. The aim of this study is to understand how the presence of two well-established biocontrol agents, a rust fungus (Puccinia myrsiphylli (Thuem) Wint [Basidiomycota: Uredinales]) and a leafhopper (Zygina sp. [Hemiptera: Cicadellidae]), might influence Crioceris sp. establishment. Crioceris sp. neonate larvae were placed on bridal creeper plants with or without the leafhopper and/or rust. The number of larvae that pupated was reduced by 38 and 65% in the presence of the rust fungus and leafhopper, respectively and by 45% in the presence of both agents. As the area infected by the rust increased the area damaged by the leafhopper decreased. The rust appeared to be negatively impacted by the presence of the leafhopper. In a second experiment, female Crioceris sp. adults were given a choice between uninfested bridal creeper plants and those infested with the rust or the leafhopper. The females preferred to lay their eggs on plants without leafhoppers but did not seem to be deterred by the presence of the rust. Consequently, the performance and impact of Crioceris sp. on bridal creeper may be reduced if populations overlap with the other biocontrol agents in the field.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

Wave-like Distribution Patterns of <Emphasis Type="Italic">Gfp</Emphasis>-marked <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Along Roots of Wheat Plants Grown in Two Soils

Culturable rhizosphere bacterial communities had been shown to exhibit wave-like distribution patterns along wheat roots. In the current work we show, for the first time, significant wave-like oscillations of an individual bacterial strain, the biocontrol agent Pseudomonas fluorescens 32 marked with gfp, along 3-week-old wheat roots in a conventionally managed and an organically managed soil. Significant wave-like fluctuations were observed for colony forming units (CFUs) on selective media and direct fluorescent counts under the microscope. Densities of fluorescent cells and of CFUs fluctuated in a similar manner along wheat roots in the conventional soil. The frequencies of the first, second, and third harmonics were similar for direct cell counts and CFUs. Survival of P. fluorescens 32-gfp introduced into organically managed soil was lower than that of the same strain added to conventionally managed soil. Thus, when root tips reached a depth of 10–35 cm below soil level, the majority of the introduced cells may have died, so that no cells or CFU”s were detected in this region at the time of sampling. As a result, significant waves in CFUs or direct counts along roots were not found in organically managed soil, except when a sufficiently long series with detectable CFUs were obtained. In this last case the wave-like fluctuation in CFUs was damped toward the root tip. In conclusion, when cells of a single bacterial strain randomly mixed in soil survived until a root tip passed, growth and death cycles after passage of the root tip resulted in oscillating patterns of population densities of this strain along 3-week-old wheat roots.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.