Machine Learning Technology Reveals the Concealed Interactions of Phytohormones on Medicinal Plant In Vitro Organogenesis.

Organogenesis constitutes the biological feature driving plant in vitro regeneration, in which the role of plant hormones is crucial. The use of machine learning (ML) technology stands out as a novel approach to characterize the combined role of two phytohormones, the auxin indoleacetic acid (IAA) and the cytokinin 6-benzylaminopurine (BAP), on the in vitro organogenesis of unexploited medicinal plants from the Bryophyllum subgenus. The predictive model generated by neurofuzzy logic, a combination of artificial neural networks (ANNs) and fuzzy logic algorithms, was able to reveal the critical factors affecting such multifactorial process over the experimental dataset collected. The rules obtained along with the model allowed to decipher that BAP had a pleiotropic effect on the Bryophyllum spp., as it caused different organogenetic responses depending on its concentration and the genotype, including direct and indirect shoot organogenesis and callus formation. On the contrary, IAA showed an inhibiting role, restricted to indirect shoot regeneration. In this work, neurofuzzy logic emerged as a cutting-edge method to characterize the mechanism of action of two phytohormones, leading to the optimization of plant tissue culture protocols with high large-scale biotechnological applicability.

Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes.

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3 -), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

Photosynthetic regulation under fluctuating light in young and mature leaves of the CAM plant Bryophyllum pinnatum.

Photosystem I (PSI) is the potential target of photodamage under fluctuating light in angiosperms. However, the response of PSI to fluctuating light in young leaves has not yet been clarified. Furthermore, the photosynthetic regulation under fluctuating light in crassulacean acid metabolism (CAM) plants is little known. In this study, we measured PSI redox state and the electrochromic shift signal in the mature and young leaves of a CAM species Bryophyllum pinnatum. The mature leaves showed stronger capacity for photo-reduction of O2 mediated by the alternative electron flow (probably the water-water cycle) when compared with the young leaves. After an increase in light intensity, both the mature and young leaves showed insufficient proton gradient (ΔpH) across the thylakoid membranes within the first seconds. Meanwhile, PSI was highly oxidized in the mature leaves but was in a more reduced state in the young leaves. Furthermore, young leaves were more susceptible to PSI photoinhibition under fluctuating light. Therefore, in the mature leaves, the alternative electron flow significantly optimized the PSI redox state under fluctuating light at relatively low ΔpH. By comparison, in the young leaves, PSI redox state was largely determined by the buildup of ΔpH. Therefore, the major photoprotective mechanism responsible for safeguarding PSI under fluctuating light can be influenced by leaf developmental stages.

Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana.

Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

Key KdSOC1 gene expression profiles during plantlet morphogenesis under hormone, photoperiod, and drought treatments.

Kalanchoe daigremontiana utilizes plantlet formation between its zigzag leaf margins as its method of asexual reproduction. In this study, K. daigremontiana SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (KdSOC1), a key intermediate in the transition from vegetative to asexual growth, was cloned. Furthermore, its expression profiles during plantlet formation under different environmental and hormone induction conditions were analyzed. The full-KdSOC1 cDNA sequence length was 1410 bp with 70% shared homology with Carya cathayensis SOC1. The conserved domain search of KdSOC1 showed the absence of I and C domains, which might indicate novel biological functions in K. daigremontiana. The full-KdSOC1 promoter sequence was 1401 bp long and contained multiple-hormone-responsive cis-acting elements. Hormone induction assays showed that gibberellins and salicylic acid mainly regulated KdSOC1 expression. The swift change from low to high KdSOC1 expression levels during long-day induction was accompanied by the rapid emergence of plantlets. Drought stress stimulated KdSOC1 expression in leaves both with and without plantlet formation. Together, the results suggested that KdSOC1 was closely involved in environmental stimulation signal perception and the transduction of K. daigremontiana plantlet formation. Therefore, future identification of KdSOC1 functions might reveal key information that will help elucidate the transition network between embryogenesis and organogenesis during plantlet formation.

Induction of wound-periderm-like tissue in Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) leaves as a defence response to high UV-B radiation levels.

BACKGROUND AND AIMS: UV-B radiation can be stressful for plants and cause morphological and biochemical changes. Kalanchoe pinnata is a CAM leaf-succulent species distributed in hot and dry regions, and is rich in flavonoids, which are considered to be protective against UV-B radiation. This study aims to verify if K. pinnata has morphological or anatomical responses as a strategy in response to high UV-B levels. METHODS: Kalanchoe pinnata plants of the same age were grown under white light (control) or white light plus supplemental UV-B radiation (5 h d(-1)). The plants were treated with the same photoperiod, photosynthetically active radiation, temperature and daily watering system. Fragments of the middle third of the leaf blade and petiole were dehydrated and then embedded in historesin and sectioned in a rotary microtome. Sections were stained with toluidine blue O and mounted in Entellan®. Microchemical analyses by optical microscopy were performed on fresh material with Sudan III, Sudan IV and phloroglucinol, and analysed using fluorescence microscopy. KEY RESULTS: Supplemental UV-B radiation caused leaf curling and the formation of brown areas on the leaves. These brown areas developed into a protective tissue on the adaxial side of the leaf, but only in directly exposed regions. Anatomically, this protective tissue was similar to a wound-periderm, with outer layer cell walls impregnated with suberin and lignin. CONCLUSIONS: This is the first report of wound-periderm formation in leaves in response to UV-B radiation. This protective tissue could be important for the survival of the species in desert regions under high UV-B stress conditions.

Transgenic perturbation of the decarboxylation phase of Crassulacean acid metabolism alters physiology and metabolism but has only a small effect on growth.

Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO(2) fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO(2) fixation. In well-watered conditions, these lines fixed all of their CO(2) in the light; they thus performed C(3) photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO(2) fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.

Anti-inflammatory effects of Bryophyllum pinnatum (Lam.) Oken ethanol extract in acute and chronic cutaneous inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Bryophyllum pinnatum (Lam.) Oken (Crassulaceae), popularly known in Brazil as "folha-da-fortuna", is a plant species used in folk medicine for the external and internal treatment of inflammation, infection, wound, burn, boil, ulcers and gastritis, and several other diseases. The present study aimed to perform the chemical characterization and the evaluation of the topical anti-inflammatory effect of the ethanol extract of Bryophyllum pinnatum leaves (EEBP) in acute and chronic mice ear edema models induced by different irritant agents. MATERIALS AND METHODS: The EEBP chemical characterization was performed by HPLC-UV DAD. Ear edema on Swiss mice was induced by the topical application of Croton oil (single and multiple applications), arachidonic acid, phenol, capsaicin and ethyl phenylpropiolate (EPP). The topical anti-inflammatory effect of EEBP was evaluated by measuring the ear weight (acute inflammation models) and thickness (chronic inflammation model). Histopathological analyses of ear tissue samples sensitized with Croton oil (single and multiple applications) were also performed. RESULTS: The flavonoids rutin, quercetin, luteolin and luteolin7-O-ß-d-glucoside were detected in EEBP. Topical application of EEBP significantly (P<0.001) inhibited the ear edema induced by Croton oil single application (inhibition of 57%), arachidonic acid (inhibition of 67%), phenol (inhibition of 80%), capsaicin (inhibition of 72%), EPP (inhibition of 75%) and Croton oil multiple application (55% after 9 days). Histopathological analyses confirmed the topical anti-inflammatory effect of EEBP since it was observed reduction of edema, epidermal hyperplasia, inflammatory cells infiltration and vasodilation. CONCLUSIONS: The results suggest that EEBP is effective as a topical anti-inflammatory agent in acute and chronic inflammatory processes possibly due to inhibition of arachidonic acid pathway, which justify the traditional use of Bryophyllum pinnatum as a remedy for skin disorders.

Ectopic expression of Kxhkn5 in the viviparous species Kalanchoe × Houghtonii induces a novel pattern of epiphyll development.

KxhKN5 (class 1 KNOX gene) was cloned from Kalanchoe × houghtonii with strong tendency to form epiphylls on leaves. KxhKN5 appear to be homologue of BP of A. thaliana on the basis of phylogeny, expression and phenotype analysis. Beside the modification of several plant and leaf traits, the appearance of epiphylls was drastically reduced by both the silencing and the over-expression of KxhKN5 in most of the generated clones. In silenced clones, epiphyll production followed the morphogenetic pathway of the WT plants: somatic embryos outbreak in the centre of each leaf-pedestal, grown in the notch between leaf indentations and were supported by a suspensor. The connection between the epiphyll and the mother plant did not include any vasculature and as a result, the epiphylls dropped easily from the mother plant. The most represented category of over expressor clones, disclosed a novel pattern of epiphyll development: the leaf-pedestals were absent and single bud outbreaks in each leaf notch. Buds developed into shoots which remained attached to the maternal plant by a strong vascular connection. The leaves supporting shoots, produced a thickened midrib and veins, and their lamina ceased expanding. Finally, the leaf/shoot structure resembles a lateral branch. The ectopic expression of KxhKN5 in K. × houghtonii induces a process comparable to the alternation of leaf and shoot formation in other species and support the idea, that it is the variation in shared molecular and developmental processes which produces the growth of specific structures.

Correlations among attributes of senescence and antioxidative status of leaf discs during epiphyllous bud differentiation in Kalanchoe pinnata Lam. (Pers.).

Leaf detachment is a common signal that triggers both the differentiation of dormant epiphyllous buds as well as the onset of foliar senescence in Kalanchoe pinnata Lam. (Pers.). The present study looked for any probable correlations among selected attributes of foliar senescence, e.g. soluble proteins, chlorophylls a and b (Chl(a+b)), and membrane stability index (MSI), and the antioxidative status, e.g. phenolics, ferric reducing ability in plasma equivalence (FRAP(eq)), and membrane protection index (MPI), during epiphyllous bud differentiation. The experimental system comprised 0.75-cm leaf discs, with or without a dormant epiphyllous bud, cultured in vitro and exposed for ten days to continuous light or dark. A steady depletion of soluble proteins and Chl(a+b), and lowering of MSI in the leaf discs were observed, the decline being relatively faster and of higher magnitude in discs exposed to dark rather than to light. The pigment loss in discs with differentiating epiphyllous buds was greater and faster than in those lacking buds, a somewhat reverse situation was observed in case of soluble proteins. Simultaneously, a time-dependent decrease in the level of phenolics was also observed. Their content was found to be lower in discs exposed to dark as compared to light, pointing to a relationship with a higher rate of senescence-related degradative processes in the dark. The change in the content of Chl(a+b) was found to be significantly correlated with the variation in the level of phenolics. The average FRAP(eq) after ten days was one half that of the initial level, which could be correlated with the decreasing levels of phenolics (intra-correlation) and maximally correlated with variations in Chl(a+b) and protein contents (inter-correlation). Aqueous alcohol foliar extracts significantly (p < 0.05) protected membranes against peroxidative stress, although the pattern was not found to be in line with that of the phenolics content or FRAP(eq). The diminishing Chl(a+b) content was found to be maximally correlated with alterations in the membrane protection.

Production of interspecific hybrids in ornamental plants.

In breeding of ornamental plants, interspecific hybridization and polyploidization have successfully been used to produce novel cultivars with blended traits of both parents and to introgress useful traits of one species to another. Embryo rescue techniques and molecular cytogenetic methods have successfully been used to produce and characterize interspecific hybrids in various genera. In this paper, recent advances in interspecific hybridization are described based on the results obtained in Primula, Cosmos, and Kalanchoe with special references to the use of embryo culture techniques for rescuing the abortive hybrid embryos. The methods for production and characterization of interspecific hybrids are categorized into three steps, i.e., (1) pollination, (2) rescue culture of immature embryo, and (3) confirmation of hybridity and ploidy level of the plants obtained. For interspecific crosses, emasculation step is usually needed to avoid self-pollination even in the genera with self-incompatibility system, such as Primula and Cosmos, since self-incompatibility is not always complete. Since interspecific crosses are usually hindered by various cross-incompatibility mechanisms, successful production of interspecific hybrids could be achieved only from limited crosses among those using many cultivars/strains of both parents, suggesting the importance of the selection of the compatible genotypes. Unilateral cross incompatibility is commonly observed in interspecific cross combinations, so reciprocal crosses should be conducted as an indispensable step. At the rescue culture step, addition of plant hormones, e.g., auxin cytokinin and gibberellin, to the culture medium at the appropriate concentrations is proved to be effective and necessary. The hybridity of the plants is efficiently confirmed at the seedling stage by DNA analysis in addition to the observation of morphological characters. The analysis of relative DNA contents by flow cytometry is an easy and rapid means to confirm hybridity and to estimate ploidy level and genomic combination.

Chemical and agronomic development of Kalanchoe brasiliensis Camb. and Kalanchoe pinnata Pers under light and temperature levels.

This study compares the development of Kalanchoe brasiliensis and Kalanchoe pinnata, which are medicinal species known as "saião" and "folha da fortuna" that are used interchangeably by the population for medicinal purposes. The experiment consisted of 20 plots/species planted in plastic bags with homogeneous substrate in a randomized design, which grown under light levels (25%, 50%, 70%, full sunlight) at environment temperature, and a treatment under a plastic with greater temperature range than the external environment. It was obtained for K. pinnata a greater plant height, total length of sprouts, stems, production and dry matter content of leaves than that obtained for K. brasiliensis, which achieved higher averages only for the length of lateral branches. The species showed increases in height, which varied in inverse proportion to the light, and it was observed the influence of temperature in K. pinnata. The production and dry matter content of leaves were proportional to the luminosity; the same occurred in the thickness of leaves for K. brasiliensis. In the swelling index and Brix degree, K. brasiliensis showed higher averages than K. pinnata. In relation to the total content of flavonoids it was not observed significant differences for both species. The analyzed parameters showed the main differences in the agronomic development of the two species.

Chemical and agronomic development of Kalanchoe brasiliensis Camb. and Kalanchoe pinnata (Lamk.) Pers under light and temperature levels

This study compares the development of Kalanchoe brasiliensis and Kalanchoe pinnata, which are medicinal species known as "saião" and "folha da fortuna" that are used interchangeably by the population for medicinal purposes. The experiment consisted of 20 plots/species planted in plastic bags with homogeneous substrate in a randomized design, which grown under light levels (25 percent, 50 percent, 70 percent, full sunlight) at environment temperature, and a treatment under a plastic with greater temperature range than the external environment. It was obtained for K. pinnata a greater plant height, total length of sprouts, stems, production and dry matter content of leaves than that obtained for K. brasiliensis, which achieved higher averages only for the length of lateral branches. The species showed increases in height, which varied in inverse proportion to the light, and it was observed the influence of temperature in K. pinnata. The production and dry matter content of leaves were proportional to the luminosity; the same occurred in the thickness of leaves for K. brasiliensis. In the swelling index and Brix degree, K. brasiliensis showed higher averages than K. pinnata. In relation to the total content of flavonoids it was not observed significant differences for both species. The analyzed parameters showed the main differences in the agronomic development of the two species.

Este estudo compara o desenvolvimento de Kalanchoe brasiliensis e Kalanchoe pinnata, espécies medicinais conhecidas como "saião" e "folha da fortuna" que são utilizadas indiferenciadamente pela população para fins medicinais. O experimento consistiu em 20 parcelas/espécie plantadas em sacos plásticos com substrato homogêneo, em delineamento ao acaso, cultivadas sob níveis de luminosidade (25 por cento, 50 por cento, 70 por cento, luz plena) em temperatura ambiente, e um tratamento sob plástico com maior amplitude térmica que o meio externo. Obteve-se para K. pinnata maiores alturas de plantas, comprimentos totais de brotos, caules, produção e teor de matéria seca de folhas que para K. brasiliensis, que alcançou maiores médias apenas para comprimentos de ramos laterais. As espécies apresentaram aumento em altura que variou em proporção inversa à incidência luminosa, e observou-se a influência da temperatura em K. pinnata. A produção e teor de matéria seca de folhas foram proporcionais à luminosidade; o que também ocorreu na espessura foliar para K. brasiliensis. No índice de intumescência e grau Brix, K. brasiliensis apresentou maiores médias que K. pinnata. Em relação ao teor de flavonoides totais, não foram observadas diferenças significativas para ambas as espécies. Os parâmetros analisados apontaram as principais diferenças no desenvolvimento agronômico das duas espécies.

Expression of KxhKN4 and KxhKN5 genes in Kalanchoë blossfeldiana 'Molly' results in novel compact plant phenotypes: towards a cisgenesis alternative to growth retardants.

Many potted plants like Kalanchoë have an elongated natural growth habit, which has to be controlled through the application of growth regulators. These chemicals will be banned in the near future in all the EU countries. Besides their structural functions, the importance of homeotic genes to modify plant architecture appears evident. In this work, the full length cDNA of five KNOX (KN) genes were sequenced from K. x houghtonii, a viviparous hybrid. Two constructs with the coding sequence of the class I and class II homeobox KN genes, KxhKN5 and KxhKN4, respectively, were overexpressed in the commercially important ornamental Kalanchoë blossfeldiana 'Molly'. Furthermore, a post-transcriptional gene silencing construct was made with a partial sequence of KxhKN5 and also transformed into 'Molly'. Several transgenic plants exhibited compact phenotypes and some lines had a relative higher number of inflorescences. A positive correlation between gene expression levels and the degree of compactness was found. However, a correlation between the induced phenotypes and the number of inserted copies of the transgene were not observed, although line '70-10' with a high copy number also had the highest expression level. Moreover, overexpression of KxhKN4 resulted in plants with dark green leaves due to an elevated content of chlorophyll, a highly desired property in the ornamental plant industry. These transgenic plants show that a cisgenesis approach towards production of compact plants with improved quality as an alternative to chemical growth retardants may be feasible.

Influence of cultivation conditions, season of collection and extraction method on the content of antileishmanial flavonoids from Kalanchoe pinnata.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) are popularly used for healing wounds. Its antileishmanial properties are established in experimental animals, and its active flavonoid components have been identified. AIM OF THE STUDY: In this study, we attempted to standardize the extract from K. pinnata leaves by evaluating the influence of season of harvest, sunlight exposure and method of extraction on antileishmanial flavonoids content. MATERIALS AND METHODS: HPLC-DAD-MS was used to identify and quantify the active antileishmanial flavonoids in different extracts. ANOVA test for analyses of variance followed by the Tukey test of multiple comparisons were used in the statistical analysis. The antileishmanial potential was assessed by the activation of nitric oxide production by murine macrophage using the Griess method. RESULTS: We demonstrated that active flavonoids were significantly more abundant when the leaves were collected in the summer, and that aqueous extraction at 50°C allowed the highest flavonoid extraction. The benefit of sunlight exposure was confirmed in plants cultivated under direct sunlight when compared with those that grown under shade. Under sunny conditions the yield of the most active antileishmanial favonoid quercitrin was increased by 7-fold. All aqueous extracts tested were capable to enhance the macrophage nitric oxide production. However, hot aqueous extract from leaves collected in summer exhibited the higher activity, in agreement with HPLC-DAD-MS analysis tendency. In addition, with the aim of reducing the individual chemical variations of the plant constituents and optimizing the production of the active extract, it was obtained in vitro monoclonal KP specimens that were easily adapted to field conditions and were able to produce antileishmanial flavonoids. CONCLUSION: Our study reports the better conditions of cultivation, harvest and extraction protocol for obtaining a K. pinnata extract exhibiting the highest antileishmanial activity. Additionally, we propose the flavonoids quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside and quercitrin, as satisfactory chemical markers for standardization purposes.

Production of compact plants by overexpression of AtSHI in the ornamental Kalanchoë.

Growth retardation is an important breeding aim and an essential part of horticultural plant production. Here, the potential of transferring the Arabidopsis short internode (shi) mutant phenotype was explored by expressing the AtSHI gene in the popular ornamental plant Kalanchoë. A 35S-AtSHI construct was produced and transferred into eight genetically different cultivars of Kalanchoë by Agrobacterium tumefaciens. The resulting transgenic plants showed dwarfing phenotypes like reduced plant height and diameter, and also more compact inflorescences, as a result of increased vegetative height. The shi phenotype was stable over more than five vegetative subcultivations. Compared with Arabidopsis, the ectopic expression of AtSHI in Kalanchoë showed several differences. None of the Kalanchoë SHI-lines exhibited alterations in leaf colour or morphology, and most lines were not delayed in flowering. Moreover, continuous treatment of lines delayed in flowering with low concentrations of gibberellins completely restored the time of flowering. These features are very important as a delay in flowering would increase plant production costs significantly. The effect of expression controlled by the native Arabidopsis SHI promoter was also investigated in transgenic Kalanchoë and resulted in plants with a longer flowering period. Two AtSHI like genes were identified in Kalanchoë indicating a widespread presence of this transcription factor. These findings are important because they suggest that transformation with the AtSHI gene could be applied to several species as a tool for growth retardation, and that this approach could substitute the use of conventional chemical growth regulation in plant production.

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation.

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.

Development of the cuticular wax during growth of Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves.

The goal of the present study was to monitor cuticular wax accumulation during leaf development of Kalanchoe daigremontiana. Leaves expanded linearly until they were 40-60 d old. Wax coverages of leaves on the third node increased steadily during initial leaf development, from 6.5 microg x cm(-2) on day 22 to 15.3 microg x cm(-2) on day 53, and then levelled off. Triterpenoids dominated the wax mixture throughout leaf development, but decreased from 74 to 40-45% in mature leaves, while very long-chain fatty acid (VLCFA) derivatives increased from 19 to 39-44%. The major VLCFA derivatives were alkanes, accompanied by fatty acids, primary alcohols, aldehydes and alkyl esters. In all compound classes, either C(34) or C(33) homologs predominated during leaf development. Eight different triterpenoids were identified, with glutinol constituting 70% of the fraction, and friedelin (20%) and germanicol (10%) as further major components of the young leaf wax. The glutinol percentage decreased, while the relative amounts of epifriedelanol and glutanol increased during development. Various leaf pairs upwards from the third node showed similar growth patterns and developmental time courses of cuticular wax amounts and composition. Based on these surface chemical analyses, the relative activities of biosynthetic pathways leading to various wax components can be assessed.

Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards compact growth.

Dwarf genotypes of the economically important flowering potted plant Kalanchoe blossfeldiana were developed by molecular breeding. Root inducing (Ri)-lines were regenerated by applying CPPU to the hairy roots, which were produced by inoculating leaf explants with a wild-type Agrobacterium rhizogenes strain ATCC15834. Amplification by polymerase chain reaction (PCR) and Southern blot analysis confirmed the presence of T-DNA in the Ri-lines. Six Ri-lines were characterised in a greenhouse trial revealing that several morphological traits changed with respect to ornamental value such as plant height, number of lateral shoots, leaf size, leaf number, flower size and number of flowers. The Ri-lines differed in their degree of Ri-phenotype, and the internodes of the Ri-lines were clearly shorter, giving a compact growth habit compared to control plants. Time to anthesis was the same in Ri-line 331 as in control plants and delayed by only 3 days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.

Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalanchoe) marnierianum: role of auxin and ethylene.

Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.