Morpho-physiological and phytohormonal changes during the induction of adventitious root development stimulated by exogenous IBA application in Magnolia biondii Pamp

Abstract Magnolia biondii Pamp is an important ornamental tree species widely grown and used as a rootstock in the propagation of different Magnolia varieties. In the current studies, anatomical, physiological and endogenous hormones were studied to check the effect of IBA 750 mg/L on the adventitious rooting and to provide theoretical and technical support for the propagation of Magnolia biondii Pamp through stem cuttings. Two thousand stem cuttings were prepared and divided into two groups i.e., IBA treated cuttings and water control. For the evaluation of antioxidant enzyme activities, and endogenous hormones levels, samples were collected on the day of planting and each 5th day and further steps were carried out in the laboratory according to the protocols and proper precautions. For the anatomical observations, samples were collected on the 13th, 15th, and 17th day for IBA treated cuttings while 21st, 23rd, and 25th day for control. Collected samples were preserved in the FAA solution and further observations were carried out in the laboratory. Anatomical observations showed that it took 13 days for the differentiation of root primordia to the appearance of young adventitious roots in IBA treated cuttings, while it took 21 days to develop primordia in the control. Antioxidant enzyme activities involved in ROS were significantly higher in the IBA treated cuttings compared to control. POD showed a peak on the 13th day before the emergence of roots in IBA treated cuttings while it showed a peak on the 21st day in the control. PPO showed a peak on the 21st day in the IBA treated cuttings while it showed a peak on the 29th day in the control. SOD showed a peak on the 17th day in IBA treated cuttings, while it showed a peak on the 25th day in the control. Exogenous application of IBA enhanced the endogenous IAA and GA3 levels compared to CK, while it reduced the levels of ABA continuously at the time of rooting and then increased gradually. Inclusively, our study suggests that IBA 750 mg/L is efficient for the rooting of Magnolia biondii Pamp cuttings, as it enhanced the process of antioxidant enzyme activities, endogenous hormones levels and reduced the time of root formation which is evident from the anatomical observations.

Resumo Magnolia biondii Pamp é uma importante espécie de árvore ornamental muito cultivada e utilizada como porta-enxerto na propagação de diferentes variedades de Magnolia. Nos estudos atuais, hormônios anatômicos, fisiológicos e endógenos foram estudados para verificar o efeito do AIB na dose de 750 mg / L no enraizamento adventício e fornecer suporte teórico e técnico para a propagação de M. biondii Pamp por meio de estacas. Duas mil estacas foram preparadas e divididas em dois grupos, ou seja, tratadas com AIB e controle de água. Para a avaliação das atividades das enzimas antioxidantes e dos níveis de hormônios endógenos, as amostras foram coletadas no dia do plantio e a cada 5 dias, enquanto as demais etapas foram realizadas em laboratório de acordo com os protocolos e os devidos cuidados. Para as observações anatômicas, as amostras foram coletadas no 13º, 15º e 17º dias para estacas tratadas com AIB e no 21º, 23º e 25º dias para o controle. As amostras coletadas foram preservadas em solução FAA, e outras observações foram realizadas em laboratório. Observações anatômicas mostraram a necessidade de 13 dias para a diferenciação dos primórdios radiculares até o aparecimento de raízes adventícias jovens em estacas tratadas com AIB e de 21 dias para o desenvolvimento dos primórdios no controle. As atividades das enzimas antioxidantes envolvidas nas ROS foram significativamente maiores nas estacas tratadas com AIB em comparação com o controle. A POD apresentou pico no 13º dia antes da emergência das raízes nas estacas tratadas com AIB, enquanto no 21º dia apresentou pico no controle. A PPO teve pico no 21º dia nas estacas tratadas com AIB e no 29º dia no controle. A SOD apresentou pico no 17º dia nas estacas tratadas com AIB e no 25º dia no controle. A aplicação exógena de AIB aumentou os níveis endógenos de IAA e GA3 em relação ao controle, enquanto reduziu os níveis de ABA continuamente no momento do enraizamento e, em seguida, aumentou gradativamente. Inclusive, nosso estudo sugere que o AIB na dose de 750 mg / L é eficiente para o enraizamento de estacas de M. biondii Pamp, visto que potencializou o processo de atividades de enzimas antioxidantes e os níveis de hormônios endógenos, além de reduzir o tempo de formação de raízes, o que fica evidente nas observações anatômicas.

Morpho-physiological and phytohormonal changes during the induction of adventitious root development stimulated by exogenous IBA application in Magnolia biondii Pamp / Alterações morfofisiológicas e fito-hormonais durante a indução do desenvolvimento radicular adventício estimulado pela aplicação exógena do AIB em Magnolia biondii Pamp

Magnolia biondii Pamp is an important ornamental tree species widely grown and used as a rootstock in the propagation of different Magnolia varieties. In the current studies, anatomical, physiological and endogenous hormones were studied to check the effect of IBA 750 mg/L on the adventitious rooting and to provide theoretical and technical support for the propagation of Magnolia biondii Pamp through stem cuttings. Two thousand stem cuttings were prepared and divided into two groups i.e., IBA treated cuttings and water control. For the evaluation of antioxidant enzyme activities, and endogenous hormones levels, samples were collected on the day of planting and each 5th day and further steps were carried out in the laboratory according to the protocols and proper precautions. For the anatomical observations, samples were collected on the 13th, 15th, and 17th day for IBA treated cuttings while 21st, 23rd, and 25th day for control. Collected samples were preserved in the FAA solution and further observations were carried out in the laboratory. Anatomical observations showed that it took 13 days for the differentiation of root primordia to the appearance of young adventitious roots in IBA treated cuttings, while it took 21 days to develop primordia in the control. Antioxidant enzyme activities involved in ROS were significantly higher in the IBA treated cuttings compared to control. POD showed a peak on the 13th day before the emergence of roots in IBA treated cuttings while it showed a peak on the 21st day in the control. PPO showed a peak on the 21st day in the IBA treated cuttings while it showed a peak on the 29th day in the control. SOD showed a peak on the 17th day in IBA treated cuttings, while it showed a peak on the 25th day in the control. Exogenous application of IBA enhanced the endogenous IAA and GA3 levels compared to CK, while it reduced the levels of ABA continuously at the time of rooting and then increased gradually. Inclusively, our study suggests that IBA 750 mg/L is efficient for the rooting of Magnolia biondii Pamp cuttings, as it enhanced the process of antioxidant enzyme activities, endogenous hormones levels and reduced the time of root formation which is evident from the anatomical observations.

Magnolia biondii Pamp é uma importante espécie de árvore ornamental muito cultivada e utilizada como porta-enxerto na propagação de diferentes variedades de Magnolia. Nos estudos atuais, hormônios anatômicos, fisiológicos e endógenos foram estudados para verificar o efeito do AIB na dose de 750 mg / L no enraizamento adventício e fornecer suporte teórico e técnico para a propagação de M. biondii Pamp por meio de estacas. Duas mil estacas foram preparadas e divididas em dois grupos, ou seja, tratadas com AIB e controle de água. Para a avaliação das atividades das enzimas antioxidantes e dos níveis de hormônios endógenos, as amostras foram coletadas no dia do plantio e a cada 5 dias, enquanto as demais etapas foram realizadas em laboratório de acordo com os protocolos e os devidos cuidados. Para as observações anatômicas, as amostras foram coletadas no 13º, 15º e 17º dias para estacas tratadas com AIB e no 21º, 23º e 25º dias para o controle. As amostras coletadas foram preservadas em solução FAA, e outras observações foram realizadas em laboratório. Observações anatômicas mostraram a necessidade de 13 dias para a diferenciação dos primórdios radiculares até o aparecimento de raízes adventícias jovens em estacas tratadas com AIB e de 21 dias para o desenvolvimento dos primórdios no controle. As atividades das enzimas antioxidantes envolvidas nas ROS foram significativamente maiores nas estacas tratadas com AIB em comparação com o controle. A POD apresentou pico no 13º dia antes da emergência das raízes nas estacas tratadas com AIB, enquanto no 21º dia apresentou pico no controle. A PPO teve pico no 21º dia nas estacas tratadas com AIB e no 29º dia no controle. A SOD apresentou pico no 17º dia nas estacas tratadas com AIB e no 25º dia no controle. A aplicação exógena de AIB aumentou os níveis endógenos de IAA e GA3 em relação ao controle, enquanto reduziu os níveis de ABA continuamente no momento do enraizamento e, em seguida, aumentou gradativamente. Inclusive, nosso estudo sugere que o AIB na dose de 750 mg / L é eficiente para o enraizamento de estacas de M. biondii Pamp, visto que potencializou o processo de atividades de enzimas antioxidantes e os níveis de hormônios endógenos, além de reduzir o tempo de formação de raízes, o que fica evidente nas observações anatômicas.

Molecular basis of differential adventitious rooting competence in poplar genotypes.

Recalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. To address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root), and used transgenic approaches to verify the role of several transcription factors in the control of adventitious rooting. Increased peroxidase activity was positively correlated with better rooting. We found differentially expressed genes encoding reactive oxygen species scavenging proteins to be enriched in OP42 compared with T89. A greater number of differentially expressed transcription factors in cambium cells of OP42 compared with T89 was revealed by a more intense transcriptional reprograming in the former. PtMYC2, a potential negative regulator, was less expressed in OP42 compared with T89. Using transgenic approaches, we demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. Our results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process.

Functional genomics by integrated analysis of transcriptome of sweet potato (Ipomoea batatas (L.) Lam.) during root formation.

BACKGROUND: Sweet potato is easily propagated by cuttings. But the molecular biological mechanism of adventitious root formation are not yet clear. OBJECTIVE: To understand the molecular mechanisms of adventitious root formation from stem cuttings in sweet potato. METHODS: RNA-seq analysis was performed using un-rooted stem (0 day) and rooted stem (3 days). Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, comparison with Arabidopsis transcription factors (TFs) of DEGs were conducted to investigate the characteristics of genes and TFs involved in root formation. In addition, qRT-PCR analysis using roots at 0, 3, 6, 9, and 12 days after planting was performed to confirm RNA-seq reliability and related genes expression. RESULTS: 42,459 representative transcripts and 2092 DEGs were obtained through the RNA-seq analysis. The DEGs indicated the GO terms related to the single-organism metabolic process and cell periphery, and involved in the biosynthesis of secondary metabolites, and phenylpropanoid biosynthesis in KEGG pathways. The comparison with Arabidopsis thaliana TF database showed that 3 TFs (WRKY, NAC, bHLH) involved in root formation of sweet potato. qRT-PCR analysis, which was conducted to confirm the reliability of RNA-seq analysis, indicated that some metabolisms including oxidative stress and wounding, transport, hormone may be involved in adventitious root formation. CONCLUSIONS: The detected genes related to secondary metabolism, some hormone (auxin, gibberellin), transports, etc. and 3 TFs (WRKY, NAC, bHLH) may have functions in adventitious roots formation. This results provide valuable resources for future research on the adventitious root formation of sweet potato.

The Rooting of Stem Cuttings and the Stability of uidA Gene Expression in Generative and Vegetative Progeny of Transgenic Pear Rootstock in the Field.

Adventitious rooting plays an important role in the commercial vegetative propagation of trees. Adventitious root formation is a complex biological process, but knowledge of the possible unintended effects induced by both the integration/expression of transgenes and in vitro conditions on the rooting is limited. The long-term stability of transgene expression is important both for original transformants of woody plants and its progeny. In this study, we used field-grown pear rootstock GP217 trees transformed with the reporter ß-glucuronidase (uidA) genes with and without intron and re-transformed with the herbicide resistance bar gene as model systems. We assessed the unintended effects on rooting of pear semi-hardwood cuttings and evaluated the stability of transgene expression in progeny produced by generative (seedlings) and vegetative (grafting, cutting) means up to four years. Our investigation revealed that: (1) The single and repeated transformations of clonal pear rootstocks did not result in unintended effects on adventitious root formation in cuttings; (2) stability of the transgene expression was confirmed on both generative and vegetative progeny, and no silenced transgenic plants were detected; (3) yearly variation in the gene expressions was observed and expression levels were decreased in extremely hot and dry summer; (4) the intron enhanced the expression of uidA gene in pear plants approximately two-fold compared to gene without intron. The current study provides useful information on transgene expression in progeny of fruit trees under natural environmental conditions.

Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings.

Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, "2101-02 MFR" and "2003 R 8", as well as in the reference cultivar "Master". We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the "2003 R 8" cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of "2003 R 8". Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.

Aeroponics for adventitious rhizogenesis in evergreen haloxeric tree Tamarix aphylla (L.) Karst.: influence of exogenous auxins and cutting type.

Tamarix aphylla (L.) Karst., a drought resistant halophyte tree, is an agroforestry species which can be used for reclamation of waterlogged saline and marginal lands. Due to very low seed viability and unsuitable conditions for seed germination, the tree is becoming rare in Indian Thar desert. Present study concerns the evaluation of aeroponics technique for vegetative propagation of T. aphylla. Effect of various exogenous auxins (indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid) at different concentrations (0.0, 1.0, 2.0, 3.0, 5.0, 10.0 mg l-1) was examined for induction of adventitious rooting and other morphological features. Among all three auxins tested individually, maximum rooting response (79%) was observed with IBA 2.0 mg l-1. However, stem cuttings treated with a combination of auxins (2.0 mg l-1 IBA and 1.0 mg l-1 IAA) for 15 min resulted in 87% of rooting response. Among three types of stem cuttings (apical shoot, newly sprouted cuttings, mature stem cuttings), maximum rooting (~ 90%) was observed on mature stem cuttings. Number of roots and root length were significantly higher in aeroponically rooted stem cuttings as compared to stem cuttings rooted in soil conditions. Successfully rooted and sprouted plants were transferred to polybags with 95% survival rate. This is the first report on aeroponic culture of Tamarix aphylla which can be utilized in agroforestry practices, marginal land reclamation and physiological studies.

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni).

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51-40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l(-1)). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l(-1)) and gibberellic acid (GA3; 0.5 mg l(-1)). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51-40%) and stem cutting (60%).

Growth and survival of Halimione portulacoides stem cuttings in heavy metal contaminated soils.

The halophytic shrub Halimione portulacoides demonstrates a high tolerance to heavy metal contamination and a capacity for accumulating metals within its tissues. On the Iberian Peninsula, this species has colonized habitats with high levels of metal pollution. The aim of this study is to analyze the response of H. portulacoides stem cuttings to this pollution. Growth, photosynthesis and metal uptake were examined in H. portulacoides through an experiment in which stem cuttings were replanted in metal-contaminated soil. This condition decreased growth and lowered both photosynthetic rate and stomatal conductance. Reduced photosynthetic performance was largely due to the reduced concentration of photosynthetic pigments. Despite these responses, there was some important evidence suggesting the phytoremediatory potential of Halimione stem cuttings. The results of our study indicate that this salt-marsh shrub may represent a biotool of value in the restoration of polluted areas.

In vitro regeneration of Eucalyptus camaldulensis.

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l(-1) benzyl aminopurine (BAP) and 0.1 mg l(-1) naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l(-1) BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l(-1) Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60-148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.

Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings

The effects of plant growth promoting rhizobacteria (PGPR) on the rooting and root growth of semi-hardwood and hardwood kiwifruit stem cuttings were investigated. The PGPR used were Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19. All the bacteria showed indole-3-acetic acid (IAA) producing capacity. Among the PGPR used, the highest rooting ratios were obtained at 47.50 percent for semi-hardwood stem cuttings from Bacillus RC03 and Bacillus simplex RC19 treatments and 42.50 percent for hardwood stem cuttings from Bacillus RC03. As well, Comamonas acidovorans RC41 inoculations indicated higher value than control treatments. The results suggest that these PGPR can be used in organic nursery material production and point to the feasibility of synthetic auxin (IBA) replacement by organic management based on PGPR.