The production of nitrogenase enzyme and auxins by free living diazotrophs has the potential to influence the growth of host plants. In this study, diazotrophs were grown in the presence of various concentrations of nitogen (N) to determine the optimal concentration of N for microbial growth stimulation, promotion of gaseous N (N2) fixation, and phytohormone production. Therefore, we investigate whether different levels of N supplied to Herbaspirillum seropedicae (Z78) have significant effects on nitrogenase activity and auxin production. The highest nitrogenase activity and the lowest auxin production of H. seropedicae (Z78) were both recorded at 0 gL(-1) of NH4Cl. Higher levels of external N caused a significant decrease in the nitrogenase activity and an increased production of auxins. In a subsequent test, two different inoculum sizes of Z78 (10(6) and 10(12) cfu/ml) were used to study the effect of different percentages of acetylene on nitrogenase activity of the inoculum via the acetylene reduction assay (ARA). The results showed that the optimal amount of acetylene required for nitrogenase enzyme activity was 5% for the 10(6) cfu/ml inoculum, whereas the higher inoculum size (10(12) cfu/ml) required at least 10% of acetylene for optimal nitrogenase activity. These findings provide a clearer understanding of the effects of N levels on diazotrophic nitrogenase activity and auxin production, which are important factors influencing plant growth.
The leaf of Gardenia jasminoides Ellis was used as explants and was cultured on MS and WPM media supplemented with various concentrations of NAA, IAA, 2,4-D, IBA, TDZ, and Kn (0 to 5 mg L(-1) with 0.5 increment). After six months, the higher percentage of callus (100%) and the best dry and fresh weight of callus were formed on WPM medium supplemented with 2,4-D and NAA (2.0-3.0 mg L(-1)) and this amount was decreased from (84%) to (69%) when this media supplemented with Kinetin and TDZ (1 mg L(-1)) respectively were used. Leaf segments cultured on WPM media added with Kn (1 mg L(-1)) and TDZ (2 mg L(-1)) yielded the least amount of callus. It was found that WPM media added with IAA (4.5-5.0 mg L(-1)) were optimum for root induction from G. jasminoides plantlets. Antibacterial screening of leaf extracts (in vivo) showed no inhibitory effect against E. coli, P. aeruginosa, S. aureus, and B. cereus, in contrast to callus extracts from leaf cultures supplemented with NAA, which showed inhibition activity against E. coli and B. cereus. The callus extracts from leaf cultures grown on both MS and WPM media showed higher antioxidant and superoxide dismutase activities than leaf extracts.
Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.
A procedure was developed for in vitro propagation of Curculigo latifolia through shoot tip culture. Direct regeneration and indirect scalp induction of Curculigo latifolia were obtained from shoot tip grown on MS medium supplemented with different concentrations and combinations of thidiazuron and indole-3-butyric acid. Maximum response for direct regeneration in terms of percentage of explants producing shoot, shoot number, and shoot length was obtained on MS medium supplemented with combination of thidiazuron (0.5 mg L(-1)) and indole-3-butyric acid (0.25 mg L(-1)) after both 10 and 14 weeks of cultures. Indole-3-butyric acid in combination with thidiazuron exhibited a synergistic effect on shoot regeneration. The shoot tips were able to induce maximum scalp from basal end of explants on the medium with 2 mg L(-1) thidiazuron. Cultures showed that shoot number, shoot length, and scalp size increased significantly after 14 weeks of culture. Transferring of the shoots onto the MS medium devoid of growth regulators resulted in the highest percentage of root induction and longer roots, while medium supplemented with 0.25 mg L(-1) IBA produced more numbers of roots.
The use of in vitro culture has been accepted as an efficient technique for clonal propagation of many woody plants. In the present research, we report the results of a number of experiments aimed at optimizing micropropagation protocol for tea (Camellia sinensis (L.) O. Kuntze) (clone Iran 100) using nodal segments as the explant. The effect of different combinations and concentrations of plant growth regulators (PGR) (BAP, TDZ, GA₃) on shoot multiplication and elongation was assessed. The influence of exposure to IBA in liquid form prior to transfer to solid media on rooting of tea microshoots was investigated. The results of this study showed that the best treatment for nodal segment multiplication in terms of the number of shoot per explant and shoot elongation was obtained using 3 mg/L BAP in combination with 0.5 mg/L GA₃. TDZ was found to be inappropriate for multiplication of tea clone Iran 100 as it resulted in hyperhydricity especially at concentrations higher than 0.05 mg/L. Healthy shoots treated with 300 mg/L IBA for 30 min followed by transfer to 1/2 strength MS medium devoid of PGR resulted in 72.3% of shoots producing roots and upon transferring them to acclimatization chamber 65% survival was obtained prior to field transfer.
The Hymenocallis littoralis, an ornamental and medicinal plant, had been traditionally used for wound healing. In the present study, an analytical method using HPLC with ultraviolet detection was developed for the quantification of lycorine in the extracts of different parts of wild plant and tissue culture samples of H. littoralis. The separation was achieved using a reversed-phase column. The method was found to be accurate, repeatable, and sensitive for the quantification of minute amount of lycorine present in the samples. The highest lycorine content was found in the bulb extract (2.54 ± 0.02 μg/mg) whereas the least was in the root extract (0.71 ± 0.02 μg/mg) of the wild plants. Few callus culture samples had high content of lycorine, comparable to that of wild plants. The results showed that plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.5 μM (2.58 ± 0.38 μg/mg) or a combination of 2,4-D at 9.00 μM with 4.5 μM of 6-benzylaminopurine (BAP), were the optimum concentrations for the production of high lycorine (2.45 ± 0.15 μg/mg) content in callus culture. The present analytical method could be of value for routine quantification of lycorine in the tissue culture production and standardization of the raw material or extracts of H. littoralis.
In vitro direct regeneration of Nelumbo nucifera Gaertn. was successfully achieved from immature explants (yellow plumule) cultured on a solid MS media supplemented with combinations of 0.5 mg/L BAP and 1.5 mg/L NAA which resulted in 16.00 ± 0.30 number of shoots per explant and exhibited a new characteristic of layered multiple shoots, while normal roots formed on the solid MS basal media. The double-layered media gave the highest number of shoots per explant with a ratio of 2 : 1 (liquid to solid) with a mean number of 16.67 ± 0.23 shoots per explant with the formation of primary and secondary roots from immature explants. In the study involving light distance, the tallest shoot (16.67 ± 0.23 mm) obtained from the immature explants was at a light distance of 200 mm from the source of inflorescent light (1000 lux). The plantlets were successfully acclimatized in clay loam soil after 8 months being maintained under in vitro conditions.
Tissue culture studies of Celosia cristata were established from various explants and the effects of various hormones on morphogenesis of this species were examined. It was found that complete plant regeneration occurred at highest percentage on MS medium supplemented with 2.0 mg/L NAA and 1.5 mg/L BAP, with the best response showed by shoot explants. In vitro flowering was observed on MS basal medium after six weeks. The occurrence of somaclonal variation and changes in cellular behavior from in vivo and in vitro grown plants were investigated through cytological studies and image analysis. It was observed that Mitotic Index (MI), mean chromosome numbers, and mean nuclear to cell area ratio of in vitro root meristem cells were slightly higher compared to in vivo values. However, in vitro plants produced lower mean cell areas but higher nuclear areas when compared to in vivo plants. Thus, no occurrence of somaclonal variation was detected, and this was supported by morphological features of the in vitro plants.
Differential effect of plant growth regulators and additives in proliferation of 18-month-old calli of Ananas comosus L. cv. Moris were assessed in vitro. The proliferation of callus relied on the growth regulators and additives. Of the different auxins supplemented in the Murashige and Skoog (MS) media, 32.22 microM alpha-naphthaleneacetic acid (NAA) gave the highest mean fresh weight of callus (46.817 g). Medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) was inferior to NAA, while b-naphthoxy acetic acid (BNOA) and p-chlorophenoxy acetic acid (4-CPA) were not effective in proliferating 18-months old callus. Addition of casein hydrolysate and coconut water to NAA supplemented medium showed better proliferation and production of callus. However, in terms of callus production, NAA at 32.22 microM was economically better.
This study represents the first paper of the effects of growth regulators on the physiochemical and phytochemical properties of the wax apple fruit, a widely cultivated fruit tree in southeast Asia. Net photosynthesis, sucrose phosphate synthase (SPS) activity, peel color, fruit firmness, juice content, pH value, total soluble solids (TSSs), and the sugar acid ratio were all significantly increased in growth regulators (PGRs) treated fruits. The application of gibberellin (GA(3)), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D) significantly reduced titratable acidity and increased total sugar and carbohydrate content compared to the control. The 50 mg/L GA₃, 10 mg/L NAA, and 5 mg/L 2,4-D treatments produced the greatest increases in phenol and flavonoid content; vitamin C content was also higher for these treatments. PGR treatment significantly affected chlorophyll, anthocyanin, and carotene content and produced higher phenylalanine ammonia lyase (PAL) and antioxidant activity levels. There was a positive correlation between peel color and TSS and antioxidant activity and both phenol and flavonoid content and PAL activity and anthocyanin formation. A taste panel assessment was also performed, and the highest scores were given to fruits that had been treated with GA₃ or auxin. The study showed that application of 50 mg/L GA₃, 10 mg/L NAA, and 5 mg/L 2,4-D once a week from bud development to fruit maturation increased the physiochemical and phytochemical properties of wax apple fruits.
This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin, 6-benzylaminopurine (BAP), and 1-naphthaleneacetic acid (NAA) on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L⁻¹ 2,4-D (70.83%). Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L⁻¹ 2,4-D and 3% sucrose (9.47 ± 0.4667 mL). The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L⁻¹ yeast extract (9.275 ± 0.082 mg L⁻¹) that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 μM tryptophan and harvested at 6 days (13.226 ± 1.98 mg L⁻¹).
Gymnema sylvestre (R.Br.) is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA). The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L) and KN (0.5 mg/L). Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w) was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w). Maximum GA production (58.28 mg/g d.w) was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors.
The oil biosynthesis pathway must be tightly controlled to maximize oil yield. Oil palm accumulates exceptionally high oil content in its mesocarp, suggesting the existence of a unique fruit-specific fatty acid metabolism transcriptional network. We report the complex fruit-specific network of transcription factors responsible for modulation of oil biosynthesis genes in oil palm mesocarp. Transcriptional activation of EgWRI1-1 encoding a key master regulator that activates expression of oil biosynthesis genes, is activated by three ABA-responsive transcription factors, EgNF-YA3, EgNF-YC2 and EgABI5. Overexpression of EgWRI1-1 and its activators in Arabidopsis accelerated flowering, increased seed size and oil content, and altered expression levels of oil biosynthesis genes. Protein-protein interaction experiments demonstrated that EgNF-YA3 interacts directly with EgWRI1-1, forming a transcription complex with EgNF-YC2 and EgABI5 to modulate transcription of oil biosynthesis pathway genes. Furthermore, EgABI5 acts downstream of EgWRKY40, a repressor that interacts with EgWRKY2 to inhibit the transcription of oil biosynthesis genes. We showed that expression of these activators and repressors in oil biosynthesis can be induced by phytohormones coordinating fruit development in oil palm. We propose a model highlighting a hormone signaling network coordinating fruit development and fatty acid biosynthesis.
This review paper discussed about publications related to micropropagation of bamboo species. In recent years, the application of tissue culture technique like in vitro micropropagation has been used to meet the demands for bamboo planting materials. In the past 30 years, protocols for micropropagation of various bamboo species have been established by researchers from all over the world. The controlling factors for cultures such as the explants, culture medium, carbon sources, combination and concentration of plant growth regulators and other additional additives are varied. The controlling factors are crucial in developing successful regeneration protocols for various bamboo species. This paper attempts to review and summarize the available and up-to-date information regarding in vitro micropropagation of bamboos.
A study was conducted to determine the effects of a plant growth regulator (paclobutrazol, PBZ) and commercial
fertilizer (Krista-K Plus) as a source of potassium nitrate (KNO3
) on the growth of Xanthostemon chrysantus. It was
also attempted to investigate the anatomical changes in the leaf and stem after the treatment. Nine treatments, i.e.
control (no PBZ and Krista-K Plus application), 0 PBZ gL-1 + 100 g Krista-K Plus, 0 PBZ gL-1 + 200 g Krista-K Plus,
0.125 PBZ gL-1 + 0 g Krista-K Plus, 0.125 PBZ gL-1 + 100 g Krista-K Plus, 0.125 PBZ gL-1 + 200 g Krista-K Plus, 0.25
PBZ gL-1 + 0 g Krista-K Plus, 0.25 PBZ gL-1 + 100 g Krista-K Plus and 0.25 PBZ gL-1 + 200 g Krista-K Plus, were
tested. PBZ was soil drenched at the commencement of the study while Krista-K Plus was applied at three-month
intervals. Plant growth performances such as tree height, diameter at breast height, canopy diameter and leaf area
were recorded monthly throughout the study period. Stem and leaf samples were collected before the application
of treatments and after six months of treatments for anatomical observation by using electron microscope. Plant
height, diameter at breast height, crown diameter and leaf area were significantly reduced with the application of
PBZ. Palisade parenchyma thickness was increased by 33.83% with 0.25 PBZ gL-1 + 200 g Krista-K Plus, while only
2.44% increment recorded in the control tree. Xylem thickness in the stem was reduced by 21.81% after treated with
the highest dosage of PBZ, while the control tree only had 1.78% increment. Spongy parenchyma thickness in the leaf
was unaffected. However, palisade parenchyma was found the thickest after combined treatment with 0.25 PBZ gL-1
+ 200 g Krista-K Plus. Micrograph images of the cross-section of leaf lamina and stem showed that the cells were
tightly arranged in response to the application of PBZ.
Lead (Pb) is one of the most abundant toxic heavy metals which adversely affected growth and yield of crop plants. Nitric oxide (NO), an endogenous signaling molecule, has been suggested to be involved in defense responses to biotic and abiotic stresses in plants. The present study was done to induce Pb tolerance in cowpea plants by exogenous NO application using two levels of Pb, 0 and 200 mg Pb (NO3)2 kg-1 soil and three NO levels, 0, 0.5 and 1 mM sodium nitroprusside (SNP), as NO donor. The results showed that Pb treatment caused a significant increase in Pb concentration in all plant parts. Roots had higher levels of Pb than the stems, leaves and seeds. Furthermore, lead toxicity reduced auxin (IAA), cytokinin and gibberellic acid (GA3) content but increased abscisic acid (ABA) level. Moreover Pb stress decreased stomatal conductance, leaf area and consequently seed yield of cowpea. Exogenous application of NO at 0.5 mM noticeably alleviated the lead toxicity by improving the leaf area, stomatal conductance and seed yield. NO increased Pb tolerance by lowering Pb uptake and translocation, enhancing the promoting phytohormone (IAA, cytokinin and GA3) level and reducing ABA content.
Rafflesia possesses unique biological features and known primarily for producing the world's largest and existing as a single flower. However, to date, little is known about key regulators participating in Rafflesia flower development. In order to further understand the molecular mechanism that regulates Rafflesia cantleyi flower development, RNA-seq data from three developmental stages of floral bud, representing the floral organ primordia initiation, floral organ differentiation, and floral bud outgrowth, were analysed. A total of 89,890 transcripts were assembled of which up to 35% could be annotated based on homology search. Advanced transcriptome analysis using K-mean clustering on the differentially expressed genes (DEGs) was able to identify 12 expression clusters that reflect major trends and key transitional states, which correlate to specific developmental stages. Through this, comparative gene expression analysis of different floral bud stages identified various transcription factors related to flower development. The members of WRKY, NAC, bHLH, and MYB families are the most represented among the DEGs, suggesting their important function in flower development. Furthermore, pathway enrichment analysis also revealed DEGs that are involved in various phytohormone signal transduction events such as auxin and auxin transport, cytokinin and gibberellin biosynthesis. Results of this study imply that transcription factors and phytohormone signalling pathways play major role in Rafflesia floral bud development. This study provides an invaluable resource for molecular studies of the flower development process in Rafflesia and other plant species.
'Bentong' ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied in many plant species to produce their disease-free planting materials. As 'Bentong' ginger is less known for its micropropagation, this study was conducted to investigate the effects of Clorox (5.25% sodium hypochlorite (NaOCl)) on explant surface sterilization, effects of plant growth regulators, and basal media on shoots' multiplication and rooting. The secondary metabolites and antioxidant activities of the micropropagated plants were evaluated in comparison with conventionally propagated plants. Rhizome sprouted buds were effectively sterilized in 70% Clorox for 30 min by obtaining 75% contamination-free explants. Murashige and Skoog (MS) supplemented with 10 µM of zeatin was the suitable medium for shoot multiplication, which resulted in the highest number of shoots per explant (4.28). MS medium supplemented with 7.5 µM 1-naphthaleneacetic acid (NAA) resulted in the highest number of roots per plantlet. The in vitro-rooted plantlets were successfully acclimatized with a 95% survival rate in the ex vitro conditions. The phytochemical analysis showed that total phenolic acid and total flavonoid content and antioxidant activities of the micropropagated plants were not significantly different from the conventionally propagated plants of 'Bentong' ginger. In conclusion, the present study's outcome can be adopted for large-scale propagation of disease-free planting materials of 'Bentong' ginger.
The SCF complex is a widely studied multi-subunit ring E3 ubiquitin ligase that tags targeted proteins with ubiquitin for protein degradation by the ubiquitin 26S-proteasome system (UPS). The UPS is an important system that generally keeps cellular events tightly regulated by purging misfolded or damaged proteins and selectively degrading important regulatory proteins. The specificity of this post-translational regulation is controlled by F-box proteins (FBPs) via selective recognition of a protein-protein interaction motif at the C-terminal domain. Hence, FBPs are pivotal proteins in determining the plant response in multiple scenarios. It is not surprising that the FBP family is one of the largest protein families in the plant kingdom. In this review, the roles of FBPs, specifically in plants, are compiled to provide insights into their involvement in secondary metabolites, plant stresses, phytohormone signalling, plant developmental processes and miRNA biogenesis.
Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid.