The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.
Monodehydroascorbate reductase (MDHAR), an important enzyme of the ascorbate-glutathione cycle, is involved in salt tolerance of plants through scavenging of reactive oxygen species (ROS). In this study, a cDNA encoding MDHAR from the mangrove plant Acanthus ebracteatus was introduced into rice to examine its role in salt tolerance. Three stable transgenic lines (MT22, MT24 and MT25) overexpressing AeMDHAR were selected in vitro using hygromycin and confirmed by PCR, quantitative reverse-transcription (qRT) PCR and enzyme assay. The transgenic line MT24 was predicted to possess a single copy of the transgene while the other two transgenic lines were predicted to have multiple transgene integrations. The AeMDHAR transcripts were detected only in transgenic rice lines but not in untransformed rice. The abundance of AeMDHAR transcripts in transgenic lines MT22 and MT25 was approximately 2.75 times the amount found in MT24. The transgenic rice lines overexpressing AeMDHAR showed a significant increase in MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions. All transgenic lines showed better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also showed tolerance to salt at germination and seedling stages. The transgenic line MT24, which harbors a single copy of AeMDHAR, displayed a lower rate of sterility, a higher number of tillers and longer panicle compared to untransformed plants when subjected to salt stress.
Genetic improvement is an important approach for crop improvement towards yield stability in stress-prone areas. Functional analysis of candidate stress response genes can provide key information to allow the selection and modification of improved crop varieties. In this study, the constitutive expression of a banana cDNA, MaRHD3 in Arabidopsis improved the ability of transgenic lines to adapt to drought conditions. Transgenic Arabidopsis plants expressing MaRHD3 had roots with enhanced branching and more root hairs when challenged with drought stress. The MaRHD3 plants had higher biomass accumulation, higher relative water content, higher chlorophyll content and an increase in activity of reactive oxygen species (ROS) scavenging enzymes; SOD, CAT, GR, POD and APX with reduced water loss rates compared to control plants. The analysis of oxidative damage indicated lower cell membrane damage in transgenic lines compared to control plants. These findings, together with data from higher expression of ABF-3 and higher ABA content of drought-stressed transgenic MaRHD3 expressing plants, support the involvement of the ABA signal pathway and ROS scavenging enzyme systems in MaRHD3 mediated drought tolerance.
Polygonum minus Huds. is a medicinal aromatic plant rich in terpenes, aldehydes, and phenolic compounds. Methyl jasmonate (MeJA) is a plant signaling molecule commonly applied to elicit stress responses to produce plant secondary metabolites. In this study, the effects of exogenous MeJA treatment on the composition of volatile organic compounds (VOCs) in P. minus leaves were investigated by using a metabolomic approach. Time-course changes in the leaf composition of VOCs on days 1, 3, and 5 after MeJA treatment were analyzed through solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). The VOCs found in MeJA-elicited leaves were similar to those found in mock-treated leaves but varied in quantity at different time points. We focused our analysis on the content and composition of monoterpenes, sesquiterpenes, and green leaf volatiles (GLVs) within the leaf samples. Our results suggest that MeJA enhances the activity of biosynthetic pathways for aldehydes and terpenes in P. minus. Hence, the production of aromatic compounds in this medicinal herb can be increased by MeJA elicitation. Furthermore, the relationship between MeJA elicitation and terpene biosynthesis in P. minus was shown through SPME-GC-MS analysis of VOCs combined with transcriptomic analysis of MeJA-elicited P. minus leaves from our previous study.
Light-harvesting complexes (LHCs) in photosystem II (PSII) regulate glutathione (GSH) functions in plants. To investigate whether LHCs control GSH biosynthesis that modifies guard cell abscisic acid (ABA) sensitivity, we evaluated GSH content, stomatal aperture, reactive oxygen species (ROS), weight loss and plant growth using a ch1-1 mutant that was defective of LHCs and compared this with wild-type (WT) Arabidopsis thaliana plants. Glutathione monoethyl ester (GSHmee) increased but 1-chloro-2,4 dinitrobenzene (CDNB) decreased the GSH content in the guard cells. The guard cells of the ch1-1 mutants accumulated significantly less GSH than the WT plants. The guard cells of the ch1-1 mutants also showed higher sensitivity to ABA than the WT plants. The CDNB treatment increased but the GSHmee treatment decreased the ABA sensitivity of the guard cells without affecting ABA-induced ROS production. Dark and light treatments altered the GSH content and stomatal aperture of the guard cells of ch1-1 and WT plants, irrespective of CDNB and GSHmee. The ch1-1 mutant contained fewer guard cells and displayed poor growth, late flowering and stumpy weight loss compared with the WT plants. This study suggests that defective LHCs reduced the GSH content in the guard cells and increased sensitivity to ABA, resulting in stomatal closure.
Adhesion of the barley husk to the underlying caryopsis requires the development of a cuticular cementing layer on the caryopsis surface. Differences in adhesion quality among genotypes have previously been correlated with cementing layer composition, which is thought to influence caryopsis cuticle permeability, the hypothesised mechanism of adhesion mediation. It is not yet known whether differences in adhesion quality among genotypes are determined by changes in caryopsis cuticle permeability. We examined changes in candidate cementing layer biosynthetic and regulatory genes to investigate the genetic mechanisms behind husk adhesion quality. We used both commercially relevant UK malting cultivars and older European lines to ensure phenotypic diversity in adhesion quality. An ethylene responsive transcription factor (NUD) is required for the development of the cementing layer. To examine correlations between gene expression, cementing layer permeability and husk adhesion quality we also treated cultivars with ethephon (2-chloroethylphosphonic acid) which breaks down to ethylene, and silver thiosulphate which inhibits ethylene reception, and measured caryopsis cuticle permeability. Differential adhesion qualities among genotypes are not determined by NUD expression during development of the cementing material alone, but could result from differences in biosynthetic gene expression during cementing layer development in response to longer-term NUD expression patterns. Altered caryopsis cuticle permeability does result in altered adhesion quality, but the correlation is not consistently positive or negative. Cuticle permeability is therefore not the mechanism that determines husk adhesion quality, but is likely a consequence of the required cuticular compositional changes that determine adhesion.
Black pepper (Piper nigrum L.) is one of the most popular and oldest spices in the world with culinary uses and various pharmacological properties. In order to satisfy the growing worldwide demand for black pepper, improved productivity of pepper is highly desirable. A primary constraint in black pepper production is the non-synchronous nature of flower development and non-uniform fruit ripening within a spike. The uneven ripening of pepper berries results in a high labour requirement for selective harvesting contributes to low productivity and affects the quality of the pepper products. In Malaysia, there are a few recommended varieties for black pepper planting, each having some limitations in addition to the useful characteristics. Therefore, a comparative study of different black pepper varieties will provide a better understanding of the mechanisms regulates fruit development and ripening. Plant hormones are known to influence the fruit development process and their roles in black pepper flower and fruit development were inferred based on the probe-based gene expression analysis and the quantification of the multiple plant hormones using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). In this study, jasmonic acid and salicylic acid were found to play roles in flowering and fruit setting, whereas auxin, gibberellin and cytokinins are important for fruit growth. Abscisic acid has positive role in fruit maturation and ripening in the development process. Distinct pattern of plant hormones related gene expression profiles with the hormones accumulation profiles suggested a complex network of regulation is involved in the signaling process and crosstalk between plant hormones was another layer of regulation in the black pepper fruit development mechanisms. The current study provides clues to help in elucidating the timing of the action of each specific plant hormone during fruit development and ripening which could be applied to enhance our ability to control the ripening process, leading to improving procedures for the production and post-harvest handling of pepper fruits.
Auxin and cytokinin regulate different critical processes involved in plant growth and environmental feedbacks. These plant hormones act either synergistically or antagonistically to control the organisation, formation and maintenance of meristem. Meristem cells can be divided to generate new tissues and organs at the locations of plant postembryonic development. The aboveground plant organs are created by the shoot apical meristem (SAM). It has been proposed that the phytohormone, cytokinin, plays a positive role in the shoot meristem function, promotes cell expansion and promotes an increasing size of the meristem in Arabidopsis, whereas it has the reverse effects in the root apical meristem (RAM). Over the last few decades, it has been believed that the apically derived auxin suppresses the shoot branching by inactivating the axillary buds. However, it has recently become clear that the mechanism of action of auxinis indirect and multifaceted. In higher plants, the regulatory mechanisms of the SAM formation and organ separation are mostly unknown. This study reviews the effects and functions of cytokinin and auxin at the shoot apical meristem. This study also highlights the merger of the transcription factor activity with the actions of cytokinin/auxin and their complex interactions with the shoot meristem in rice.
Bacteria under the operational group Bacillus amyloliquefaciens (OGBa) are all Gram-positive, endospore-forming, and rod-shaped. Taxonomically, the OGBa belongs to the Bacillus subtilis species complex, family Bacillaceae, class Bacilli, and phylum Firmicutes. To date, the OGBa comprises four bacterial species: Bacillus amyloliquefaciens, Bacillus siamensis, Bacillus velezensis and Bacillus nakamurai. They are widely distributed in various niches including soil, plants, food, and water. A resurgence in genome mining has caused an increased focus on the biotechnological applications of bacterial species belonging to the OGBa. The members of OGBa are known as plant growth-promoting bacteria (PGPB) due to their abilities to fix nitrogen, solubilize phosphate, and produce siderophore and phytohormones, as well as antimicrobial compounds. Moreover, they are also reported to produce various enzymes including α-amylase, protease, lipase, cellulase, xylanase, pectinase, aminotransferase, barnase, peroxidase, and laccase. Antimicrobial compounds that able to inhibit the growth of pathogens including non-ribosomal peptides and polyketides are also produced by these bacteria. Within the OGBa, various B. velezensis strains are promising for use as probiotics for animals and fishes. Genome mining has revealed the potential applications of members of OGBa for removing organophosphorus (OPs) pesticides. Thus, this review focused on the applicability of members of OGBa as plant growth promoters, biocontrol agents, probiotics, bioremediation agents, as well as producers of commercial enzymes and antibiotics. Here, the bioformulations and commercial products available based on these bacteria are also highlighted. This review will better facilitate understandings of members of OGBa and their biotechnological applications.
Multifunctionalities linked with the microbial communities associated with the millet crop rhizosphere has remained unexplored. In this study, we are analyzing microbial communities inhabiting rhizosphere of kodo millet and their associated functions and its impact over plant growth and survival. Metagenomics of Paspalum scrobiculatum L.(kodo millet) rhizopshere revealed taxonomic communities with functional capabilities linked to support growth and development of the plants under nutrient-deprived, semi-arid and dry biotic conditions. Among 65 taxonomically diverse phyla identified in the rhizobiome, Actinobacteria were the most abundant followed by the Proteobacteria. Functions identified for different genes/proteins led to revelations that multifunctional rhizobiome performs several metabolic functions including carbon fixation, nitrogen, phosphorus, sulfur, iron and aromatic compound metabolism, stress response, secondary metabolite synthesis and virulence, disease, and defense. Abundance of genes linked with N, P, S, Fe and aromatic compound metabolism and phytohormone synthesis-along with other prominent functions-clearly justifies growth, development, and survival of the plants under nutrient deprived dry environment conditions. The dominance of actinobacteria, the known antibiotic producing communities shows that the kodo rhizobiome possesses metabolic capabilities to defend themselves against biotic stresses. The study opens avenues to revisit multi-functionalities of the crop rhizosphere for establishing link between taxonomic abundance and targeted functions that help plant growth and development in stressed and nutrient deprived soil conditions. It further helps in understanding the role of rhizosphere microbiome in adaptation and survival of plants in harsh abiotic conditions.
The importance of plant secondary metabolites for both mankind and the plant itself has long been established. However, despite extensive research on plant secondary metabolites, plant secondary metabolism and its regulation still remained poorly characterized. In this present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling was applied to generate the expression profiles of Polygonum minus in response to salicylic acid (SA) and methyl jasmonate (MeJA) elicitations. This study reveals two different sets of genes induced by SA and MeJA, respectively where stress-related genes were proved to lead to the expression of genes involved in plant secondary metabolite biosynthetic pathways. A total of 98 transcript-derived fragments (TDFs) were up-regulated, including 46 from SA-treated and 52 from MeJA-treated samples. The cDNA-AFLP transcripts generated using 64 different Mse1/Taq1 primer combinations showed that treatments with SA and MeJA induced genes mostly involved in scavenging reactive oxygen species, including zeaxanthin epoxidase, cytosolic ascorbate peroxidase 1 and peroxidase. Of these stress-related genes, 15 % of other annotated TDFs are involved mainly in secondary metabolic processes where among these, two genes encoding (+)-delta cadinene synthase and cinnamoyl-CoA reductase were highlighted.
The present study investigates the effects of different concentrations, as well as type of plant growth regulators (PGRs) and medium (MS, Duchefa) on the growth and development of Centella asiatica in semi-solid culture. In addition, a protocol for successful sterilization of C.asiatica explants prepared from field-grown plants highly exposed to fungal and bacterial contamination was determined. Results for sterilization treatments revealed that applying HgCl₂ and Plant Preservative Mixture (PPM) with cetrimide, bavistin and trimethoprim which were included after washing with tap water, followed by the addition of PPM in the medium, produced a very satisfactory result (clean culture 90 ± 1.33%) and TS5 (decon + cetrimide 1% + bavistin 150 mg/L + trimethoprim 50 mg/L + HgCl₂0.1% + PPM 2% soak and 2 mL/L in medium) was hence chosen as the best method of sterilization for C.asiatica. The synergistic combination of 6 benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in concentrations of 2 mg/L and 0.1 mg/L, respectively, in Duchefa medium compared with MS induced the most optimal percentage of sprouted shoots (93 ± 0.667), number of shoots (5.2 ± 0.079) and nodes (4 ± 0.067) per explant, leaf per explant (14 ± 0.107) and shoot length (4.1 ± 0.67 cm). Furthermore, optimum rooting frequency (95.2 ± 0.81%), the number of roots/shoot (7.5 ± 0.107) and the mean root length (4.5 ± 0.133 cm) occurred for shoots that were cultured on full-strength MS medium containing 0.5 mg/L indole-3-butyric acid (IBA). In this study, the acclimatized plantlets were successfully established with almost 85% survival. The findings of this study have proven an efficient medium and PGR concentration for the mass propagation of C.asiatica. These findings would be useful in micropropagation and ex situ conservation of this plant.
Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.
The rise in the World's food demand in line with the increase of the global population has resulted in calls for more research on the production of sustainable food and sustainable agriculture. A natural biopolymer, chitosan, coupled with nanotechnology could offer a sustainable alternative to the use of conventional agrochemicals towards a safer agriculture industry. Here, we review the potential of chitosan-based agronanochemicals as a sustainable alternative in crop protection against pests, diseases as well as plant growth promoters. Such effort offers better alternatives: (1) the existing agricultural active ingredients can be encapsulated into chitosan nanocarriers for the formation of potent biocides against plant pathogens and pests; (2) the controlled release properties and high bioavailability of the nanoformulations help in minimizing the wastage and leaching of the agrochemicals' active ingredients; (3) the small size, in the nanometer regime, enhances the penetration on the plant cell wall and cuticle, which in turn increases the argochemical uptake; (4) the encapsulation of agrochemicals in chitosan nanocarriers shields the toxic effect of the free agrochemicals on the plant, cells and DNA, thus, minimizing the negative impacts of agrochemical active ingredients on human health and environmental wellness. In addition, this article also briefly reviews the mechanism of action of chitosan against pathogens and the elicitations of plant immunity and defense response activities of chitosan-treated plants.
A crude methanol extract of Goniothalamus andersonii J. Sinclair strongly inhibited elongation of lettuce (Lactuca sativa L.) radicles. We conducted bioassay-guided purification of G. andersonii bark extract and obtained goniothalamin as the major bioactive compound. Its EC50 values against elongation of lettuce radicles and hypocotyls were 50 and 125 micromol L(-1), respectively. Among the six species tested, timothy was the most sensitive to goniothalamin. Quantification of this compound in other Goniothalamus species suggested that the plant inhibitory activity of this genus is explainable by goniothalamin, with G. calcareus as an exception.
Efficient plant regeneration of Saintpaulia ionantha (African violet) has been obtained in the present study. MS medium supplemented with 1.0 mg L(-1) IAA and 2.0 mg L(-1) Zeatin resulted in 100% shoot regeneration and induced the highest number of shoots (average 15.0 +/- 0.8 shoots per explant) after being cultured for 8 weeks. The above hormone combination was optimum for shoot regeneration. Most of Saintpaulia ionantha plantlets derived from tissue culture system could be hardened and transferred to the greenhouse conditions with 84.0 +/- 1.6% success rate. However, regenerated plantlets of Saintpaulia ionantha (even after 12-months-old) failed to flower. Morphological characters of regenerated plantlets of Saintpaulia ionantha were observed and compared with in vivo (intact) plants. Regenerated plantlets showed some differences in morphological characters, such as height and leaf size, texture and colour, but the plantlets showed no variation in leaf arrangement and leaf margin. However, the morphological characters of the regenerated plantlets were found to be unstable.
Regeneration potentials in Gerbera jamesonii Bolus ex. Hook f. from tissues culture system was studied using leaf, petiole and root explants. In vitro regeneration, callus induction and root formation were optimized by manipulation of growth regulators during organogenesis. Various kinds of plant growth regulators such as 6-Benzylaminopurine (BAP), alpha-Naphthalene acetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA), Indole-3-Butyric acid (IBA), N6-[2-Isopentenyl]adenine (2iP), Kinetin and Zeatin were used to initiate cultures. These plant growth regulators were added to Murashige and Skoog medium in different combinations and concentrations. Adventitious shoots were obtained from petiole explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L(-1) BAP and 0.5 mg L(-1) NAA. Effectiveness of shoot regeneration medium, type of growth regulator used and duration of induction period were investigated. Leaf explants cultured on MS medium supplemented with 1.0 mg L(-1) BAP and 2.0 mg L(-1) 2, 4-D showed the best results for callus induction. Root explants were found to be non-regenerative in all experiments conducted. Petiole segment was identified as the best explant for regeneration of this species. Regenerated plants were rooted on Murashige and Skoog basal medium. Plantlets were then transferred to field with 75% survival rate.
Seven different hormone treatments, namely 6-benzylaminopurine (BAP) at 2, 3 mg L(-1) was applied singly and in combination with Indole Acetic Acid (IAA) at 0.18, 0.8 and 1.8 mg L(-l), BAP at 3.3 mg L(-l) in combination with IAA at 1.8 and 3.3 mg L(-l) and triple combination of BAP at 2.3, IAA at 1.8 and Gibberellic acid (GA3) at 1.0 mg L(-1) were tested, over four different incubation periods of 30, 45, 60 and 75 days, for their effect in the proliferation and growth of Smooth cayenne pineapple shoot-tip culture. Combined application of BAP at 3.3 and IAA at 1.8 mg L(-1) induced the highest proliferation of 19 shoots/explant and the highest total of 121 and 125 shoots over 4 cycles of multiplication. Raising the IAA to 3.3 mg L(-1) resulted in the lowest proliferation and stunted shoots. Incorporation of GA3 improved the shoot length but caused drastic reduction in proliferation. The other treatments showed an intermediate effect.
Intact immature flower buds of African violet (Saintpaulia ionantha H. Wendl.) were used as explant sources for in vitro studies. The effect of exogenous hormones, NAA and BAP on the indirect organogenesis of this species was observed. Callus was formed on the cut end (base) of pedicels of floral buds where they were in contact with the medium. When maintained on the same medium, callus was differentiated into adventitious shoots after 10 weeks in culture. MS media supplemented with 2.0 mg L(-1) NAA and 1.0 mg L(-1) BAP gave the highest number of sterile or vegetative floral buds from the surface of callus of the explants, but these buds failed to develop further. The floral buds were expanded as abnormal flowers. The floral structures were smaller in size compared to intact flowers. Petals (corolla) were white to purple in colour but did not form any reproductive organs, i.e., stamens or pistils. All sterile or vegetative floral buds and abnormal flowers survived for 3 months in culture but failed to reach anthesis.
In the present study, various explants of Murraya paniculata (Jack) Linn., such as cotyledons, shoots and young stems were cultured on MS medium supplemented with various concentrations of Benzyl Amino Purine (BAP) under 25 +/- 1 degree C with 16 h light and 8 h dark and also 8 h light and 16 h dark to obtain complete plant regeneration. In vitro flowering was observed from shoot explants cultured on MS supplemented with 0.5-2.0 mg L(-1) Naphthalene Acetic Acid (NAA) and also on MS basal medium under similar conditions. The leaves and flowers obtained from both in vivo and in vitro conditions were examined and compared. Morphological studies such as leaf clearing, epidermal peeling were studied using light and scanning electron microscope. Macromorphological studies of the flowers produced from in vivo and in vitro conditions were also examined. Morphologically, there were no differences between in vivo and in vitro flowers except the flowers produced from tissue culture systems were smaller in size with protruding stigmas. Differences were also found in the number of layers of palisade cells and the presence or absence of epicuticle layer of the leaves. Leaves produced from tissue culture system were smaller in size with membranous texture. Stomata were present only on the abaxial surfaces of both in vivo and in vitro leaves but the stomata were raised above the epidermis in the latter.