Leaf Disc Explants Research Articles

Effects of different explant and growth regulator combinations on in vitro propagation of Poinsettia (Euphorbia pulcherrima Willd.)

The aim of the research is to develop a suitable micropropagation protocol for the Poinsettia (Euphorbia pulcherrima Willd.) plant, which is used as an ornamental plant. In the sterilization step, mercuric chloride together with sodium hypochlorite is approved as the best option. Thus, contamination of explants is eliminated. At the shoot propagation stage, BA is absolutely necessary as all doses of BA increased shoot propagation compared to the control. The highest number of shoots per explant (2.62 shoots/explant) and the longest shoots (2.16 cm) were obtained from 1.0 mg.L-1 BA medium. The highest number of leaves (20.41 leaves/explant) was obtained from 2.0 mg.L-1 BA medium. Kinetin was also effective when adding different concentrations to the culture medium compared to the control. In general, BA is more effective than Kinetin in these parameters. Kinetin, on the other hand, performed much better than BA in terms of shoot number. On the other hand, the highest rooting percentage (58.2%) was obtained from the addition of 0.3 mg.L-1 NAA. The highest root number (5.10 roots/explant) was obtained by adding 0.1 mg.L-1 NAA. Good performance was found in the acclimatization phase with plantlets transferred to the soil with a high survival rate reaching 100%. Most of the plantlets started growing well. The plantlets grew well and did not show morphological abnormalities. In addition, a successful plant regeneration was achieved by adding 1.0 mg.L-1 BA and 0.5 mg.L-1 NAA on the callus produced in leaf disc explants and a very good organogenesis was determined in terms of roots and shoots.

Somatic Embryogenesis of Myrciaria dubia (Kunth.) McVaugh

Myrciaria dubia (caçari or camu-camu) is a species with great relevance because of its high levels of bioactive compounds and antioxidant activity. The species is propagated mainly by seed, which leads to high genetic diversity. Producing plants that maintain their characteristics on a large scale at a low cost is a challenge for fruit species, especially in the Myrtaceae family. Therefore, this study aimed at assessing the effects of different plant growth regulators at various concentrations for the induction of somatic embryogenesis from caçari nodal segments and leaf disk explants. Two independent experiments were performed using nodal segments and leaf disks from plants grown in the greenhouse. In the first experiment, the combined effect of auxin and cytokinin at different concentrations was evaluated: 2,4-dichlorophenoxyacetic acid at 0, 1, 2, and 4 mg⋅L–1; and benzylaminopurine at 0, 0.25, 0.5, and 1 mg⋅L–1). In the second experiment, the application of different plant growth regulators (benzylaminopurine, kinetin, thidiazuron, and isopentenyl adenine) and their concentrations (0, 2, 4, and 6 mg⋅L–1) were evaluated. In both experiments, the basic culture medium was woody plant medium. Callus formation via nodal segments and leaf disks occurred in the first 30 d from cultivation and proved to be responsive to induction by 2,4-dichlorophenoxyacetic acid and benzylaminopurine. Auxin proved to be essential for somatic embryogenesis induction in nodal segments using 1 and 4 mg⋅L–1 of 2,4-dichlorophenoxyacetic acid alone. The results of this experiment will help advancing protocols for regeneration of somatic embryos and elucidating the physiological, molecular, and genetic mechanisms involved in the process of somatic embryogenesis for M. dubia. The development of an efficient protocol for in vitro clonal propagation of this species also lays the groundwork for further optimization of the system for genetic transformation.

Optimizing nutrient media conditions for continuous production of shoot biomass enriched in major medicinal constituents, amarogentin and mangiferin of endangered medicinal herb, Swertia chirayita.

Biosynthesis and accumulation of Amarogentin and Mangiferin from shoot culture of endangered herb Swertia chirayita helped in rescuing its natural population along with continuous production of quality rich herbal material. Although, presence of Amarogentin and Mangiferin had already been reported, but such studies did not elaborate the significant developmental stages at two varying temperature (15 ± 1 °C and 25 ± 1 °C) in shoot cultures of S. chirayita. Different developmental stages involved throughout from callus induction to complete regeneration of plant by using shoot cultures of S. chirayita, reveal different amounts of significant medicinal compounds having high pharmacological importance like bearing anti-diabetic and anti-cancerous properties. So in the present study, different developmental stages i.e. plant segment as leaf disc explants, initiation of callus formation, callus mass development, shoots primordial, manifold shoot formation and shoot elongation with complete growth were explored for accumulation of Amarogentin and Mangiferin. The Amarogentin content was 4.72 µg/mg at 15 ± 1 °C and 4.41 µg/mg at 25 ± 1 °C whereas Mangiferin content was 15.54 µg/mg at 15 ± 1 °C and 9.70 µg/mg at 25 ± 1 °C in leaf discs provided with the medium MS + 2,4D = 1 mg/L, 6BAP = 0.5 mg/L, TDZ = 0.5 mg/L, respectively. The accumulation of Amarogentin and Mangiferin started from callus cultures differentiating into shoots and reached to the detectable amount equivalent to actual leaf explants in fully grown shoots with content of 5.79 µg/mg at 15 ± 1 °C and 5.35 µg/mg at 25 ± 1 °C whereas 15.56 µg/mg at 15 ± 1 °C and 13.15 µg/mg at 25 ± 1 °C provided with the medium MS + IBA = 3 mg/L, KN = 1 mg/L, respectively. Maximum accumulation of bioactive compounds was observed in ≈3 months old in-vitro grown shoots at 15 ± 1˚ C wherein, the content of Amarogentin was ≈8.51 folds higher and Mangiferin was ≈4.09 folds higher than the ≈3 months old green house grown shoots. So, the in-vitro raised shoots of S. chirayita enriched with marker medicinal compounds would be utilized as ready to use raw material for pharmaceutical industries for herbal drug formulations and can be utilized to transfer under natural habitats for conserving its diminishing population.

Organogenesis from Leaf-derived Calli of S. potatorum L. f.: A Vulnerable Medicinal Tree Species

An efficient protocol for in vitro regeneration of Strychnos potatorum L. f. through indirect organogenesis was developed using mature leaf disc explants. The explants were selected from twenty years old tree. Cultured on Murashige and Skoog’s medium supplemented with 2,4-dichloro phenoxy acetic acid (2,4-D) was used for the induction of organogenic calli. A pale yellow coloured friable callus developed at 2 mg/L-1 2,4-D where showed higher morphogenic potential. Further de-novo shoots formation and multiplication in optimal concentrations of cytokinins with different combinations of auxins. An average mean shoots number (14.2±0.42) and shoots height (3.5±0.27cm) was obtained from piece of calli in 6-benzylaminopurine (BAP) 1 mg/L-1 with 0.5 mg/L-1 α-naphthalene acetic acid (NAA). BAP in combinations with NAA was found to be superior to shoot development than other combination used. The well developed shoots were transferred to root induction medium for effective rooting. The rooting medium consisted of in half-strength MS medium augmented with 0.5 mg/L-1 NAA was used. The maximal mean number of roots per shoots 6.5±0.53 was recorded in 4 weeks. The rooted plantlets were removed from the culture tubes, gently washed with sterile double distilled water to remove agar adhering on the plant surface. The regenerated plants were transferred to plastic cups filled with a potting mixture containing sterile sand and soilrite (1:1 ratio) and allowed to grow in the greenhouse. The slowly acclimatized plants were further transferred to substrate containing red soil and farmyard manure (1:1) for effective hardening. 75% survival rate was recorded. This protocol can be useful for the ex-situ conservation and rehabilitation of this vulnerable medicinal tree S. potatorum.
 Keywords: Indirect organogenesis, friable callus, shoot development, 6-benzylaminopurine, α-naphthalene acetic acid

Root-preferential expression of Newcastle virus glycoproteins driven by NtREL1 promoter in tobacco hairy roots and evaluation of oral delivery in mice.

Newcastle disease virus (NDV) is a lethal virus in avian species with a disastrous effect on the poultry industry. NDV is enveloped by a host-derived membrane with two glycosylated haemagglutinin-neuraminidase (HN) and Fusion (F) proteins. NDV infection usually leads to death within 2-6days, so the preexisting antibodies provide the most critical protection for this infection. The HN and F glycoproteins are considered the main targets of the immune system. In the present study, two constructs harboring the HN or F epitopes are sub-cloned separately under the control of a root-specific promoter NtREL1 or CaMV35S (35S Cauliflower Mosaic Virus promoter) as a constitutive promoter. The recombinant vectors were transformed into the Agrobacterium tumefaciens strain LBA4404 and then introduced to tobacco (Nicotiana tabacum L.) leaf disk explants. PCR with specific primers was performed to confirm the presence of the hn and f genes in the genome of the regenerated plants. Then, the positive lines were transformed via non-recombinant A. rhizogenes (strain ATCC15834) to develop hairy roots.HN and F were expressed at 0.37% and 0.33% of TSP using the CaMV35S promoter and at 0.75% and 0.54% of TSP using the NtREL1 promoter, respectively. Furthermore, the mice fed transgenic hairy roots showed a high level of antibody responses (IgG and IgA) against rHN and rF proteins.

A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis\u2009\xd7\u2009Passiflora edulis f. flavicarpa)

BackgroundPassion fruit (Passiflora edulis Sims) is an important horticultural crop in the tropics and subtropics, where it has great commercial potential due to high demand for fresh edible fruits and processed juice as well as source of raw materials in cosmetic industries. Genetic engineering shows great potential in passion fruit improvement and can compensate for the limitations of conventional breeding. Despite the success achieved in genetic modification of few passion fruit varieties, transgenic passion fruit production is still difficult for farmer-preferred cultivars. Therefore, it is important to establish a simple and fast Agrobacterium-mediated cell transformation of commercial hybrid passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa).ResultsIn the present study, we have developed a simple and fast Agrobacterium-mediated transformation system for hybrid passion fruit KPF4 using leaf disc explants. Factors affecting the rate of transient beta (β)-glucuronidase (gusA) expression and consequently transformation efficiency were optimized as follows: Agrobacterium cell density with an OD600 of 0.5, 30 min infection time, 3 days of co-cultivation duration and the incorporation of 200 µM acetosyringone into Agrobacterium infection suspension medium. Using the optimized conditions, transgenic plants of KPF4 were produced within 2 months with an average transformation efficiency of 0.67%. The β-glucuronidase (GUS) histochemical staining confirmed the expression and integration of an intron-containing gusA gene into transformed leaf discs and transgenic plant lines of KPF4. The presence of gusA gene in the transgenic plants was confirmed by polymerase chain reaction (PCR). The results confirmed that the gusA gene was efficiently integrated into the passion fruit genome.ConclusionsThe developed transformation protocol is simple and rapid and could be useful for functional genomic studies and transferring agronomically important traits into passion fruit hybrid KPF4. This study developed a method that can be used to transfer traits such as resistance to viral diseases, low fruit quality and short storage life. To the best of our knowledge, this is the first report on genetic transformation system for commercial passion fruit hybrid KPF4.

Challenges in micropropagation of Bael [Aegle marmelos (L.) Corr.] from leaf Disk Explants and methods for its adoption amongst progressive farmers and farm women

The present study was performed to define tissue culture techniques for micropropagation of bael cvs. through leaf disk explants from 25 days old in vitro grown plantlets on Knop’s medium supplemented with 0.5 mgl-1 BAP. Leaf disk explants were cultured on various concentrations of growth regulators (BAP, KIN and NAA). Knop’s medium supplemented with 1.0 mgl-1 BAP, 0.5 mgl-1 KIN and 0.5 mgl-1 NAA produced maximum percentage of shoot regeneration (46.66%) and maximum per cent multiple shoot (33.33%) was found in cv. Mirzapuri on Knop’s medium supplemented with 1.0 mgl-1 BAP+0.5 mgl-1 KIN +0.5 mgl-1NAA. Maximum mean number of shoots per explants (1.20) was recorded in cv. Mirzapuri at 1.0 mgl-1 BAP+0.5 mgl-1 KIN+0.5 mgl-1NAA whereas maximum shoot length (0.95cm) was observed in cv. Mirzapuri on Knop’s medium supplemented with 1.0mgl-1 BAP+0.5mgl-1KIN+0.5mgl-1NAA.

An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants

Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy. Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.

Responses of Different Explants of Sweet Potato on Modified MS and LS Based Nutrient Media in vitro

A good number of reports on sweet potato (Ipomoea batatas L.) tissue culture using different tissues of various cultivars with varying level of efficiency and reproducibility are available but callus induction and plantlet regeneration are recalcitrant and limited to a few genotypes in response to different treatments in vitro. Reported in this paper is a procedure in which several explants of three high yielding and drought tolerant sweet potato cultivars (SK010, WHCH005 and PRAP496), mostly grown and cultivated in the highlands of Papua New Guinea (PNG) were used to induce embryogenic callus and regenerate plantlets. To achieve a reliable and an efficient system for inducing embryogenic callus on stem, petiole and leaf disc explants, a modified form of Murashige and Skoog (MS) and Linsmaier and Skoog (LS) media, supplemented with 1 gL-1 picloram and 8 gL-1 agar was used. This procedure resulted in large amount of embryogenic calli that were potentially capable of regenerating whole plantlets on all the different types of explants tested. Further attempts made in plantlet regeneration failed, however ways in which improvement of the tested regeneration media can be made for plant regeneration in similar studies were discussed.

Thidiazuron and Explant Type Effects on High-frequency In Vitro Mass Propagation of Cherry Tomato

Cherry Tomato is one of the most important vegetable crops cultivated for export in Egypt. In vitro culture response was assessed in tomato (Solanum lycopersicum L. var. cerasiforme) cv. (Summer Cherry) for optimum callus induction and plantlet regeneration. Callus induction was achieved within eight to 12 days directly on the cut surfaces of hypocotyl, cotyledon and leaf disc explants cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations of Thidiazuron (TDZ)] and benzyl adenine (BA) alone, but not in hormone free-medium. The highest callusing index (3.9 and 3.7) was obtained on hypocotyl explants cultured on MS medium supplemented with TDZ(1.0 and 2.0 mgl-1) followed by an index of 3.5 obtained from the same explant by using 0.5mgl-1 BA. However, for the leaf disc explants, the highest callusing index (3.1) was obtained on MS medium supplemented with BAat 2.0 mgl-1. After 8 weeks of culture, organogenesis was observed only on the explants cultured on medium containing different concentrations of TDZ and BA. The best shoot formation (93%) was obtained from leaf disc explant callus induced on MS medium containing TDZ. The highest number (13.4) of shootsexplant-1 was found when cotyledon explant callus was sub cultured on MS medium supplemented with 2.0 mgl-1 TDZ. Half strength of MS was found to be the best rooting medium, however, addition of IAA at 1.0 mgl-1 and IBA at 2.0 mgl-1 were found necessity to induce highest number of roots (22.5) and longer roots (11.0 cm), respectively. Acclimation of in vitro rooted plant is important for testing the post culture behavior of tissue culture regenerated plants. Cherry tomato derived from different explant sources under different concentration of TDZ and BA were not significantly different in their vegetative characters to those obtained from seed. However number of (raceme plant-1, flower raceme-1, fruit raceme-1, fruit plant-1) which produced by seed-derived plants was significantly less than those derived from in vitro propagated plants. This protocol would be valuable to create somaclonal variation and develop transgenic approaches for varietal improvement of cherry tomato.

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

In vitro propagation of Bergenia ciliata Sternb: A valuable medicinal and ornamental plant of Sikkim Himalaya

Bergenia ciliata is an important ornamental medicinal plant of Sikkim Himalaya with wide range of pharmaceutical applications. In the present study, an efficient in vitro propagation protocol has been developed for B. ciliata using leaf disc explants. The highest number of regenerated shoots per explant was obtained on solid Murashige and Skoog (MS) medium supplemented with a combination of 5 μM 6-benzylaminopurine (BA) and 5 μM indole-3-acetic acid (IAA). The 100% rooting was observed in shoots after 6 weeks of culture on liquid MS medium. The plantlets were successfully acclimatized (100%) in a garden soil-sand-farmyard manure mixture (1: 1: 1 v/v/v) in the greenhouse. This micropropagation protocol would facilitate rapid propagation of B. ciliata for its conservation, and could help pharmaceutical demands sustainably.

Screening and optimizing media constituents for enhanced production of medicinal N-alkylamide Deca-2E,6Z,8E-trienoic acid isobutylamide from dedifferentiated in vitro cell lines of Spilanthes paniculata

Abstract Spilanthes paniculata is a rich source of therapeutic polyunsaturated fatty acid amides. This report established the cell cultures from leaf-disc explants of Spilanthes paniculata as an alternative method of cell biomass utilization and conservation of natural plant resources. Furthermore, a methodology to maximize the production of a prime N -alkylamide, Deca-2 E ,6 Z ,8 E -trienoic acid isobutylamide (spilanthol), from in vitro cell cultures was optimized using statistical tools. Placket-Burman (PB) design was applied to identify the significant nutrient media constituents. Among the five variables tested, Murashige and Skoog (MS) major salts, sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzylaminopurine (BAP), were found to have significant effect on spilanthol production. The optimal concentrations of the four variables were determined using central composite design (CCD), which is widely used response surface methodology (RSM) design. The most suitable concentration of variables for spilanthol production were, 1½ MS, 5% sucrose, 4.82 µM BAP and 1.8 µM 2,4-D. At these optimal parameters, the maximum yield of spilanthol was obtained experimentally as 2.81 mg/g DW, which was found to be very close to its predicted value of 3.72 mg/g DW. The developed mathematical model was found to fit well with the experimental data by which the higher production of metabolite was achieved as compared to non-optimized media constituents. Before optimization, the callus cultures and leaves from parental plant (control) yielded 1.75 mg/g DW and 0.26 mg/g DW spilanthol, respectively. Thus, by using statistical model, it is possible to understand the existence of interactions between variables and their effect on maximizing the production of therapeutic compounds.

High-frequency plant regeneration and histological analysis of callus in Cichorium intybus: An important medicinal plant

&lt;p&gt;An efficient in vitro propagation and in vitro flowering protocols were developed for the medicinal plant Cichorium intybus (Asteraceae) using leaf disc explants. Media supplemented with the growth regulator naphthalene acetic acid (NAA) (1.5 mg/l) + 6-benzyle adenine (0.25 mg/l) was used for the initial induction of the callus and further subcultured to the same media for the proliferation of the callus. Pale yellow and green calli were noticed, which depends on incorporation of the growth hormones and their varying concentrations. Murashige and Skoog medium in addition with 2 mg/l kinetin+ 0.5 mg/l indole-3-acetic acid (IBA) + 500 mg/l casein hydrolysate resulted in maximum regeneration. Media supplemented via IBA (0.5 mg/l) and NAA (0.5 mg/l) (98%) was found to be&lt;br /&gt;optimum for rhizogenesis for in vitro regenerated plants. For acclimatization 5-6 weeks mature in vitro regenerated plants were transferred into the greenhouse for acclimatization. The histological study revealed the presence actively dividing meristematic cells in callus. The occurrence of the peripheral meristematic zone associated with callus was noticed in after 20 days, which formed the shoot meristems after 45 days of incubation. To our knowledge, this is the first report on high-frequency plant regeneration which was carried out indirectly from the&lt;br /&gt;leaf explants which was grown in controlled environment with varying concentration of the growth regulators and histology of callus of different stages from leaf explants of C. intybus.&lt;/p&gt;

Effect of collection season, type and length of explant on callus induction and its survival in peach

It takes 5–6 weeks to complete callus formation over callus surface in peach. During the course of investigations effect of explants collection season, type and length of explants for callusing in peach were standardized. Among various combinations best response in terms of maximum callus induction (97.78%) was noted in spring x internodal segments x 0.5 cm length of explants closely followed by (95.55%) in spring x leaf disc x 0.5 cm length of explants however maximum callus culture survival (100%) was noted in spring x leaf disc as well as in internodal segments x 0.5 cm length of explants. Observations on callus morphology indicate that the leaf disc explants collected during winter season formed white, watery type callus with poor callus growth while no callus formation was noticed in internodal segments in winter season. However, yellowish green, friable type callus with excellent growth was observed during spring season in leaf disc and green colour callus along with compact callus type were observed with internodal segment. During summer season brown colour callus with friable type were noticed in leaf disc explants with good callus growth and whitish green colour callus with semi-compact type were observed in internodal segment explants along with excellent callus growth.

Effect of explant type and surface sterilants on asepsis and survival culture for callus induction in peach

For the optimization of surface sterilization of explants and their in vitro establishment for callus, aseptic culture and survival of explants were measured. The present study was carried out to investigate the influence of explant type, various surface sterilants and their exposure time on the establishment of explants for callus induction in peach (Prunus persica L. Batsch). The leaf disc and intermodal segment explants taken from newly growing twigs of peach cultured on MS medium supplemented with BAP (0.5 mg/L) + 2, 4-D (2 mg/L) for callus induction after surface sterilization with various surface sterilents. The overall results suggested that the best sterilization treatment for culture establishment in peach explants was sequential exposure of explants to ethanol (70%) 30 second + HgCl2 (0.1%) 3 minute + NaOCl (1.0%) 3 minute resulting 63.34 per cent aseptic culture with 80.00 per cent survival of explants in both leaf disc and internodal segment comparatively suitable among all the five treatments. Further increase in exposure time to various sterilants resulted in significant reduction in survival of explants. Hence, ethanol (70%) 30 second + HgCl2 (0.1%) 3 minute + NaOCl (1.0%) 3 minute was used for surface sterilization of explants in peach for establishment of explants for callus induction.

In vitro propagation and conservation of Satureja avromanica Maroofi—an indigenous threatened medicinal plant of Iran

An efficient and rapid in vitro propagation system for Satureja avromanica, a rare and endangered folk medicinal plant of Iran was developed through the shoot tip and leaf disc explants. Nodal and leaf explants from wild plants were established on MS and WPM media supplemented with BA, BAP and TDZ (0, 0.1, 0.5, 1, 1.5, 2, 5 and 10 mgl(-1)) alone or by application of BA and TDZ (0, 2, 5 and 10 mgl(-1)) in combination with IBA and 2,4-D (0, 0.1, 0.5 and 1 mgl(-1)), respectively. Based on results, the highest mean shoot number (6.21) was obtained on MS medium supplemented with 2 mgl(-1) BA. Regarding the shoot elongation, MS supplemented with 2mgl(-1) TDZ and MS containing 5mgl(-1) BA showed the longest shoots (4.82 and 4.39cm, respectively) after 6weeks of culture. As a matter of fact, increasing all three tested cytokinins levels led to enhancement of explant response frequency and regenerated shoot number. On the other side, WPM medium supplemented with 0.1 mgl(-1) IBA was found suitable for rooting of regenerated shoots. RAPD molecular analysis revealed genetic stability of in vitro raised plants. In conclusion, individual application of BA, BAP and TDZ were in favor of S. avromanica direct shoot regeneration while treatment media with a combination of IBA and BA as well as 2,4-D and TDZ resulted in callogenesis in most explants. Finally, the in vitro raised plantlets were acclimatized and successfully established in the greenhouse conditions. Our developed protocol can be employed for the large-scale micropropagation and conservation of S. avromanica as a threatened medicinal plant.

Effect of seaweed liquid extracts and plant growth regulators on in vitro mass propagation of brinjal (Solanum melongena L.) through hypocotyl and leaf disc explants

The effect of seaweed liquid extracts (SLEs) made from Gracilaria salicornia, Padina gymnospora, Padina boergesenii, Gelidiella acerosa and plant growth regulators (PGRs) were examined on in vitro mass propagation using hypocotyls and leaf disc explants of brinjal (Solanum melongena L.) cultivar Pusa purple long. For the germination bioassay, seeds germinated with 20–40 % SLEs exhibited enhanced germination. Initially, hypocotyls and leaf discs were cultured on Murashige and Skoog (MS) medium containing 6-benzylaminopurine, zeatin and thidiazuron for shoot induction. The best responding cytokinin, 6-benzylaminopurine, was employed with different auxins (indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid) for shoot proliferation. In a second experiment, all the four SLEs (10–60 %) combined with MS medium were studied for shoot propagation. Augmented shoots transferred to half-strength MS medium and supplemented with auxins and SLEs (10–70 %) individually to induce rooting. In these experiments high rate of shoot induction (96.2 %), proliferation (6 cm) and rooting (95.3 %) was found with 20–40 % of SLEs. Well-matured plantlets were transferred to soil cups, maintained in a growth chamber for a week to control humidity and then shifted to a greenhouse. This study demonstrated that SLEs could serve as an alternative to phytohormones as they were easy to extract and gave quick and high-frequency mass propagation.

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l−1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.

English

Microrhizomes were produced from leaf disc derived callus of a threatened endemic medicinal plant - Decalepis hamiltonii. Murashige and Skoog (MS) medium supplemented with 2 mM 6-benzyleaminopurine (BAP) and 6 mM 1-napthaleneacetic (NAA) acid was found to be optimum for rapid callus induction and establishment from leaf disc explants. Further differentiation of callus into microrhizome was conquered in MS medium supplemented with 4 mM indole-3-butyric acid (IBA) and 8 mM NAA. A maximum of 20 microrhizomes in a cluster was produced within 90 days. Yeast extract (0.05%) and polyvinylpyrrolidone (0.05%) further enhanced the microrhizome formation when supplemented along with plant growth regulators (PGRs). &nbsp; Key words: Decalepis hamiltonii, microrhizome, herbal medicine, secondary metabolites.