Highest Callus Induction Rate Research Articles

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0mg/L TDZ. Meanwhile, the MS medium containing 1.0mg/L 6-BA and 0.4mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n=3x=27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Callus Derived from Petals of the Rosa hybrida Breeding Line 15R-12-2 as New Material Useful for Fragrance Production.

Rose (Rosa hybrida) is a major flower crop worldwide and has long been loved for its variety of colors and scents. Roses are mainly used for gardening or cutting flowers and are also used as raw materials for perfumes, cosmetics, and food. Essential oils, which are extracted from the flowers of plants, including roses, have various scents, and the essential oil market has been growing steadily owing to the growing awareness of the benefits of natural and organic products. Therefore, it is necessary to develop a system that stably supplies raw materials with uniform ingredients in line with the continuous increase in demand. In this study, conditions for the efficient induction of callus were established from the petals of the rose breeding line 15R-12-2, which has a strong scent developed by the National Institute of Horticultural and Herbal Science, Rural Development Administration. The highest callus induction rate (65%) was observed when the petals of the fully open flower (FOF) were placed on the SH11DP medium so that the abaxial surface was in contact with the medium. In addition, the VOCs contained in the petals of 15R-12-2 and the petal-derived callus were analyzed by HS-SPME-GC-MS. Thirty components, including esters and alcohols, were detected in the petal-derived callus. Among them, 2-ethylhexan-1-ol, which showed 59.01% relative content when extracted with hexane as a solvent, was the same component as detected in petals. Therefore, petal-derived callus is expected to be of high industrial value and can be suggested as an alternative pathway to obtaining VOCs.

Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.)

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 μmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

Effects of mammalian sex hormones on in vitro organogenesis of common bean (Phaseolus vulgaris L.)

Beans are an important plant species and are one of the most consumed legumes in human nutrition, especially as a protein, vitamin, mineral, and fiber source. Common bean (Phaseolus vulgaris L.) is a plant that also has an important role in natural nitrogen fixation. Currently, in vitro regeneration and micropropagation applications are limited in relation to genetic factors in bean Accordingly, there is great need to optimize micropropagation and tissue culture methods of the bean plant. To date, the effect of mammalian sex hormones (MSH) on in vitro conditions in P. vulgaris L. is poorly understood. This study examined the effects of different types of explants (embryo, hypocotyl, plumule, and radicle), MSH type (progesterone, 17 β-estradiol, estrone, and testosterone), and MSH concentration (10−4, 10−6, 10−8 and 10−10 mmol L−1) on the responding explants induction rate (REI), viability of plantlets rate (VPR), shoot proliferation rate (SPR), root proliferation rate (RPR), and callus induction rate (CIR). The effects of mammalian sex hormones, concentrations, explant type, and their interactions were statistically significant (p ≤ 0.01) in all examined parameters. The best explants were embryo and plumule. Our results showed that the highest REI rate (100%) was recorded when 10−10 mmol L−1 of all MSH was applied to MS medium using the plumule explant. The highest VPR (100%) was obtained when 10−10 mmol L−1 of all MSH was applied to MS medium using the plumule explant. The highest root proliferation rates (77.5%) were recorded in MS medium supplemented with 10−8 mmol L−1 17β-estradiol using embryo explant. The highest percentage of shoot-forming explants (100%) generally was obtained from embryo and plumule cultured in the MS culture medium with low MSH concentration. In addition, the highest CIR (100%) was obtained from embryo and plumule explant cultured in MS medium containing 10−10 mmol L−1 of all MSH types. In conclusion, we observed that mammalian sex hormones may be used in bean in vitro culture.

Establishment of in vitro regeneration system and molecular analysis of early development of somatic callus in Capsicum chinense and Capsicum baccatum.

Regeneration is extremely important to pepper genetic development; however, the molecular mechanisms of how the callus reactivates cell proliferation and promotes cell reprogramming remain elusive in pepper. In the present study, C. baccatum (HNUCB81 and HNUCB226) and C. chinense (HNUCC22 and HNUCC16) were analyzed to reveal callus initiation by in vitro regeneration, histology, and transcriptome. We successfully established an efficient in vitro regeneration system of two cultivars to monitor the callus induction of differential genotypes, and the regenerated plants were obtained. Compared to C. chinense, there was a higher callus induction rate in C. baccatum. The phenotype of C. baccatum changed significantly and formed vascular tissue faster than C. chinense. The KEGG enrichment analysis found that plant hormone transduction and starch and sucrose metabolism pathways were significantly enriched. In addition, we identified that the WOX7 gene was significantly up-regulated in HNUCB81 and HNUCB226 than that in HNUCC22 and HNUCC16, which may be a potential function in callus formation. These results provided a promising strategy to improve the regeneration and transformation of pepper plants.

Biotransformation of stevioside to rebaudioside – A in suspension cells of Stevia: Applications of leaf extract in functional biscuits

The suspension culture of Stevia was conceived as a means of establishing a stevioside-producing cell line in vitro. The highest callus induction rate (90%) was achieved using the Stevia leaf explants in modified MS medium supplemented with 1 mg/L BAP and 2 mg/L NAA. Friable and rapidly growing suspension cells were established in this media. The growth curve of suspension culture was studied by determining its biomass accumulation in PCV where the maximum biomass was obtained after 3 weeks. The suspension culture was preferred over callus culture for the biotransformation. The results indicated the simplicity, relatively inexpensive, eco-friendly nature of the process by showing 37.5% conversion of stevioside to rebaudioside A. As a result, it appears to be a potential method for the large-scale industrial synthesis of rebaudioside-A. This high biotransformation efficiency will be useful in substituting sucrose in functional foods. In the current study, the sensory characteristics and energy values of the biscuits containing Stevia, as a sugar replacement, were investigated. According to our findings, Stevia extract may be utilized as a natural sweetener in a variety of food products such as biscuits. In addition, these functional biscuits may be beneficial in maintaining good health, managing obesity and diabetes in patients.

An Optimized Protocol for Indirect Organogenesis from Root Explants of Agapanthus praecox subsp. orientalis ‘Big Blue’

Agapanthus praecox has become a burgeoning variety in the flower market due to its high ornamental value with unique large blue-purple inflorescence. For rapid entering into the market, tissue culture technology or organogenesis has an attractive application over the conventional reproduction approach. In this study, a highly efficient protocol based on indirect organogenesis has been successfully established for A. praecox subsp. orientalis ‘Big Blue’. Two types of explants, root tips versus root segments, were compared for callus induction frequency in response to the induction culture media. The induction media contain Murashige and Skoog’s (MS) Basal Salt supplemented with various concentrations of picloram (PIC), 2,4-Dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), kinetin (KT) and naphthalene acetic acid (NAA). Of the two types of explants, root tips were found to be more effective for callus induction than root segments. Among the induction media tested, the highest callus induction rate (100.00%) was achieved when cultured on MS supplemented with 2.0 mg/L PIC, 1.5 mg/L KT and 0.1 mg/L NAA, which was probably accredited to higher endogenous phytohormone contents, especially of 3-indoleacetic (IAA). The optimal medium for callus proliferation was MS + 1.0 mg/L PIC + 1.0 mg/L 6-BA + 0.4 mg/L NAA, and the fresh weight increased by 72.74%. After being transferred onto the adventitious bud induction medium for 25 days, shoots were dedifferentiated from the surface of the flourishing callus, which then developed to the plantlet with roots in 90 days. The plantlets were transplanted in a greenhouse with a survival rate of 92.86%. This study innovatively established an indirect organogenesis tissue culture system of A. praecox with roots as explants, which provided a practical reference in its application.

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus caryophyllus semperflorens flore pleno) Cultivars

Abstract Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm−3 of 2,4-D and 200 mg·dm−3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm−3 of BA, 0.6 mg·dm−3 of NAA, 5 mg·dm−3 of AgNO3, and 40 mg·dm−3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm−3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Callus Formation and Regeneration Tendency of Justicia vasica Nees under Saline Conditions

Salinity is one of the major environmental threats to plants because a large number of plants are intolerant to NaCl stress. One such plant, Justicia vasica, which is the subject of our study, is said to have great medicinal properties. However, the natural occurrence and cultivation of this species has been constrained due to various factors including its poor seed germination rate under its normal conditions of habitat, and a low availability of seeds. This study was undertaken to examine the efficiency of callus formation and regenerative tendencies of Justicia vasica under saline conditions. The leaf, shoot tips and inter nodal cuttings were employed for this study using MS media formulated with several concentrations of different kinds of hormonal combinations. This study also tested the effects of different concentrations of NaCl on callus induction and growth. Each experiment was conducted thrice with 12 replicates. A superior level of callus formation was achieved by treating shoot tips with 2.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenine purine (BAP) in combination. From this study, it was concluded that there was no inhibitory action from the medium supplemented with 0.05 mg/l NaCl. The optimal hormonal combination also exhibited highest rate of callus induction and growth with 0.05 mg/l NaCl added. There was no callus necrosis at this saline concentration after 60 days of inoculation. Furthermore, some somatic embryogenic callus displayed the organogenesis of leaf like structures after 75 days of inoculation. The development of salinity tolerance of plants by genetic application has been a vital area on modern agriculture as well as medicinal plant research because more land has been salinized through poor local irrigation practices. The present investigation assumed that the NaCl tolerance of this plant could be effectively obtained in vitro.

Determination of antioxidant activity, total phenolics and fatty acids in essential oils and other extracts from callus culture, seeds and leaves of Argania spinosa (L.) Skeels

Argan (Argania spinosa (L.) Skeels) is an endangered and endemic agroforestry species of Morocco highly appreciated for its nutraceutical properties. Herein, the antioxidant activity, total phenolic content and fatty acids were evaluated in different extracts obtained from callus culture, seeds and leaves of four argan genotypes: G25, G36, G41 and G84. Callus induction, proliferation and morphology varied depending on genotype and explant type. The highest callus induction rate (97.5%) was observed in the cotyledon explants of genotype G84. The radical scavenging activity values ranged from 76.5 to 98.1%. The essential oils extracted from calli induced from seedling-derived leaves exhibited a slightly higher radical scavenging activity (91.7%) than those extracted from field-grown leaves (90–91.1%). The total phenolic content ranged from 0.72 mg/g dry weight gallic acid equivalent in the methanolic extracts of G84 callus obtained from seedling-derived leaves to 198.26 mg/g dry weight gallic acid equivalent in the essential oils of G84 seeds. The fatty acid composition varied significantly among the different samples. The essential oils extracted from seeds and callus obtained from cotyledon explants have high contents in oleic and linoleic acids (26–37.9% and 25–36.8%, respectively), while the major fatty acid found in the essential oils of leaves and callus obtained from seedling-derived leaves was eicosenoic acid (18.8–45.4%). The present study showed that argan callus culture could be envisaged for sustainable and continuous production of bioactive compounds, and that each extract analyzed had unique and distinct characteristics. The essential oils and other extracts obtained from argan callus induced in vitro were characterized and were compared with those obtained from seeds and field-grown leaves

Studies on <i>In Vitro </i>Response to Callus Induction and Regeneration of Five High Yielding <i>Indica </i>rice Varieties

Due to growing population, there is an increasing demand of rice production but the productivity of rice is lessened day by day. To overcome this problem various biotechnological tools can be used for developing various rice varieties. However, the lack of a simple and efficient protocol for callus induction, embryogenic callus formation and quick plant regeneration in this cereal crop. In this study embryogenic calli from mature seeds of five indica rice varieties viz. Binadhan-5, Binadhan-6, BRRI dhan-48, BRRI dhan-58 and IR-64 were observed that is done in four different types of media composition. The highest callus induction were observed in media containing Sucrose as a carbon source. Among those varieties Binadhan-6 and BRRI dhan-48 showed highest rate of callus induction respectively. This study will be useful for selecting suitable callus induction medium for callus induction in future that will be useful for not only national but also international plant breeders for producing new variety and so on.

Somatic embryogenesis and regeneration of Kenyan wheat (Triticum aestivum L.) genotypes from mature embryo explants

Wheat (Triticum aestivum L.) improvement via genetic transformation depends on an efficient regeneration system for recovery of transgenic events. This study reports a somatic embryogenesis-based regeneration system for two Kenyan wheat genotypes (Eagle 10 and Njoro bread wheat II) from mature embryos. The study investigated the efficiency of mercuric chloride, commercial bleach, and chlorine gas in surface sterilizing explants prior to in vitro culture. Callus induction and somatic embryogenesis were done by culturing wheat mature embryos in Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) used either singly or in combination with 1-naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid (4-CPA) or 6-benzylaminopurine (BAP). Embryo germination and plantlet recovery were done by culturing embryogenic callus in MS medium without plant growth regulators (PGRs). Chlorine gas was significantly (p<0.001) the most effective in surface sterilization and maintenance of explant viability. All 2, 4-D concentrations tested (1, 2, 4 and 8 mg/l) induced embryogenic callus. A significantly higher callus induction rate and callus fresh weight were obtained when 2,4-D was used in combination with either NAA or 4-CPA than when it was used singly. Combining 2,4-D with BAP led to a significantly lower callus induction frequency. Somatic embryo germination was achieved in MS medium without plant growth regulators. These findings have the potential to inform future efforts in the application of modern biotechnology for accelerated wheat cultivar improvement. Key words: Wheat, somatic embryogenesis, mature embryos.

English

Haploid breeding is an effective method of shortening the breeding period of plants. In this study, to develop a haploid breeding technique, optimum conditions of the culture medium were investigated for anther-derived callus induction in the strawberry cultivar Seolhyang. The effects of the culture medium type, myo-inositol, auxin and cytokinin combination treatment, silver nitrate (AgNO3), and ferric ethylenediaminetetraacetic acid (Fe-EDTA) on anther-derived callus induction were analyzed. Anthers were incubated in Murashige and Skoog (MS) medium, Gamborg B5 medium (B5), and Lichter medium (NLN) for 8 weeks. Each culture medium had 0.4 mg∙L–1 of 6-benzyladenine (BA), 0.1 mg∙L−1 of indole-3 acetic acid (IAA), and 2.0 mg∙L−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) added to it. Results showed that MS medium was most effective in callus induction. When 100 mg∙L−1 of myo-inositol was added to each medium, the callus induction rate increased. Auxin and cytokinin combination treatment was more effective with the addition of 0.4 mg∙L−1 of BA, 0.1 mg∙L−1 of IAA, and 2.0 mg∙L−1 of 2,4-D to MS medium compared to the addition of 0.1 mg∙L−1 of BA, 2.0 mg∙L−1 of IAA, and 0.4 mg∙L−1 of 2,4-D. With AgNO3 treatment, the highest callus induction rate was found at a concentration of 25 mg∙L−1. The callus induction rate increased as the AgNO3 concentration increased; however, a significant decrease was seen at 30 mg∙L−1. In the case of Fe-EDTA, the most effective concentration for callus induction was 25 mg∙L−1. Therefore, supplementing myo-inositol, AgNO3 and Fe-EDTA can help in anther-derived callus induction in Seolhyang strawberries. Key words: Anther culture, auxin, B5 medium, callus induction, cytokinin, haploid breeding, NLN medium.

Somatic embryogenesis of East Kalimantan local upland rice varieties

Somatic embryogenesis is the formation, growth and development of embryos from somatic cells. Somatic embryo induction is one of the in vitro plant propagation techniques that is very important for plant developmental purposes. Four local upland rice varieties of East Kalimantan, Mayas Pancing, Gedagai, Siam and Serai, were used in this study. A total of 200 explants (mature rice grains) for each varieties were inoculated on MS solid medium supplemented with 1 mg L-1 2,4 Dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L-1 6-Benzylaminopurine (BAP). The results showed that response of each variety differed to embryosomatic induction, indicated by callus induction rate and callus quality, in terms of callus color and structure. The fastest callus formation was sobserved in Gedagai variety (8 days) while Mayas Pancing (13 days) was the latest one. The rate of callus induction varied from 60 to 98.5 %, and Serai variety has the highest callus induction rate. The highest friable callus structure was found in Siam variety (89.1%) and the lowest was in Gedagai (62.5%). Callus color was dominated by the yellowish-white (transparent) on all varieties tested. Most of the callus was potential as embryogenic callus characterized from the nodular and globular of friable callus structure and its yellowish-white color.

Experimental Paper. In vitro synthesis of mucilage in Plantago ovata Forsk affected by genotypes and culture media

SummaryIntroduction: Psyllium (Plantago ovata Forsk) is medicinally used mainly for its mucilage content. Objective: In the present study, an attempt was made to improve mucilage yield under in vitro callus culture using different genotypes, explants and culture media. Methods: The effects of a range of concentrations of plant growth regulators including 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) were evaluated on mucilage synthesis under in vitro culture using cotyledon, hypocotyl and seed explants. Fourteen genotypes originating from different geographical regions of Iran were used to evaluate their response to in vitro mucilage synthesis. Results: The highest rate of callus induction (76%) and callus growth rate CGR (0.38 mm/day) were induced on MS medium supplemented with 0.5 mg/l 2,4-D and 1 mg/l Kin and the hypocotyl explant. The results of analysis of variance showed significant genotypic differences for callus induction, CGR and mucilage content of callus and seeds. The mucilage content ranged from 0.38 to 0.08 (g/g DW) and 0.13 to 0.042 (g/g DW) for callus and seed, respectively. The superior callus induction (73%), CGR (0.45 mm/day) and mucilage content of callus (0.38 g/g DW) was denoted to Po1 genotype. The callus produced nearly three times more mucilage than the seeds using superior genotype (Po1). Conclusion: The results of this study revealed that high efficiency of callus culture of P. ovata using hypocotyl explant accompanied by the exploration of genetic diversity are important to improve the yield of mucilage synthesis by in vitro callus culture.

Embryo and callus induction by different factors in ovary culture of cucumber

Haploid and doubled haploid lines can be obtained in a short time using in vitro methods. In this study, unfertilized ovaries of cucumber were harvested and placed on semi-solid MS media as explants in Petri dishes. The thermal shock pretreatment, cucumber genotypes, hormonal combinations and AgNO 3 were evaluated as experimental factors in three consecutive experiments. The results of first experiment revealed that the thermal shock pretreatment had a significant influence on embryo induction, and the highest rate of embryogenesis was produced in presence of thermal shock pretreatment and the NAA+2,4-D+KIN+BAP (0.5+0.7+1+1.8) mg/L hormonal combination. The highest rate of callus induction was recorded in combination of absence thermal shock and the NAA+2,4-D+KIN+BAP (1.5+0.7+1+1.8) mg/L hormonal combination. According to the second experiment results, genotypes and the other hormonal combinations in media culture had highly significant effects on embryo and callus induction. The NBDC6 * 6/32441 genotype had the highest effects on these traits. A combination of BAP (4 mg/L) and 2,4-D (1.5 mg/L) was found to be optimal for embryogenesis. In third experiment, the highest level of embryogenesis (24.93%) and callus (24.19%) induction were found in the local Iranian cultivar in comparison to NBDC6 * 6/32441 genotype. Silver nitrate treatment had significant effects on the embryo and callus induction. The highest rate of embryo induction was recorded in presence of silver nitrate; however the absence of AgNO 3 had a positive effect on the callus induction. In conclusion, the thermal shock pretreatment, silver nitrate, genotype and hormonal combination factors could play key roles in embryo and callus production, independently and simultaneously.

Investigation of explants sterilization process, effect of light intensity and concentration of agar for callus induction of Kappaphycus alvarezii (Doty) doty (Rhodophyta) in vitro

Objective of this study was to ascertain the optimum condition for callus induction of K. alvarezii (Doty) in vitro such as to determine the explants sterilization process, the effect of intensity of light and the concentration of agar. Fresh thalli treated with 0.5% - 1% detergent for 5 mins followed by 0.5% - 1% betadine for 2 – 3 mins and incubated with 0.5% - 1% broad spectrum antibiotic mixture in PES medium for 1 day produced 95 – 98% bacteria free healthy explants. Two independent experiments with light intensity and agar contentration of the environment were carried out at 5 different levels of 0, 5, 25, 50, 70 µmol photon.m2.s-1 and 9 agar concentrations of 0.5%, 0.75%; 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.5%, 3.0%. The highest callus induction rate was (96 ± 3.5 – 98 ± 2.1%) at 5 - 25 µmol photon.m-2.s-1 and (87 ± 5.8% – 90 ± 5.0%) in 1% - 3% agar concentration after 2 weeks of explants. The highest callus living rate was 98% at the light intensity of 25 µmol photon.m-2.s-1 and (75 ± 5.7 – 84 ± 1.1%) in 0.75 – 1.5% agar concentration after 2 months of explants. The highest callus re-induction rate was 50 – 55% at the light intensity of 5 – 25 µmol photon.m-2.s-1 and 60 – 65% in 1 – 1.5% agar concentration. Callus was not observed in dark condition (0 µmol photon.m-2.s-1). These calluses, that were strong, big and had filamentous type, will be a good material for the next production stage of embryonic callus production and seedling regeneration from micropropagules.

Anther Culture and Plant Regeneration of Tetraploid Purple Coneflower (Echinacea purpurea L.)

Anthersisolated from tetraploid purple coneflowerplants were cultured in vitro. The highest callus induction rate was obtained when the medium was consisted of N6 basal elements, 4% sucrose, 0.5 mg?L?1 BA, and 0.10 mg?L?1 NAA. Various morphogenesis such as globular, heart-shape, torpedo-shapeand final state embryos as well asvarious texture calluses around were observed. Out of 110 plantlets regenerated, 104 were confirmed as diploid and the rest were as tetraploid. Plants of one diploid offspring strain presented aspecialcharacter in pot: unlike the original tetraploid plants, it grown tubular, bisexual ray florets. The results obtained in the present studies indicated that although the tetraploid purple coneflower plants produced only diploid microspores, the recovery of some useful mutants through in vitro anther cultures might be reasonably expected.

Androgenic potential and anther &lt;i&gt;in vitro&lt;/i&gt; culture of &lt;i&gt;Lagenaria siceraria&lt;/i&gt; (Molina) Standl an edible-seed cucurbit

Lagenaria siceraria Molina has a high productive potential of fruits but the seeds weight remain low. The aim of this study is to regenerate doubled haploids from androgenesis in order to create genotypes with good agronomic characteristics. For this purpose, flower buds of six accessions namely NI185, NI202, NI215, NI276, NI356 and NI271 were pre-treated at 4 °C for 0, 1, 2, 4, 6, 7 days. To assess the ability of these accessions to induce callus, the anthers excised from the collected floral buds were cultured following two standard protocols referred to as Metwally et al. and Kumar and Murthy, respectively. Histological sections were performed with buds of different sizes in order to select pollen at uni-nucleate stage, the most appropriate for the androgenesis. The histological sections showed that the pollen is at uni-nucleate stage when the bud size varied from 5 to 7 mm. No callus production was observed with the protocol of Metwally et al. whereas a significant formation of callus were expressed on medium corresponding to the protocol of Kumar and Murthy. The best rate (79%) of callus induction was obtained after one and two days of pre-treatment at 4 °C. The highest rate of callus induction was observed with the accessions NI271, NI276 and NI356. © 2015 International Formulae Group. All rights reserved. Keywords: Cucurbitaceae, anther culture, thermal shock, androgenesis, plant regeneration

Mucilage Synthesis in Callus Cultures of Plantago ovata Forsk

The seed husk of Plantago ovata known as psyllium (Isabgol) yields medicinally important mucilage. The amount of mucilage produced is about 25 % (by weight) of the total seed yield. In the present study, an attempt was made to increase the amount of mucilage through callus cultures of P. ovata. The first step involved establishment of callus cultures in P. ovata. Leaf explants from 10 to 20 day old seedlings were cultured on MS basal medium supplemented with various concentrations of different plant growth regulators. The highest rate of callus induction (89 %) was obtained on MS medium containing 0.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 Kinetin. The mucilage content was estimated from the callus obtained in different media. The best mucilage production was obtained in the MS medium supplemented with 0.25 mg l−1 2,4-D and 0.25 mg l−1 thidiazuron. Significant differences with regard to the total mucilage content were recorded. Overall, the callus produced nearly five times more mucilage than the seeds. The present technology provides an alternative route to production of large quantities of mucilage without plants.