Leaf Callus Research Articles

De Novo Regeneration of Cannabis sativa cv. Cheungsam and Evaluation of Secondary Metabolites of Its Callus

Cannabis sativa L. cv. ‘Cheungsam’ is an industrial hemp plant of Republic of Korea origin, primarily cultivated for fiber and seed production. In vitro seed germination and tissue culture are valuable tools for developing various biotechnological techniques. In the present study, we aimed to develop a tissue culture process for hemp plants using Cheungsam as a model plant and examine the secondary metabolites produced from its callus. We also developed a method to prepare pathogen-free seedlings from field-derived seeds using hydrogen peroxide (H2O2) solution as a liquid germination medium. Treating seedlings with removed seed coat in 3% H2O2 significantly reduced the contamination rate. Callus formation and de novo organogenesis of shoots and roots from callus were successfully achieved using cotyledon and leaf tissues prepared from the pathogen-free seedlings. The most effective in vitro regeneration results were obtained using the Murashige and Skoog (MS) medium supplemented with certain targeted growth regulators. An optimal combination of 0.5 mg/L thidiazuron (TDZ) and 1.0 mg/L 1-naphthalene acetic acid proved highly effective for callus induction. The addition of 0.5 mg/L TDZ in the MS medium significantly stimulated shoot proliferation, while robust root development was best supported by MS medium supplemented with 2.5 mg/L indole-3-butyric acid for both cotyledon and leaf explants. Finally, gas chromatography–mass spectrometry (GC–MS) analysis of ethanol extract from Cheungsam leaf callus revealed the presence of different secondary metabolites, including 9-octadecenamide, methyl salicylate, dodecane, tetradecane, and phenol, 2,4-bis-(1,1-dimethylethyl). This study provides a comprehensive de novo regeneration protocol for Cheungsam plants and insight into the secondary metabolite profiles of its callus.

Insights into the bZIP gene family in Osmanthus fragrans and the role of OfbZIP98 in the callus and flower

Osmanthus fragrans has important ornamental and economic value. As the callus re-differentiation process is difficult, a genetic transformation system has not been successfully established in O. fragrans. The basic leucine zipper (bZIP) transcription factors play an important role in plant growth and development. A total of 116 OfbZIP genes were identified in O. fragrans using bioinformatics methods. According to the evolutionary relationships, these genes were divided into 12 subfamilies and were found to be unevenly distributed on 23 chromosomes of O. fragrans. The quantitative real-time PCR results showed that the expression trend of OfbZIP98 was consistent with the proliferation of O. fragrans callus. The subcellular localization and yeast self-activation results showed that OfbZIP98 was localized in the nucleus and had self-activation activity. Further, phenotypic observation of over-expressed tobacco showed that compared with the wild type (WT), the transgenic strain formed callus 4 d earlier, and callus re-differentiation began on day 15. Analysis of the callus differentiation rate showed that after 20 d of culture, the differentiation rate of the leaf callus of the transgenic strain was about twice that of the WT plants. In addition, measurements of corolla diameter, corolla circumference, corolla area, and corolla tube diameter indicated that the petals of the transgenic strain were significantly larger than the WT plants. Our results showed that OfbZIP98 has the important function of promoting callus growth, re-differentiation, and flower organ enlargement. These findings provide new insights into the potential role of OfbZIP98 in the growth and development of O. fragrans.

FORMULATION OF NANOSTRUCTURED LIPID CARRIER GEL FROM CALLUS EXTRACT OF MULBERRY LEAF (MORUS ALBA L.) WITH 2, 4-DICHLORO PHENOXY ACETIC ACID AND BENZYL AMINO PURINE AS CALLUS GROWTH FACTOR

Objective: This research aimed to formulate the callus extract of mulberry leaf in the form of a Nanostructured lipid Carrier (NLC) gel. Methods: Dichlorophenoxyacetic acid (2,4–D) and Benzyl Amino Purine (BAP) was used as a callus growth factor. Callus leaf extracted with ethanol 96% by maceration-sonication method. An amount of 0.5% callus leaf extract was formulated into NLC. The NLC is then evaluated for its particle size and polydispersity index. The NLC gel is evaluated for its organoleptic, homogeneity, viscosity, flow ability, and pH. The callus extract and the NLC gel were also evaluated for their tyrosinase inhibitor activity. Results: The best formulation of NLC showed a particle size of 189.8 nm and a polydispersity index of 0.578. The NLC is a semi-solid, yellowish, odorless, homogeneous gel, with viscosity of 26,666.67 cPs, plastic-thixotropic type, pH of 5.26. The evaluation of tyrosinase inhibitory activity of the callus extract and the NLC gel showed IC50 value of 217.64 and 248.12 ug/ml. Conclusion: It can be concluded that leaf callus extract of mulberry can be formulated into an NLC gel that is physically and chemically stable and has good skin-lightening activity.

An Efficient In Vitro Shoot Organogenesis and Comparative GC-MS Metabolite Profiling of Gaillardia pulchella Foug

Gaillardia pulchella Foug. is a widely studied plant because of its high pharmacological and ornamental value. The leaves of G. pulchella were used for inducing callus and subsequent plant regeneration as it is the primary source of phytocompounds. The purpose of the present investigation was to formulate an in vitro propagation method for Gaillardia by using leaf explants in MS (Murashige and Skoog) medium. The best callus induction was observed on high (2.0 mg/L) α-naphthalene acetic acid (NAA) and a low (0.5 mg/L) 6-benzylaminopurine (BAP) with callus induction frequency of 91.66%. The leaf callus also demonstrated high caulogenesis ability (95.83%), with an average 5.2 shoots/callus mass at 0.5 mg/L BAP and 2.0 mg/L NAA. Indole Acetic acid (IAA) at 1.0 mg/L had the maximum rooting percentage (79.17%) with 12.4 roots per shoot. Rooted plantlets were later transferred to greenhouse conditions, showing a survivability rate of 75–80%. The physiological parameters, i.e., phenolic compounds and the flavonoids’ level, in the DPPH assay were higher in leaves obtained in vitro compared to callus formed from leaves and field-obtained (mother) leaves. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of leaves (in vivo and in vitro) and leaf callus presented a wide array of compounds. In callus extract, some 34 phytocompounds were identified. Some of them were 3-hydroxy-2,3-dihydromaltol (25.39%), isoamyl acetate (11.63%), palmitic acid (11.55%), 4-methyloxazole (7.54%), and 5-methoxypyrrolidin-2-one (7.49%). Leaves derived in vivo and in vitro had 45 and 28 phytocompounds, respectively, belonging to different classes like lignans, phenols, terpenoids, alkaloids and fatty acids, etc. Those findings demonstrated that the leaf derived callus and the leaves are the potential stable source of several compounds with medicinal importance. The developed protocol may provide an alternative source of compounds without affecting wild flora.

Antioxidant and Anticancer Activity of Tannins Isolated from Callus Cultures of Achyranthes aspera L.

In the current study, the impact of oxidative stress induced by free radicals generated during metabolic processes was investigated. It was found to be linked to a variety of diseases due to their detrimental effects on nucleic acids and proteins. The primary focus remained on investigating the free radical-scavenging properties and potential anticancer activities of tannins found in callus cultures of A. aspera. It was observed that the induction of callus formation was notably successful when using leaf and root explants, resulting in a callus index as high as 160, as compared to stem explants in the presence of auxins. The extraction yield of callus tannins was the most abundant in chloroform extracts, although the overall antioxidant activity was comparable among chloroform, methanol, and petroleum ether callus extracts. Among these extracts, the chloroform extract from stem callus cultures grown on the MSDN medium showed the highest total antioxidant activity. Notably, tannins extracted from A. aspera leaf callus extracts displayed significant anticancer effects against the Jurket cell line. These effects were evaluated through measures such as cell viability and colony formation. The anticancer activity was notably higher in callus culture extracts as compared to the control. In summary, the results showed that in vitro biomass production can be a valuable approach for augmenting the production of bioactive compounds, such as tannins, from selected medicinal plants including A. aspera. Tannins extracted from A. aspera hold promise for their potential role in the development of new medicines.

Somatic embryogenesis and plant regeneration from leaf callus with genetic stability validation using SCoT markers in Paramignya trimera, a medicinal plant native to Vietnam

Xao tam phan (Paramignya trimera (Oliv.) Guillaum) is a medicinal plant native to Vietnam, that is renowned for its therapeutic properties, particularly for the treatment of various ailments, including cancer. This study investigated in vitro propagation of P. trimera through somatic embryogenesis and shoot organogenesis using leaf callus. Various culture media, plant growth regulators, malt extract, and carbon sources were evaluated to optimize callus induction and somatic embryo formation from leaf explants. DNA barcoding confirmed 96.96% to 100% homology with P. trimera specimens from Khanh Hoa province, Vietnam. The highest callus formation rate, reaching 100%, was observed in one-year-old explants cultured in Woody Plant Medium (WPM) supplemented with 2.0 mg L-1 naphthaleneacetic acid (NAA) and 0.2 mg L-1 benzylaminopurine (BAP) in the dark for over six weeks. In WPM supplemented with 30 g L-1 sucrose, 4.0 mg L-1 BAP, and 500 mg L-1 malt extract, globular stage embryos developed into embryoids and shoots and buds clumped at 10 and 18 weeks, respectively. Shoot organogenesis was observed in WPM supplemented with 30 g L-1 sucrose and 0.07 mg L-1 thidiazuron (TDZ) after 18 weeks of culture. Genetic fidelity assessments using 12 SCoT markers indicated that in vitro plantlets were homologous to the mother plant. This study provides a viable method for the conservation and sustainable cultivation of Xao tam phan, ensuring a stable supply of this valuable medicinal resource.

Callus cell and explants regeneration, glucose, mineral, antioxidant and flavonoid content development using broccoli root tip and leaf cutting in vitro.

The study was conducted to evaluate the root, shoot and leaf callus cell regeneration and its biochemical properties like antioxidant, carbohydrate, pigment and mineral content from broccoli root, shoot and leaf cutting in vitro. An in vitro factorial experiment was carried out based on a Completely Randomized Design (CRD) with 5 replicates in tissue culture applying different IBA (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mg/l) and BAP (1 mg/l) concentrations using broccoli root tip and leaf cutting. The results showed that a higher callus weight was found in the cultured leaf cutting than in root tip cutting in the concentration of 1.0, 1.5 & 2.0 mg/l IBA + 1.0 mg/l BAP combination. The highest callus weight was found in the cultured leaf cutting than root tips cutting at the concentration of 1.5mg/l IBA+1.0 mg/l BAP. Furthermore, the highest inverted sugar and glucose, chlorophyll and nutrient content (K+, NO3- & Ca++), total phenol, flavonoid and total antioxidant were found in the concentration of 1.5mg/l IBA+1.0 mg/l BAP combination in both broccoli leaf and root cutting. The results seemed that it was best to use the combination of the IBA and BAP in the concentration of 1.0-2.0 mg/l and 1mg/l to regenerate root, leaf and callus cell proliferation of broccoli from the root tip and leaf cutting.

Effects of plant growth regulators on the callus of rose leaves

In the present study effects of different plant growth regulator combinations on leaf callus growth in two Rosa hybrida cultivars, Rosa hybrida ‘Ingrid Bergman’ and Rosa hybrida ‘Xindongfang’, were analyzed. The plant growth regulators were added into MS, MS + 2,4-D (1.5 mg/l, 2.5 mg/l, 3.5 mg/L, and 4.5 mg/l) and MS + 2.0 mg/l NAA + 1.0 mg/l ZT, MS + 2.0 mg/l NAA + 1.0 mg/l KT, MS + 2.0 mg/l NAA + 1.0 mg/l 6-BA and MS + 2.0 mg/l NAA. The leaves were treated as broken and cultured on these nine different media types. Results showed significant differences in color, morphology, and callus quantity. In the first medium, the callus of rose leaves was the best on MS + 4.5 mg/l 2,4-D medium, followed by MS + 2.5 mg/l 2,4-D and MS + 3.5 mg/l 2,4-D, and finally MS + 1.5 mg/l 2,4-D. No callus was found in MS medium. In the second kind of medium, the leaves grow callus, and the callus grew best on MS + 2.0 mg/l NAA + 1.0 mg/l ZT medium, followed by MS + 2.0 mg/l NAA + 1.0 mg/l KT, finally MS + 2.0 mg/l NAA + 1.0 mg/l 6-BA, MS + 2.0 mg/l NAA medium, the callus grew loose and dry, and lost luster after a few days. Therefore, 2,4-D, KT, ZT, 6-BA, and NAA can promote the formation of callus. With the increase of concentration of 2,4-D, the callus induction rate increased, and the color of the callus also changed significantly. The combination of KT, ZT, and NAA had an obvious effect on callus induction and expansion. Bangladesh J. Bot. 52(3): 867-873, 2023 (September)

Shoot Organogenesis and Regeneration from Leaf Seedlings of Diospyros oleifera Cheng.

Persimmons (Diospyros) are economically important trees that are widely cultivated for wood production in China, and Diospyros oleifera Cheng is the main persimmon grafting stock. However, an efficient tissue culture system has not been perfected for D. oleifera due to the limits of proliferation and rooting cultures. Therefore, this study examined the effects of different plant growth regulators and concentrations on the primary culture of young embryos, induction of leaf callus, differentiation of adventitious shoots, and rooting culture of D. oleifera. The optimal formula for young embryo germination was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg/L gibberellic acid (GA3); after 25 days, the sprouting rate of the young embryos was 67.3%. The best medium for leaf callus induction was 1/2MS medium containing 2.0 mg/L of 6-benzylaminopurine (6-BA) and 0.5 mg/L of naphthaleneacetic acid (NAA), and the callus induction rate was 88.9%. Then, the callus was transferred to 1/2MS medium containing 2.0 mg/L of zeatin (ZT), 0.5 mg/L of NAA, and 2.0 mg/L of thidiazuron (TDZ) to induce adventitious shoots; after 25 days, 5.4 buds were produced per explant, and the induction rate of the adventitious shoots was 88.3%. The adventitious shoots were transferred to 1/2MS medium containing 2.0 mg/L of ZT, 2.0 mg/L of 6-(γ,γ-dimethylallylamino)purine (2iP), and 0.1 mg/L of indole acetic acid (IAA) for the proliferation culture, for which the multiplication coefficient approached 7.5. After multiplication, the adventitious shoots were inoculated into 1/2MS medium containing 1.0 mg/L of indole butyric acid (IBA), 0.5 mg/L of NAA, and 1.0 mg/L of kinetin (KT); the rooting rate was 60.2%, and the average number of roots was 6.9.

Screening of Preliminary Phytochemicals and In vitro Antibacterial Activity of Methanolic Extracts of Stem, Leaf, Root and Leaf Callus of Corynandra felina (L.f.) Cochrane & Iltis

Screening of Preliminary Phytochemicals and In vitro Antibacterial Activity of Methanolic Extracts of Stem, Leaf, Root and Leaf Callus of Corynandra felina (L.f.) Cochrane & Iltis

In vitro establishment of cell suspension culture of Ceropegia bulbosa for improved production of cerpegin content through elicitation of engineered carbon and ZnO nanoparticles.

The present investigations aimed to conserve C. bulbosa a threatened plant species and for production of cerpegin through cell culture technology using ENP elicitation. Leaf explants were aseptically cultured with normal MS medium-supplemented PGRs BA and NAA various concentrations, and the best callus induction response was recorded on 4.5 + 4.5 μM. The prospective special effects of the ENPs on plant cell cultures are the key part of our study and used to evaluate leaf callus culture proliferation with the reduction of browning, establishment, biomass, and metabolite formation. The CNP concentrations (0, 2, 4, 8, 12 mg/l) are used for the callus proliferation and browning reduction. The cell suspension cultures are also established, and they were elicited with EZnONPs (0, 25, 50, 100, 150 mg/l) for evaluation of biomass, antioxidant, non-antioxidant enzyme activation, toxicity, ROS defense activation, and metabolite development in cell cultures. The metabolite extraction, UV, and NMR characterization confirm that the toxic and nontoxic effect of ENPs on leaf cell cultures varies with high to low concentration.

Effect of abiotic elicitor on the production of camptothecin from Nothapodytes nimmoniana (J. Graham) Mabb

The plant Nothapodytes nimmoniana belongs to the Icacinaceae family, and is a significant source of camptothecin (CPT), an effective quinoline alkaloid with an array of pharmacological benefits including anti-cancer, anti-HIV, antibacterial, and antioxidant etc. The elicitation method is the most effective strategy to increase and maintain the synthesis of particular compounds in vitro culture. In this study, to understand the impact of various abiotic elicitors on CPT production by using established N. nimmoniana cell suspension culture obtained from friable leaf callus grown in Murashige & Skoog basal media with sucrose and agar augmented with 2 mg/L of 2,4-dichloro phenoxy acetic acid (2,4-D). A total of six abiotic elicitors were used such as cadmium chloride, ethanol, salicylic acid, tryptophan, copper chloride, and calcium chloride. The impact of these abiotic elicitors on the CPT yield at three different concentrations by cell suspension was evaluated for up to 15 days. High-performance liquid chromatography (HPLC) was used to quantify the CPT production. A maximum of 9.43 and 6.26-fold increment was recorded in CPT production in suspension culture elicitated with 2 mM of tryptophan and 1 mM of tryptophan on the 12th day of inoculation followed by ethanol 7.5% treated cell suspension which enhanced the CPT production up to 1.44-fold increase while compared to control. The cell viability of the suspension culture treated with calcium chloride and cadmium chloride was observed in decreased biomass as per the increment in the concentration. The findings of the current study indicate the potential use of abiotic elicitation as another possible option for boosting up the synthesis of CPT in N. nimmoniana cell suspension cultures.

The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis.

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

An Efficient Transformation System for Fast Production of VcCHS Transgenic Blueberry Callus and Its Expressional Analysis.

The Agrobacterium tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of cv. 'Legacy' and 'Northland' were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with A. tumefaciens was operated on WPM plus 100 μM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L-1 2,4-D, 0.4 mg·L-1 6-BA, 50 mg·L-1 hygromycin, and 200 mg·L-1 cefotaxime. The VcCHS transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of cv. 'Northland'. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.

Novel R2R3-MYB Transcription Factor LiMYB75 Enhances Leaf Callus Regeneration Efficiency in Lagerstroemia indica

Lagerstroemia indica is an important woody ornamental plant worldwide. However, the application of many technologies, such as transgenic breeding and genome editing, has been severely hampered due to the lack of efficient calli induction and regeneration technology. Here, we discussed a reliable and efficient calli induction and regeneration protocol using whole-leaf explants. This protocol’s effectiveness for the calli induction and regeneration systems in crape myrtle were up to 70.33% and 44.33%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was created from leaf calli, and the green fluorescent protein (GFP) was able to detect up to 90% of its positive frequency. Meanwhile, two positive lines’ transfer DNA insertion sites and directions were identified using whole genome sequencing. LiMYB75, a novel R2R3-MYB transcription factor, was identified and transferred to the L. indica genome to enhance the leaf calli regeneration frequency. Surprisingly, overexpressing LiMYB75 increased the frequency of calli regeneration in the leaf by 1.27 times and the number of regenerated plantlets per callus by 4.00 times compared to the wild type, by regulating the expression levels of genes involved in callus formation, such as SHOOT MERISTEMLESS (STM). Overall, our findings revealed a simple, reliable, and highly efficient transformation approach and identified the desirable candidate gene LiMYB75, which improves L. indica’s calli regeneration efficiency.

PROPAGATION PROTOCOL OF THE MEDICINAL PLANT – ALOE VERA USING TISSUE CULTURE

Aloe vera is one of the most popular cactus-type plants in the global market due to its widespread uses in pharmaceutical, cosmetic, food, and decorative purposes. The present study derived callus cultures from the Aloe vera plant leaves, then reproduced on agar-solidified MS medium from June to December 2021 at the University of Mosul, Iraq. Results revealed that the MS medium + 3.0 mg L-1 benzyl adenine (BA) proved suitable for induction of leaf callus up to 85%, while the MS medium supplement with 1.0 and 2.0 mg L-1 BA reached 70%. The MS medium with 1.0 mg L-1 BA showed the best results for growing apical shoots of A. vera plants and producing vegetative branches. The formation of roots emerged within two weeks after placing them on the rooting medium. The shoots regenerated from the growing apices and were rooted easily in agar-solidified MS medium. The obtained plants attained successful acclimatization in terms of their growth and length, afterward, transferred to the peat-moss mixture.

Establishment of a Highly Efficient In Vitro Propagation System of Diospyros lotus

Persimmon (Diospyros) is an economically important tree widely cultivated for woody grain production in China, and Diospyros lotus is mainly used as the grafting stock of persimmon. However, the breeding of stress-resistant rootstocks of D. lotus using molecular means has yet to be achieved; in particular, an efficient blade-regeneration system has not been perfected to date. This study examined the effects of different plant-growth regulators and concentrations on the primary culture of stems with buds, the induction of leaf callus, the differentiation of adventitious shoots, and rooting culture of D. lotus. The optimal formula for inducing axillary buds from stems with buds was 1/2 Murashige and Skoog (MS) medium containing 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L naphthaleneacetic acid (NAA), in which the induction rate of axillary buds approached 67.1%. The best medium formula for leaf callus induction was 1/2 MS medium containing 2.0 mg/L 6-BA and 0.5 mg/L NAA. Then callus was transferred to 1/2 MS medium containing 2.0 mg/L 6-(γ,γ-dimethylallylamino)purine (2iP), 2.0 mg/L thidizuron (TDZ), and 40 g/L sucrose to induce adventitious shoots after dark culture for 48 h, resulting in 7.9 shoots per explant and a 75.2% induction frequency of adventitious shoots. In addition, it was difficult to induce adventitious shoots from callus after six times of continuous transfer and differentiation. The adventitious shoots were transferred to 1/2 MS medium containing 2.0 mg/L zeatin (ZT) and 2.0 mg/L 2iP for proliferation culture, in which the multiplication coefficient approached 7.6. The adventitious shoots after multiplication were inoculated into 1/2 MS + 1.0 mg/L IBA + 0.5 mg/L NAA medium, the rooting rate was 70.2%, and the average number of heels was 9.6. Thus, studies in this area are expected to facilitate rapid and excellent growth, as well as theoretical support for factory saplings’ care and molecular breeding.

High Efficiency Regeneration System from Blueberry Leaves and Stems.

The main propagation approach is tissue culture in blueberries, and tissue culture is an effective and low-cost method with higher economic efficiency in blueberries. However, there is a lack of stable and efficient production systems of industrialization of tissue culture in blueberries. In this study, the high-efficiency tissue culture and rapid propagation technology system were established based on blueberry leaves and stems. The optimal medium for callus induction was WPM (woody plant medium) containing 2.0 mg/L Forchlorfenuron (CPPU), 0.2 mg/L 2-isopentenyladenine (2-ip) with a 97% callus induction rate and a callus differentiation rate of 71% by using blueberry leaves as explants. The optimal secondary culture of the leaf callus medium was WPM containing 3.0 mg/L CPPU with an increment coefficient of 24%. The optimal bud growth medium was WPM containing 1.0 mg/L CPPU, 0.4 mg/L 2-ip, with which the growth of the bud was better, stronger and faster. The optimal rooting medium was 1/2 Murashige and Skoog (1/2MS) medium containing 2.0 mg/L naphthylacetic acid (NAA), with which the rooting rate was 90% with shorter rooting time and more adventitious root. In addition, we established a regeneration system based on blueberry stems. The optimal preculture medium in blueberry stem explants was MS medium containing 2-(N-morpholino) ethanesulfonic acid (MES) containing 0.2 mg/L indole-3-acetic acid (IAA), 0.1 mg/L CPPU, 100 mg/L NaCl, with which the germination rate of the bud was 93%. The optimal medium for fast plant growth was MS medium containing MES containing 0.4 mg/L zeatin (ZT), 1 mg/L putrescine, 1 mg/L spermidine, 1 mg/L spermidine, which had a good growth state and growth rate. The optimal cultivation for plantlet growth was MS medium containing MES containing 0.5 mg/L isopentene adenine, with which the plantlet was strong. The optimal rooting medium for the stem was 1/2MS medium containing 2.0 mg/L NAA, with which the rooting rate was 93% with a short time and more adventitious root. In conclusion, we found that stem explants had higher regeneration efficiency for a stable and efficient production system of industrialization of tissue culture. This study provides theoretical guidance and technical support in precision breeding and standardization and industrialization in the blueberry industry.

Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 μM NAA + 5 μM kinetin (ODK3) or 5 μM NAA + 0.5 μM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 μM IBA + 2.5 μM kinetin (ODY1) or 5 μM NAA + 0.5 μM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

Cretan Dittany (Origanum dictamnus L.), a Valuable Local Endemic Plant: In Vitro Regeneration Potential of Different Type of Explants for Conservation and Sustainable Exploitation

Origanum dictamnus L. is a medicinal local endemic to the Island of Crete, Greece. Its propagation through biotechnological tissue culture techniques is essential due to its augmented multi-industrial sector demand. For direct organogenesis, among different culture media variants (MS, Gamborg B5), and cytokinins [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyl adenine (2-iP)], the MS + added with BA (2.2 μM) was the most effective treatment for shoots and roots formation. For indirect organogenesis, all explant types (leaves, petioles, roots) showed a 100% callusing rate after 2 months in all media variants tested; ODK1: 20 μM thidiazuron (TDZ) + 5 μM indole-3-butyric acid (IBA) or ODK2: 0.5 μM kinetin + 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D). The leaves and petiole explants assured a low rate of shoot regeneration (20%) in ODK1. Afterwards, leaf-, petiole-and root-callus derived from both media were transferred to four new media plant growth regulators-free or with BA + IBA + gibberellic acid (GA3). After 10 months from callus transferring, the petiole callus gave rise to roots (20-75%) while the leaf callus exhibited 10-30% shoot or 30% root regeneration. In this study, indirect organogenesis of O. dictamnus was carried out for the first time, thus various organs can be used for plant regeneration, and the developed protocol may be applicable in the horticulture industry.