Efficient Micropropagation System Research Articles

Establishment of a Highly Efficient Micropropagation System of Aquilaria crassna Pierre ex Lecomte

Aquilaria crassna Pierre ex Lecomte is a principal species renowned for its production of agarwood. However, the active components of agarwood are not universally in compliance with the standards set by the Chinese Pharmacopoeia. We have identified an elite A. crassna tree with agarotetrol and alcohol extract levels that exceed these standards and have successfully established a stable in vitro micropropagation system using stem segments from this elite tree. The effects of auxins and minerals on axillary-bud induction, shoot multiplication, and rooting were investigated. The most effective medium for axillary-bud induction was a half-strength Murashige and Skoog (1/2MS) medium supplemented with 0.50 mg/L 6-benzylaminopurine (6-BA), achieving an induction rate of 53.33% with minimal hyperhydricity. The optimal shoot proliferation medium was an MS medium with 0.40 mg/L 6-BA, yielding a propagation coefficient of 2.96 without hyperhydricity. The best rooting medium comprised quarter-strength MS (1/4MS) macroelements and 1/2MS microelements with 0.10 mg/L naphthaleneacetic acid (NAA), resulting in an 82.54% rooting rate. Substrate effects on transplant survival and growth were also evaluated, and peat soil was identified as the best substrate, achieving a survival rate of 96.67%. This study introduces a straightforward and efficient in vitro micropropagation system utilizing mature A. crassna as explants. It holds significant importance for the consistent production of agarwood that complies with the standards of the Chinese Pharmacopoeia and provides a model for the targeted breeding of medicinal plants.

Development of an efficient micropropagation system for Dioscorea bulbifera L. and phytochemical profile of regenerated plants

BackgroundThe present study was designed to develop an improved in vitro regeneration system for Dioscorea bulbifera using mature nodal explants. Direct organogenesis from nodal segments was achieved by culturing the nodal explants on Murashige and Skoog medium supplemented with 3.5 mgl−1 6-benzylaminopurine (BAP). Shoot multiplication was widely affected by the kind and concentration of plant growth regulators, and the sub-culturing of in vitro regenerated shootlets on fresh medium. After incubation for 4 weeks at optimum BAP concentration, cultures were transferred to secondary medium with BAP (optimized concentration) supplemented with different auxins. ResultsOn medium with 3.5 mgl−1 6-benzylaminopurine, maximum regeneration (87 ± 1.85%) with an average shoot number of 2.0 ± 0.08, and length of 3.5 ± 0.04 cm were observed after 4 weeks of incubation. Maximum number of shoots (3.9 ± 0.21) was observed with 3.5 mgl−1 BAP in combination with 0.75 mgl−1 indole-3-acetic acid. The best root formation was observed on ½ MS medium supplemented with 0.75 mgl−1 α-naphthalene acetic acid, with 4.7 ± 0.03 roots per shoot. The well-rooted plantlets were successfully acclimatized in with 100% survival rate. The plantlets grew well with normal growth, flowering and showed, by High performance thin layer chromatography, almost same number of phytoconstituents compared with the mother plant. ConclusionsThis is the first study on comparative phytochemical analysis of wild growing and in vitro regenerated plants of D. bulbifera which validates the medicinal and nutritional properties of in vitro raised plants.

Micropropagation of Vaccinium corymbosum L.: An Alternative Procedure for the Production of Secondary Metabolites

In vitro culture has become a dependable approach for the mass production of plant material as the market for innovative plant-derived medicinal approaches has grown significantly. Furthermore, because it permits manipulation of biosynthetic routes to boost the production and accumulation of certain compounds, this technology has enormous potential for the manufacture of natural bioactive chemicals. As a result, the goal of this study was to develop an efficient micropropagation system for biomass production and to investigate the accumulation of bioactive compounds from Vaccinium corymbosum L., Duke and Hortblue Petite cultivars. Two in vitro plant tissue culture systems were used for shoots production: a solid medium (5 g/L Plant agar) and liquid medium (Plantform bioreactor). The culture medium used was Woddy Plant Medium (WPM) supplemented with two growth regulators: 0.5 mg/L and 1 mg/L zeatina (Z) and 5 mg/L N6-(2-Isopentenyl) adenine (2iP). The content of phenolic compounds, carotenoids, and chlorophylls of the in vitro shoot extracts were examined via the HPLC-DAD-MS/MS technique. The results showed that cv. Hortblue Petite produced a higher amount of biomass compared with cv. Duke, on all variants of culture media in both systems (solid and liquid), while the shoots extract of the Duke variety in the liquid culture system (under all concentrations of growth regulators) had the highest content of total phenolic compounds (16,665.61 ± 424.93 μg/g). In the case of the lipophilic compounds analysed (chlorophylls and carotenoids), the solid medium reported the highest values, whereas media supplemented with 0.5 mg/L Z was proved to have the richest total content for both cultivars.

Impact of Stevia rebaudiana Culturing in Liquid Medium: Elevation of Yield and Biomass, Mitigation of Steviol Glycosides

An efficient micropropagation system is developed by culturing nodal segments containing the axillary shoot buds on liquid Murashige and Skoog (MS) medium. Direct shoot and root formation are highly effective in both solid and liquid MS media without any plant growth regulators (PGRs). Interestingly, a significant difference in yield is obtained between solid and liquid cultures. It is revealed that a relatively higher amount of plant biomass is obtained after culturing for 4 weeks in a liquid MS medium. However, the shoots produced on solid MS medium produce a remarkable decline in all physiological parameters. On contrary, the bioactive steviol glycosides (rebaudioside-A and stevioside) content is higher in shoots grown in solid MS on a comparative basis, which could be compensated by higher yield. After hardening off, all the regenerants are effectively grown in the field with a negligible loss (<1 %), and steviol glycosides spectra is again obtained by conducting high-performance liquid chromatography (HPLC) analysis after 10 weeks of the plantation. This method has great potential to be applied on large scale in bioreactors.

Establishment of an Efficient Micropropagation System for Humulus lupulus L. cv. Cascade and Confirmation of Genetic Uniformity of the Regenerated Plants through DNA Markers

The demand for virus-free hop planting material has increased in the last few years due to its multipurpose uses. The present study aimed to establish an effective protocol for clonal propagation of cv. Cascade using only the cytokinins as PGRs in all stages of micropropagation: (i) in vitro culture initiation using single-node micro-cuttings inoculated on modified Murashige and Skoog (MSm) medium solidified with Plant agar and supplemented with 0.5 mg L−1 6-benziyladenine (BA) with 76% recorded viability of nodal explants; (ii) in vitro multiplication of multinodal shoots on MSm medium gelled with Plant agar and supplemented with different types and concentrations of cytokinins: 2 mg L−1 kinetin (KIN), 0.7 mg L−1 1-(2-Chloro-4-pyridyl)-3-phenylurea) (1 CPPU), 2 mg L−1 meta-topoline (mT) and 0.5 mg L−1 BA, which was the best variant for shoot proliferation (9.48 ± 0.78 shoots/explant); (iii) rooting and acclimatization with the best results obtained by ex vitro rooting and acclimatization of plants in the same stage in perlite (96.00 ± 0.60% acclimatized rooted plants with 100% survival under greenhouse conditions). The true-to-type nature of in vitro raised plants with the mother plant was assessed by Random Amplified Polymorphic DNA (RAPD) and Start Codon Target Polymorphism (SCoT) molecular markers, and then their genetic uniformity were confirmed.

Asymbiotic seed germination and in vitro seedling development of Orchis militaris, an endangered orchid in Siberia

BackgroundTerrestrial orchids belonging to the Orchis genus are difficult to propagate and are under great pressure in their natural habitats. Studies regarding the influence of photoperiod and temperature regimes on Orchis militaris germination and morphological changes during immature seed development in vitro are scarce. Our aim was to identify photoperiod, temperature, and different nutrient media requirements for optimization of O. militaris seed germination and vigorous seedling production. ResultsPost-germination morphological changes were recorded with O. militaris seeds collected from 32-day-old fruits, where the percentage of O. militaris seeds without embryo was 38.4%. The highest rate of O. militaris seed germination (82.6%) was obtained on Malmgren modified terrestrial orchid medium (mM), enriched by 5% coconut water, 5% birch sap, and 0.1% AC. Nine percent of seedlings were able to reach the advanced seedling stage (stage 6) after 12 months of maintenance on this medium. In all 3 modified media (Harvais, Knudson С and Malmgren), regeneration was via the production of protocorms and seedlings without callus formation. It was proved that more abundantly vigorous protocorms were formed on the modified Harvais 2 under continuous darkness, while the subculture in Knudson C medium with AC addition could be necessary to stimulate their further development. The regeneration success of the species for in vitro conditions was increased by following its natural seasonal cycle. ConclusionThis study demonstrated an efficient micropropagation system for O. militaris using immature seeds and thus widely opened the perspectives for its conservation in nature. The favorable conditions of seed germination periods for in vitro culture, identified as the definite shift of temperatures and photoperiod regimes intrinsic to the species in nature, could improve seedling survival of this medicinally important orchid.

Establishment of an efficient micropropagation system in enhancing rooting efficiency via stem cuttings of apple rootstock M9T337

Rootstocks play a vital role in regulating the environmental adaptability and controlling the growth and development of apple trees. M9T337, an excellent apple rootstock widely used in commercial orchards, could confer dwarf tree architectures, early fruiting and suitability for high-density planting. However, the rooting ability of M9T3337 is low when it is vegetatively propagated, and researchers have not yet established an efficient micropropagation system. The present study systematically evaluated the multiplication in adventitious shoots and the in vitro formation of adventitious roots to determine the effects of the culture media and plant growth regulators of M9T337 and a rapid micropropagation system was developed. For the shoot multiplication, the highest multiplication index of 3.93 was obtained on Murashige and Skoog (MS) media supplemented with 2.0 mg/L 6-BA, 0.1 mg/L NAA and 0.3 mg/L GA3 from 12 combinations of 6-BA and NAA. Stronger and taller adventitious shoots were grown on MS supplemented with 1.8 mg/L 6-BA and 0.5 mg/L NAA. The optimal media with 100% rooting was obtained using 1/2 MS supplemented with 0.3 mg/L IBA or MS supplemented with 0.6 mg/L IBA for the rooting induction, resulting in mean rooting numbers of 13.00 and 11.33, respectively. Additionally, the effect on rooting of adding 0.3 mg/L IBA or not on the 1/2 MS and MS media was compared; the results suggested that an appropriate IBA concentration was the key to successful rooting. The rooted plantlets were acclimatised in a shaded greenhouse with an 84% survival rate. The established micropropagation system could be used for the rapid propagation of M9T337 for commercial production.

The effect of humic acid (HA) and zinc oxide nanoparticles (ZnO-NPS) on in vitro regeneration of date palm (Phoenix dactylifera L.) cv. Quntar

Date palm (Phoenix dactylifera L.) micropropagation still faces many problems, such as reduced growth and development of callus, low multiplication efficiency and low rooting rate. The effect of Humic Acid (HA; 0, 0.5, 2.5, and 5.0 mg L−1) and Zinc oxide nanoparticles (ZnONPs; 0, 50, 75 and 150 mg L−1) at different stages of micropropagation (i.e., callus establishment, shoot multiplication and rooting) was studied in date palm cv. Quntar. Media supplemented with 2.5 mg L−1 HA in combination with 50 m L−1 ZnO-NPs gave the highest callus induction (274 mg/jar), the highest percentage of callus producing buds (83.34%), and average bud formation (14.2) per callus. The macronutrients Nitrogen (N), Phosphorus (P), Potassium (K), Sulfur (S) as well the micronutrient Zinc (Zn), significantly increased in in vitro shoots regenerated in medium containing of 2.5 mg L−1 HA and 50 mg L−1 ZnO-NPs compared to other treatments. The addition of HA and ZnO-NPs to the medium was most effective in root regeneration and the number of roots per shoot, where the best result (83.34%, 4.4 roots per shoot, respectively) was obtained using 2.5 mg L−1 humic acid and 75 mg of L−1 zinc oxide (ZnO-NPs), compared with other treatments. These results also indicate that using 2.5 mg L−1 HA combined with 75 m L−1 ZnO-NPs as a supplement can increase antioxidant enzymes CAT and POD compared with other treatments. The data revealed that maximum chlorophyll content of shoots occurred in a medium supplemented with 2.5 mg L−1 HA and 75 mg L−1 ZnO-NPs. Furthermore, we found that the combined application between 2.5 mg L−1 HA + 75 mg L−1 ZnO-NPS resulted in the highest shoots content of endogenous IAA (3.644 µg.kg−1), compared with other treatments. We have established a highly efficient micropropagation system for date palm by using (HA) and zinc oxide nanoparticles (ZnO-NPS). The initiation, broad optimization, and comprehensive phytochemical studies of tissue cultures were elaborated.

Highly efficient rapid micropropagation and assessment of genetic fidelity of regenerants by ISSR and SCoT markers of Solanum khasianum Clarke

An efficient method of rapid micropropagation of Solanum khasianum Clarke was successfully established from the leaf, petiole, and nodal explants. The morphogenetic response of different concentrations of TDZ and BAP individually or in combination with auxins (IAA/IBA/2,4-D) was tested. Friable callus was obtained on different concentrations of BAP alone or in combination with IAA/IBA/2,4-D. Rapid multiple shoot induction was achieved from friable callus on MS medium supplemented with varying concentrations of TDZ and IBA. The leaf explants exhibited a high frequency of multiple shoots than petiole and nodal explants with an optimal percentage of response (92.73%), mean shoot number (53.5 ± 0.47), and shoot length (11.2 ± 0.53 cm) on MS medium augmented with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1). Maximum rooting efficiency was achieved on MS medium with 1.5 mg l−1 IBA with 12.8 ± 0.36 mean number of roots. The in vitro rooted plants were acclimatized with a survival rate of 80%. The genetic fidelity of the regenerants assayed by the ISSR and the SCoT markers showed no genetic variation. The study examined the micropropagation responses of S. khasianum in the presence of various growth regulators and provided a simple and more suitable protocol adapted for the mass propagation of clones in this species. We have established a highly efficient micropropagation system for large scale production in Solanum khasianum. Evaluation of clonal fidelity by using ISSR and SCoT markers detected no somaclonal variations. The present study helps to the enhancement of potential alkaloids (solasodine) with the help of biotechnological tools.

Retraction Note to: In vitro somatic embryogenesis from immature female flower of Musa AAB cv. Chenichampa and molecular analysis of transcript factors (TFs) during somatic embryogenesis

Somatic embryogenesis (SE) is a process where somatic embryos can form differentiated tissues and regenerate into new plants. Efficient SE and liquid culture system for large-scale production of banana cv. Chenichampa is presented. The embryo maturation media (M3) was modified with different organic additives and high-frequency somatic embryos were observed in M3 media supplemented with CH: Gln (100:150 mg/L) resulting in a tenfold increase in somatic embryo formation as compared to the control. The generated plantlets were hardened and polymerase chain reaction (PCR) with SSR primers were used to confirm the genetic fidelity of the plantlets. To understand the molecular mechanism of SE, expression patterns of transcript factors (TFs) in respect of embryogenic calli (EC) and embryogenic cell suspension (ECS) was also studied. Transcript factor (TF) such as BBM has been reported to play a significant part in converting explants to EC. The higher expression of TFs MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the EC and MaBBM2 and MaWUS2 in ECS, suggested that these genes could play a crucial role in SE. The protocol developed in this commercially important banana cv. Chenichampa could be highly useful for large-scale micropropagation and genetic manipulation studies which can be adapted to other cultivars lacking in male flowers. An efficient micropropagation system using SE for banana cultivar Chenichampa (Musa AAB) is presented. Differential expression profiles of TFs during various stages of SE studied. Organic additives enhanced the SE and this optimized protocol will serve as a platform for developing efficient propagation system for banana cultivars that lack male flowers.

A novel regeneration system through micrografting for Argania spinosa (L.) Skeels, and confirmation of successful rootstock-scion union by histological analysis

A novel regeneration system through micrografting is reported for the endangered Argania spinosa (L.) Skeels. Rootstocks were obtained from in vitro germinated seeds of the argan genotype G27. It was found that the storage time significantly affects the seed germination capacity, and that the seeds cultured immediately after harvest exhibit the highest germination percentage (91.6%). Besides, transferring seedlings to half-strength Murashige and Skoog (½MS) medium supplemented with 1 mg l−1 gibberellic acid (GA3) and 1 mg l−1 6-benzylaminopurine (BAP) resulted in the highest shoot and root lengths (2.05 and 5.73 cm, respectively). Scions were taken from axillary shoots developed in vitro from microcuttings of genotype G41. Micrografting was performed by using the wedge technique. Afterwards, the micrografted plants were transferred to media supplemented with different plant growth regulators (PGRs). After 2 months of culture, 65–100% of the micrografted plants survived, and no difficulties were observed during the formation of the rootstock-scion union. Interestingly, the use of GA3 at concentrations ranging from 0.1 to 1 mg l−1 was essential for successful micrografting (85–100%) and subsequent growth and development of shoots (2.40–2.72 cm length). After micrografting, scions produced one, two or multiple shoots, depending on PGRs. Histological analysis clearly demonstrated the successful union between rootstocks and scions, with active cell division and vascular tissue formation in the grafting region. After transferring the micrografted plants to the glasshouse, a survival rate of 80% was observed, and the plants showed normal growth and development. An efficient micropropagation system through in vitro grafting is reported for the first time ever for Argania spinosa (L.) Skeels, and successful micrografting is confirmed by histological analyses.

Antimicrobial and antioxidant potential of Tylophora indica (Burm. f.) Merrill and development of an efficient micropropagation system for its therapeautic use

Infectious diseases caused by pathogenic microorganisms are the leading cause of human mortality worldwide. Emergence of multidrug resistance in these pathogens and negative effects of available antibiotics have necessitated the search for new potent antimicrobial agents of plant origin. Tylophora indica commonly known as “Antamool” or “Asthama herb” is a valuable medicinal plant traditionally used as a folk remedy for the treatment of a variety of diseases and disorders. An efficient and reproducible in vitro protocol was established for the mass cloning of this valuable plant through de novo adventitious shoot formation directly from the leaf explant. The antimicrobial activity of different solvent extracts of leaves of both native and in vitro plants was tested against bacterial strains (Staphylococcus aureus, Escherichia coli) and fungal strains (Aspergillius niger, Penicillium chrysogenum) using agar well diffusion method. The leaf extracts prepared by Rao and Book's method and methanolic leaf extracts exhibited a significant microbial inhibition at all tested concentrations (25, 50, 100 μg/ml), whereas very less activity was observed with acetone extract. Likewise the methanolic root extract of in vitro plants exhibited significant antioxidant activity. The highest free radical scavenging activity i.e. 75% was seen at 1000 μg/ml concentration depicting strong antioxidant effect. The present study suggests that T. indica may prove to be a potential natural source of antimicrobial and antioxidant agents which needs to be further explored.

MICROPROPAGATION STUDIES AND ANTIOXIDANT ANALYSIS OF THE ENDANGERED PLANTS OF BULGARIAN YELLOW GENTIAN (Gentiana lutea L.)

In order to develop an efficient micropropagation system, it is essential to establish the appropriate concentration of growth regulators for seed germination, shoot formation and rooting. Nodal segments from in vitro obtained seedlings of Gentiana lutea L. were cultured in vitro in Murashige and Skoog’s medium supplemented with BAP, Thidiazuron and Zeatin (0.5, 1.0 and 2.0 mg L−1). A maximum number of shoots with the highest height was recorded at 2.0 mg L−1 BAP. For further optimization of the process, we used nutrient media containing BAP and Zeatin with a combination of low concentration of Indoleacetic acid. MS medium containing 2.0 mg L−1 Zeatin and 0.2 mg L−1 IAA resulted in maximum numbers of shoots 94.3) with shoot height 2.5 cm. The multiple plants were successfully ex vitro acclimatized with 65% survival. The presence of growth regulators (2.0 mg L−1 Zeatin and 0.2 mg L−1 IAA) in the nutrient media resulted in an effective antioxidant activity in G. Lutea determined by the low molecular antioxidant metabolites such as phenols and flavonoids and activities of antioxidant enzymes – catalase, ascorbate peroxidase, guaiacol peroxidase, and superoxide dismutase. The described protocol allows the establishment of numerous micropropaged plants of rare and endangered G. lutea.

Influence of meta-topolin on in vitro organogenesis in Tecoma stans L., assessment of genetic fidelity and phytochemical profiling of wild and regenerated plants

Comparative effect of meta-topolin and other cytokinins was assessed to develop an efficient and reliable regeneration protocol for Tecoma stans, using mature nodal explants. The morphogenic effect of benzyl adenine (BA), kinetin (Kin), meta- topolin (mT) and 2-iP (2-iso pentenyl adenine) at various concentrations (1.0–10 µM) was studied individually or in combination with auxins (IAA, IBA or NAA). Superior multiplication rates were achieved on MS medium supplemented with mT and NAA. Of the tested combinations, maximum shoot regeneration (95%), mean shoot number (19.6 ± 0.60) and length (5.26 ± 0.73 cm) was recorded on MS medium supplemented with 7.5 µM mT + 0.5 µM NAA after 8 weeks of incubation. Among the different auxins employed for in vitro root induction, 92.5% microshoots rooted on MS medium enriched with 1.0 µM IBA with 10.8 ± 0.20 mean root number and 5.62 ± 0.17 cm length after 4 weeks of incubation. The acclimatized plants grew well in green house with 90% survival rate. The gas chromatography–mass spectrometry (GC–MS) analysis of ethanol leaf extract of in vitro-raised plants yielded a higher number of compounds than control plant. The assessment of genetic fidelity among regenerants, using ISSR markers did not reveal any somaclonal variation. Therefore, the protocol developed appears to be simple and reliable for mass production of clones with higher diversity of secondary metabolites. Our studies based on interaction of mT with auxins for better shoot proliferation will undoubtedly be helpful in understanding many developmental processes in plant tissue culture. The regenerated plantlets survived well under field conditions and no somaclonal variation was detected. This study proved that in vitro raised plants are superior for the production of higher diversity of secondary metabolites than in vivo plant. Thus, our findings are helpful in generating an efficient micropropagation system for mass scale production of plants with higher diversity of bio-active molecules without losing their genetic stability.

Meta-topolin improved micropropagation in Syzygium cumini and acclimatization to ex vitro conditions

An efficient micropropagation system was developed for a recalcitrant woody tree Syzygium cumini (L.) Skeels using nodal explants excised from 15-d-old aseptic seedlings. The explants were employed on an Murashige and Skoog (MS) medium supplemented with different concentrations (1.0 - 10.0 μM) of cytokinins, such as benzyladenine (BA), kinetin (Kin), meta-topolin (mT), or 2-isopentyl adenine (2ip), either alone or in combination with different concentrations (1.0 - 3.0 μM) of auxins, such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Of the cytokinins tested, mT proved to be best for shoot bud induction and proliferation. Among the tested combinations, a maximum regeneration (90 %) with a mean shoot number of 25.33 ± 0.33 and a shoot length of 5.20 ± 0.11 cm were recorded on the MS medium containing 5.0 μM mT + 2.0 μM NAA after 12 weeks of incubation. Further 4-week incubation on optimum 5.0 μM mT before transfer to a secondary medium consisting of MS + 5.0 μM BA + 2.0 μM NAA yielded up to 51 microshoots with an average length (6.53 cm). For in vitro rooting, healthy shoots (about 5 cm) were excised and incubated on the half or full strength MS medium enriched with different concentrations (1 - 7.5 µM) of NAA. A substantial increase in rhizogenic competency (15 %) was observed in shoots raised on a medium with mT with a mean root number of 6.33 ± 0.10 and a mean length of 5.13 ± 0.21 cm on the half MS supplemented with 5.0 µM NAA after 4 weeks. A maximum of 95 % plantlets regenerated on the medium with mT was successfully acclimatized and established in earthen pots under field conditions. The consistent increases in activities of superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase during acclimatization indicate that mT raised plantlets response well to the stress induced by ex vitro conditions.

In vitro propagation of Rosa ‘Konstancin’ (R. rugosa × R. beggeriana), a plant with high nutritional and pro-health value

Abstract The aim of the study was to develop an efficient micropropagation system for Rosa ‘Konstancin’, an interspecific hybrid between R. rugosa and R. beggeriana, whose fruits have high pro-health value. Shoot cultures were initiated from shoot buds collected in May and August from 15-year-old field-grown Rosa ‘Konstancin’ shrubs. The effect and interaction of different concentrations of phytohormones, sucrose and iron sources on in vitro initiation, multiplication and rooting of shoots were studied. The time of collecting explants from donor plants significantly affected the initiation of shoot culture of Rosa ‘Konstancin’. Considerably higher frequency of bud break (100%) was obtained in explants isolated in August as compared to those collected at the end of May (30%). All buds developed into single shoots after 2-4 weeks of growing on the basal Murashige and Skoog medium containing 2.2 µM BAP, 0.3 µM GA3 and 88 mM of sucrose. The highest multiplication rate (4.8 shoots/explant) in a 5-week period was obtained on MS medium containing 50% of nitrogen salts, 3.1 µM BAP, 0.9 µM GA3 and 58 mM sucrose. High rooting frequency (100%) and quality of rooted plantlets was obtained on a medium containing 0.5 µM IBA, 138 µM Fe-EDDHA and 88 mM sucrose. Fe-EDDHA had a beneficial effect on the growth and photosynthetic activity of Rosa ‘Konstancin’ plantlets, which were successfully acclimatized ex vitro, with a more than 90% survival rate.

Comparison of Phytochemical and Antioxidant Activities in Micropropagated and Seed-derived Salvia miltiorrhiza Plants

Salvia miltiorrhiza (commonly known in China as Danshen) is widely used in traditional Chinese medicine, and it is applied in the treatment of many diseases, particularly cardiovascular disease. Commercial propagation of Danshen is carried out either through seed germination or in vitro regeneration (micropropagation). However, it is not clear if the different propagation methods affect the chemical properties of the derived plants. In the present study, we first established a highly efficient tissue culture system for Danshen propagation. The addition of 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 α-naphthalene acetic acid (NAA) to Murashige and Skoog (MS) medium was optimal for inducing adventitious shoots; the highest rate of rooting was recorded on MS medium with 0.2 mg·L−1 NAA, on which the survival rate of transplanted plantlets was 95%. Next, we assessed antioxidant properties in the different tissues of plants of the same age, derived from micropropagation or seed germination, and measured tanshinone, total phenol, and total flavonoid contents. Our results showed that tissues of micropropagated plantlets had higher antioxidant activities than tissues of seed-derived plantlets; the micropropagated plantlets also had higher tanshinone contents in their roots. Thus, a rapid and efficient micropropagation system was established for Danshen, and it can be used for cultivating this plant to obtain therapeutic compounds.

ESTABLISHMENT OF AN IMPROVED, EFFICIENT AND ECO-FRIENDLY MICROPROPAGATION SYSTEM IN SALACIA CHINENSIS L. AN ENDANGERED ANTI-DIABETIC MEDICINAL PLANT

An efficient micropropagation system via indirect organogenesis was developed in Salacia chinensis L., an endangered anti-diabetic medicinal plant. Accurate investigation of the various plant sources (Leaf, node, shoot tip) and plant growth regulators (PGRs) impacts were accomplished in this study. Maximum rate of callus induction (93.43 ± 2.75%) was achieved from nodes inoculated on MS medium fortified with 1.0 mg/l NAA+ 2.0 mg/l BAP. Maximum shoot induction (93.33 ± 2.02%), number of shoots/explant (5.12 ± 0.09) and shoot length (3.17 ± 0.00 cm) were obtained from nodal explants inoculated on MS medium with 1.5 mg/l BAP + 1.0 mg/l NAA. IBA (2.0 mg/l) in ½ MS medium was observed to be the best rooting treatment, which promoted the highest frequency of rooting (91.66 ± 2.33%). The results suggested an efficient regeneration system for conservation and large scale production for pharmaceutical industry demands.

Establishment of a Rapid and Efficient Micropropagation System for Succulent Plant Haworthia turgida Haw.

In the present study, the effect of plant growth regulators (PGRs) on callus regeneration, adventitious shoot differentiation, and root formation of Haworthia turgida Haw. was investigated. The greatest callus induction percentage (95.6%) was achieved with leaf explants inoculated on Murashige and Skoog (MS) medium with 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 1-naphthaleneacetic acid (NAA), and this callus induction medium supplemented with 2.5 mg·L−1 thidiazuron (TDZ) was optimal for callus proliferation. The maximum number of shoots (25.7) was obtained when the callus was cultured on MS medium supplemented with 1.0 mg·L−1 BA and 0.2 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The highest number of roots per shoot (6.2) and highest rooting frequency (82.0%) were obtained when adventitious shoots were inoculated on MS medium with 0.05 mg·L−1 NAA. Regenerated plantlets were transferred to a mixture of vermiculite and soil and acclimated in a greenhouse. The survival rate of the transplanted plantlets was about 91.6%. The rate of ex vitro rooting was 83.3%, indicating that this technique is effective for root induction in H. turgida. This study has established a rapid and efficient micropropagation system that can be beneficial for commercial cultivation and germplasm conservation of H. turgida.

Establishment of an efficient micropropagation and callus regeneration system from the axillary buds of Bambusa ventricosa

Based on the screening of various hormone combinations, we have established an efficient micropropagation and callus regeneration system from the axillary buds of B. ventricosa. We found that 6-benzyladenine (6-BA) had a dominant role in promoting bud sprouting, multiple bud induction and proliferation in B. ventricosa. Meanwhile α-naphthaleneacetic acid (NAA) was found to be an effective factor of inducing rooting in proliferated buds. The Murashige and Skoog medium containing 22.2 µM 6-BA was optimal for bud initiation, and the MS medium containing 26.6 µM 6-BA provided a good result for multiple bud induction. However, for buds proliferation MS medium containing 22.2 µM 6-BA, 0.23 µM Thidiazuron (TDZ: N-phenyl-N-[(1, 2, 3-thidiazol-5-yl) urea]) and 0.27 µM NAA was found to be very effective. The optimal medium for rooting of proliferated bud was MS medium containing 2.7 µM NAA, 4.9 µM indole butyric acid and 4.4 µM 6-BA. Based on the establishment of an efficient micropropagation system, we investigated the frequencies of callus formation from buds of in virto raised plantlets under different culture conditions. We showed that media containing 27 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2.7 µM NAA and 0.0045 µM TDZ effectively produced callus. The callus induction rate varied between it to 60 percent. TDZ was found to be a main factor influencing the callogenesis of B. ventricosa. Medium containing 22.6 µM 2, 4-D, 2.2 µM 6-BA and 5.4 µM NAA was preferred for callus amplification among the four tested subculture media. Plants were successfully regenerated on a MS medium containing 13.3 µM 6-BA and 2.7 µM NAA, subsequently acclimatized and transplanted to an experimental pod.