Liquid Murashige And Skoog Research Articles

Ultrastructure of somatic embryo development and plant propagation for Lachenalia montana

Embryogenesis for plant propagation by cell suspension culture was achieved for the first time using friable embryogenic callus (FEC) from leaf explants of Lachenalia montana Schltr. ex W.F. Barker. FEC was established with solid (8gL−1 agar) MS (Murashige and Skoog, 1962) medium containing various concentrations and combinations of sucrose and plant growth regulators (PGRs). The somatic embryos (SEs) were developed from FEC in liquid MS (MSL) medium with or without PGRs. A higher number of SEs of different developmental stages (26.0–19.4, globular to cotyledonary-stages respectively) were obtained on MSL medium with 0.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1μM thidiazuron (TDZ). The germination frequency (33.7%) was highest in MSL medium containing 1μM 2,4-D and 2μM TDZ. The different stages of SEs germinated (92%) on solid full-strength MS medium containing 15gL−1 sucrose and 10μM phloroglucinol (PG). The plantlets were successfully acclimatized in the greenhouse. Developmental stages and features of embryoids were confirmed by histological and ultrastructural studies using light and transmission electron microscopy (TEM). Many mitochondria, lipid bodies together with starch grains, chloroplasts, Golgi apparatuses, vacuoles and nuclei were detected. The system developed offers controlled large-scale clonal propagation which ensures germplasm conservation, confirms viability of embryogenesis by ultrastructural studies and provides a system for genetic transformation studies for horticultural and ornamental improvement.

Cryopreservation of in Vitro Grown Shoot Tips of Sweet Potato (Ipomoea batatas L.) by the Encapsulation-Vitrification Method

Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulation-vitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at <TEX>$25^{\circ}C$</TEX>. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

Rapid propagation of Mondia whitei by embryonic cell suspension culture in vitro

We have developed an efficient in vitro mass propagation system for Mondia whitei, an endangered South African medicinal plant of significant traditional importance, using somatic embryogenesis in cell suspension culture. Leaf explants generated friable embryogenic calli (FEC) when cultured on embryogenic callus induction medium (ECIM), which contained Murashige and Skoog (MS) medium, 8gL−1 agar with 30–50gL−1 sucrose and different plant growth regulators [PGRs: 2,4-dichlorophenoxyacetic acid (2,4-D) and picloram]. Different stages (globular/heart/torpedo/cotyledonary) of somatic embryos (SEs) were produced from FEC using liquid MS medium supplemented with different concentrations and combinations of PGRs. Formation of SEs (torpedo and cotyledonary stages) were significantly higher in liquid MS medium containing 0.5μM naphthaleneacetic acid (NAA) and 1μM meta-topolin riboside (mTR). The highest embryo germination rates achieved in liquid (half-strength MS+0.5μM NAA) and solid (MS+40gL−1 sucrose +2μM NAA) media were 98.3% and 97.6%, respectively. The synthetic seeds were prepared with sodium alginate (3% w/v) and calcium chloride (100mM) from SEs (torpedo and cotyledonary stages) and stored in darkness at 25±2°C and 4°C for 10–60days. Germination frequency was 68.6% at 25±2°C after 60days of storage. All the plantlets from SEs and synthetic seeds were successfully acclimatized (100%) with vermiculite in the greenhouse. These results provide high-frequency, rapid production and germination of SEs and synthetic seeds, and provides further strategies that can be used for overcoming extinction threats, ensuring germplasm conservation, production of bioactive compounds, evaluation of bioactivity and genetic transformation studies.

Evaluation of Micropropagation System of Bacopa monnieri L. in Liquid Culture and Its Effect on Antioxidant Properties

Nodal explants of Bacopa monnieri were cultured in a solid Murashige and Skoog (MS) medium containing 1 mg L−1 kinetin, transferred after 30 days to a liquid MS medium of the same composition in 100 mL and 250 mL Erlenmeyer flasks (with 10 mL and 50 mL medium, respectively) and Phyta Jars (with 10 mL medium) and subcultured every 7 d. Shoot doubling time was estimated and the total antioxidant capacity of aqueous extracts from liquid-cultured and acclimatized plants was studied. In the solid medium, 5 rooted shoots/explant were produced after ~20 days, with shoot doubling time of 7.99 days; in liquid media, 36–75 rooted shoots were produced with shoot doubling time of 0.91–1.17 days. Aqueous extracts of acclimatized plants showed 10-fold higher total antioxidant capacity compared to in vitro liquid cultured plantlets.

Enhanced micropropagation protocol of Morinda citrifolia L. through nodal explants

We report an improved in vitro propagation protocol for Morinda citrifolia L. in the present communication using nodal segments as explants from a 4 year old plant. Murashige and Skoog (MS) medium was used to inoculate the sterilized explants. Under laboratory conditions, 4.6±0.48 shoots were regenerated from the nodal meristem of an explant on the combination of MS medium fortified with 4.0mg/L 6-benzylaminopurine (BAP). Semi-solid (with agar) and liquid MS medium was used for the multiplication of shoots in vitro. Combined effect of BAP and kinetin in shoot multiplication was reported favorable in semi-solid (10.6±0.17 shoots per vessel) and liquid MS medium (11.4±0.47 shoots per vessel). The shoots were rooted in vitro on half-strength agar-gelled MS medium supplemented with 1.0mg/L indole-3 butyric acid (IBA) and 44.30 roots per shoot were observed from the cut ends of the shoots. All the shoots were also rooted by ex vitro method and maximum 36.12 roots per shoot were induced by treating the shoots with 300mg/L IBA for 5min. The in vitro rooted and ex vitro treated plantlets were transferred to the soilrite containing paper cups for hardening in the greenhouse. The plantlets were shifted to nursery bags after 4 weeks. Finally the hardened plantlets were planted in the field with 100% rate of survival under natural conditions. The ex vitro rooting was reported more advantageous than in vitro rooting in terms of cost, time and percent survival of plantlets.

Use of coconut water and fertilizer for in vitro proliferation and plantlet production of Dendrobium ‘Gradita 31’

To improve the micropropagation of Dendrobium ‘Gradita 31’, coconut water (CW) and fertilizer media were used to enhance growth, proliferation, and germination of protocorm-like bodies (PLBs). PLBs formed when shoots approximately 0.4 cm long were cultured on semi-solid half-strength Murashige and Skoog (MS) basal medium containing 1 mg L−1 thidiazuron (TDZ) and 0.5 mg L−1 N6-benzyladenine (BA). After four rounds of 15-d subcultures, PLBs were initially proliferated in liquid half-strength MS medium supplemented with 0.3 mg L−1 TDZ and 0.1 mg L−1 α-naphthalene acetic acid (NAA) and subcultured monthly into fresh medium. When 15% (v/v) CW was added to half-strength MS medium, optimal growth and proliferation of PLBs resulted with the following principal findings: a maximum of 2.86 g total fresh weight (FW) of PLBs formed from 1 g of PLBs, an average of 0.46 g FW of PLBs added per subculture period, a total of 157.1 PLBs formed, an average of 25.3 PLBs added per subculture period, and a low percentage of PLB browning (20.7%). Half-strength MS medium containing 15% (v/v) CW with Rosasol® medium (liquid fertilizer; 1.5 g L−1 18 N:18P:18 K + 1.5 g L−1 25 N:10P:10 K + TE) resulted in a similar organogenic outcome with 25.5% PLB browning. PLBs germinated easily and rooted on Rosasol-supplemented plant growth regulator-free medium. In total, 180 Dendrobium ‘Gradita 31’ plantlets from all treatments were successfully acclimatized with 100% survival on a Cycas rumphii bulk substrate and grew well after repotting in a mixture of wood charcoal and C. rumphii bulk (1:1, v/v).

Thidiazuron induced efficient in vitro multiplication and ex vitro conservation of Rauvolfia serpentina – a potent antihypertensive drug producing plant

An efficient system for in vitro propagation of the endangered medicinal plant Rauvolfia serpentina has been developed. Proliferation of shoots was achieved from nodal segment explants, excised from a field grown plants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–2.5 μmol/L) although with low regeneration response and few number of shoots per explant. Greater number of shoots was achieved from nodal explants pretreated with higher concentrations of TDZ (5–100 μmol/L) in liquid MS medium for different time periods (4, 8, 12 and 16 days), followed by their transfer on a growth regulator-free medium. The highest response in terms of percent regeneration (90%), average number of shoots/explant (23.17 ± 2.15) and maximum shoot length (5.3 cm ± 0.83 cm) were achieved by pretreating the nodal explants with 50 μmol/L TDZ for 8 days. Rooting was achieved in MS medium supplemented with 0.5 μmol/L indole-3-butyric acid with an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transferred to soil, where 90% of them grew well and exhibited a normal development. Chlorophyll a and b, carotenoids, net photosynthetic rate and antioxidative enzymes, viz., superoxide dismutase and catalase activities were measured in leaf tissues during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Most of the activities showed a decreasing trend but subsequently increased after 7 days of acclimatization. These changes suggested the adjustment of the in vitro propagated plantlets to ex vitro conditions.

Morphohistological and molecular profiles during the developmental stages of somatic embryogenesis of Musa acuminata cv. ‘Berangan’ (AAA)

The present study established a protocol for generating embryogenic cell suspension (ECS) cultures from an early stage embryogenic callus of Musa acuminata cv. ‘Berangan’. The necessary baseline data for morphohistological recognition and the selected gene expression for differentiating embryogenic and non-embryogenic cells were defined to enable early selection, as well as for quality assessment, which could aid in scaling up the production of these cells. In this study, male bud-derived callus on Murashige and Skoog (MS) medium containing 23 μM 2,4-dichlorophenoxyacetic acid (2,4-D) was transferred to M1 medium containing different concentrations of 2,4-D and picloram for somatic embryo initiation. ECSs established in liquid MS medium (M2a) containing 4.1 μM biotin, 10 mg/l ascorbic acid, 100 mg/l glutamine, 100 mg/l malt extract, 4.5 μM 2,4-D, 1.0 μM zeatin, and 20 g/l sucrose were used to study embryogenic development. The frequency of embryo development and maturation was influenced by the culture media. The highest frequency of embryo development (96.4 %) and maturation (66.4 %) was achieved on solid MS hormone-free medium. Almost 69 % of embryos were induced to form shoots on the MS medium containing 34 μM 6-benzylaminopurine (BAP). Histological study indicated that only embryos with distinct layer of protoderm having cells with prominent nucleus and dense cytoplasm were able to regenerate into plantlets. Endochitinase and phenylalanine ammonia-lyase (PAL) genes were expressed at all the developmental stages, and their expression level was tightly regulated during embryo development and reached its maximum in germinating embryos. Expression of these genes in embryogenic cells was higher than in non-embryogenic cells. The study findings suggested that these genes play an important role in the developmental stage and regeneration pathway of somatic embryos of banana.

Iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of Rehmannia glutinosa Libosch.

Spontaneous shoot regeneration was obtained from the hairy roots of Rehmannia glutinosa cultured in liquid woody plant medium for 4 weeks in darkness after induction by Agrobacterium rhizogenes strain A4. These shoots were proliferated on solid Murashige and Skoog (MS) medium in 300 mL glass jars and in liquid MS medium in a 5 L nutrient sprinkle bioreactor. These media were supplemented with 0.1 mg L−1 of indole-3-acetic acid (IAA) and 1.0 mg L−1 of 6-benzylaminopurine. All shoots became rooted on MS agar medium containing IAA or indole-3-butyric acid within 4 weeks. The plantlets were then transferred into soil and grew in a greenhouse. The transgenic nature of the plants was confirmed by polymerase chain reaction (PCR) and reverse-transcriptase PCR analyses. The pRi-regenerated transformants demonstrated a few phenotypical alterations. Production of six iridoid (catalpol, aucubin, loganin, catalposide, harpagide, harpagoside) and two phenylethanoid (verbascoside and isoverbascoside) glycosides was determined in shoots cultured in vitro and leaves and roots of 6- and 12-month-old plants by ultra high performance liquid chromatography. The shoot culture was able to synthesize low amounts of catalpol, verbascoside and isoverbascoside. Leaves and roots of pRi-regenerants accumulated similar (harpagoside, isoverbascoside), higher (catalposide, aucubin, harpagide, verbascoside) or lower (catalpol, loganin) levels of the metabolites in comparison with untransformed R. glutinosa plants and their content varied with the age of the plants and the organ analyzed. However, the increase in shoot and root biomass of transformed plants resulted in a significantly greater yield of all analyzed compounds.

PROPAGATION OF Portulaca oleracea L. IN LIQUID MEDIUM: IMPLICATIONS OF PLANT GROWTH REGULATORS IN CULTURE

Portulaca oleracea L. is a medicinal plant, growing in warm and moist regions of north hemisphere of the world. A protocol for in vitro propagation using nodal shoot segments as explants has been outlined. The percent shoot response with shoot induction rate, 6.4 ± 0.7 shoots per explant, was achieved when cultured on agar-gelled Murashige and Skoog (MS) medium containing 2.0 mg/L of BAP (6-benzylaminopurine). The cultures were amplified by passages on MS medium with 1.0 mg/L each BAP and kinetin (Kn). The best shoot amplification (37.5±0.9 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clumps on liquid MS medium. Shoots regenerated in vitro by both the processes were rooted on ½ strength of MS medium + 2.0 mg/L of indole-3 butyric acid (IBA). Ninety six percent of the shoots rooted in vitro. The in vitro rooted plantlets were hardened under different regimes of temperature and humidity in the greenhouse. The hardened plantlets were transferred to mixture of soil and manure in polybags.

Embryogenesis and synthetic seed production in Mondia whitei

An effective plant regeneration system via somatic embryogenesis and synthetic seeds was developed for Mondia whitei, an endangered medicinal plant. Friable embryogenic callus was induced by culturing leaf explants on solid Murashige and Skoog (MS) medium containing various concentrations and combinations of sucrose and plant growth regulators. The highest frequency of somatic embryogenesis (100 %) and production of all developmental stages of somatic embryos were obtained on MS medium with 40 g l−1 sucrose, 8 g l−1 agar, 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM thidiazuron. This was followed by establishment in MS medium with 20 g l−1 sucrose, 8 g l−1 agar, 0.5 μM meta-topolin riboside (mTR) and 0.25 μM indole-3-acetic acid (IAA). All the embryos germinated and produced healthy plantlets on the same medium. Somatic embryos at the heart, torpedo and cotyledonary-stages were collected from media (EDM) containing MS medium plus 20 g l−1 sucrose, 8 g l−1 agar, 0.5 μM mTR and 0.25 μM IAA. The embryos were encapsulated with liquid MS medium plus different concentrations of sodium alginate (SA) and calcium chloride (CaCl2·2H2O) with a 10 min exposure. A combination of 3 % SA and 100 mM CaCl2·2H2O provided higher survival (95.7 %) and germination (73 %) frequencies of synthetic seeds. Germination frequency of synthetic seeds was 51.6 % after 50 days of storage at 4 °C. Somatic embryos and synthetic seed-developed plantlets were successfully acclimatized in the greenhouse with 90 % survival ex vitro. Application of the protocol provides a relatively simple and rapid system for conservation of natural populations for germplasm conservation. Analysis of bioactive compounds and genetic transformation studies can also be performed.

Effect of copper and zinc on the in vitro regeneration of Rauvolfia serpentina

The present study exemplifies morphogenic roles played by copper and zinc during micropropagation of Rauvolfia serpentina, an important medicinal shrub. Incorporation of 20 μM CuSO4 or 25 μM ZnSO4 to a Murashige and Skoog (MS) medium with optimized concentrations of auxins and cytokinins induced a maximum number of shoots per explant (40.67 ± 1.76 and 45.47 ± 0.24, respectively). However, higher concentrations of both the micronutrients negatively affected the morphogenic potential. The pigment content of the regenerants increased up to the optimal concentrations of both metals and thereafter decreased, whereas the maximum proline content was at the highest concentrations used. In vitro rooting of healthy shoots was accomplished using 0.5 μM IBA in a half strength liquid MS medium with 8.20 ± 0.37 roots, and root length of 5.50 ± 0.14 cm per microshoot. The plants survived a hardening procedure and were successfully acclimatized to field conditions with 95 % survival.

In vitro germination and propagation of a threatened medicinal orchid, Cymbidium aloifolium (L.) Sw. through artificial seed

ABSTRACT Objective To study the in vitro germination and plantlet regeneration from artificial seeds of Cymbidium aloifolium (C. aloifolium), a highly threatened medicinal orchid of Nepal. Methods Artificial seeds were produced in vitro by encapsulation of protocorms with 4% sodium alginate and 0.2 mol/L calcium chloride solution. In vitro germination and plantlet regeneration of the artificial seeds were tested by culturing them on different strength of Murashige and Skoog (MS) liquid media (0.25, 0.5 and 1.0) and MS liquid medium supplemented with 0.5 mg/L benzyl amino purine and 0.5 mg/L naphthalene acetic acid. Freshly produced artificial seeds were stored up to 28 d at 4 oC. In order to check the viability, storage artificial seeds were treated with five different sterilization techniques (T1, T2, T3, T4, T5) and inoculated on full strength (1.0) of MS liquid medium after each 7 d of interval upto 28th days. Results The highest percentage of germination (100%) of artificial seed was obtained on quarter (0.25), half (0.5) and full (1.0) strength of MS liquid medium. Experimentally, full strength of MS liquid medium was more effective for earlier seedling development of C. aloifolium. Artificial seeds were successfully stored at 4 oC till 28th days. Treatments T1 and T2 showed 97.5% viability of storage artificial seeds and hence considered as the most effective sterilization techniques to recover the plant from storage artificial seeds. Plantlets developed from artificial seeds were successfully acclimatized in potting mixture containing cocopeat, litter and sphagnum moss with 85% survival rate. Conclusions The present study revealed that artificial seeds are the good alternative explants for in vitro mass propagation and short term conservation of C. aloifolium.

Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation

This study was carried out to optimize a modified droplet-vitrification procedure for the cryopreservation of shoot tips from different carnation genotypes. The best procedure was developed by applying orthogonal tests to the experimental data and by further investigation of the effects on the regrowth percentage. It consisted in preculturing shoot tips in liquid Murashige and Skoog (MS) medium with 0.3 M sucrose for 2 days, pretreating them in liquid MS medium with 5 % Dimethyl sulfoxide +5 % glycerol + 0.3 M sucrose for 10 min, osmoprotecting in Loading solution for 20 min at 25 °C, cryoprotecting with Plant vitrification solution No.2 (PVS2) for 60 min at 0 °C, transferring in drops of fresh PVS2 over aluminum strips and finally storing them in Liquid nitrogen. With the application of the optimized protocol, four carnation cultivars (‘Master’, ‘Calibra’, ‘Lamour’ and ‘Ofcar’) achieved regrowth percentage after cryopreservation ranging from 41 to 73 %. Ultrastructural observations investigated by using transmission electron microscopy showed that the cells encountered the stress during cryopreservation and the main damages occurred during the dehydration step. For surviving cells, the most of the damaged cells could be repaired after recovery growth. This modified protocol will aid in the long-term conservation of carnation germplasm and the ultrastructural studies will benefit for understanding the damage and recovery of the cells during cryopreservation.

Optimization of adventitious root culture for production of biomass and secondary metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l(-1)) and naphthalene acetic acid (NAA; 1.5 mg l(-1)). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5-2.0 mg l(-1)). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l(-1) NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l(-1) NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l(-1) NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.

English

The present study was designed to understand seed germination barriers and to develop an efficient micropropagation system in Drypetes roxburghii. Fresh seeds with coats removed, cultured on MS medium supplemented with + 1.4 &mu;M gibberellic acid (GA3) + 0.01% activated charcoal (AC) resulted in maximum (83.30 &plusmn; 0.63%) seed germination with healthy seedling development. Cotyledonary nodal explants showed best shoot proliferation on Murashige and Skoog (MS) medium supplemented with 1 &mu;M 6-benzylaminopurine (BAP) with maximum number of shoots (1.80 &plusmn; 0.24) and shoot length (4.10 &plusmn; 0.34 cm). The combination of hormones did not show any significant effect compared with the individual cytokinins tested. The maximum number of shoots (1.70 &plusmn; 0.26) with shoot length 3.44 &plusmn; 0.22 cm was observed on MS medium supplemented with 1 &mu;M BAP + 2 &mu;M kinetin (Kn). In encapsulation experiments, the nodal explants encapsulated with liquid MS medium + 2 &mu;M BAP + 3% sucrose + 3% sodium alginate showed maximum shoot sprouting (1.70 &plusmn; 0.33) on MS medium supplemented with 1 &mu;M BAP + 3% sucrose. The in vitro derived shoots were best rooted on MS medium supplemented with 2 &mu;M IBA with maximum number of roots (2.10 &plusmn; 0.34) and root length (6.21 &plusmn; 0.24 cm) per shoot. The plantlets developed in vitro were successfully established under field conditions with 90% survival rate. &nbsp; Key words: Seed germination, micropropagation, phytohormones, synthetic seeds.

Effect of BA Treatments on Morphology and Physiology of Proliferated Shoots of &amp;lt;i&amp;gt;Bambusa vulgaris&amp;lt;/i&amp;gt; Schrad. Ex Wendl in Temporary Immersion

Axillary buds, collected from greenhouse-grown plants of Bambusa vulgaris Schrad. ex Wendl (B. vulgaris), were incubated on a static liquid culture medium, Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 12.0 μM 6-benzyladenine (BA). They were transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 6.0, 12.0, 18.0 μM BA. The morphological and anatomical indicators were measured. The BA influenced in vitro multiplication of B. vulgaris. The best results were achieved in the SIT with a concentration of 6.0 μM of BA, which increased the number of shoots (5.1 shoots/explant) in the absence of hyperhydric shoots. Results demonstrated that the water content in the sprouts increased with 12.0 and 18.0 μM BA every four hours. Furthermore, these high levels of BA contributed to a lower accumulation of phenolic compounds and lignin content. The total chlorophyll significantly increased when using 6.0 uM BA, but decreased both parameters with other treatments. These results favor to increase the number of shoots/explants during in vitro multiplication. They will also optimize the in vitro culture conditions, leading to an improvement of in vitro propagation methods for this species.

Cryopreservation of cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens) shoot tips by droplet-vitrification technique

ABSTRACT The droplet-vitrification technique was applied to in vitro shoot tips of cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens). Explants were precultured in the dark at 23 °C, in liquid MS medium with a progressively increasing sucrose concentration (0.3 M for 15 h, then 0.7 M for 5 h). Loading involved a 30 min incubation of explants in a solution comprising 1.9 M glycerol and 0.5 M sucrose. Explants were dehydrated at room temperature using a solution PVS A3 [Murashige and Skoog (MS) liquid medium, 22.5% (w/v) sucrose, 37.5% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide] for 30, 40 and 50 min and the PVS3 solution [MS liquid medium, 50% (w/v) sucrose, 50% (w/v) glycerol] for 60, 90 and 120 min. Explants were cooled by direct immersion in liquid nitrogen (LN) in 10 μl droplets of vitrification solution placed on aluminum foil strips. The foil strips were retrieved from LN and immersed in preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, and an equal volume of unloading solution at room temperature was added for further incubation for 30 min. Shoot tips were transferred onto the regrowth medium, cultivated in the dark for 7 days before being incubated under standard conditions. Three weeks after transferring the shoot tips onto the regrowth medium, the survival rate of control and cryopreserved explants of Gisela 5 dehydrated with PVS A3 was 100%, regardless of the treatment duration. After dehydration with solution PVS3, the survival varied between 70 and 100% for control explants and 78 and 95% for cryopreserved shoot tips. Gisela 5 shoot tips dehydrated for 40 min with PVS A3 vitrification solution demonstrated the best regrowth (38%). When using the PVS3 solution, survival of cryopreserved shoot tips was the highest (95%) after 60 min treatment followed by 40% regrowth. After three successive subcultures on shoot multiplication, medium shoots recovered viability, multiplication ability and morphology equal of that prior to cryopreservation.

Micropropagation and Polyploid Induction of Acer platanoides ‘Crimson Sentry

Protocols were developed for micropropagation and induction of autopolyploids in a fastigiate cultivar of Norway maple (A. platanoides L. ‘Crimson Sentry’). Murashige and Skoog (MS) medium, woody plant medium (WPM), and Quoirin and Lepoivre medium were supplemented with 2 μM 6-benzylaminopurine (BA), meta-Topolin, 6-(γ,γ-dimethylallylamino) purine, kinetin, or thidiazuron to evaluate microshoot proliferation. Murashige and Skoog medium with 2 μM BA yielded the most microshoots (3.2) and longest microshoots (30.6 mm) per subsample after 5 weeks. The influence of BA concentration on proliferation was evaluated at 0, 2, 4, 8, or 16 μM. Optimal multiplication rate was achieved at 2 or 4 μM BA producing approximately 2.8 microcuttings per subsample after 5 weeks. To induce in vitro rooting, half-strength WPM was supplemented with 0, 5, 10, 20, 40, or 80 μM indole-3-butyric acid (IBA). Optimal in vitro rooting (70%), number of roots (2.5), and root length (15 mm) per subsample were achieved with 10 μM IBA after 8 weeks. To induce polyploidy, microcuttings were pretreated for 7 days on MS medium with 4 μM BA alone or combined with 1 μM IBA, indole-3-acetic acid (IAA), or 1-naphthaleneacetic acid prior to treatment in liquid MS medium containing 15 μM oryzalin for 3 days. Homogenous tetraploids were only obtained from shoots pretreated with IAA. This research provides optimized protocols for micropropagation and autopolyploid induction of A. platanoides ‘Crimson Sentry’ and demonstrates the development of tetraploid lines for use in future improvement programs.

Shoot Induction, Biochemical Changes During in vitro Rooting in Paeonia lactiflora Pall 'Hortensis'

Background: Peony is an ornamental as well as medicinal plant which is difficult to propagate in vitro . Its major problem during in vitro culture is leaching out of phenols in the medium. It causes hindrance in growth and development. Rooting is a complex process and main step for vegetative propagation. The main objective is to propagate this plant and produce multiple copies and understand rooting phenomenon which is usually complicated. Methods: The underground rhizomatous buds of Paeonia lactiflora Pall. ‘Hortensis’ were selected as the explants. In vitro micropropagation was carried out along with biochemical studies during rooting process. Results: Shoot induction, axillary shoot proliferation and rooting were established. The best initial medium for ‘hortensis’ was the half-strength Murashige and Skoog (MS) medium (double-strength Ca ) supplemented with 1 mg L N +2 1 6 benzyl-adenine (BA) and 0.5 mg L Gibberellic Acid (GA ). Shoot induction was 100% with 6.2 shoots per explant. 1 3 Rooting was also observed on the half-strength MS medium supplemented with 1.0 mg L Indole-3-butyric Acid 1 (IBA) and indole-3-acetic acid (IAA). The highest percentage of rooting (15%) was observed when in vitro shoots were dipped in the full-strength liquid MS medium supplemented with 10.0 mg L IBA without agar for five days and then 1 transferred to the quarter strength MS medium without any Plant Growth Regulator (PGR). Conclusion: The cultivar P. lactiflora hortensis is hard to root in vitro. The activities of peroxidase, indole acetic oxidase, polyphenolic oxidase and phenolic content have been estimated prior to and after rooting. These enzyme activities and phenolics have been reported to play a major role in rooting.