Axillary Shoot Multiplication Research Articles

Influences of Thidiazuron Concentrations and Exposure Duration on Axillary Shoot Proliferation of Faba Bean [Vicia faba (L.)] Genotypes

Background: Faba bean is one of the most important feed grain legume crops in the world. However, this crop is a recalcitrant species for in vitro regeneration. Methods: In this study, in vitro shoot proliferation of ten faba bean genotypes, that are classified according to their seed size into small, medium and large, were investigated in response to different light regimes and thidiazuron (TDZ) treatments. Result: Incubation under different light regimes, explant type as well as genotypes influenced seedling growth and axillary shoot multiplication of faba bean. The explants with single cotyledon and incubated under continuous darkness for 6 days resulted higher number of shoot than nodal explants. Medium size seeded genotype (ILB4347) produced the highest shoot and root length and shoot proliferation as compared with large (Luz) or small (Triple White) seeded genotypes. The effects of TDZ concentrations (2-10 mM/L) and exposure duration (10 and 20 days) on axillary shoot multiplication were investigated for the ten faba bean using cotyledonary explants. The overall mean of TDZ time exposure showed that treatment of 20 days exposure had higher number of shoot compare to 10 days exposure treatment with 3.77 shoot per explant. On the basis of genotype used, the three most responsive genotype in tissue culture were medium sized seeds genotypes (ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot per explant, respectively). ILB4347 were the most responsive genotype in tissue culture. Treatment of 8 mM TDZ after 20 days followed by 2 weeks on MS medium without PGRs generated the highest proliferation with 6.63 shoot per explant. These findings contribute towards effective in vitro propagation of faba bean.

Multiplication of apical and axillary shoots of coffee arabica using various types of cytokinins

Abstract In-vitro shoot multiplication presents a promising avenue for replicating the genetic characteristics of parent plants, commonly utilized in various plant biotechnological techniques such as mutation replication, protoplast fusion, anther culture, and genetic engineering. The primary aim of this research was to assess the impact of different cytokinins on the proliferation of apical and axillary shoots in Arabica coffee multiplication. A Completely Randomized Factorial Design was employed, considering two factors: explant source and cytokinin type. Explants were sourced from both apical and axillary branches of AS2K Arabica coffee plantlets. Three cytokinins, namely 6-Benzyl adenine purine (BAP), isopentenyl adenine (2-IP), and kinetin, were incorporated into the Murashige and Skoog (MS) 1962 basal medium at concentrations of 0, 1, 2, and 3 mg/l. The findings revealed significant effects of all three cytokinins on shoot number, plantlet height, and leaf growth, albeit without influencing root development. Notably, axillary shoot-derived explants demonstrated a more pronounced effect on shoot count compared to those from apical shoots. Among the cytokinin treatments assessed, BAP at a concentration of 2 mg/l in the MS medium yielded optimal results for propagating the AS2K variety of Arabica coffee, thus representing the recommended medium for this purpose.

Plant Growth Regulator- and Elicitor-Mediated Enhancement of Biomass and Andrographolide Production of Shoot Tip-Culture-Derived Plantlets of Andrographis paniculata (Burm.f.) Wall. (Hempedu Bumi).

Andrographis paniculata (Burm.f.) Wall. (Acanthaceae) is revered for its medicinal properties. In vitro culture of medicinal plants has assisted in improving both the quantity and quality of their yield. The current study investigated the effects of different surface sterilization treatments, plant growth regulators (PGRs), and elicitors on culture establishment and axillary shoot multiplication of A. paniculata. Subsequently, the production of andrographolide in the in vitro plantlets was evaluated using high-performance liquid chromatography (HPLC) analysis. The shoot-tip explant was successfully sterilized using 60% commercial bleach for 5 min of immersion with a 90% survival rate and 96.67% aseptic culture. The optimal PGR for shoot growth was 6-benzylaminopurine (BAP) at 17.76 µM, supplemented into Murashige and Skoog (MS) media, producing 23.57 ± 0.48 leaves, 7.33 ± 0.10 shoots, and a 3.06 ± 0.02 cm length of shoots. Subsequently, MS medium supplemented with 5 mg/L chitosan produced 26.07 ± 0.14 leaves, 8.33 ± 0.07 shoots, and a 3.63 ± 0.02 cm length of shoots. The highest andrographolide content was obtained using the plantlets harvested from 5 mg/L chitosan with 2463.03 ± 0.398 µg/mL compared to the control (without elicitation) with 256.73 ± 0.341 µg/mL (859.39% increase). The results imply that the protocol for the shoot-tip culture of A. paniculata was developed, and that elicitation enhanced the herbage yield and the production of andrographolide.

Ex Situ Conservation of Atriplex taltalensis I.M. Johnst. via In Vitro Culturing of Its Axillary Shoots

Atriplex taltalensis is an endangered shrub endemic to northern Chile. Sparse populations of this species can be found in areas with extreme edaphoclimatic conditions in the Atacama desert, and its seeds have a poor germination rate. Consequently, mass-cultivating it can be challenging. In this study, the vegetative propagation mechanisms of A. taltalensis were evaluated using an in vitro culture to aid in their conservation. A. taltalensis shoot explants were treated with two phytoregulators, indole-3-butyric acid (IBA) and 6-benzylaminopurine (BAP), to assess the morphogenic responses and their effects on the propagation of the species, based on shoot multiplication, elongation, and rooting, through subculturing. During multiplication, the treatment with IBA alone efficiently promoted explant elongation, lateral root formation, and axillary shoot proliferation, allowing for the rapid development of shoots into whole plants. Alternatively, treatment with IBA and BAP in combination stimulated the proliferation of basal shoots with little elongation and rooting and promoted shoot hyperhydricity at 0.25–1 mg L−1 BAP concentrations. Thus, we conclude that A. taltalensis propagation is viable through in vitro plant tissue culture using a rapid axillary shoot multiplication system, and this method could aid in the conservation of this species through in vitro propagation and rescue programs.

In Vitro Propagation of Pyracantha angustifolia (Franch.) C.K. Schneid.

Narrow-leaf firethorn or pyracantha (Pyracantha angustifolia (Franch.) C.K. Schneid.), from the family Rosaceae, is a species of large and thorny evergreen shrub. In this study, a procedure is presented for efficient axillary shoot multiplication and root induction in P. angustifolia using Murashige and Skoog (MS), woody plant (WPM), and Linsmaier and Skoog (LS) culture media supplanted with 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA). The disinfection of the axillary buds was performed with a 70.23% success rate on a basal MS medium augmented with 0.5 mg·L−1 gibberellic acid (GA3). Uniform and axenic explants were then cultured on MS, WPM, and LS media enriched with different concentrations of BAP, 0.3 mg·L−1 GA3, and 0.1 mg·L−1 IBA. The highest multiplication coefficient (2.389) was obtained for the MS medium supplemented with 2.5 mg·L−1 BAP. After one month, newly formed micro-shoots were transferred to rooting media (MS, WPM, and LS) containing different concentrations of IBA, together with a constant concentration of 0.1 mg·L−1 BAP. The micro-shoots were kept in the dark for one week and then cultured in a 16/8 h light/dark regime. The MS medium supplemented with 1 mg·L−1 IBA was the most effective in stimulating rooting (88.76% of micro-shoots). The highest number of roots (3.5 per micro-shoot) was produced in the MS medium enriched with 1.5 mg·L−1 IBA. The rooted plantlets were transferred into pots filled with perlite and peat moss in a 2:1 proportion and acclimatized to ambient greenhouse conditions, with a resultant mean 92.84% survival rate. Thus, this protocol can be successfully applied for the in vitro mass propagation of P. angustifolia.

EXPLANT STERILIZATION AND IN VITRO CULTURE OF PURPLE SWEET POTATO TO OPTIMIZE NORMAL SHOOT FORMATION

<p class="abstrakinggris">Sweet potato (<em>Ipomoea batatas</em> L.), a tuber-producing plant, is a functional food that produces carbohydrates while meeting nutritional needs. Propagation of sweet potato through tissue culture is often hampered in the initial culture due to contamination. The study aimed to optimize sterilization of explants and growth of <em>in vitro</em> culture of purple sweet potato. Tubers of purple sweet potato cv. Antin 2 were <em>ex vitro</em> cultured through a semi-immersion system to produce shoots. The shoots as explants were sterilized with alcohol, fungicide, and sodium hypochlorite (P1); alcohol and sodium hypochlorite (P2); sodium hypochlorite (P3), and washed on sterile distilled water as control (C). The explants were then cultured on MS solid medium in tubes and jars. Growth and multiplication of shoots were carried out on MS solid medium added with cytokinins (BA and kinetin) at different concentrations. The results showed that the best sterilization method was obtained in the sodium hypochlorite (P3) and alcohol-sodium hypochlorite (P2) treatments, with sterile shoots reaching 100%. Planting the explants in jars gave higher normal shoot formation (85–100%) than that in tubes (12.5–48%). The use of kinetin at 0.5–1 mg L<sup>-1</sup> gave good shoot vigor. The best axillary shoot multiplication was found on media with 0.5 mg L<sup>-1</sup> BA. Growing explants on the semi-immersion system and sodium hypochlorite sterilization produced the highest sterile ones, whereas culturing three explants promotes normal growth straight after sterilization.</p>

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR’s) and concentrations tested. The aforesaid PGR’s combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542–1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Energy-Saving LED Light Affects the Efficiency of the Photosynthetic Apparatus and Carbohydrate Content in Gerbera jamesonii Bolus ex Hook. f. Axillary Shoots Multiplied In Vitro.

Simple SummaryGerbera is one of the most important ornamental plants on the cut flower market. The basic reproduction methods of numerous cultivars of this species are in vitro techniques. In in vitro cultures, all plant growth conditions are controlled, including the light (intensity, quality, and duration). In tissue cultures, light quality is the most important factor that influences plant morphogenesis (growth and development). Light emitting diodes (LEDs), in contrast to the commonly used fluorescent lamps, allow for adjusting the light quality to the specific requirements of plants. LEDs are also energy-efficient and contain no harmful substances (e.g., mercury). The aim of the study was to analyze the effect of different light qualities emitted by LEDs during in vitro multiplication of Gerbera on its metabolic and physiological development. We compared endogenous carbohydrate content in the tissues and the condition of the photosynthetic apparatus in plants grown under fluorescent lamps and LED light. The study showed that the use of LEDs did not disturb the secondary metabolism of carbohydrates and the multiplied shoots were of high quality. The mixture of red and blue LED light in a 7:3 proportion is recommended for gerbera micropropagation. This light quality positively influenced the functioning of the photosynthetic apparatus.An energy-saving light emitting diode (LED) system allows for adjustment of light quality, which affects plant development and metabolic processes in in vitro cultures. The study investigated the content of endogenous carbohydrates and the condition of the photosynthetic apparatus of Gerbera jamesonii Bolus ex Hook. f. Our aim was to analyze the effects of different LED light qualities—100% red light (R LED), 100% blue (B LED), a mixture of red and blue (7:3) (RB LED), and a fluorescent lamp as a control (Fl)—during the multiplication of axillary shoots. After 40 days, the culture measurements were performed using a non-invasive pulse amplitude modulation (PAM) fluorimeter. Sugar content was assessed with high performance liquid chromatography (HPLC). Two forms of free monosaccharides (glucose and fructose), two sugar alcohol derivatives (inositol and glycerol), and seven forms of free oligosaccharides were identified. Of those, glucose content was the highest. LEDs did not disturb the sugar metabolism in multiplied shoots. Their monosaccharides were three times more abundant than oligosaccharides; the same results were found in plants grown under control light. R light depleted the performance of the photosynthetic apparatus and caused its permanent damage. The RB LED spectrum ensured the most efficient non-photochemical quenching of the photosystem II (PS II) excitation state and high shoot quality.

Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don.

This study aimed to investigate the impact of plant growth regulators, sucrose concentration, and the number of subcultures on axillary shoot multiplication, in vitro flowering, and somaclonal variation and to assess the phytochemical composition, antioxidant capacity, and enzyme inhibitory potential of in vitro-established callus, somaclonal variant, and normal green shoots of Catharanthus roseus. The highest shoot induction rate (95.8%) and highest number of shoots (23.6), with a mean length of 4.5 cm, were attained when the C. roseus nodal explants (0.6–1 cm in length) were cultivated in Murashige and Skoog (MS) medium with 2 µM thidiazuron, 1 µM 2-(1-naphthyl) acetic acid (NAA), and 4% sucrose. The in vitro flowering of C. roseus was affected by sucrose, and the number of subcultures had a significant effect on shoot multiplication and somaclonal variation. The highest levels of phenolics and flavonoids were found in normal green shoots, followed by those in somaclonal variant shoots and callus. The phytochemicals in C. roseus extracts were qualified using liquid chromatography–tandem mass spectrometry. A total of 39, 55, and 59 compounds were identified in the callus, somaclonal variant shoot, and normal green shoot tissues, respectively. The normal green shoot extracts exhibited the best free radical scavenging ability and reducing power activity. The strongest acetylcholinesterase inhibitory effects were found in the callus, with an IC50 of 0.65 mg/mL.

An approach for the mass propagation of Cupressus sempervirens L. (Cupressaceae), for quality propagule production

The scenarios of climate change in Mediterranean regions bring back the need for tree species tolerant to drought and disease, for agro- and urban-forestry purposes, landscape rehabilitation, as well as for post-fire and quarry restoration plans. Therefore, forest industries focus on selecting, propagating and growing superior trees. The Cupressus sempervirens L. is such a coniferous tree species, with a fundamental ecological, financial and ornamental role in the Mediterranean region. The aim of this study was to develop an efficient macropropagation protocol, which would lead to mass-selected genotypes or phenotypes of C. sempervirens f. sempervirens. For this purpose the axillary shoot multiplication method was used, by adopting both cutting propagation and intermittent mist methods. These methods were used for the first time in the macropropagation of C. sempervirens f. sempervirens. For shoot proliferation, 24 different modified macropropagation treatments at different concentrations and combinations were tested: shoot cuttings, concentrations of K-IBA and rooting systems. The elongation and root induction was carried out in an intermittent mist system for 50 days, then the new plantlets were moved for acclimatization for 15 days in a greenhouse. The current workflow presents an effective preliminary protocol with clear steps and treatments for macropropagation of C. sempervirens f. sempervirens. The developed protocol for macropropagation of tree species ensures cost- and time-efficient propagation. Produced plantlets were developed efficiently under in vivo conditions, allowing to propagate and store genetic material for conservation and domestication. This protocol can be generated for other tree species of the Cupressus genus and the Cupressaceae family.

Optimizing axillary shoot culture as a short-term conservation method for American chestnut (Castanea dentata)

Protocols were investigated in the short-term conservation of Castanea dentata (Marsh.) Borkh. to maximize axillary shoot multiplication rates at the genotype level and to reduce the incidence of shoot tip necrosis (STN). Factors investigated included explant growth stage (embryo, seedling, and mature stages) of the source material and two culture incubation temperatures (21 ± 1 °C and 31 ± 1 °C). Multiplication rate was measured across 22 genotypes. The role of 6-benzylaminopurine (0.5, 1.0, 2.0, and 4.0 μmol·L–1), calcium (3.0 and 6.0 mmol·L–1), boron (0.025 mmol·L–1), magnesium (3.0 mmol·L–1), and gelling agent (agar, 6.0, 7.0, and 8.0 g·L–1; gellan gum, 3.0, 3.5, and 4.0 g·L–1) on STN was investigated. Genotypes derived from 4-month-old seedlings had the greatest overall multiplication rate (7.87 ± 0.38 at 6 weeks). Temperature affected multiplication rate in three of eight genotypes tested, with multiplication rate greater at 21 ± 1 °C for one genotype and greater at 31 ± 1 °C for two genotypes. Multiplication rates differed significantly between genotypes and ranged from 1.45× at 12 weeks to 13.25× at 6 weeks. Incidence of STN was decreased to 1.40% ± 0.02% at 4.0 μmol·L–16-benzylaminopurine. The development of an efficient axillary shoot culture system for C. dentata greatly benefits current conservation efforts.

Rooting of <i>Vaccinium corymbosum</i> L. microshoots cv. «Blue-Berry» in culture <i>in vitro</i> and <i>ex vitro</i>

This paper discusses a method of micropropagation of Vaccinium corymbosum L. cv. Blue-Berry. The results showed that WPM supplemented with 1,0 mg/l of zeatin in combination with 0,1 mg/l of indolyl-3-butyric acid was more effective for the multiplication of blueberry axillary shoots. The maximum increase in the number of healthy axillary shoots was observed in the fourth subculture, whereas the phenomenon of hyperhydration (vitrification) began to appear in the fifth subculture. In addition, it was established that the presence of indolyl-3-butyric acid and 1,0 g/l of activated charcoal in the nutrient medium lead to the development of good root system of the Vaccinium corymbosum cv. Blue-Berry. The highest mean number of roots formed per explant was obtained on WPM medium, supplemented with 0,5 mg/l indolyl-3-butyric acid after 10 weeks. Acclimatization of in vitro regenerated plantlets of Vaccinium corymbosum with a developed root system in ex-vitro conditions (pH 3,54) showed a 100% survival rate.

EFFECT OF SUCROSE AND MEDIA STRENGTH ON IN VITRO MULTIPLICATION IN SWERTIA CHIRATA BUCH.-HAM EX WALL: AN ENDANGERED MEDICINAL HERB

Objective: The present study was performed to investigate the role of varying concentrations of carbohydrate source and strengths of nutrient medium in growth and development of in vitro shoots of Swertia chirata-an endangered medicinal plant.Methods: MS medium supplemented with 6-Benzylaminopurine (4.44 µM), Indole-3 acetic acid (2.85 µM) and Adenine sulphate (271.45 µM) was used to test the efficiency of of sucrose at concentrations of 1-5% and of media strength varying from full to one-fourth. The data were analysed using analysis of variance (ANOVA) of Completely Randomized Design (CRD) in GenStat 5 Edition 3.2 for PC/Windows NT (Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station))Results: Observations on axillary shoot multiplication indicated that sucrose at a concentration of 3% and MS medium in its full strength proved to be most optimal for in vitro culture multiplication. On this medium combination mean number of 11.80 shoots (after 4 w) and 18.50 shoots (after 8 w) could be obtained On sucrose free medium the shoots exhibited necrosis while at lower concentrations of 1-2% sucrose, the shoots developed were thin and unsuitable for further growth in vitro. At higher levels of sucrose in the medium, the shoots became thick and stunted. Similarly, reduction in medium strength resulted in a decline in shoot number and shoot length to an average of 6.50 shoots (1.33 cm mean length) on half strength medium and 5.60 shoots (0.88 cm mean length) on one-fourth strength; as observed after 4 w.Conclusion: The experimental findings suggest that any decline from the standard had a significant effect on the number, size and overall health of shoots developed in vitro. The conditions so standardized augment the production of healthy shoots that shall aid in subsequent rooting and survival after transplantation of tissue-culture raised plantlets.

Factor affecting in vitro propagation of some genotypes of Himalayan cedar [Cedrus deodara (Roxb. ex Lamb) G. Don.

Four genotypes of Himalayan cedar were grown in vitro for assessing shoot proliferation. This experiment consist three parts. Initially, the explants (leafy and defoliated shoot-tips) of mature plants were disinfected and cultured on different basal media (LP, MS and WPM) that supplemented with benzyladenine (BA) at different concentrations for 6 weeks. Leafless explants produced the highest number of shoots and the longest shoots for four genotypes. There was no significant difference between the culture mediums and benzyladenine concentrations. In second phase, the influence of benzyladenine (2.5, 5, 10, 20 µM) and thidiazuron (TDZ) (0.4, 0.8, 1.6 µM) with combination of different auxin (NAA) concentrations (0, 1, 2, 3 µM) was determined on axillary shoot proliferation of the leafless explants of four genotypes grown on WPM. For all thidiazuron concentrations, significant differences between genotypes were detected. In general, with all genotypes, the use of 0.8 μM thidiazuron in the absence or presence of auxin (2 μM) led to the highest length and number of axillary shoots per explant, respectively. Finally, in another experiment, the following cytokinin treatments were investigated for axillary shoot multiplication of the CD1 genotype: thidiazuron (0, 0.1, 0.2, 0.4, 0.8 μM) and N6-[2-Isopentenyl] adenine (2iP) (0, 0.1, 0.2, 0.3, 0.4, 0.5 μM) in combination with benzyladenine (2.5 µM). The best results were obtained in thidiazuron (0.8 µM) with combination of benzyladenine (2.5 µM). This protocol is considered as the first successful report on culture establishment of some genotypes of mature C. deodara trees.

Sources of silicon influence photosystem and redox homeostasis-related proteins during the axillary shoot multiplication of Dianthus caryophyllus

The present endeavor has demonstrated the impacts of different sources of silicon (Si) such as potassium silicate (K2SiO3) and calcium silicate (CaSiO3) during the in vitro axillary shoot multiplication of carnation. For the Si treatments, nodal explants were cultured onto the Murashige and Skoog’s medium fortified with 1.0 mg L−1 of 6-benzyladenine and 0.5 mg L−1 indole-3-acetic acid with or without K2SiO3 and CaSiO3 in three different concentrations (0, 1.8, or 3.6 mM). After six weeks, the shoot induction ratio, number of shoots produced per explant, expression of photosystem (PS) I and II core proteins, and activities of antioxidant enzymes were examined. Among the Si sources, K2SiO3 application enhanced the axillary shoot multiplication and the uptake of Si on comparison with CaSiO3. Both forms of Si resulted in the enhancement of stomatal density, and PS-related protein such as PsaA and PsbA illustrating the apparent involvement of Si on the photosynthetic process. Nevertheless, addition of Si improved the antioxidant capacity during the in vitro shoot multiplication. Overall, the outcomes of the present study suggested that Si can be utilized as a supplementary source during the in vitro propagation of carnation.

Efficient in vitro Clonal Propagation of Fast Growing Salix gracilistyla

This study was determined optimal condition as culture medium, pH, carbohydrate source and growth regulators through axillary buds cultures of Salix gracilistyla. S. gracilistyla shoots were obtained from axillary bud on 1/2MS basal medium after 2 weeks of culture. Highest shoot growth was showed in the MS medium(GI= 2.43). Shoot growth was highest in 9% sucrose compared to other concentration. BAP affects axillary shoot multiplication in of S. gracilistyla. Among nutrients, Ca2+ was not affected shoot number of S. gracilistyla. However, NH4+, NO3 -, and K+ is greatly influenced shoot number. However, Ca2+ was affected in shoot length of S. gracilistyla. Feeding of Mg2+ was not affected in shoot length. Root number is greatly influenced by NH4 +, NO3 -, and K+, however NH4 + and K+ was not affected root number of S. gracilistyla. NH4 + was affected root length, however Mg4 + was not affected root length. Treatment of 0.1mg/L IBA promoted in vitro rooting. In vitro rooted plantlets were successfully transferred to pots for 4weeks hardening process. Later, these plants were transferred to soil with above 90% survival rate in BS+SD(2:1). These results could be easily adopted for commercial large scale cultivation.

The effect of in vitro culture conditions on the pattern of maximum photochemical efficiency of photosystem II during acclimatisation of Helleborus niger plantlets to ex vitro conditions

The influence of in vitro conditions including sucrose and nitrogen content in the culture medium as well as temperature on the pattern of chlorophyll a photochemical efficiency of photosystem II (PSII) of Helleborus niger L. plantlets upon transfer to ex vitro conditions were examined. In the experiment, the micro-shoots which were proliferated and rooted in vitro on the media with different levels of sucrose (10, 20, 30, 40, 50, 60, 70, and 80 g L−1) and nitrogen salts KNO3 and NH4NO3 (both 25, 50, 100 % according to MS medium), as well as temperature (15 and 20 °C) were used. The multiplication of axillary shoots were performed on modified MS medium supplemented with cytokinins (2iP, BAP and kinetin, each at a concentration of 1.0 mg L−1) and GA3 (2.5 mg L−1) added together. For the rooting of shoots, the medium was supplemented with IBA 1 mg L−1 and NAA 0.1 mg L−1 added together. The maximal photochemical efficiency of PSII immediately upon removal of microplants from in vitro culture ranged from 0.24 to 0.81 depending on the treatment. High concentration of sucrose (above 50 g L−1) and standard (100 %) or nitrogen salts decreased to 25 % in the MS medium as well as 20 °C temperature decrease the photochemical efficiency of microplants at transplanting time to ex vitro conditions as well as hinder acclimatisation and further growth out of culture. During acclimatisation to ex vitro conditions, a gradual increase of the maximal photochemical efficiency of in vitro-derived leaves was observed in all treatments. The maximal photochemical efficiency of PSII ranged from 0.79 to 0.83 after 4 weeks and from 0.81 to 0.83 after 20 weeks, indicating that H. niger plantlets gradually adapted to the greenhouse conditions and recovered their photosynthetic performance.

Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

AbstractStudy describes protocols for

Influence of Benzyladenine and Thidiazuron on Shoot Regeneration from Leaf and Shoot tip Explants of Sedum sarmentosum Bunge

Effects of N6-benzyladenine (BA) or thidiazuron (TDZ) on adventitious shoot regeneration and axillary shoot multiplication of Sedum sarmentosum was investigated. Leaf and shoot tip explants obtained from in vitro-grown shoots of S. sarmentosum were cultured on Murashige and Skoog (MS) medium supplemented with 0, 2.0, 4.0 or 8.0 µM BA or TDZ. Of the two cytokinins studied, BA was found to be more responsive as compared to TDZ with respect to number of shoots produced per explant. High frequency of shoot regeneration (92.2%) with a mean of 12.3 shoots was produced when the leaf explants were cultured on MS medium supplemented with 8.0 µM BA. The highest number of shoots (25.4) was obtained when shoot tip explants were cultured on MS medium devoid of cytokinins after 35 days of culture. For root induction, regenerated shoots were cultured on MS medium supplemented with 0, 2.0, 4.0 or 8.0 µM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest number of (17.6) roots per shoot was obtained on MS medium supplemented with 2.0 µM IBA after 30 days of culture. Regenerated plantlets were successfully acclimatized in the greenhouse with 100% survival rate.

Assessing the effects of basal media on the in vitro propagation and nutritional status of Eucalyptus dunnii Maiden

In tissue culture, essential aspects of growth and development can be modulated by managing mineral nutrition and growth regulators. Therefore, basal medium selection is one of the key factors for success with this technique since the nutritional status of tissue cultures relies on it. Although Murashige and Skoog (MS), JADS, and woody plant medium (WPM) media are among the most common basal media used for the in vitro propagation of eucalypts, they have shown limited success for use in Eucalyptus dunnii micropropagation, where excessive callus formation, hyperhydricity, and chlorosis are frequent issues. These morpho-physiological responses could be related to different nutritional conditions provided by each media, showing that none of them was competent for E. dunnii micropropagation. To prevent these problems, a modified version of a novel basal medium for E. dunnii (EDMm), based on chemical analysis of E. dunnii young stump shoots was developed. The morpho-physiological responses and mineral nutrient contents of four E. dunnii clones cultured in EDMm, JADS, MS, and WPM basal media were assessed. Multivariate analysis suggested that Cu, Fe, Mn, and Zn concentrations in tissues, all of which are at higher concentrations in EDMm medium than the other media, were associated to higher rooting rates. On the other hand, higher K and S concentrations were related to higher multiplication and growth rates. The results showed that all basal media tested support axillary shoot multiplication, but only the shoots grown in EDMm media presented no Fe chlorosis or oxidation symptoms and showed higher rooting rates.