Survival Of Explants Research Articles

Establishment of an Open, Sugar-Free Tissue Culture System for Sugarcane Micropropagation

Tissue culture has been used for many years to produce a large number of plant species by micropropagation. Unfortunately, microbial contamination and production costs are major problems associated with this technology. We have developed a low-cost, open tissue culture system for rapid sugarcane micropropagation that uses a combination of broad-spectrum antimicrobial compounds, a sugar-free medium, and CO2 gas fertilizer as carbon source. A novel antimicrobial compound called “QX1” that contains nicotine, garlic extract, carbendazim, and “Yipeilong” was developed in this study. Non-autoclaved induction medium containing 0.5% (v/v) QX1 had a significantly lower microbial contamination and a significantly higher explant survival rate in comparision to that of autoclaved induction medium. Sugar-free medium containing 0.5% (v/v) QX1 and 1.0 g/L CO2 gas fertilizer was used for multiplication and rooting under the open tissue culture environment. Propagation efficiencies were similar in the newly developed and conventional tissue culture systems, but contamination was significantly less in the new system during both multiplication and rooting. Also, plantlet survival rate, fresh weight, and net photosynthetic capacity were increased significantly, and production costs decreased dramatically using the new tissue culture system compared with the conventional method. Thus, the low-cost, open, sugar-free tissue culture system can be adapted to produce sugarcane plantlets on a commercial scale.

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos.

Wheat is the most widely grown staple food crop in the world and accounts fordietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2days, 30min and 48h, respectively. The bacterial inoculum concentration of OD of1at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4mg/l along with antibiotics (cefotaxime 500mg/l and timentin 300mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

In vitro micropropagation in tropical short day onion (Allium cepa L.)

For in vitro micropropagation, short day tropical varieties of onion ‘Agrifound Dark Red’, ‘Punjab Naroya’ and ‘PRO-6’ were exposed to different concentrations and combinations of growth hormones. Pre-sterilization of basal plate of onion in 0.5% solution of bavistin followed by treatment with 0.1% mercuric chloride for 10 min. produced the highest rate of survival of explants (47.9%). Survival was further enhanced to 53.17% with the addition of 750 ppm cefotaxime in MS medium. Varietal differences were observed for in vitro establishment, multiplication and root induction. Among various combinations of growth hormones, MS medium supplemented with 4.0 mg/l BAP + 0.5 mg/l NAA, 2 mg/l BAP and 0.5 mg/l NAA and half MS carrying 1.0 mg/l IBA + 0.5 mg/l NAA produced the highest in vitro establishment (53.07%), multiplication (64.46%) and rooting (66.37%) respectively, in ‘Agrifound Dark Red’.

In vitro tetraploidization for the augmentation of wedelolactone in Sphagneticola calendulacea (L.) Pruski

A practical and reliable method for in vitro tetraploidization of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] has been established to enhance the production of wedelolactone. Shoot tip and nodal explants from in vitro-grown culture (2n = 50) were exposed to the antimitotic chemical, i.e., colchicine, at various concentrations (0, 0.025, 0.05, 0.1, 0.3, and 0.5%; w/v) for 12, 24, 36, 48, and 60 h. The treated explants were then incubated and proliferated on Murashige and Skoog (MS) medium fortified with 0.2 mg l−1 thidiazuron and 0.05 mg l−1 naphthalene acetic acid, followed by root induction in 1.0 mg l−1 indole-3 acetic acid enriched ½MS medium. Treatment of shoot tips with 0.05% colchicine for 24 h supported the maximum rate of survival (63.33%) of explants as well as tetraploid induction (42.93%). Morphological, stomatal, and cytological characteristics along with the secondary metabolite content of the in vitro tetraploids were compared to that of diploids. The recovered tetraploid plants possessed superior plant height, stem diameter, leaf size, root number, and increased length and width of stomata but decreased stomatal frequency. The tetraploid plants demonstrated twice the chromosome number (2n = 4x = 100) than the diploids as confirmed through cytology, spectrophotometry and flow cytometry. High-performance thin-layer chromatography showed a significant enhancement in the wedelolactone content of tetraploid plants (541.48 µg g−1 of dried sample) in comparison to diploid plants (325.43 µg g−1 of dried sample), signifying the prospective of this technique for the trade value improvement.

Leptin protects placental cells from apoptosis induced by acidic stress.

Development of the human placenta is critical for a successful pregnancy. The placenta allows the exchange of oxygen and carbon dioxide and is crucial to manage acid-base balance within a narrow pH. It is known that low pH levels are a risk of apoptosis in several tissues. However, there has been little discussion about the effect of acidic stress in the placenta. Leptin is produced by the placenta with a trophic autocrine effect. Previous results of our group have demonstrated that leptin prevents apoptosis of trophoblast cells under different stress conditions such as serum deprivation and hyperthermia. The purpose of the present work is to evaluate acidic stress consequences in trophoblast explant survival and to determine leptin action in these conditions. For this objective, term human trophoblast explants were cultured at physiological pH (pH 7.4) and at acidic pH (pH 6.8) in the presence or absence of leptin. Western blot assays were performed to study the abundance of active caspase-3 and the p89 fragment of PARP-1. Pro-apoptotic and pro-survival members of Bcl-2 family, as Bax, t-Bid, and Bcl-2, were studied. Moreover, p53 pathway was also evaluated including Mdm-2, the main p53 regulator. Active caspase-3 and cleaved PARP-1 abundances were increased at low extracellular pH. Moreover, t-Bid levels were also augmented as well as p53 expression and phosphorylation on S46. Leptin treatment prevents the consequences of acidosis, decreasing p53 expression and increasing Mdm-2 expression. In summary, this work demonstrated for first time that low pH induces apoptosis of human trophoblast explants involving apoptotic intrinsic pathway, and leptin impairs this effect.

A novel microfloating culture system for the in vitro rooting of Echinacea angustifolia D.C.: photosynthetic performance and production of caffeic acid derivatives

The in vitro rooting phase of Echinacea angustifolia DC. was studied through an unconventional approach, where micropropagated shoots from adult plants were grown under autotrophic conditions. The system employed artificial supports (cellulose plugs) soaked with a liquid sugar-less MS medium that contained 1 mg L−1 indole-3-butyric acid (microfloating). The culture vessels were provided with passive ventilation (PV) or forced ventilation (FV) by bubbling air into the liquid medium. Using this microfloating system, the E. angustifolia plantlets reached about 65% rooting capacity. The cultures were analyzed for their photosynthetic rates (PN), carbohydrate content and phytochemical profile. When the ventilation of the vessels was forced, the net photosynthetic rate was enhanced improving explant quality and survival during acclimatization, but the content of caffeic acid derivatives (CADs) was negatively affected. On the contrary, at the end of the multiplication phase in mixotrophic conditions, the photosynthesis was impaired and the production of CADs was improved. The mixotrophy or autotrophy status experienced by the E. angustifolia microplants during the micropropagation process can direct the cultures towards either the accumulation of active compounds or the production of good quality transplants suitable to undergo the acclimatization process.

In vitro response of three contrasting cassava (Manihot esculenta Crantz) varieties to mannitol-induced drought stress

Abstract In vitro selection of drought-tolerant cassava varieties is essential for rapid breeding for drought tolerance. The objectives of this study were to determine the response of three contrasting cassava varieties to mannitol-induced drought stress to establish its suitability for in vitro screening and examine relationships among growth parameters. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing 0 (control), 5, 10, 15, 20, 25 and 30 g/l mannitol. Variety CH 140 had the highest survival of explants and frequency of root formation, while MV 99/0395 recorded the highest number of chlorotic leaves and the lowest survival of explants. The lowest numbers of leaves were produced at 25 and 30 g/l mannitol by the three varieties. In CH 140, the highest number of leaves was produced in medium free of mannitol, while the highest number of leaves was produced at 5 and 10 g/l mannitol in MV 99/0395 and TMS 01/1206, respectively. In TMS 01/1206, number of roots produced decreased as the concentration of mannitol in culture media increased, whereas in CH140, number of roots increased as the concentration of mannitol increased before decreasing; while in MV 99/0395, number of roots was not affected by an increase in mannitol concentration. As the concentration of mannitol in the culture media increased shoot height of plantlets decreased with a sharp decline at 20 mg/l mannitol. Concentration of mannitol and survival of explants had significant negative correlation with all parameters. However, frequency of root formation only had significant positive correlation with shoot length. The study concluded that differential responses were expressed by the three varieties to mannitol-induced drought stress and mannitol at 20 g/l concentration was a suitable in vitro drought inducing-agent for screening cassava varieties for drought tolerance.

In vitro response of three contrasting cassava (Manihot esculenta Crantz) varieties to mannitol-induced drought stress

Abstract In vitro selection of drought-tolerant cassava varieties is essential for rapid breeding for drought tolerance. The objectives of this study were to determine the response of three contrasting cassava varieties to mannitol-induced drought stress to establish its suitability for in vitro screening and examine relationships among growth parameters. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing 0 (control), 5, 10, 15, 20, 25 and 30 g/l mannitol. Variety CH 140 had the highest survival of explants and frequency of root formation, while MV 99/0395 recorded the highest number of chlorotic leaves and the lowest survival of explants. The lowest numbers of leaves were produced at 25 and 30 g/l mannitol by the three varieties. In CH 140, the highest number of leaves was produced in medium free of mannitol, while the highest number of leaves was produced at 5 and 10 g/l mannitol in MV 99/0395 and TMS 01/1206, respectively. In TMS 01/1206, number of roots produced decreased as the concentration of mannitol in culture media increased, whereas in CH140, number of roots increased as the concentration of mannitol increased before decreasing; while in MV 99/0395, number of roots was not affected by an increase in mannitol concentration. As the concentration of mannitol in the culture media increased shoot height of plantlets decreased with a sharp decline at 20 mg/l mannitol. Concentration of mannitol and survival of explants had significant negative correlation with all parameters. However, frequency of root formation only had significant positive correlation with shoot length. The study concluded that differential responses were expressed by the three varieties to mannitol-induced drought stress and mannitol at 20 g/l concentration was a suitable in vitro drought inducing-agent for screening cassava varieties for drought tolerance.

Conserved thermal performance curves across the geographic range of a gametophytic fern.

Species-level responses to environmental change depend on the collective responses of their constituent populations and the degree to which populations are specialized to local conditions. Manipulative experiments in common-garden settings make it possible to test for population variation in species’ responses to specific climate variables, including those projected to shift as the climate changes in the future. While this approach is being applied to a variety of plant taxa to evaluate their responses to climate change, these studies are heavily biased towards seed-bearing plant species. Given several unique morphological and physiological traits, fern species may exhibit very different responses from angiosperms and gymnosperms. Here, we tested the hypothesis that previously detected population differentiation in a fern species is due to differentiation in thermal performance curves among populations. We collected explants from six populations spanning the species’ geographic range and exposed them to 10 temperature treatments. Explant survival, lifespan and the change in photosynthetic area were analysed as a function of temperature, source population and their interaction. Overall results indicated that explants performed better at the lowest temperature examined, and the threshold for explant performance reflects maximum temperatures likely to be experienced in the field. Surprisingly, explant fitness did not differ among source populations, suggesting that temperature is not the driver behind previously detected patterns of population differentiation. These results highlight the importance of other environmental axes in driving population differentiation across a species range, and suggest that the perennial life history strategy, asexual mating system and limited dispersal potential of Vittaria appalachiana may restrict the rise and differentiation of adaptive genetic variation in thermal performance traits among populations.

Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.).

Saffron (Crocus sativus L) is a triploid (2n = 3x = 24), sterile geophyte which can only be propagated by means of underground vegetative corms. Since corm multiplication does not induce genome variations, therefore, the entire saffron population is expected to have a similar genetic makeup. Keeping in view the economic importance of the plant and the factors responsible for its low yield, the present investigation has been undertaken to establish an in vitro ethyl methanesulfonate (EMS) mutagenesis protocol followed by characterization of the induced variability in the advanced generations. The present report is limited to standardization of in vitro mutagenesis protocol only. Among the mutagenic treatments tested, concentrations ranging from 0.1 to 0.5% EMS showed a varied survival of explants. Based on various growth parameters, the LD50 was calculated to be 0.3% EMS for 3h. Among the two types of explants analyzed, the corm explant gave better results for in vitro survival and the growth parameters than callus explant. An average of 57.33 and 92.00 daughter cormlets in all EMS treatments as compared to 47.67 and 57.67 daughter cormlets in control, obtained from callus and corm explants respectively, were transferred to the field. The maximum, average daughter cormlet weight was obtained in control (3.01g, corm explant) followed by 0.1% EMS (2.8g, corm explant). In general, the growth parameters showed decreasing trend with an increase in EMS concentration in both the explants. The present study has been a significant achievement in the sense that the first mutagenesis protocol for C. sativus has been standardized.

Long-term experimental in situ farming of Crambe crambe (Demospongiae: Poecilosclerida).

BackgroundThe marine sponge Crambe crambe was chosen as an experimental model of sustainable shallow-water mariculture in the Sardinian Sea (Western Mediterranean) to provide biomass with high potential in applied research.MethodsExplants were cultured in four long-term experiments (19 and 31 months at ca. 2.5 m depth), to determine the suitability of new culture techniques by testing substrata and seeding time (season), and monitoring survival and growth. Explants were excised and grown in an experimental plant close to the wild donor sponge population. Percentage growth rate (GR%) was measured in terms of surface cover area, and explant survival was monitored in situ by means of a digital photo camera.ResultsExplant survival was high throughout the trial, ranging from 78.57% to 92.85% on travertine tiles and from 50% to 71.42% on oyster shells. A few instances of sponge regression were observed. Explant cover area correlated positively with season on two substrata, i.e., tiles and shells. The surface cover area and GR% of explants were measured in the starting phase and monitored up to the end of the trial. High GR% values were observed both on tiles (>21%) and on oyster shells (>15%).DiscussionThe data on the behaviour and life-style of cultured fragments, together with an increase >2,400% in cover area, demonstrate that in situ aquaculture is a viable and sustainable method for the shallow-water biomass supply of Crambe crambe.

Multiplicação in vitro e aclimatização de Melocactus sergipensis

Melocactus sergipensis is a newly discovered species, endemic to the state of Sergipe and critically endangered. The objective of this research was to establish a protocol for micropropagation and acclimation of M. sergipensis plants. The experimental setting was a completely randomized design, with four treatments, 10 replicates and in each experimental unit an explant was introduced. Explants were obtained from medial sections of the stem of approximately 5 mm. The nutrient medium was ½ salts of MS supplemented with 30 g L -1 of sucrose, gelled with 7 g L -1 of agar and with the following concentrations of phytoregulators: 6-benzylaminopurine (BAP) [0,0; 1,5; 3,0 and 6,0 mg L -1 ] and naphthalenoacetic acid (ANA) [0,0; 1,5; 3,0 and 6,0 mg L -1 ] and the BAP/ANA combinations [0,0; 1,0/0,5; 2,0/1,0 and 4,0/2,0 mg L -1 ]. The mean values of (shoots per explant, stem height and stem diameter) and percentage of calogenesis, rooting explants, survival of explants and shoots and weight of fresh matter were evaluated. Data were submitted to analysis of variance and means were compared by the Tukey test, 5% significance. There were no significant differences between treatments in relation to bud formation, callus, shoot and bud survival, stem height, stem diameter, and cracked weight. Nutritional supplementation with 1,0/0,5 BAP/ANA mg L -1 promoted a higher shoot formation. During the ex vitro phase, 70% of the normal shoots and 0,1% of the hyperhydric shoots survived.

Micropropagation protocol for Antigonon leptopus an important ornamental and medicinal plant.

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3 min followed by MC 0.1% for 2 min while, using each of them individually (Clorox 20% or MC 0.1%) for 5 min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2 mg/l. The highest numbers of shootlets/explant were obtained when 2.0 mg/l of BAP or 0.5 mg/l BA + 0.2 mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0 mg/l each combined with 0.2 mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36 bp, micropropagated plants showed 95.2% similarity in relation to mother plant.

Effects of various preculture, pretreatment, and recovery conditions on the morphogenetic response of cryopreserved Lady Orange chrysanthemum shoot tips

The aim of this research was to optimize the encapsulation-dehydration cryopreservation protocol of Lady Orange chrysanthemum explants on the preculture, pretreatment, and post-rewarming recovery steps. Shoot tips were precultured on MS medium with various abscisic acid concentrations (0-30 µM). Next, the encapsulated explants were osmotically dehydrated for 3 or 5 days and desiccated for 3-4 h and, after rewarming, they were subcultured on recovery media of various compositions (control and cytokinin/auxin-supplemented). A high explant survival rate, even up to 100%, was observed. The value of this parameter, however, changed depending on the post-rewarming culture duration. Moreover, not all the viable explants were capable of forming shoots. A lower ABA concentration (15 µM) during preculture and the presence of 4.65 µM kinetin in the post-rewarming recovery medium enhanced cryopreservation efficiency with a high survival rate and typical microshoot formation. A higher ABA concentration and the presence of 6-benzylaminopurine in the recovery medium resulted in shoot multiplication, abundant callus formation, and root formation inhibition.

English

Cassava starch from nine varieties, namely, NR 8082, TMS 97/2205, TMS 97/0162, TMS 92/0057, TMS 98/0505, TMS 92/0326, TMS 30572, TMS 82/0058, and TME 419 were evaluated for their suitability as gelling substitute to conventional gelling agents (gellan gum and agar) in medium using cassava shoot tips and nodal segments as explants. Explants were seeded singly into a 15 ml cassava multiplication medium gelled either in 0.2% gellan gum, 0.7% agar or 7% starch from the nine cassava varieties. Cultures were maintained at 28 ± 2°C, 16 h photoperiod and 30 to 40 μEm-2 s-1 flux intensity supplied by white fluorescent tubes on shelves for four weeks. Percentage survival of explants irrespective of type ranged from 61.5 to 100 with NR 8082 and TMS 97/2205 cassava starch-gelled medium recording the highest score while the mean number of nodes produced per explant ranged between 3.6 ± 1.43 and 5.33 ± 0.87 for shoot tips and 2.73 ± 0.96 and 4.79 ± 0.97 for nodal segments. The nodal segments from TME 419 starch-gelled medium had the highest mean number of nodes though not significantly different (p>0.05) from those from gellan gum and agar media. TME 419 was the most consistent in influencing regeneration of cassava plantlets.   Key words: TME 419, cassava starch, explants, gelling agent, micropropagation.

Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum\xa0\xd7\xa0grandiflorum /Ramat./Kitam.

Cryopreservation is widely applied to many economically important species excluding chimera plants which are problematic for long-term conservation. Their storage problems can be circumvented only by cryopreserving meristems. This study looked at the morphogenetic response of shoot tips of periclinal chimera chrysanthemum ‘Lady Orange’ and ‘Lady Salmon’, as well as the solid mutant ‘Richmond’, that were cryopreserved by encapsulation-dehydration technique. By applying 10 µM ABA in the preculture medium followed by 4-day-long dehydration treatment, the explant survival reached up to 67%. Besides the stimulation of typical single shoot recovery, cryopreservation led to direct or indirect multiple shoot formation, shoot malformation, as well as inhibited their spontaneous rooting. Microscopic analysis revealed three types of structural damages of shoot tips which can correspond with their morphogenetic response in recovery culture. No influence of cryostorage on the acclimatisation efficiency of the recovered chrysanthemums was observed.

Mother plant luminescence and zeatin concentration in the in vitro establishment of an olive plant

This study aimed to evaluate the effect of the presence and absence of light provided to the mother plant and zeatin concentrations in vitro establishment, to reduce oxidation and promote the establishment of olive explants. The trials were carried out at the Fruit Tree Propagation Lab, Federal University of Pelotas, Brazil. In the first experiment, plants of olive 'Arbequina' were subjected to the absence and presence of light and established on WPM with 0 and 2mg.L-1 zeatin. In the second experiment, plants of olive 'Leccino', 'Arbequina' and 'Frantoio' 'were submitted to the absence of light, and established in vitro on WPM with 0,4 and 8 mg.L-1 Zeatin. Mother plants of olive tree 'Arbequina' in conditions of absence of light are the most suitable in vitro establishment. The concentration of 2 mg.L-1 of zeatin promotes survival of explants of olive 'Arbequina'. The concentrations 0,4 and 8 mg.L-1 of zeatin) results no difference between Leccino and Arbequina cultivars for the variable fungal contamination.

Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

Summary In vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

Compatible solutes improve regrowth, ameliorate enzymatic antioxidant systems, and reduce lipid peroxidation of cryopreserved Hancornia speciosa Gomes lateral buds

Hancornia speciosa is a native fruit species from Brazil with important economic applications. However, the use of this species has been challenged by difficulties in its storage. Cryopreservation is a safe and efficient tool for long-term storage. Nevertheless, cryopreservation may promote reactive oxygen species (ROS) formation with subsequent lipid peroxidation, which causes membrane damage. The use of in vitro antioxidant compounds could help eliminate ROS and consequently improve explant survival. In the current study, the lateral buds of H. speciosa were precultivated in proline or glycine betaine (GB)-containing solutions, and the effect of these compounds on the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) and on lipid peroxidation was measured after sample warming. The present findings indicated that oxidative stress induced by cryopreservation was significantly reduced by the precultivation of lateral buds with proline and GB. SOD activity increased, and subsequently, hydrogen peroxide (H2O2) production increased followed by the immediate activation of CAT and APX. The enzyme activity increased for 7 following rewarming, indicating that precultivation with 0.1 M proline was fundamental to achieving high levels of shoot regeneration from H. speciosa lateral buds.

Plantlet regeneration of adult Pinus massoniana Lamb. trees using explants collected in March and thidiazuron in culture medium

A protocol for micropropagation using nodal explants from mature Pinus massoniana trees has been developed. Time of explant collection is crucial for the initial success of aseptic culture. Explants collected in early March gave the highest percentage of explant survival (64.5%) and shoot-forming percentage (52.3%). Thidiazuron (TDZ) concentration significantly influenced shoot formation; 4 μM TDZ was optimum, with 4.8 shoots produced per explant with a mean length of 7.1 cm after 120 days of culture. Regenerated shoots rooted for 60 days in basic medium with 1 μM NAA were ready for growth in pots. This is the first report on plantlet regeneration in vitro from mature trees of P. massoniana that provides a reliable method for propagating selected elites.