Use Of Explants Research Articles

Effect of Explants Type and Plant Growth Regulators on the Initiation and Multiplication of Vegetative Shoots of Stevia rebaudiana Bertoni in vitro

This study was carried out in the Plant Tissue Culture Laboratory of the College of Agriculture - Karbala Unaiversity for the period from 2022 to 2023. The study included the use of explants and different combinations in the sterilization, initiation and multiplication of the vegetative shoots of the Stevia plantiv vitro, the study was carried out in three phases: the first included sterilization of explants (shoot tips and lateral buds) using sodium hypochlorite at concentrations of (0, 0.5, 1, 1.5, 2)% for periods of (5, 10, 15) minutes. The second included the initiation of shoots cultures by cultivating the shoot tips and lateral buds on the MS medium with different concentrations (0, 0.5, 1, 1.5) mg l-1 BA and (0, 0.1, 0.2, 0.3) mg l -1 NAA, the third stage was carried out by cultivating the growths resulting from the shoot tips cultivated in the previous stage on the MS medium prepared with different concentrations (0, 1, 2, 3, 4) mg l-1 BA and (0, 0.1, 0.2, 0.3, 0.4) mg l-1NAA. The results of the study showed the superiority of the developing apical in achieving the lowest contamination rate of 38% compared to the side buds that achieved a contamination rate of 40.67%, and the concentration of 2% of NaOCl achieved the lowest pollution rate of 8.33%, and the duration of 15 minutes achieved the lowest pollution rate of 24%. The results of the initiation experiment showed the superiority of the shoot apical as a explants that achieved the highest response rate to emergence of 70% compared to the lateral shoots that recorded a response rate of 54.69%, and the concentration of 1 mg l-1 BA was significantly superior and achieved a response rate of 82.5% compared to the comparison treatment that gave a rate of 82.5%. A response of 32.5%. As for the results of the vegetative multiplication experiment, the results showed the superiority of BA at a concentration of 3 mg l-1 in achieving the highest rate in the number and length of branches was 33.35 branches Plant-1, 8.20 cm respectively. compared to the comparison treatment that achieved the lowest rate, and the concentration of 0.3 mg l-1 NAA was superior in achieving the highest average of the number and length of the vegetative total reached 30.84 plant branch-1, 7.65 cm respectively, while the comparison treatment achieved the lowest mean for the studied traits.

Bud Dormancy Release Dynamics in the Olive Tree and Validation of the Use of Explants to Determine Chilling Requirement Fulfillment

Bud Dormancy Release Dynamics in the Olive Tree and Validation of the Use of Explants to Determine Chilling Requirement Fulfillment

Organogenesis on apical buds in common fig (Ficus carica) var. Black Jack

Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 µM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 µM BAP + 8 µM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. In the present study the observations indicate that WPM supplemented with 20 µM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant. How to cite: Parab AR, Chew BL, Yeow LC, et al. Organogenesis on apical buds in common fig ( Ficus carica ) var. Black Jack. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2020.01.010

MRNA transcription profile of potato (Solanum tuberosum L.) in response to explant cutting

The explant is an important parameter in plant tissue culture. Most plant tissue culture studies in the literature have focused almost exclusively on the use of explants as a tool for organogenesis or (micro)propagation, and there is limited information that documents the changes that occur in explants in response to cutting during explant preparation just prior to or during culture. To better understand the molecular changes occurring during the wounding process that accompanies the preparation of an explant, this study examined the mRNA transcription profile of 4-week-old potato (Solanum tuberosum L. cv. Desirée) single-node stem segments. To achieve this, RNA-seq analysis was performed to identify significantly differentially expressed genes (DEGs) that existed between uncut in vitro plantlets and cut explants. DEGs corresponding to specific enzyme profiles were broadly connected to cellular processes in the extracellular region, nucleus, and plasma membrane, and were associated with biosynthesis, carbohydrate metabolism and catabolism, cellular protein modification, growth and development, and response to stress. The RNA-seq data of eight highly up- or down-regulated DEGs was validated by RT-qPCR, showing high positive Spearman and Pearson correlation coefficients (0.73 and 0.95, respectively) between SeqMonk LFC and RT-qPCR LFC. These results provide a novel perspective of the changes taking place within explants when they are cut and serve as a valuable basis for the study of explant-related stress in plant tissue culture in other species. In addition, our findings may contribute to an understanding of the success or failure of the tissue culture protocol and subsequent establishment of in vitro cultures by better appreciating the stress-related molecular changes taking place.

Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia

Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.

Using blubber explants to investigate adipose function in grey seals: glycolytic, lipolytic and gene expression responses to glucose and hydrocortisone

Adipose tissue is fundamental to energy balance, which underpins fitness and survival. Knowledge of adipose regulation in animals that undergo rapid fat deposition and mobilisation aids understanding of their energetic responses to rapid environmental change. Tissue explants can be used to investigate adipose regulation in wildlife species with large fat reserves, when opportunities for organismal experimental work are limited. We investigated glucose removal, lactate, glycerol and NEFA accumulation in media, and metabolic gene expression in blubber explants from wild grey seals. Glycolysis was higher in explants incubated in 25 mM glucose (HG) for 24 h compared to controls (C: 5.5 mM glucose). Adipose-derived lactate likely contributes to high endogenous glucose production in seals. Lipolysis was not stimulated by HG or high hydrocortisone (HC: 500 nM hydrocortisone) and was lower in heavier animals. HC caused NEFA accumulation in media to decrease by ~30% relative to C in females, indicative of increased lipogenesis. Lipolysis was higher in males than females in C and HG conditions. Lower relative abundance of 11-β-hydroxysteroid dehydrogenase 1 mRNA in HG explants suggests glucose involvement in blubber cortisol sensitivity. Our findings can help predict energy balance responses to stress and nutritional state in seals, and highlight the use of explants to study fat tissue function in wildlife.

Adventitious shoot organogenesis from leaf and petiole explants of European hazelnut

Adventitious shoot organogenesis and somatic embryogenesis are the basis for implementing new genetic variability and biotechnological approaches in woody species, particularly if mature tissues from valuable cultivars are used. To date, these technologies are only applied to few tree species due to the absence of efficient regeneration protocols. In hazelnut, adventitious shoot organogenesis and somatic embryogenesis have only been carried out with zygotic embryo tissues up to now. Here we report plant regeneration from explants of somatic origin by using in vitro rejuvenated mature tissues (leaves, petioles and stipules). A histological analysis carried out on calli grown on various media, showed significant evidence of shoot regeneration, as proved by the presence of vascular elements such as tracheids with annular or helical secondary wall thickening. Subsequently, the optimization of the regeneration protocol performed by pre-treating the explants with antibiotics (carbenicillin, vancomycin, and cefotaxime) as molecules with auxin-like effects enabled us to achieve shoot organogenesis in hazelnut. The organogenic competence strictly depended on the explants and antibiotics used in the experiments. Following an antibiotic pre-treatment of cv Tonda Gentile Romana explants in the proliferation stage, organogenic responses (frequencies of 40 %) were obtained. According to the results obtained, the best protocol for inducing shoot organogenesis in hazelnut should include the use of explants (leaves and petioles) conditioned in a double-liquid layer of cefotaxime 1000 mg L−1 added to the proliferation medium, cultured on the induction medium, consisting in solid MS medium supplemented with 3 % sucrose, 6-benzylaminopurine 1 mg L−1, indole-3-butyric acid 1 mg L−1 and kinetin 2 mg L−1, and then by sub-culturing the newly-formed calli to regeneration medium, consisting of half-strength solid MS medium with sucrose (30 g L−1) and 6-benzylaminopurine (0.5 mg L−1).

DOMESTIC EXPLANTS BASEKS IN RECONSTRUCTIVE SURGERY OF AORTA AND MAGISTRAL ARTERIES

Purpose. The use of explants in cardiovascular surgery is often accompanied by infectious, thrombotic, hemorrhagic complications and the most severe complication of postoperative period represents the infection of the explants itself, which develops at a frequency of 1.5 to 6 % followed by a mortality rate of 75–100 %. Thrombosis of the explants after surgery occur with a frequency of 6 to 42 %. Materials and methods. From 1996 to 2015, explants of BASEKS were used in 3 600 patients. Implants of BASEKS were used in 75 patients with aortic valve malformation in combination with an aneurysm of the ascending aorta and prosthetic endocarditis and in 1 944 patients with reconstruction of the aorta and arteries. Results. Bacteriological studies have demonstrated a wide range of antimicrobial action of the modifying components covering the explant and patches. The most promising results were obtained for micro-organisms which most commonly caused infections of these explants. All the sample explants had antimicrobial property and the zone of lysis around these explants were maintained for a longer time (up to 30 days or more). The residual anti-microbial explants after surgery were studied in vitro in 5 patients who died from peritonitis, heart, lungs and kidney failure. Explants after sampling were placed on growth media seeded with clinical strains of microorganisms. Within 12–24 hours, zones (15–18 mm) that caused the delay of the growth of microorganisms were formed around the studied fragments, confirming the preservation of the residual antimicrobial activity of the explants after implantation. During the period of follow-up, in 1.9 % of the patients in immediate postoperative period and in 4.1 % of patients in later postoperative period occurred thrombosis of the explants. Surface festering of the postoperative wound was observed in 155 (4.3 %) patients, deep festering – in 51 (1.4 %), 98 (2.7 %) patients succumbed to postoperative complications. Conclusion. The explants and patches of BASEKS which is antimicrobial, thromb-resistant and impermeable, could be widely used in cardiovascular surgery.

The Diagnostic Performance of Dynamic Contrast-enhanced MR Imaging for Detection of Small Hepatocellular Carcinoma Measuring Up to 2 cm: A Meta-Analysis.

To determine the performance of dynamic contrast material-enhanced magnetic resonance (MR) imaging in the diagnosis of small (≤2-cm) hepatocellular carcinoma (HCC) and to identify factors that influence this performance. Medline and Embase databases were searched for studies performed from January 2000 to March 2014 in which the performance of MR imaging was reported for the detection of HCC up to 2 cm on either a lesion- or patient-based level, with sufficient data to construct 2 × 2 contingency tables. Diagnostic performance was quantitatively pooled for all studies by using a bivariate random-effects model with exploration involving subgroup analysis, meta-regression, and determination of study heterogeneity. Twenty-two studies with 1387 small HCC lesions in 1908 patients met inclusion criteria. Heterogeneity was higher for sensitivity (range, 30%-99%) than specificity (range, 61%-100%). Overall sensitivity was 78% (95% confidence interval [CI]: 68%, 85%; I(2) = 89%), and overall specificity was 92% (95% CI: 88%, 95%; I(2) = 69%). The primary potential source of bias was use of explant as the reference standard in only 13% of studies, although lower sensitivity in such studies was not significant (59% vs 80%, P = .165). Sensitivities were significantly higher for studies that originated from Asia compared with those that originated elsewhere (89% vs 71%, P = .028), those performed with hepatobiliary phase imaging compared with those without (87% vs 65%, respectively; P = .004), and those in which gadoxetate disodium was used versus an extracellular agent (92% vs 67%, P ≤ .001). Specificity was not significantly different between subgroups (P ≥ .122). At pairwise meta-regression analysis with either study origin from Asia or performance of hepatobiliary phase imaging, only gadoxetate disodium contrast agent showed significant independent association with higher sensitivity (P = .002-.007). Results of this meta-analysis suggest that dynamic contrast-enhanced MR imaging has moderate sensitivity and excellent specificity in the detection of HCC up to 2 cm. Gadoxetate disodium contrast agent showed the strongest association with increased sensitivity.

Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker & JC-1). Output of this sperm assay (obtained after 30min) was compared to cell viability (ATP-content, data obtained after 24–48h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P<0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r=0.559) and proportions of motile (r=0.55) or progressively motile (r=0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

Early-flowering sweet orange mutant ‘x11’ as a model for functional genomic studies of Citrus

BackgroundThere had been many reports on genetic transformation of Citrus for functional genomic studies but few included genes associated with flower or fruit traits. A major reason for this might derive from the extensive juvenile stage of Citrus plants when regenerated from juvenile explants (epicotyls, cotyledon or calli), which delays the observation of the resulting phenotype. Alternatives include the use of explants from adult tissues, which sometimes may be recalcitrant to regeneration or transformation, or of early-flowering genotypes. However, there is no report about the use of early-flowering sweet orange mutants for functional genomic studies.ResultsHere, we propose a sweet orange spontaneous early-flowering mutant, named ‘x11’, as a platform for Citrus functional genomic studies, particularly for genes associated with flower or fruit traits. We report a procedure for efficient regeneration and transformation using epicotyl segment explants of ‘x11’ and Agrobacterium tumefaciens as a proof-of-concept. The average transformation efficiency was 18.6%, but reached 29.6% in the best protocol tested. Among 270 positive shoots, five were in vitro micrografted and acclimatized, followed by evaluation of transgene expression by quantitative amplification of reversed transcripts (RT-qPCR) and determination of the number of copies inserted. Four of these plants, containing from one to four copies of the transgene, exhibited the first flowers within three months after ex vitro establishment, and the other, two months later, regardless of the period of the year. Flowers of transgenic plants displayed fertile pollen and gynoecium, with self-pollination inducing fruit development with seeds. Histochemical staining for β-glucuronidase activity using stem segments, flowers and fruits from 5 to 7 month-old acclimatized transgenic plants confirmed the constitutive transgene expression in these organs.ConclusionThe ‘x11’ sweet orange is suitable for functional genomics studies with a satisfactory transformation rate, and it can be considered a good model for functional genomic studies in commercial sweet oranges, for traits related to flower and fruit.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-511) contains supplementary material, which is available to authorized users.

Explantes teciduais: um modelo redescoberto na experimentação animal

The ex vivo technique was developed in the 30’s and in recent years, due to ethical and legal issues in laboratory experiments, was rediscovered as effective technique. Pigs are often used as animal model in this technique due to the high similarity in various organ systems with humans. Explants of intestine, skin, joint, lung and bronchus of pigs have been used in animal experiments, with different purposes, such as evaluating the effects of toxic, carcinogenic, therapeutic, and biological agents or sensitivity tests. The use of explants minimizes ethical problems and lack of human samples, that often affect researches. A major limitation of the technique is time cell viability, due to hypoxia that occurs during incubation. Moreover, the method allows more controlled experimental procedures associated with a reduction in the number of animals, expanding the areas of scientific research.

In vitro protection of auditory hair cells by salicylate from the gentamicin-induced but not neomycin-induced cell loss

Salicylate has been shown to protect animals and people from the gentamicin-induced hearing loss. The objective of our study was to determine if salicylate is otoprotective in vitro. In this fashion, we wanted to validate the use of explant culture system for future studies on the ototoxicity prevention. In addition, we wanted to find out if salicylate protects from the ototoxicity of other aminoglycosides. As a model, we used the membranous cochlear tissues containing the organ of Corti, spiral limbus and spiral ganglion neurons dissected from the cochleas of p3–p5 Wistar pups. The explants were divided into apical, medial and basal parts and cultured in presence or absence of 100 μM gentamicin, 100 μM neomycin and 5 mM salicylate. Following the tissue fixation and staining with phalloidin–TRITC, the number of inner and outer hair cells (IHCs, OHCs) was scored under the fluorescent microscope. Presence of 5 mM salicylate in explants cultures exposed to 100 μM gentamicin significantly reduced the loss of IHCs and OHCs, as compared to explants exposed to gentamicin alone. In contrast, neomycin-induced auditory hair cell loss remained unaffected by the presence of salicylate. Our results corroborate earlier in vivo findings and validate the use of cochlear explants for future studies on ototoxicity and its prevention. Moreover, the inability of salicylate to prevent neomycin-induced ototoxicity implies possible differences between the mechanisms of auditory hair cell loss induced by gentamicin and neomycin.

Efficient Callus Induction, Plant Regeneration and Estragole Estimation in Tarragon (Artemisia dracunculus L.).

Our research studied the fast-breeding technology of Artemisia dracunculus using tissue culture in order to provide the scientific foundation for industry production. The susceptibility of callus formation and plant regeneration was studied under different conditions of basic medium, hormones and additives. The use of explants of different origins as the leaf, stem and root explants was also studied for best callus induction rate, regeneration ability and estragole concentration. The best culture medium for the induction of calli was MS + 2.4.D 1.0 mgl−1, which started with the leaves as explants, and the best medium for stem regeneration was MS + BA 0.5 mgl−1+ NAA 0.1 mgl−1. A new HPLC method was developed to provide a specific procedure for the rapid and facile analysis of the major constituent of tarragon volatile oil estragole. Estragole concentration was estimated at all the experiment treatments including the induced calli under different medium conditions, as well as the regenerated plants. Estragole concentration was found to be 9.8% dry weight in the in vitro regenerated plantlets. Using this method estragole was found to be absent in different plant extracts of the field grown plants (acclimatized after in vitro regeneration)

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented. Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture. The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower. The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates.

Application of tissue culture to cashew ( Anacardium occidentale L.) breeding: An appraisal

Summary of the previous works on the in vitro culture of cashew is highlighted with emphasis on the critical factors that influence the explants response and plantlet regeneration. The recalcitrant nature of cashew has been attributed to the limited success recorded so far in the in vitro culture of the crop and abnormal development has been reported in the calli derived from its explants.Browning of explants in cashew was found to be due to the presence of high secondary metabolites and it has been reduced through frequent transfer of explants, addition of activated charcoal and dark treatment. Explants necrosis has also been traced to the effect of strong sterilization. Meanwhile, the use of explants from in vitro germinated seedlings or fungicidal treated young flush has been found to improve the success rate significantly. The use of MS base salt supplemented with two-step treatment of cytokinins enhances the response of cashew explants and development of derived plantlets. Key words: Cashew, Invitro Culture, Breeding, Explant Browning, Growth Regulators

How to produce large sized microtubers of potato cv. Desiree

In vitro tuberization was induced on explants with different number of nodes layered on a medium with high sucrose (8%) content: 30, 15, 10, 7 and 6 explants per jar were cultured containing 1, 2, 3, 4 or 5 nodes, respectively. Microtubers developed were graded by their smallest diameter, and the number of tubers per jar, their size distribution, their fresh weight and the multiplication rate were recorded. The highest multiplication rate (1.98) was obtained for explants with 5 nodes. The size distribution of tubers was markedly affected by treatments. The majority of microtubers (49.4%) were 6-8 mm in the case of the smallest explants (with I node). When explants with 2 to 5 nodes were used, the most microtubers were 8-10 mm but with an increase of explant size, more and more microtubers were produced with larger diameter up to 16 mm and average fresh weight of tubers also increased with the increase of explant size. For the microtuber production of Desiree the use of explants with two nodes can be suggested because in this treatment the average fresh weight of microtubers was high enough (250 mg) and the number of large sized microtubers was very high (79% was larger than 6 mm and 53% was larger than 8 mm).

Micropropagation of Limonium cordatum (L.) Mill. for conservation purposes

SummaryL. cordatum (L.) Mill. is a Ligurian endemism of the Northern Mediterranean area. Human use of the coast and seaside resorts has modified the areas where this plant has its habitat. Reviews of the literature and field investigations have enabled a progressive decrease in the population and in the number of specimens in the areas occupied by the species to be measured. Micropropagation tests pointed out the possibility of ex situ multiplication activities. The use of explants from the basal part of the inflorescence stems was very successful in obtaining many new vegetative buds producing shoots with a normal appearance and development. In L. cordatum, low BA concentrations increase the rate of production of new shoot but had an inhibitory effect on rooting; on the contrary, the addition of auxin to the medium leads to increased root formation but inhibits shoot development. According to our experience, a micropropagation procedure based on the use of a simple MS medium without addition of growth regulators, is suggested for L. cordatum. In situ planting could be possible, with good results. Once young plants had survived in vivo transplanting they incurred no further adaptation problems.

DECONTAMINATION OF KAVA (PIPER METHYSTICUM) FOR IN VITRO PROPAGATION

The major constraint to kava (Piper methysticum) tissue culture is endogenous contaminants. Despite stringent surface sterilisation procedures, decontamination has been unsuccessful in the past. In view of this problem, which prevents the establishment of kava in vitro, different approaches to decontamination have been investigated. The first approach involved growing kava plants in a glasshouse and applying 8-hydroquinoline with various concentrations of BAP to induce new shoots. Once new shoots had been initiated the plants were sprayed weekly with a solution containing a fungicide (Benlate) and an antibiotic (rifampicin). It was thought that the combination of chemicals would alleviate the problems of endogenous contaminants. However, the use of explants from treated plants and new shoots were both unsuccessful. The second approach involved the use of antibiotics in sterilization procedures and the incorporation of antibiotics in culture medium. Various fungi, bacteria, and yeast contaminants were identified and were tested against a range of antibiotics that were added to the culture medium. There were always a few cultures that remained clean for 3 - 5 weeks and when contaminants started to appear, an attempt to rescue these cultures by resterilizing and transferring to new medium failed due to phytotoxicity effects. The use of explants from the treated plants is preferred as they showed a limited degree of contamination and delayed the appearance of contaminants. The decontamination strategies suggested are based on the results of these decontamination procedure investigations and the knowledge of the phytotoxicity effects of the disinfectants and antibiotics on kava.

Photoautotrophic growth of potato plantlets as affected by explant leaf area, fresh weight and stem length

Photoautotrophic growth in vitro of potato ( Solanum tuberosum L. cv. Benimaru) explants varied with their initial leaf area and stem length. Photoautotrophic growth was much greater in leafy than in leafless explants. Variability in photoautotrophic growth was smallest in the explants with the greatest leaf area. The results indicated that use of explants with a large leaf area is important to maximize photoautotrophic growth and to minimize variation in photoautotrophic growth of explants in vitro.