Adventitious Bud Differentiation Research Articles

Establishment of an efficient regeneration system of ‘ZiKui’ tea with hypocotyl as explants

Zikui (Camellia sinensis cv. Zikui) is a recently discovered cultivar of local purple tea in Guizhou, China. It is a purple leaf bud mutation material of Meitan Taicha (Camellia sinensis cv. ‘Meitan-taicha’) ‘N61’ strain, which is an important local germplasm resource in Guizhou. It is also a model plant for the study of anthocyanins, but the limited germplasm resources and the limitation of traditional reproduction hinder its application. Here, an efficient regeneration system is established by using hypocotyl as explants for the first time. Different plant growth regulators (PGRs) are evaluated during different regeneration processes including callus and root induction. According to our findings, using the optimal disinfection conditions, the seed embryo contamination rate is 17.58%. Additionally, the mortality rate is 9.69%, while the survival rate is measured as 72.73%. Moreover, the highest germination rate of 93.64% is observed under MS + 2.40 mg/L GA3 medium conditions. The optimal callus induction rate is 95.19%, while the optimal adventitious bud differentiation rate is 20.74%, Medium with 1.6 mg/L IBA achieved 68.6% rooting of the adventitious shoots. The survival rate is more than 65% after 6 days growth in the cultivated matrix. In summary, our research aims to establish a regeneration system for Zikui tea plants and design a transformation system for tea plant tissue seedlings. This will enable transfer of the target gene and ultimately facilitate the cultivation of new tea varieties with unique characteristics.

In vitro shoot regeneration from organogenic callus culture and rooting of Carpathian endemic Aconitum bucovinense Zapał.

Aconitum bucovinense Zapał. is the European species of monkshood, endemic to the Eastern and Southern Carpathians. A protocol has been developed for the in vitro regeneration of adventitious shoots by indirect organogenesis from leaf explants. An initiation of cultures carried out on a medium (B5 macronutrients, MS micronutrients) with picloram and kinetin allowed obtaining a callus. More than a 200% FM increase of the callus and at the same time differentiation of adventitious buds were obtained on IBA and BAP supplemented medium. Excised buds were used to establish shoot cultures and multiplied after a transfer to nutrient media with an addition of BAP with IBA, IAA or NAA. Almost 70% of rooted shoots were obtained on a 1.5 mg L−1 IBA and 1.0 mg L−1 BAP supplemented medium with simultaneous efficient multiplication. An analysis of peroxidase activity revealed its gradual increase in shoots until the appearance of roots. For the first time, an efficient way to regenerate, multiply and root A. bucovinense shoots has been developed and can be used for ex situ conservation of this species.

Integrated transcriptome and endogenous hormone analysis provides new insights into callus proliferation in Osmanthus fragrans

Osmanthus fragrans is an important evergreen species with both medicinal and ornamental value in China. Given the low efficiency of callus proliferation and the difficulty of adventitious bud differentiation, tissue culture and regeneration systems have not been successfully established for this species. To understand the mechanism of callus proliferation, transcriptome sequencing and endogenous hormone content determination were performed from the initial growth stages to the early stages of senescence on O. fragrans calli. In total, 47,340 genes were identified by transcriptome sequencing, including 1798 previously unidentified genes specifically involved in callus development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) was significantly enriched in plant hormone signal transduction pathways. Furthermore, our results from the orthogonal projections to latent structures discrimination analysis (OPLS-DA) of six typical hormones in five development stages of O. fragrans calli showed jasmonic acid (JA) could play important role in the initial stages of calli growth, whereas JA and auxin (IAA) were dominant in the early stages of calli senescence. Based on the weighted gene co-expression network analysis, OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b were selected as hub genes from the modules with the significant relevance to JA and IAA respectively. The gene regulation network and quantitative real-time PCR implied that during the initial stages of callus growth, the transcription factors (TFs) OfERF4 and OfMYC2a could down-regulate the expression of hub genes OfSRC2 and OfPP2CD5, resulting in decreased JA content and rapid callus growth; during the late stage of callus growth, the TFs OfERF4, OfMYC2a and OfTGA21c, OfHSFA1 could positively regulate the expression of hub genes OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b, respectively, leading to increased JA and IAA contents and inducing the senescence of O. fragrans calli. Hopefully, our results could provide new insights into the molecular mechanism of the proliferation of O. fragrans calli.

Establishment of regeneration system of callus pathway for Iris sanguinea Donn ex Horn

Iris is a perennial flowering plant, usually cultivated through seeds or bulbs. However, due to the limitations of traditional reproduction, the establishment of a callus regeneration system is particularly important, and callus induction and plant regeneration are the keys to transgenic technology. The callus regeneration system was studied using the I. sanguinea flower stem as explants. The experiment investigated the impact of different disinfectant solution concentrations, disinfection exposure times, and hormone concentrations on the explant growth status at various culture stages. The research analyzed the impact of the experimental variables on explant regeneration to determine the optimum callus regeneration system for I. sanguinea. In this research, it was determined that when I. sanguinea flower stems, that were used for explants, were treated with 75% alcohol for 30 s and 2% NaClO for 8 min for disinfection, there was a 54.48% callus induction rate. The callus induction medium was Murashige and Skoog medium (MS) + 6-benzylaminopurine (6-BA) 1.0 mg/L + 1-naphthylacetic acid (NAA) 0.2 mg/L + 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg/L for 40 d. The optimum medium for callus multiplication was MS + 6-BA 0.5 mg/L + 2,4-D 0.5 mg/L, while the optimum medium for callus adventitious bud induction was MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L + kinetin (KT) 0.5 mg/L, 60 d. The adventitious bud differentiation rate from flower stem calli was 46.03%.

Effects of Hormones and Epigenetic Regulation on the Callus and Adventitious Bud Induction of Fraxinus mandshurica Rupr.

Fraxinus mandshurica Rupr. (hereafter “F. mandshurica”) is known as one of northeast China′s important, valuable hardwood timber species. However, tissue culture and micropropagation of the species are difficult and have low efficiency, limiting asexual propagation. In this manuscript, stem explants were utilized to establish an effective regeneration system through adventitious bud organogenesis. The factors influencing callus regeneration in vitro were determined, and callus regeneration technology was established. The mechanism of adventitious bud formation was analyzed. Thidiazuron (TDZ) played a crucial role in the formation of adventitious buds. Elevated concentrations of TDZ were beneficial to callus induction and low concentrations of 6-benzyladenine (BA) led to loose state callus formation. The order of callus induction rates for different explants was stem cotyledon (100%) > segment (98.54%) > hypocotyl (92.56%) > root (50.71%). The effects of exogenous addition of 6-BA and TDZ on the endogenous hormone content of plants during the regeneration of adventitious buds were also assessed, as well as the expression characteristics of genes related to the regeneration pathway. The comprehensive analysis results showed that the suitable medium for callus induction and adventitious bud differentiation was c12 medium (MSB5 + 30 g/L sucrose + 7 g/L Agar + 5 mg/L 6-BA + 8 mg/L TDZ + 2 mg/L glycine + 0.1 mg/L IBA + 5% coconut water). The induction rates of callus and adventitious buds were 99.15% and 33.33%. The addition of 2.4 mg/L of the DNA demethylation reagent 5-azacytidine (5-aza) and 0.15 mg/L of the histone deacetylase inhibitor trichostatin A (TSA) increased the rates of adventitious bud induction by 17.78% over the control. This further laid the foundation for large-scale cultivation of excellent varieties and genetic transformation techniques.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant. In this study, seeds of D. spiculifolius were used as explants for callus induction, adventitious bud differentiation, and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (6-BA), and naphthaleneacetic acid (NAA) to Murashige and Skoog medium. The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min, and transgenic regenerated plants were obtained by kanamycin (30 mg · L−1) screening. RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D. spiculifolius. The β-glucuronidase (GUS) histochemical staining confirmed GUS gene expression in transgenic calli, adventitious buds, and regenerated plants of D. spiculifolius. The green fluorescent protein (GFP) visual analysis showed GFP gene expression in transgenic calli. Furthermore, subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D. spiculifolius callus cells. These results indicated a successful establishment of a reliable and efficient A. tumefaciens-mediated genetic transformation system, which will contribute to functional gene research and genetic improvement of D. spiculifolius.

Study on tissue culture system of Polygonatum cyrtonema

In order to accelerate the breeding of the excellent seedlings of Polygonatum cyrtonema,tissue culture system of P. cyrtonema was established through the comprehensive regulation of key factors( leaf age,leaf location,basic media and plant growth regulators) and cytological basis of callus formation and differentiation was analyzed through paraffin section. The results showed that the 30-day-old leaf base explanton medium MS+6-BA 1. 50 mg·L~(-1)+2,4-D 0. 20 mg·L~(-1) had the highest induction rate( 80. 00%). The callus was initiated from cells on leaf base epidermis and near cortex,formed by the differentiation of middle vascular bundle cells. The optimal medium for adventitious bud differentiation was MS+ 6-BA 4. 00 mg·L~(-1)+ 2,4-D 0. 20 mg·L~(-1) with the differentiation rate of90. 33%,and the average number of buds was 5. 16. The adventitious buds had two origin types: exogenous and endogenous origin,formed by callus proximal cells and callus internal meristemoid. The adventitious bud proliferation medium was screened by orthogonal design,which determined the optimum combination was MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 10 mg·L~(-1) and MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 20 mg·L~(-1). The tubers with three leaves were cut and inoculated in the medium 1/2 MS+IBA 2. 00 mg·L~(-1),showing the highest rooting rate of 94. 00%. The rooting seedlings transplanted into the peat-vermiculite( 1 ∶ 1) matrix grew healthy and the survival rate was over 85. 00%. This research provided a novel solution for large-scale cultivation of P. cyrtonema seedling.

Physical state of the culture medium triggers shift in morphogenetic pattern from shoot bud formation to somatic embryo in Solanum khasianum.

Solanum khasianum is a rich source of steroidal alkaloids that are important secondary metabolites with enormous pharmaceutical uses. Development of plantlets from somatic tissues, under in vitro conditions, takes place both through adventitious shoot bud differentiation or somatic embryogenesis (SE) pathway. We observed that the physical state of medium, solid or liquid, determined the regenerant differentiation patterns from root segment explants in S. khasianum. In the solidified medium, the root segments developed adventitious shoot buds whereas somatic embryos were regenerated in the liquid medium. Varying gradients from liquid to solid medium were further used to confirm the effect of solidified condition on regeneration pathway. Histological analysis of developing shoot buds and somatic embryos was also performed to confirm their development and differentiation patterns. In order to further confirm the developmental pathways, qRT-PCR analysis of the marker genes of SE and shoot regeneration was also performed. While SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SkSERK1) expression was significantly up-regulated during the early embryogenic stage, the LATE EMBRYOGENESIS ABUNDANT (SkLEA) proteinwas found to be highly expressed in the mature embryos. Expression of the HISTONE DEACETYLASE (HDA6), a repressor of SE related genes, was highly decreased during embryogenesis in the liquid culture. Furthermore, expression of the ENHANCER OF SHOOT REGENERATION (ESR) gene was comparatively increased during shoot regeneration in the culture usingsolid medium. Our results point out that the physical state of the medium in S. khasianum plays a decisive role in differentiation pattern which was independent of hormonal supplements.

Effects of Different Growth Regulating Substance on the Actinidia deliciosa Leaves Excised Direct Regeneration

Using the leaves of test-tube seedlings of Actinidia deliciosa as expiants, the effect of different growth regulating substance on the Actinidia deliciosa leaves excised regeneration system was researched, including the differentiation of adventitious buds, the regeneration and rooting of regenerated shoots. The results indicated that the maximum adventitious buds regeneration frequency achieved 76.6% on the medium of Murashige and Skoog medium(MS) containing 5.0 mg·L−1 6-benzylaminopurine(6-BA) and 0.2 mg·L−1 1-naphthaleneacetic acid (NAA); the most suitable medium for shoot multiplication was MS with 2.0 mg·L−1 6-BA, 0.2 mg·L−1 NAA and 0.1 mg·L−1 gibberellin acid(GA3) and the multiplication coefficient was up to 4.76; 1/2MS with 0.7 mg·L−1 1h-indolo-3-butanoic acid(IBA) was the most suitable for the rooting of regenerated shoots, and the rooting rate was 100%.

Construction of genetic transformation system of Salix mongolica: in vitro leaf-based callus induction, adventitious buds differentiation, and plant regeneration

Songnen meadow grassland is a typical saline-alkaline land majorly comprised of carbonate soil. Salix mongolica, a woody species with high adaptability to carbonate soil, is an important supplementary feed in the grassland. Therefore, it is necessary to cultivate new varieties of S. mongolica by using genetic engineering methods to reveal the functions of the plant’s related genes and to construct a plant regeneration and genetic transformation system. In this study, we used leaves of S. mongolica as the explants for induction of leaf-based callus, differentiation of adventitious buds and rooting of adventitious by adding different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine and naphthaleneacetic acid into the Murashige and Skoog medium. Under the screening conditions of 7.5 mg L−1 hygromycin B and transformation period of 2–5 min using a specific Agrobacterium containing pCXSN-gus plasmids infection concentration (ODλ600 = 0.5), we obtained transgenic strains. PCR detected exogenous gus gene integrated into the chromosome of S. mongolica, Southern blot analysed the T0 transgenic strains single copy inserted into the chromosome, Northern hybridization signals indicated that gus gene mRNA was expressed in the five contemporary transgenic strains. The infected callus, adventitious buds, and regenerated plants displayed a blue color through detection by GUS staining, which reflected the activity of s-glucuronidase enzyme. This result demonstrated the successful establishment of an Agrobacterium-mediated genetic transformation system from the callus (S. mongolica leaf as a transformation receptor).

The Efficient Tissue Culture System of <i>Orostachys fimbriata</i>

This study aimed to investigate the tissue culture and rapid propagation techniques of Orostachys fimbriata, a medicinal and ornamental herbaceous perennial herb belonging to the family Crassulaceae. The leaves of O. fimbriata were used as explants to investigate the effects of plant growth regulators such as thidiazuron (TDZ), 6-benzylamino purine (6-BA) and 1-naphthaleneacetic acid (NAA), on the induction of callus, differentiation of adventitious buds and rooting of shoots. Our results showed that optimum callus induction medium was MS medium supplemented with 0.5 mg·L-1 TDZ and 0.2 mg·L-1 NAA. 1.5 mg·L-1 6-BA and 0.2 mg·L-1 NAA was the optimum hormone combination for differentiation of adventitious buds. And 0.1 mg·L-1 NAA could efficiently promote rooting. The survival rate of transplants reached about 90%. In this study, using leaves of O. fimbriata as explants, high efficient tissue culture and regeneration system of O. fimbriata were established, and the period from leave to transplant plantlet was about 3 months. The presently developed protocol could be used for large scale clonal propagation and germplasm conservation of O. fimbriata. The efficient tissue culture system of O. fimbriata would provide technical support for its utilization.

Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens

Using epicotyl fragments of the actinorhizal tree Casuarinacunninghamiana and the disarmed strain of Agrobacterium tumefaciens C58C1 (pGV2260) containing the pBIN19-35S-GUSINT binary vector, a method for the genetic transformation of C. cunninghamiana was established. Transformed cells were initially selected for 2 weeks on nutrient medium supplemented with kanamycin 20 mg L−1 during callus induction, and then subcultured with 50 mg L−1 until adventitious bud and shoot differentiation. Different factors involved in the early stages of the T-DNA transfer process were studied. Agrobacterium-mediated DNA delivery was most successful when epicotyl fragments excised from 45-day-old seedlings were co-cultivated with an exponentially growing culture of A. tumefaciens at an OD600nm of 0.3, for 5 days in the presence of 50 μM of acetosyringone. Kanamycin resistant calli were observed on 88.89 % of the explants and transgenic rooted C. cunninghamiana plants were obtained in 6 months. Evidence of genetic transformation was demonstrated by s-glucuronidase histochemical assays and polymerase chain reaction analyses. The possibility to obtain transgenic nitrogen-fixing nodules after inoculation by the soil actinomycete Frankia was established.

Study on Tissue Culture of Sweet Broad Pea

s. The cotyledons, Internodes, leaves and stems of sweet broad pea were studied on tissue culture. Research results show that: The ability of different explants for callus formation and adventitious bud differentiation in different culture medium is different. The callus formation rate and sprouting rate of Internodes is significantly higher than other explants, which is a ideal material for tissue culture. The callus formation rate of Internodes was 100% in MS +BA1.0 mg/L+NAA 1.0 mg/L and MS+ 2, 4-D 0.5 mg/L; The bud differentiation is best at the medium of MS+ 6-BA 2 mg/L, which reached 86.7%; the rooting rate was 83.3% at the medium of MS+ NAA 3mg/L. The study provides a experimental basis for further study on the plant regeneration in the sweet broad pea.

A urea-type cytokinin, 2-Cl-PBU, stimulates adventitious bud formation of Eucalyptus urophylla by repressing transcription of rboh1 gene

An efficient regeneration protocol is the prerequisite of genetic modification of eucalyptus. However, adventitious bud differentiation from calli of eucalyptus is difficult because of rapid callus browning. By comparing three cytokinins in tissue culture of Eucalyptus urophylla, it was found that N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (2-Cl-PBU), a home-made urea-type cytokinin, induced more green callus than N-phenyl-N′-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ) or 6-benzylaminopurine (BAP). Only green calli were regarded as organogenic calli because they were the only one capable of differentiating adventitious buds in subsequent procedures. Six physiological indexes of green calli, including the contents of hydrogen peroxide, superoxide anion radicals, cellulose and lignin, and the activities of peroxidase and superoxide dismutase, were higher than that of white calli. Furthermore, the transcription levels of four respiratory burst oxidase homolog (rboh) genes declined significantly in organogenic green calli, especially that of EgrRboh01 (rboh1). Among the three treatments by 2-Cl-PBU, BAP and a combination of 2-Cl-PBU and BAP, the primary callus induced by BAP showed the highest rboh1 transcription level, 12.7–20 times higher than that by 2-Cl-PBU or by the combination of BAP and 2-Cl-PBU. The lowest rboh1 transcription level was observed in the calli induced by 2-Cl-PBU. The results indicated that 2-Cl-PBU restrained callus browning and promoted adventitious bud differentiation of E. urophylla by repressing the transcription of rboh1 gene.

In vitro selection of resistant mutant garlic lines by using crude pathogen culture filtrate of Sclerotium cepivorum

In vitro treatment of tissue cultures of two garlic cultivars with crude culture filtrates from Sclerotium cepivorium was used to select somaclonal variants with enhanced resistance to white rot disease. The crude culture filtrate had a negative effect on callus induction and growth. It also inhibited the differentiation of adventitious buds. The inhibitory effects were greater at higher culture filtrate concentration. With increase in concentration of crude pathogen culture filtrate, rate of callus induction, callus survival, relative growth rate of callus, callus differentiation and number of divided shoots per callus decreased. The inhibitory effect for all the parameters was more visible when crude pathogen culture filtrate was used at 50% concentration or more. However, the performance of disease resistant cultivar Hangzhong Red Skin was better than Gailiang (disease susceptible cultivar) at higher concentrations of crude pathogen culture filtrate. Some somaclonal lines and plantlets (predominantly from Hangzhong Red Skin) that showed resistance to Sclerotium cepivorum were obtained at 50%concentration level of crude pathogen culture filtrate.

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l −1 α-naphthaleneacetic acid (NAA) and 1.5 mg l −1 2,4-D with 0.5 mg l −1 NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l −1 NAA and 0.5 mg l −1 1-phenyl-3-(1,2,3-thiadiazol-5- yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l −1) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.

In vitro morphogenetic studies on three apomictic species of Garcinia

Comparison of morphogenetic potential of three important Indian species of Garcinia—G. indica, G. cambogia and G. xanthochymus has been reported. Apomictic seeds of G. indica were found to be morphogenetically most potential followed by G. cambogia. The explants of G. xanthochymus were highly recalcitrant towards in vitro conditions and failed to induce adventitious buds on any of the media tested. High frequency direct shoot bud differentiation was induced in aseptic seed cultures of G. indica and G. cambogia on MS medium supplemented with cytokinins (BAP, kinetin or TDZ). Amongst the three cytokinins tested, TDZ (0.1–0.5 μM) was most effective for adventitious bud differentiation in both G. indica and G. cambogia, however, the proliferating buds failed to elongate. Substantial number of buds induced on BAP supplemented media elongated into shoots after subculture on elongation medium. Addition of NAA along with cytokinins in the induction medium enhanced callusing without improvement in bud induction response. The induced adventitious buds were elongated on MS basal medium containing 0.2% activated charcoal. Direct rooting was achieved in both G. indica and G. cambogia on auxin supplemented media with best response at 10 μM IBA concentration in both the species. The in vitro raised plantlets showed 90% survival in the field when transferred after hardening and acclimatization.

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO3 on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ½ macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L−1 sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO3 1 mg L−1. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.

In vitro regeneration of Centaurium erythraea Rafn from shoot tips and other seedling explants

Various explants from 30-day-old seedlings of <em>Centaurium erythraea </em>Rafn were evaluated for their morphogenetic capacity under in vitro culture conditions. Shoot formation from shoot tip explants was achieved mainly through adventitious bud differentiation. The highest number of shoots (up to 43.3 ± 2.2 from a single shoot tip) was obtained on Murashige and Skoog medium (MS) supplemented with indole-3-acetic acid (IAA) (0.57 μM) and 6-benzylaminopurine (BAP) (4.4 μM). Adventitious shoot regeneration was also achieved through organogenesis from calluses obtained from hypocotyls, cotyledons, roots and leaves on MS medium containing IAA (2.85 μM) and BAP (0.88 μM). Significant differences were noted between explant types in their effects on shoot regeneration. In the primary culture, the best response was obtained either from calluses derived from roots or leaves (44.4 ± 4.5 and 40.2 ± 6.0 shoots per callus, respectively). The number of subcultures of inoculated calluses affected both the multiplication rate (the number of shoots/explant) and shoot morphology (the frequency of shoot hyperhydricity). Shoots rooted with the frequency of 94-100% after culture on MS medium without growth regulators. Plantlets were successfully acclimatized (97%) under high relative humidity and then moved to the greenhouse.

Culture <I>in vitro</I> and bud morphogenesis of <I>Valeriana officinalis</I> L. var. <I>latifolia</I> Miq.

Amplification protocol in vitro was developed for multiplication of Valeriana officinalis L. var. latifolia Miq. by using leaves, petiols, rhizome and roots derived from wild plants grown in pot as explants. Effects of different plant growth regulators on callus induction, adventitious bud differentiation and bud anatomy were studied. The results showed that it was optimal for the explants to be disinfected with 70% ethanol for 30 s and then soaked with 0.1% mercuric chloride for 6 min, the optimum medium for callus induction was MS+2,4-D 2 mg/L +6-BA 0.5 mg/L, and callus induction rate of leaves was up to 80%. However, buds were hard to regenerate from callus, and the highest differential rate was 25% on the medium of MS+6-BA 4 mg/L+NAA 0.5 mg/L. Buds regenerated exogenous from callus epidermal cells.