μM NAA Research Articles

<b>Somatic embryogenesis in anthurium (<i>Anthurium andraeanum</i> cv. Eidibel) as affected by different explants</b> - doi: 10.4025/actasciagron.v36i1.16557

This study establishes a protocol for the induction of somatic embryogenesis in Anthurium andraeanum cv. Eidibel. The experiment was arranged in a completely randomized 5 x 5 x 5 factorial design using five explant types (whole leaves, half leaves; petiole; nodal segments and root segments) from in vitro plantlets; five auxins: indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 4-amino-3,5,6-trichloropicolinic acid (Picloram); at five concentrations (0, 2.5, 5, 7.5 and 10 μM), with five replications using five Petri dishes. The cultures were maintained in a growth room at 25 ± 2oC in the dark. The explant type was investigated for the induction of somatic embryogenesis in anthurium cv. Eidibel, and nodal segments were shown to be the most suitable explant for this process. After 60 days in culture, the highest number of embryogenic calli was recorded for the nodal segments cultured in NAA (5, 7.5 and 10 μM), 2,4-D (10 μM) and Picloram (7.5 and 10 μM). The histological analysis confirmed the presence of embryos with established polarization, procambium, ground meristem and protoderm in the nodal segments cultured in Pierik medium containing 10 μM NAA. After the conversion of the somatic embryos into plantlets, these plantlets were acclimatized transferred to in vivo conditions and grown into normal plants.

Cytokinin, Carbon Source, and Acclimatization Requirements for <i>in Vitro</i> Propagation of <i>Scutellaria barbata</i> D. Don and <i>Scutellaria racemosa</i> Pers.

Micropropagation protocols to minimize hyperhydricity were optimized for medicinal Scutellaria barbata and Scutellaria racemosa. Six cytokinins and eight different carbon sources at two different incubation periods of 14 and 21 days were studied for adventitious shoot bud induction using nodal explants. In S. barbata, 5 μM meta-Topolin and 0.1 μM NAA supplemented shoot induction medium produced four shoots each after 14 and 21 day incubation. Observation of S. racemosa nodal explants recorded four and five shoots after 14 and 21 day incubation. In both species, control explants (no plant growth regulators in the medium) consistently resulted in the bud break with two shoots in both 14 and 21 day incubation. The effect of carbon source on shoot regeneration was studied by supplementing eight different sugars at 0.1 M concentration to the optimized shoot induction medium (5 μM meta-Topolin and 0.1 μM NAA). S. barbata nodal explants cultured on shoot induction medium supplemented with fructose and glucose for 14 days produced 10 and nine adventitious shoots respectively; and after 21 day incubation adventitious shoot count reached 19 in glucose supplemented medium. S. racemosa explants in the same experiment produced five shoots in maltose and four shoots in sorbitol supplemented medium after 14 day incubation; whereas after 21 day incubation, sucrose and maltose produced five shoots; fructose, glucose, and sorbitol produced four shoots. Regenerated plants were successfully acclimatized and Scanning Electron Microscopy of the leaf surface revealed differences in stomatal behavior and cuticle deposition between in vitro and acclimatized plants. The antioxidant assay conducted on both Scutellaria species showed considerable total polyphenol content, TEAC activity and flavonoid content in fresh and dried leaf samples attributing to their medicinal potential.

Hormones Regulate in Vitro Organ Regeneration from Leaf-Derived Explants in <i>Arabidopsis</i>

Plant in vitro organogenesis is well-controlled and thus provides an ideal system for plant propagation and studying mechanisms of plant development. However, the data on systematic in vitro organogenesis from leaf explant with various concentrations and combinations of hormones are limited. Arabidopsis is a very useful model plant species for many aspects of plant biological study. Here, we reported a simple, fast and efficient one-step process for evaluating leaf explant-derived in vitro Arabidopsis organogenesis involving the application of various concentrations and combinations of exogenous hormones. The central portion of the fourth rosette leaf was harvested from the 21-days-old seedling and cultured in vitro on the media containing 216 combinations of exogenous hormones. Different types of organs, including roots, shoots, inflorescences, and leaf-like organs were initiated from leaf explants. Several optimal experimental combinations were selected. A hormone combination, 1.00 μM NAA + 10.00 μM ZT, was considered as the most efficient one for adventitious shoot regeneration. And for adventitious root regeneration, six hormone combinations, [(NAA + ZT: 1.00 + 0.10 μM; 10.00 + 0.01 μM; 20.00 + 0.10 μM; 20.00 + 1.00 μM) and (NAA + 6-BA: 10.00 + 0.10 μM; 20.00 + 10.00 μM)], were thought to be the best ones. Further, both auxin and cytokinin ratios and concentrations were crucial for efficient in vitro organogenesis. Our study provides the important information for hormone-regulated organogenesis.

English

High frequency shoot regeneration from in vitro derived leaf explants of Wattakaka volubilis (L.f.) Stapf was achieved through callus mediated organogenesis. Organogenic calli were induced from 20 day old aseptic seedling explants on Murashige and Skoog medium fortified with various concentrations and combinations of plant growth regulators, benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), indole 3-butyric acid (IBA) and gibberellic acid (GA3). A mean of 8.6 shoots developed from organogenic callus induced from a 2 x 2 cm leaf explants on MS medium with 3% sucrose having 5.37µM NAA in combination with 2.22 µM BAP with 60% induction capacity. Further development of adventitious shoots could be achieved by sub culturing the callus to the same medium with 4.40 µM BAP and 0.288 µM GA3. Organogenesis could not be achieved from the calli of ex vitro derived leaf explants. The developed shoots were rooted on half-strength MS medium with 1% sucrose, 4.90 µM IBA and 0.93 µM kinetin at a frequency of 85%. Well rooted plantlets were then transplanted to vermiculite soil (3:1) mixture in polythene covered pots kept under culture room conditions. Approximately 60% plantlets survived and grew into whole plants. Key words: Adventitious shoots, caulogenesis, organogenic callus-histology.

Somatic embryogenesis in Manchurian ash: performance of various explant stock trees, optimisation of the basal medium, and simplified plant growth regulator combinations

SummaryManchurian ash (Fraxinus mandshurica Rupr.) is an ecologically and economically important hardwood tree species distributed in Northeast China. Somatic embryogenesis has been achieved in this species. However, to improve the efficiency of somatic embryogenesis in this species, the effects of explant stock tree, basal medium type, and plant growth regulator combination were investigated. Somatic embryos (SEs) of Manchurian ash were induced on cotyledons from young zygotic embryos, and the highest rate of induction was obtained from zygotic cotyledons cultured on 0.5× Murashige and Skoog (MS) medium. When cultured on 0.5× MS medium containing 8.1 µM αnaphthaleneacetic acid (NAA) plus 2.2 µM 6-benzyladenine (BA), the cotyledon explants derived from different stock trees had varying potentials to produce somatic embryogenic tissue. Somatic embryos were formed on only 33 of the 46 stock trees (numbered 1 – 46) tested, and the mean rates of induction ranged from 3 – 67%. Significant differences were observed in the combination of plant growth regulators required for maximum induction of SEs on cotyledon explants obtained from four additional stock trees (numbered 59 – 62), for which the optimal combination was 5.4 – 10.7 µM NAA plus 2.2 µM BA. These tree-specific results imply that breeding source needs to be considered before the potential application of somatic embryogenesis for rapid and large-scale propagation of superior lines of Manchurian ash.

Micropropagation of Scutellaria baicalensis Georgi

Plant propagation via nodal explants of <em>Scutellaria baicalensis</em> Georgi (Lamiaceae) was studied. Murashige and Skoog (MS) medium supplemented either with NAA and kinetin or NAA and BA in different concentrations was used for this purpose. The most favourable for axillary branching was MS medium supplemented with 0.5 µM NAA and 2.5 µM kinetin. The number of regenerated shoots per explant reached 3.8 ± 1.3. To increase the multiplication rate a two stage procedure of propagation was also elaborated. First, an induction of organogenic calli was achieved using thidiazuron (TDZ) as a sole growth regulator in the nutrient medium (concentration range: 0.1-0.5 µM). Next, regeneration of shoots on the medium containing 2.2 µM BA was performed. Over 20 shoots per explant could be obtained following the described procedure. The regenerated shoots rooted easily on a modified MS medium (1/2 macronutrients) and were successfully adapted to growth in pots.

In vitro regeneration through direct shoot organogenesis in Honey Orange (<i>Citrus tangerina</i>)

Rapid propagation of Honey orange (Citrus tangerina) was achieved by induction of shoots from epicotyl and cotyledonary node explants and rooting of cotyledonary node derived shoots. Significant explant differences were observed in the induction of direct shoots. Cotyledonary node explant is the most efficient in regeneration frequency followed by epicotyl explant. Cotyledonary node explants cultured on Murashige and Skoog (MS) medium supplemented with 8.88 µM N6-benzyladenine (BA) and 0.54 µM α-naphthaleneacetic acid (NAA) developed more than five shoots per explant. The isolated shoots transferred onto the MS medium supplemented with 5.4 µM NAA rooted 100% within 30 days.

Shoot organogenesis and somatic embryogenesis from leaf explants of Valeriana jatamansi Jones

The in vitro leaves of Valeriana jatamansi Jones were used as explants to induce adventitious shoots and somatic embryos from embryogenic callus. Both morphogenic processes could be induced on Murashige and Skoog (MS) basal medium supplemented with different concentrations and combinations of plant growth regulators. Embryogenic callus, which was induced exclusively in the presence of auxins, namely 1.0–7.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0μM α-naphthaleneacetic acid (NAA), differentiated into adventitious shoots and somatic embryos, respectively when left on the same medium. Callus, when placed on MS medium containing 5.0μM indole-3-butyric acid (IBA), 5.0μM NAA or 1.0–5.0μM 2,4-D, induced adventitious roots. Cytokinins that were added singly to callus induction medium, including 6-benzyladenine, kinetin and thidiazuron, could not induce callus at 5.0μM except when combined with 0.25–1.0μM NAA.

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum, an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0-4.0 μM) alone or in combination with IBA (0.2-0.6 μM) or 2, 4-D (0.5-1.5 μM). The optimum frequency (94%) of callus induction was recorded on MS medium supplemented with 3.0 μM Kn and 0.4 μM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0-7.0 μM) of BA or TDZ alone or in combination with NAA (0.2-1.0 μm) was employed. The highest frequency of shoot regeneration (91%) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 μM BA and 0.7 μM NAA. The shoots were excised and cultured on MS medium with 4.0 μM IBA produced 3.4 roots per shoot in 88% cultures. Of the 65 plants transferred to soil 54 survived (83%). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.

Rapid in vitro multiplication and ex vitro establishment of Caribbean copper plant (Euphorbia cotinifolia L.): an important medicinal shrub

An efficient micropropagation protocol was developed for E. cotinifolia by utilizing mature nodal segments for axillary shoot proliferation. The nodal explants from a 2-year-old plant were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5 and 10.0 μM) of 6-benzyladenine (BA), Kinetin and 2-isopentenyl adenine singly as well as in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA) (0.1, 0.5 and 1.0 μM). The highest regeneration frequency (92 %) with multiple shoots (13.0 ± 1.15) and shoot length (4.23 ± 0.14 cm) was achieved on MS medium supplemented with 5.0 μM BA and 0.1 μM NAA after 8 weeks of culture. Further experiments were performed to test the effects of medium type, medium strength, pH and subculture passages on shoot induction and proliferation. An enhancement in average number of shoots (16.6 ± 0.45) per explant was obtained after four subculture passages. Micro shoots exhibited in vitro rooting on half strength MS medium containing 2.5 μM Indole-3-butyric acid (IBA) after 4 weeks of culture. The in vitro raised healthy plantlets with well-developed roots and shoots were successfully acclimatized in plastic cups containing sterile soilrite for 8 weeks under culture room conditions (150 PPFD) prior to field transfer. Through the acclimatization period (0–56 days), photosynthetic pigments (Chlorophyll a, b and Carotenoid content) decreased during the initial 2 weeks followed by significant increase during the successive period (21–56 days) of acclimatization. At the same time, all the tested antioxidant enzymes (SOD, CAT, APX and GR) exhibited an increasing trend throughout the acclimatization period. The culture room acclimatized plantlets were successfully established in earthen pots containing garden soil in greenhouse with 70 % survival rate.

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with s-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.

Fitorreguladores e posição de explantes foliares na indução à calogênese em cerejeira-do-mato

O presente trabalho objetivou avaliar o efeito de diferentes formas de inoculação, no meio nutritivo, de explantes foliares de Eugenia involucrata DC., uma espécie florestal nativa com diversas potencialidades econômicas. Foram avaliadas as posições abaxial, adaxial, com e sem cortes no limbo foliar. Foi utilizado o meio de cultura MS acrescido de 10 µM de ANA isolado ou das combinações, em µM, de 2,4-D e BAP: 5-5 e 5-10. A posição dos explantes afeta a calogênese e a organogênese em segmentos foliares de E. involucrata, sendo mais adequada a abaxial sem cortes na região do limbo. A associação dos reguladores de crescimento 2,4-D + BAP na concentração de 5-10 µM foi mais promissora para a obtenção de calos, especialmente os nodulares, putativos à embriogênese somática.

Regeneración de plantas a partir de cultivos de callos de <i>Vitex trifolia</i> (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Volatile organic compounds obtained by in vitro callus cultivation of Plectranthus ornatus Codd. (Lamiaceae).

Plectranthus spp (Lamiaceae) are plants of economic importance because they are sources of aromatic essential oils and are also cultivated and several species of this genus are used as folk medicines. This paper describes the effects of different concentrations of the 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) on the induction of callus from nodal segments of Plectranthus ornatus Codd and in the production of volatile organic compounds (monoterpenes and sesquiterpenes). The 20 and 40 day calli were subjected to solid phase micro extraction (HS-SPME) and submitted to GCMS analysis. Variations in VOCs between the samples were observed and, a direct relationship was observed between of the major constituent detected (α-terpinyl acetate) and the monoterpenes α-thujene, α-pinene, β-pinene, camphene, sabinene and α-limonene that were present in the volatile fractions. Besides α-terpinyl acetate, isobornyl acetate and α-limonene were also major constituents. Variations were observed in VOCs in the analyzed periods. The best cultivation media for the production of VOCs was found to be MS0 (control). Moderate success was achieved by treatment with 2.68 µM and 5:37 µM NAA (Group 2). With 2,4-D (9.0 µM), only the presence of α-terpinyl acetate and isocumene were detected and, with 2.26 µM of 2,4-D was produced mainly α-terpinyl acetate, α-thujene and β-caryophyllene (16.2%). The VOC profiles present in P. ornatus were interpreted using PCA and HCA. The results permitted us to determine the best cultivation media for VOC production and, the PCA and HCA analysis allowed us to recognize four groups among the different treatments from the compounds identified in this set of treatments.

Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)

Digitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.

Shoot regeneration from various explants of horse chestnut (Aesculus hippocastanum L.)

A protocol is described for the adventitious shoot bud regeneration from leaf and stem explants in Aesculus hippocastanum. Effect of different media (Heller, WPM, Rugini and DKW) and growth regulators (BA, TDZ and NAA) combinations on shoot organogenesis were examined. Culture medium content had a significant effect on shoot regeneration in Aesculus. The highest shoot number and length was achieved on Rugini and WPM medium. Adventitious shoot formation was observed in all types of explants. Regeneration frequency and shoot number depended on explant type and growth regulators. The highest regeneration (100%) and the highest shoot bud number (9.7) were recorded in stem explants on WPM medium with 8.9μM BA+0.5μM NAA. Generally, TDZ was less effective on shoot regeneration in stem and leaf explants compared to BA. The addition of NAA to the culture medium improved shoot regeneration from stem discs. Rooting of elongated shoots was significantly increased (68.1%) on full-strength WPM medium supplemented with 2.7μM NAA. Rooted plantlets were acclimatized successfully under greenhouse conditions.

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, 'Malas Saveh' and 'Yousef Khani'. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4μM) of kinetin along with 0.54μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7μM for 'Malas Saveh' and 9.2μM for 'Yousef Khani', resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6μM IBA and 0.3μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9μM BAP and 0.1μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.

Adventitious shoot formation and plant regeneration from leaf explants of carnation (Dianthus caryophyllus L.)

Shoot multiplication and regeneration system was developed for two carnation (Dianthus caryophyllus L.) cultivars. For shoot multiplication, shoots of White Sim cultivar were cultivated onto MS media containing different levels of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA). High multiplication frequency was obtained on media containing 0.54 μM NAA combined with 4.4 or 8.8 μM BA. For regeneration study, leaf explants from White Sim and Red Sim cultivars were cultured onto media containing different concentrations of N-1,2,3-thiadiazol-5-y-N’-N’-phenyl urea (TDZ) combined with different concentrations of NAA. Shoot regeneration was obtained only on media containing high concentrations of the cytokinin, up to 85% shoot regeneration was obtained with 20 μM TDZ and 2.7 μM NAA in ‘White Sim’ and 75% in ‘Red Sim’. Similar trend was observed with shoot number in both cultivars. High root regeneration was obtained when the explants were cultured on medium with NAA only. Shoot regeneration was associated with callus formation. Regenerated shoots were rooted ex vitro, acclimatized and grown normally in the greenhouse.