Adventitious Shoot Formation Research Articles

Plant Regeneration via Adventitious Shoot Formation from Immature Zygotic Embryo Explants of Camelina.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos' hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

Increase in ENHANCER OF SHOOT REGENERATION2 expression by treatment with strigolactone-related inhibitors and kinetin during adventitious shoot formation in ipecac.

Increase of ENHANCER OF SHOOT REGENERATION 2 expression was consistent to treatment with kinetin, TIS108, and KK094 in adventitious shoot formation of ipecac. Unlike many plant species, ipecac (Carapichea ipecacuanha (Brot.) L. Andersson) can form adventitious shoots in tissue culture without cytokinin (CK) treatment. Strigolactone (SL) biosynthesis and signaling inhibitors stimulate adventitious shoot formation in ipecac, suggesting their potential use as novel growth regulators in plant tissue culture, but the molecular mechanism of their action is unclear. In this study, we compared the effects of SL-related inhibitors (TIS108 and KK094) and CKs (2iP, tZ, and kinetin) on adventitious shoot formation in ipecac. Exogenously applied SL-related inhibitors and CKs stimulated adventitious shoot formation. Combinations of SL-related inhibitors and kinetin also promoted adventitious shoot formation, but without additive effects. We also analyzed the expression of CK biosynthesis genes in ipecac. TIS108 increased the expression of the ipecac homolog of ISOPENTENYL TRANSFERASE 3 (CiIPT3) but decreased that of LONELY GUY 7 homolog (CiLOG7), presumably resulting in no change in 2iP-type CK levels. KK094 and kinetin increased CiLOG7 expression, elevating 2iP-type CK levels. Among pluripotency- and meristem-related genes, TIS108, KK094, and kinetin consistently increased the expression of ENHANCER OF SHOOT REGENERATION 2 homolog (CiESR2), which has a key role in shoot regeneration, in the internodal segment region that formed adventitious shoots. We propose that CiESR2 might be a key stimulator of adventitious shoot formation in ipecac.

Введение in vitro и регенерационная способность реликтового эндемика недзвецкия семиреченская Niedzwedzkia semiretschenskia B. Fedtsch

В статье приведены экспериментальные данные по получению асептической культуры редкого реликтового вида недзвецкия (инкарвиллея) семиреченская Niedzwedzkia semiretschenskia от исходных семян из природных популяций на юге Казахстана. Изучено влияние гормонального состава питательной среды Мурасиге и Скуга на всхожесть семян, адвентивное побегообразование и укоренение in vitro. Проведена оценка коэффициента размножения в ходе многократного микрочеренкования асептических побегов. Установлено, что in vitro всхожесть семян не превышала 25 % на среде Кнопа, максимальная всхожесть отмечалась при их посадке в почвенный субстрат. Внесение в среду цитокинина 6-бензиламинопурина (БАП) приводило к образованию каллуса из тканей семядолей, к адвентивному побегообразованию у апикальных эксплантов проростков с настоящими листьями. Выявлено, что оптимальной средой для микроклонального размножения является среда с внесением 0,5 мг/л БАП с максимальным коэффициентом размножения 7,84 на втором пассаже. Установлено, что при депонировании в условиях низкой положительной температуры в течение 6 месяцев пробирочная культура инкарвиллеи сохраняет жизнеспособность и восстаналивает рост при переносе в стационарные условия световой комнаты. В результате работы проведено введение в культуру тканей, микроклональное размножение и создана депонированная коллекция in vitro реликтового вида N. semiretschenskia.

Rhizome Segments Form Shoots, Whereas Leaf Cuttings Form Shoots and Rhizomes in Eucodonia ‘Adele’ Treated with Benzyladenine

Eucodonia ‘Adele’ initiates seasonal shoot growth from a scaly rhizome. Larger rhizome segments (>2.5 cm) produced shoots at a greater percentage compared with smaller rhizome segments. Shoots produced on larger segments were initiated sooner and had a longer length. However, when shoot formation efficiency was calculated as the number of potential shoots per original stock rhizome, smaller rhizome segments were more efficient, producing three to four times as many shoots. Rhizome segments (2.5 cm) soaked overnight in benzyladenine (BA) produced three to four times more shoots per rhizome (four shoots) compared with untreated or water-soaked rhizomes (0.3 and 0.7 shoot, respectively). The scaly rhizome consists of a central stem-like core surrounded by numerous leaf-like scales. Scales appear to be storage leaf tissue based on anatomy and presence of numerous amyloplasts. New shoots initiate as axillary shoots formed from the central core at the scale attachment. Isolated individual scales also have the capacity for adventitious shoot formation, but only form in about 25% of isolated scales. Leaf cuttings were capable of producing adventitious shoots, roots, and rhizomes. Untreated petiole and lamina cut leaf cuttings formed approximately three rhizomes per leaf cutting compared with less than one adventitious shoot per leaf cutting. Benzyladenine-treated leaf cuttings did not show an increase in rhizome initiation, but soaking lamina cut leaf cuttings in water or BA increased shoot formation to ∼1.5 shoots per cutting. This work with isolated rhizome segments and leaf cuttings presents efficient systems for regenerating rhizomes that can be used to produce stock plants for a stem cutting system for Eucodonia ‘Adele’ as a seasonal pot plant.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Effect of cytokinins and light quality on adventitious shoot regeneration from leaflet explants of peanut (Arachis hypogaea)

ABSTRACT Plant tissue culture and novel breeding techniques such as genome editing in the economically important crop Arachis hypogaea constitute potential for genetic improvement. Therefore, in this study, high-frequency in vitro regeneration via formation of adventitious shoots was described for different A. hypogaea lines. The leaflets of the primary leaves of 5-day-old seedlings were cultured for 16 weeks on Murashige and Skoog’s medium supplemented with equimolar concentrations (22.19 µM) of the cytokinins 6-benzylaminopurine (BAP); thidiazuron (TDZ); zeatin (Zea) or meta-topolin (MT), which was reduced to 7.40 µM after eight weeks plus 2.3 µM kinetin. Highest shoot regeneration percentages of 89.1% and 81.3% for BAP and TDZ containing media, respectively, were achieved in the line ’Jimmy’s pride’ with the maximum shoot number obtained on BAP. The regeneration percentages for three further lines were lower than 50%. Furthermore, the effect of different light qualities, applied using LEDs, and compared to fluorescent lamps was investigated. Shoot regeneration percentages did not improve significantly under the different LEDs, instead a pronounced effect on the regeneration efficiency of the culture temperature was observed which differed among the light treatments. However, fluorescent lamps can be replaced by red or blue LEDs without negative effects on shoot regeneration.

Phytosulfokine promotes cell division in protoplast culture and adventitious shoot formation in protoplast-derived calluses of Nicotiana benthamiana

Phytosulfokine promotes cell division in protoplast culture and adventitious shoot formation in protoplast-derived calluses of Nicotiana benthamiana

Gene expression profiling before and after internode culture for adventitious shoot formation in ipecac

BackgroundIn ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), adventitious shoots can be induced simply by placing internodal segments on phytohormone-free culture medium. The shoots form locally on the epidermis of the apical region of the segments, but not the basal region. Levels of endogenous auxin and cytokinin transiently increase in the segments after 1 week of culture.ResultsHere, we conducted RNA-seq analysis to compare gene expression patterns in apical and basal regions of segments before culture and after 1 week of culture for adventitious shoot formation. The results revealed 8987 differentially expressed genes in a de novo assembly of 76,684 genes. Among them, 276 genes were upregulated in the apical region after 1 week of culture relative to before culture and the basal region after 1 week of culture. These genes include 18 phytohormone-response genes and shoot-formation-related genes. Validation of the gene expression by quantitative real-time PCR assay confirmed that the expression patterns were similar to those of the RNA-seq data.ConclusionsThe transcriptome data show that expression of cytokinin biosynthesis genes is induced along with the acquisition of cellular pluripotency and the initiation of cell division by wounding in the apical region of internodal segments, that trigger adventitious shoot formation without callusing.

Transcriptome analysis reveals the effects of strigolactone on shoot regeneration of apple

We have demonstrated that strigolactone inhibitor, Tis108, could be used to improve shoot regeneration of apple, and provided insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Lack of an efficient transformation system largely stagnated the application of transgenic and CRISPR technology in apple rootstock. High shoot regeneration ability is an important basis for establishing an effective transformation system. In this study, we first demonstrated the inhibitory effects of strigolactones on the adventitious shoot formation of apple rootstock M26. Next, we successfully verified that strigolactone-biosynthesis inhibitor, Tis108, could be used to improve the shoot regeneration of woody plants. Our results also suggest strigolactone-biosynthesis gene, MdCCD7, can be a target gene for biotechnological improvements of shoot regeneration capacity. Furthermore, we have employed transcriptome analysis to reveal the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Differentially expressed genes associated with photosynthesis, secondary growth, and organ development were identified. WGCNA suggests SLs might affect shoot regeneration through interaction with other hormones, especially, auxin, cytokinin, and ethylene. We were able to identify important candidate genes mediating the cross-talk between strigolactone and other hormones during the process of adventitious shoot formation. Overall, our findings not only propose a useful chemical for improving shoot regeneration in practice but also provide insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation.

Regeneration of Red Palestine FIG (Ficus carica L.) from Formation of Adventitious Shoots in Murashige & Skoog Media with Additional of BAP/TDZ and NAA

The research is aim to determine the regeneration of Red Palestine fig plant by direct or indirect organogenesis; to get the best combination of auxin & cytokinin concentration to induce callus formation; and the best combination of BAP/TDZ and NAA concentrations to regenerate fig plant from adventitious shoot formation. Experimental Treatment I: TDZ 5 mg.l-1; TDZ 7 mg.l-1; 2.4D 4 Kin. 0 mg.l-1; 2.4D 4 Kin. 0.25 mg.l-1; 2.4D 4 Kin. 0.5 mg.l-1; IBA 2 TDZ 1 mg.l-1; IBA 2 TDZ 2 mg.l-1; IBA 4 TDZ 1 mg.l-1; and IBA 4 TDZ 2 mg.l-1. Experimental Treatment II : BAP 2 NAA 0.25 mg.l-1; BAP 2 NAA 0.5 mg.l-1; BAP 3 NAA 0.25 mg.l-1; BAP 3 NAA 0.5 mg.l-1; TDZ 5 mg.l-1 NAA 0.25 mg.l-1; TDZ 5 NAA 0.5 mg.l-1; TDZ 7 NAA 0.25 mg.l-1; and TDZ 7 mg.l-1 NAA 0.5 mg.l-1. The research used randomized block design with 4 replications. The data were analyzed for variance and then Tukey’s HSD at 5% level. The results showed about Red Palestine fig regeneration through indirect organogenesis; the best combination for callus induction were IBA 2 TDZ 1 mg.l-1; and the best treatment for regeneration of adventitious shoots were BAP 2 NAA 0.25 mg.l-1 followed by TDZ 7 NAA 0.25 mg.l-1.

Rare and endemic plant species of Mountain Crimea flora: a morphogenic response to conservation in the gene bank in vitro

Biotechnological approaches have been actively used in recent years to preserve rare and endemic plant species under slow growth conditions. This is reached by lowering the temperature and light intensity, increasing the osmotic concentration, introducing retardants into the culture medium. For conservation, we used microshoot segments of Lamium glaberrimum (K. Koch) Taliev (Lamiaceae), Crepis purpurea L. (Asteraceae), Scrophularia exilis Popl. (Scrophulariaceae), Silene jailensis N. I. Rubtzov (Caryophyllaceae) cultured under standard in vitro conditions for 8‒10 months. The explants were placed on ¼ MS culture medium supplemented with plant growth inhibitors: 0.2 g/L 2-chloroethyl-trimethyl-ammonium chloride (CCC) and 60 g/L sucrose. Conservation conditions were: the temperature 4–6 °C, light intensity 1.25–3.75 µM m-2 s-1, photoperiod 16 hours. Screening of the morphometric parameters indicated high viability and a decrease in growth kinetics after 12 and 24 months of deposition in the gene bank in vitro. Along with the microshoot slow growth, the formation of adventitious shoots, leaves, and roots was observed.

Transcriptome analysis reveals the regulatory mechanism by which MdWOX11 suppresses adventitious shoot formation in apple.

Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants. We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2–13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.

Strigolactone signaling inhibition increases adventitious shoot formation on internodal segments of ipecac.

SL inhibited adventitious shoot formation of ipecac, whereas the SL-related inhibitors promoted adventitious shoot formation. SL-related inhibitors might be useful as new plant growth regulators for plant propagation. In most plant species, phytohormones are required to induce adventitious shoots for propagating economically important crops and regenerating transgenic plants. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Here we evaluated the effects of GR24 (a synthetic strigolactone, SL), SL biosynthetic inhibitors, and an SL antagonist on adventitious shoot formation during tissue culture of ipecac. We found that exogenously applied GR24 suppressed indole-3-acetic acid transport in internodal segments and decreased the number of adventitious shoots formed; in addition, the distribution of adventitious shoots changed from the apical to middle region of the internodal segments. In contrast, the SL-related inhibitors promoted adventitious shoot formation on both apical and middle regions of the segments. In particular, SL antagonist treatment increased endogenous cytokinin levels and induced multiple shoot development. These results indicate that SL inhibits adventitious shoot formation in ipecac. In ipecac, one of the shoots in each internodal segment becomes dominant and auxin derived from that shoot suppresses the other shoot growth. Here, this dominance was overcome by application of SL-related inhibitors. Therefore, SL-related inhibitors might be useful as new plant growth regulators to improve the efficiency of plant propagation in vitro.

Half-high blueberry plants from bioreactor culture display elevated levels of DNA methylation polymorphism

Blueberry (Vaccinium spp. L.) plants exhibit high potential of regeneration via adventitious shoot formation on a semi-solid medium followed by shoot elongation in a liquid medium under bioreactor systems. To find out whether DNA methylation plays a role during shoot elongation, we compared DNA methylation level in the regenerants of two in vitro-grown half-high blueberry (V. corymbosum L. × V. angustifolium Ait.) cultivars Patriot and Chippewa on a semi-solid medium (SSM) in glass bottles and in a liquid medium in temporary immersion bioreactors (TIB), via methylation-sensitive amplification polymorphism (MSAP) technique. The SSM was separately fortified with various combinations of two plant growth regulators, zeatin and thidiazuron for shoot regeneration but elongation was carried out using the same medium under both SSM and TIB systems with only zeatin. Zeatin at 9.2 µM produced the maximum shoots in SSM and TIB for both cultivars, which varied from 30 to 33 in SSM and from 28 to 29 in TIB for Patriot and Chippewa, respectively. However, shoots proliferated in TIB were more vigorous. Noticeable changes in the methylation profiles were detected using MSAP in the regenerants grown in each type of culture system. The TIB system exhibited a significant increase in total methylation percentage (47–50%) in comparison to SSM (25–32%) in both cultivars. Similarly, the plants regenerated from TIB system were more polymorphic than those from SSM system. We describe here the effects of culture in vitro on DNA methylation that induced during the process of shoot elongation in two half-high blueberry cultivars. In vitro shoot culture was achieved in half-high blueberry cultivars using a semi-solid and a liquid medium; plantlets developed from liquid medium showed higher level of DNA methylation alterations.

Induction of adventitious shoots and tetraploids in Antirrhinum majus L. by treatment of antimitotic agents in vitro without plant growth regulators.

We examined the effects of five antimitotic agents using Antirrhinum majus L. 'Maryland True Pink' on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.

Hormonal Regulation for <i>de novo</i> Organogenesis in <i>in vtro</i> Derived Leaf and Internode of <i>dAegle marmelos</i> L.

Adventitious shoot formation from internode explants of Aegle marmelos was achieved. Leaf and internode explants were procured from shoots obtained from in vitro germinating seedlings. Callogenesis was induced from leaf and internode explants on Murashige and Skoog (MS) semisolid medium variously supplemented with 6-Benzyladenine (BA) and 2, 4-Dicholophenoxyacetic acid (2, 4-D) while the organogenic response was found remarkable only in BA supplemented medium. Both types of explant markedly influenced callus formation while organogenesis was noticed only in internode explants. The number of shoots formed in internode explants was greater in the medium supplemented with 0.5µM BA and registered 6.86 numbers of shoot. The regenerated shoots transferred to half MS medium supplemented with 2mg/l IBA produced 75% rooting followed by 66% in IAA supplemented medium. The plantlets raised in vitro were successfully hardened to ex vitro conditions.

In vitro large scale clonal propagation of Achyranthes aspera L.: A high value medicinal plant

Achyranthes aspera is an important medicinal plant widely used in indigenous systems of medicine for the treatment of a variety of diseases and disorders. Efficient and reproducible protocols were established for in vitro clonal propagation of A. aspera through axillary shoot proliferation from nodal segments and de novo adventitious shoot formation directly from the leaf and stem explants. Multiple shoot proliferation from nodal segments was achieved on Murashige and Skoog’s (MS) medium supplemented with different concentrations of cytokinins like benzyl aminopurine and kinetin and the best results were achieved on MS medium supplemented with 8.88 μM benzyl aminopurine where 15-16 shoots were initiated after 5-6 weeks. The present material exhibited a high propensity of de novo adventitious root and shoot formation directly from the leaf and stem segments without the intervening callus phase. Direct regeneration of shoots was observed on MS medium supplemented with naphthlene acetic acid (10.74-21.48 μM) alone or in conjunction with benzyl aminopurine or kinetin producing 18-20 shoots. The regenerated shoots were rooted on basal MS medium and the plantlets were successfully acclimatized and established in the field with 80% survival.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

Regeneration of Adventitious Shoots from Induced Callus of Lantana Camara L. Plant by in Vitro Culture Technique

The results showed that the MS medium supplemented with a concentration of 1.4 mg L-1 of NAA and 0.1 mg L-1 BA resulted in the highest significant value in the percentage of response to callus induction of 96.67% compared with the other combinations of growth regulators. The results showed that a combination of 9.5 mg L-1 BA and 0.2 mg L-1 NAA which was added to the MS medium recorded the highest percentage of response to indirect adventitious shoot formation from callus of Lantana Camara, several shoots per 100 mg callus and shoot length reached 96.67%, 10 shoots per 100 mg callus and 3.00 cm, respectively, after 12 weeks of culturing. The adventitious shoots of indirect organogenesis of Lantana Camara were rooted when cultured on the MS medium supplemented with a concentration of 1.0 mg L-1 of NAA and 0.1 mg L-1 BA after 8 weeks of planting.