Callus Pieces Research Articles

Induction and multiplication of callus of AS2K clones Coffea arabica L. through 2,4-D and BAP combination

Abstract Arabica coffee (Coffea arabica L.) has dominated international trade with more than 70% of global coffee production because of it’s taste was more preferred than the other types of coffee. The AS2K clone of C. arabica is a superior clone with high productivity, resistance to pests and diseases, and has a good taste. To maintain these characters, the appropriate propagation is by vegetatively. The aim of this research was to induce callus formation from C. arabica leaves, and to multiplicate the callus on twelve (12) combinations of 2,4-D and BAP as an effort to develop a method for propagating Arabica coffee. Callus induction was conducted on Murashige and Skoog (MS) medium containing 2,4-D 2 mg/L combined with BAP 1 mg/l, and then the multiplication of these callus on MS medium with 12 combinations of 2,4-D (2, 3, or 4 mg/L) and BAP (0, 0.25, or 1 mg/L). The research design used a completely randomized design with 3 replications, and each replication contained 5 callus pieces. The results showed that the combination of 2 ppm 2,4-D and 1 ppm BAP was able to induce callus formation from leaf explants by 49% at week 3 culture, yellowish white in color and friable in texture. Combination of 2,4-D 3 mg/L and BAP 0.5 mg/L provide the best result for callus multiplication.

Regeneration of Transgenic Ficus lyrata via Indirect Somatic Embryogenesis and Isolation of Variants for Development of New Cultivars

Ficus lyrata is a popular ornamental foliage plant with unique violin- or guitar-shaped green leaves. In our previous study, a grapevine gene VvMybA1 was introduced into F. lyrata via Agrobacterium-mediated transformation, which resulted in the availability of purple-leaved plants. Since VvMybA1 is a transcription factor, the regeneration of transgenic purple-leaved plants might potentially produce variants with multicolored leaves. The objective of this study was to establish a method for regenerating purple-leaved F. lyrata and determine if variants with different coloration or variegation could be isolated from regenerated populations. Leaf explants derived from a completely purple-leaved transgenic plant were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA). Callogenesis occurred in leaf explants, and a subculture of callus-borne explants on the same medium resulted in callus proliferation and the occurrence of somatic embryos. Somatic embryos were more effectively induced from callus pieces cultured on MS medium supplemented with 8.88 μM of BA and 0.27 μM of NAA. More than 30 embryos were induced per callus piece, and the embryos matured and converted to plantlets. MS medium supplemented with 4.92 μM of indolyl-3-butanoic acid (IBA) greatly improved root development. Plantlets were transplanted into soilless substrate and grown in a shaded greenhouse for morphological evaluation. Nine variants with different degrees of coloration and variegation were isolated from the regenerated populations. Our results suggest that the regeneration of transgenic plants that harbor a transcription factor, such as VvMybA1, could be an additional way of isolating novel variants for the development of new cultivars of ornamental plants.

Callus Induction and Plant Regeneration from Carum copticum and Assessment of Antioxidant Responses and Phytochemical Profiling by In Vitro Salinity Stress

Higher production of secondary metabolites is one of the adaptive responses to alleviate the impact of environmental injuries. In the present investigation, the production of these metabolites with medicinal importance induced by salinity in Carum copticum was investigated. To develop a better way for the production of medicinal substances, callogenesis and plant regeneration were analyzed, and seeds, calli, and/or regenerated seedlings were exposed to different concentrations of NaCl under in vitro culture conditions. The maximum frequency of callus induction was obtained on a medium supplemented with 0.25 mg L−1 2, 4-dichlorophenoxyacetic (2,4-D) and 1 mg L−1 benzyl amino purine (BAP) from stem explants. Plant regeneration with multiple shoots was obtained from pieces of callus transferred to MS medium fortified with 0.25 mg L−1 2,4-D and 1.5 mg L−1 BAP. Four weeks after treatment, salinity induced a substantial increase in the accumulation of reducing sugars and proline as compatible osmolytes and the activity of antioxidant enzymes. Total phenolics and anthocyanin significantly increased in all samples with increasing NaCl concentrations; however, the regenerated seedlings showed a reduction in these compounds at severe NaCl concentration compared to the control. Moreover, NaCl enhanced thymol, γ-terpinene, sabinene, and myrcene in the seedlings and calli, as well as carvacrol, limonene, and α-terpinene in the regenerated seedlings. These results suggest that salinity has a marked impact on improving the content of antioxidant metabolites and essential oils in C. copticum, whose callus might be the most salt tolerant in all tested samples.

Optimization of micropropagation protocol and elicitation of luteolin and rutin biomolecules using phenylalanine and chitosan in root callus of Rumex hastatus D. Don

Rumex hastatus D. Don, a traditional medicinal plant is known for its antitumorous, antiangiogenic and antibacterial potential. HPLC analysis of different plant parts showed that root had the maximum quantity of luteolin and rutin and thus was selected for the establishment of aseptic cultures. Murashige and Skoog’s medium supplemented with 5 µM TDZ proved best for developing greenish-brown and compact calli in 98% root cultures within 30 days. Upon subculturing such callus pieces differentiated an average of 10.50 ± 1.29 shoots per culture. The excised shoots were transferred on MS + 0.1 µM IBA medium where they developed an average of 12.03 ± 2.49 roots per shoot. These plantlets were successfully acclimatized under greenhouse conditions and their genetic fidelity was evaluated employing SRAP and SCoT markers which revealed their high similarity with mother plant proving their true-to-type nature. Interestingly, the in vitro regenerants showed elevated levels of phenols, flavonoids, luteolin and rutin contents and higher antioxidant activities over the mother plant as revealed by phosphomolybdenum and DPPH assays. For elicitation, 100 mg/L phenylalanine was best for enhancing luteolin content (407.18 ± 10.58 μg/g d.w.) up to 17.42-fold over control (22.10 ± 0.57 μg/g d.w.) whereas, 25 mg/L chitosan proved most effective for accumulation of rutin content (1169.07 ± 46.49 μg/g d.w.) up to 2.74-fold over control (312.41 ± 12.42 μg/g d.w.). To the best of our knowledge, this is the first report on micropropagation and elicitation of luteolin and rutin in R. hastatus through root culture. The present investigation focused on selection of an elite plant part, regeneration through in vitro root callus, genetic fidelity analysis, evaluation of secondary metabolites, antioxidant activity and enhancement of luteolin and rutin using elicitors in Rumex hastatus.

Micropropagation of Atropa acuminata Royle ex Lindl. (a critically endangered medicinal herb) through root callus and evaluation of genetic fidelity, enzymatic and non-enzymatic antioxidant activity of regenerants

Atropa acuminata Royle ex Lindl. (family Solanaceae) is a critically endangered therapeutic herb, known for its immense therapeutic potential against innumerable ailments. In view of protecting this species from brink of extinction, present work focuses upon development of an effectual micropropagation protocol via indirect organogenesis using root-derived callus. Best callusing was observed on Murashige and Skoog (MS) medium augmented with 10 µM Thidiazuron (TDZ), where 99% of root explants induced whitish-green and compact calluses within 45 days. Upon sub-culturing on same medium (10 µM TDZ), these callus pieces differentiated an average of 5.54 ± 0.65 shoots having 2.38 ± 0.37 cm of length in 71.5% of the explants, after 28 days. MS + 1 µM indole-3-butyric acid (IBA) resulted in optimum rooting response where an average of 8.08 ± 0.19 roots having an average length of 7.28 ± 0.11 cm were produced. Micropropagated plants with well-developed and thick roots were acclimatized under greenhouse conditions. Genetic fidelity of ex vitro acclimatized plants using start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) markers, revealed 96% similarity between mother plant and micropropagated plants. These plants showed higher yield of phenols, flavonoids and exhibited better antioxidant (DPPH and phosphomolybdenum assay) activities in comparison with mother plant. Higher levels of photosynthetic pigments and antioxidant enzymes were also observed in these plants as compared to the mother plant. Besides, quantification of atropine and scopolamine using high-performance liquid chromatography (HPLC) in different plant parts of acclimatized plantlets revealed higher amount of both the alkaloids in leaves. The present protocol shall be gainfully employed for large-scale micropropagation, conservation and extraction of biotherapeutic molecules for commercial purposes.

Determination of Effective Mutagen Dose for Carrot (Daucus carota ssp. sativus var. atrorubens alef and D. carota) Callus Cultures

Recep Tayyip Erdogan Universitesi, Doga Bilimleri Fakultesi, Bahce Bitkileri Bolumu, TAEK, RHTDB / AR-GE Projeleri Subesi Uludag Universitesi Ziraat Fakultesi Bahce Bitkileri Bolumu, Ankara Universitesi Ziraat Fakultesi BAhce Bitkileri Bolumu

Regeneration of Gynura aurantiaca ‘Purple Passion’ via indirect shoot organogenesis

Gynura aurantiaca (Blume) DC ‘Purple Passion’ is an important ornamental foliage plant. Its purple-colored leaves contain acylated anthocyanins which have potential to be natural food colorants. Production of this crop requires healthy and uniform starting materials or propagules. This study developed simple and efficient protocols for regenerating ‘Purple Passion’ through indirect shoot organogenesis. Reddish, purple colored callus was induced from leaf explants cultured on MS basal medium supplemented with 2.0 mg L−1 TDZ or 2.0 mg L−1 BA in combination with 0.5 mg L−1 2,4-D. Callus pieces (5 mm3) were cultured on the same medium with the same concentrations of TDZ or BA with 2,4-D for inducing adventitious shoots. More than 25 adventitious shoots were produced per callus piece. Microcuttings derived from the shoots were easily rooted in vitro, and rooted cuttings were transplanted into a peat-based substrate and grown in a shaded greenhouse. Plants grew vigorously in the shaded greenhouse with a high survival rate. They were morphologically stable without any phenotypic variation. Chromosome numbers of randomly selected regenerated plants and stock plants were the same with 2n = 40. Additionally, reddish, purple callus had significantly higher content of anthocyanins than leaves of regenerated plants grown in the shaded greenhouse, suggesting that callus induced through the established protocols could be used to produce acylated anthocyanins as natural food colorants through bioreactor culture.

Somatic embryogenesis and plant regeneration from cell suspension cultures of Gentiana kurroo Royle.

Gentiana kurroo Royle, native to North- Western Himalayas, is one of the critically endangered perennial herbs, which is being overexploited due to its multiple medicinal uses. The goal of this work was to develop a protocol for somatic embryogenesis and efficient plant regeneration through cell suspension culture. Selected ranges of plant growth regulators were experimented for somatic embryogenesis. Light micrographs of the established suspension cell cultures of somatic embryos at different developmental stages were also observed under light microscope. Somatic embryogenesis was induced from the leaf explants and the maximum induction frequency (97.2%) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg/l each of NAA, BAP and TDZ. Cell suspension cultures were established in MS liquid medium containing IAA (0.5 mg/l) + BAP (1.0 mg/l) with 89-93% viable cells. Transfer of cotyledonary somatic embryos to the MS agar medium containing 2, 4-D (0.4 mg/l) and KN (1.5 mg/l) resulted in somatic embryogenesis at high frequencies (80%) with an average of 28 ± 0.2 embryos/ callus piece. After transfer onto the half strength MS medium without growth regulators approximately 80-90% somatic embryos developed into complete plantlets. The protocol described here could be used for somatic hybridization, genetic transformation, isolation of protoplasts and large-scale propagation of G. kurroo.

THE PECULIAR PETIOLE CALLUSES GROWTH OF Amorphophallus titanum (Becc.) Becc. ex Arcang AND ITS IMPLICATIONS FOR Ex situ CONSERVATION EFFORTS

Amorphophallus titanum is a plant species endemic to Sumatera. Land degradation and illegal capture of hornbills (a distributor of A. titanum seeds) is leading to potential extinction of A. titanum in the wild. In order to conserve the species and save it from extinction, there is an urgent need to develop methods to propagate it both in situ and ex situ. The aim of this research was to discover environmental factors triggering callus growth from petiole cuttings of A. titanum in its natural habitat in Sumatera and to determine the viability of callus pieces as a propagation material. A completely randomized design with a single factor, i.e., callus size, was employed on five callus replicates. Each replicate consisted of four callus samples. The treatments consisted of three callus piece sizes i.e. 0.5 x.0.5 cm2, 1 x 1 cm2, 2 x 2 cm2. For each replicate, the following parameters of growth were assessed: the time of appearance of shoots; the shoot height when the first leaf fully opened; the petiole diameter; the diameter of the leaf lamina; and the number of young shoots. The results of our field observations showed that environmental factors such as temperature, humidity, and soil influence the formation of the callus in the wild. The size of the callus affected the shoot and root growth. The best result was obtained from callus 2 cm², which could produce 2–3 shoots with an average height of 18.8 cm at the time of first fully opened leaf. The collection of  petiole calluses of A. titanum that formed in its natural habitat are recommended instead of carrying the tuber.This ex vitro calluses can be used as propagation materials and then it could be planted in the botanic gardens as one of ex situ conservation effort.

Transformation of pomegranate (Punica granatum l.) a difficult-to-transform tree

Abstract In this study, an Agrobacterium-mediated transformation method was developed for pomegranate, a difficult-to-transform plant. We first optimized callus induction and shoot regeneration efficiency. Induction of calluses was best achieved from the intermodal stem sections on WPM medium supplemented with 12 μM BA and 8 μM NAA. The highest frequency of shoot regeneration (69.33%) and number of shoots (7.16) per piece of callus were obtained when calluses were incubated on WPM medium supplemented with 12 μM BA and 2 μM NAA. Callus pieces were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBin19 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and green fluorescent protein (GFP) gene as a reporter. After 4 weeks in WPM selection medium (the best callus induction medium supplemented with 50 mg L−1 kanamycin), putative transgenic calluses were obtained. From such calluses transformed shoots began to appear approximately 3 week after the kanamycin resistance transgenic callus pieces had been placed on the best shoot regeneration medium containing 50 mg L−1 kanamycin. GFP fluorescence was observed in putatively transformed calluses and shoots. Molecular analysis confirmed the integration of the nptII transgene in transformed shoots. The transgenic shoots were rooted and successfully acclimatized.

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100% in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree

The present study develops the highly reliable and reproducible protocol for indirect organogenesis of Helicteres isora L., a medicinally important multipurpose plant of Sterculiaceae family. Murashige and Skoog (MS) medium supplemented with different plant growth regulators induced callus with different type and texture. The combination of 10.74μM naphthalene acetic acid (NAA) and 5.71μM indole-3-acetic acid (IAA) fortified with MS medium resulted best response (100%) in callus induction with fresh weight from leaf (3.75±0.11g) and internodal (3.30±0.14g) explants. Green compact nodular callus (GCNC) was transferred to shoot regeneration medium for shoot regeneration. The multiplication rate of adventitious shoots was influenced by various factors like explant type, media composition, plant growth regulator combinations and concentrations. MS medium supplemented with 2.69μM NAA, 0.57μM IAA and 4.92μMN6-(2-isopentenyl) adenine (2ip) combination resulted in 80.95±4.76 percentage of response with 8.43±0.43 number of shoots per piece of callus derived from leaf explant. This combination was expressed 76.19±4.76 percentage of response with 7.28±0.28 number of shoots per internode-derived callus respectively. The addition of 50 mg l−1 glutamine in 2.69μM NAA, 0.57μM IAA and 4.92μM 2iP combination enhanced the shooting frequency with 12.14±0.83 and 9.14±0.51 shoots from leaf and internodal explant-derived callus. Elongated shoots [4.92μM 2iP and 1.44μM of gibberellic acid (GA3)] were achieved rooting in half-strength MS medium fortified with 4.90μM indole-3-butyric acid (IBA) with 582.84μM activated charcoal. The genetic fidelity between mother plant and in vitro plants was assessed through SCoT and ISSR marker systems. Both these analysis revealed that all the samples were found monomorphic in nature. This suggests that the current study standardised the true-to-type culturing protocol and authenticated the in vitro raised plantlets are remain free from somaclonal variations.

Ex situ conservation of the endangered species Androcalva perlaria (Malvaceae) by micropropagation and cryopreservation

Micropropagation and cryopreservation protocols were developed for the threatened Australian species Androcalva perlaria. Vegetative shoots were brought into culture using a simplified surface sterilisation process with between 26 and 100 % of shoots successfully initiated across all genotypes. Shoots were multiplied on ½ MS basal salts medium (BM) with 1.25 µM 6-furfurylaminopurine (K) + 0.125 µM 6-benzylaminopurine (BAP). Cryopreservation was then developed for a single genotype to facilitate long-term ex situ storage for conservation purposes. Highest survival (>80 %) of shoot tips was achieved by preculture on 1.2 M glycerol for 48 h, incubation in PVS2 solution at 0 °C for 30 min, followed by rapid LN immersion then recovery. Application of this cryogenic approach to shoot tips from a range of genotypes gave variable post-cryopreservation regeneration results; survival for one genotype was only 3 %, while for four other genotypes survival varied between 60 and 80 % which compared favourably with post-cryopreservation regeneration (85 %) of the genotype used to develop the protocol. Callus production was achieved by culturing stem segments on ½ MS BM with 2.5 µM α-naphthaleneacetic acid + 2.5 µM BAP. Adventitious shoots were best regenerated from callus through incubation on BM only. Small callus pieces were successfully cryopreserved from 16 genotypes (1–88 % regeneration). Using a callus tissue pathway plant material was placed into LN storage after 6–8 weeks from the time of collection (compared to ~6 months using shoot tips). Plants derived from cryogenically preserved callus tissues were re-established in soil 28 weeks after removal from LN. This study demonstrates how biotechnology can be effectively utilised for the rapid ex situ conservation of endangered flora while ensuring that a significant range of genetically diverse samples can be conserved for long-term biosecurity.

Cryopreservation Technique of Jiangxi Yanshan Red Bud Taro(Colocasia esculenta L. Schott var. cormosus CV.Hongyayu) Embryogenic Callus by Droplet-vitrification

To explore the cryopreservation of Jiangxi Yanshan red bud taro( Colocasia esculenta L. Schott var. cormosus CV. Hongyayu) embryogenic calli by droplet vitrification,which will provide the technical and theoretical basis for the cryopreservation of its germplasm resources. Plant tissue culture and single factor test were applied. A cryopreservation system of Jiangxi Yanshan red bud taro embryogenic calli by dropletvitrification was established: About 0. 2 g embryogenic calli was pre-cultured in liquid MS medium supplemented with 2 mg·L- 1TDZ,1 mg·L- 1NAA and 0. 5 mmol·L- 1sucrose and maintained under a 14 h photoperiod at 25℃ for 3 d. Pre-cultured embryogenic calli was loaded with MS + 2 mmol·L- 1Gly + 0. 4 mmol·L- 1sucrose for 20 min at 25℃ and then dehydrated with PVS2 at 0℃ for 40 min. Five drops of PVS2 were absorbed and dropped to aluminum foil strip,the dehydrated red bud taro embryogenic callus pieces were transferred to the PVS2 droplet on the aluminum foil strip. The aluminum foil strips were dipped in the liquid nitrogen using tweezers,then quickly transferred to 2 mL freezing tubes filled with liquid nitrogen. After keeping for 1 d,the aluminum foil strips were removed from freezing tubes using tweezers and immersed into the preheated 40℃ washing solution( MS + TDZ 2 mg·L- 1+ NAA 1 mg·L- 1+ 1. 2 mmol·L- 1sucrose),the embryogenic callusblocks dropped from the aluminum foil strips, and then washed three times with liquid MS medium supplemented with 2 mg· L- 1TDZ,1 mg· L- 1NAA and 1. 2 mmol·L- 1sucrose and each kept for 10 min and then post-cultured on solidified MS medium supplemented with 2 mg·L- 1TDZ,1 mg·L- 1NAA in the dark for 7 d and then transferred to a 14 h photoperiod. After 30 d,the differentiated embryoids were again transferred onto fresh solidified MS medium supplemented with 2 mg·L- 1TDZ,1 mg·L- 1NAA under 14 h photoperiod to developed into the whole plantlets. The average survival rate of embryogenic calli after cryopreservation by droplet-vitrification amounted to about 80%. No significant difference was observed in the morphological,physiological and cytological index of plantlets coming from control and cryopreserved embryogenic calli. Red bud taro embryogenic calli cryopreservation by droplet-vitrification could guarantee the stability of its genetic resources, which provided a new way for in vitro conservation of Jiangxi Yanshan red bud taro germplasm resources.

The antimicrobial activity of leaves and callus extracts of Thevetia peruviana In vitro

The antimicrobial activity for Thevietia peruviana was evaluated by measuring inhibition zone diameter in agar using well diffusion assay. The aim of the present study was to evaluate the antimicrobial potential of Thevietia peruviana leaf extract as compared with callus extract against some bacterial strains and fungi. The results showed that the addition of 2,4-D at the concentration of 9 mg/l, and 0.1 mg/l of kinetin led to obtain callus weight reached 800 mg. It was noticed that the reduction of 2,4-D concentration up to 6 mg/l resulted in compact and green pieces of callus. The optimal weight and friable callus was obtained at 9 mg/l. Among the susceptible bacteria are the gram negative pseudomonas aeruginosa seemed to be sensitive against all concentration of Thevietia peruviana leaf and callus extracts, While Escherichia coli showed resistance with all concentrations of extracts. It was noted that the extracts were more active against gram positive staphylococcus aureus, as compared with other bacterial species. Results of this study revealed that callus extract of Thevetia peruviana possess higher activity in comparison with leaf extract against gram positive bacteria (Staphylococcus aureus, Bacillus cereus) and gram negative (Pseudomonas aeruginosa). Finally all the bioextracts were well stable at room temperature during the period of the study and did not show any reduction of activity against the bacterial strains used in this study experiments.

Regeneration of Anthurium andraeanum from Leaf Explants and Evaluation of Microcutting Rooting and Growth under Different Light Qualities

This study established a new method for regenerating Anthurium andraeanum Lind. and evaluated effects of different wavelengths from light-emitting diodes (LEDs) on rooting and growth of adventitious shoots. Callus occurred in leaf explants of A. andraeanum ‘Alabama’ and ‘Sierra’ cultured on a modified Murashige and Skoog (MS) basal medium supplemented with four concentrations of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ). Adventitious shoots were induced from callus pieces (≈1 cm3) cultured on the modified MS medium containing 6-benzyladenine (BA) with kinetin (KN), BA, and/or KN with 3-indolebutyric acid (IBA) or α-naphthalene acetic acid (NAA). Results showed that 1.82 μM TDZ induced 83.3% and 77.8% of leaf explants of ‘Alabama’ and ‘Sierra’ to produce callus and 24.9 and 24.7 adventitious shoots were produced per callus piece of ‘Alabama’ and ‘Sierra’ cultured on the modified MS medium containing 0.89 μM BA, 2.32 μM KN, and 0.98 μM IBA, respectively. Adventitious shoots were cut and rooted in the modified MS medium containing 0.98 μM IBA and grown under the same light level but with different light qualities. All adventitious shoots rooted; root numbers, root lengths, root fresh and dry weights, and leaf area of plantlets grown under red plus blue light were comparable to those grown under conventional fluorescent white light. Shoot height was the greatest in monochromic blue light followed by red light. Shoot fresh and dry weights of plantlets grown under red plus blue light, however, were significantly greater than those grown under the other light qualities. Plantlets grown under red plus blue light had 22.7% greater total dry weight and more balanced root-to-shoot ratio than those grown under fluorescent white light. These results suggested the use of complex of red plus blue LED could be an option for improving growth of A. andraeanum plantlets in vitro.

Regeneration of Chlorophytum amaniense ‘Fire Flash’ Through Indirect Shoot Organogenesis

Chlorophytum amaniense Engl. ‘Fire Flash’ is a popular exotic ornamental foliage plant as a result of its unique coral-colored midribs and petioles and tolerance to interior low light levels. Currently, demand for propagative materials exceeds the availability of seeds. This study was intended to develop an in vitro culture method for rapid propagation of this cultivar. Leaf and sprouted seed explants were cultured on a Murashige and Skoog basal medium supplemented with different cytokinins with 1.1 μM α-naphthalene acetic acid (NAA) or 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Leaf explants showed poor responses in callus production and no adventitious shoots were obtained. Callus formation frequencies from sprouted seeds were 71% and 85% when induced by 9.8 μM N6-(2-isopentyl) adenine (2iP) with 1.1 μM NAA and 9.1 μM N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with 1.1 μM NAA, respectively. Adventitious shoots occurred after the induced calluses were subcultured on the same concentrations of TDZ or 2iP with NAA. Shoot formation frequencies from calluses cultured on TDZ with NAA and 2iP with NAA were 92% and 85%, and the corresponding mean shoot numbers were 37 and 31 per piece of callus (1 cm3), respectively. Adventitious shoots rooted at 100% after transferring to the basal medium containing 4.4 μM 6-benzylaminopurine (BA) with 2.7 μM NAA. Plantlets, after transplanting to a soilless substrate were easily acclimatized in a shaded greenhouse under a photosynthetic photon flux (PPF) density of 200 μmol·m−2·s−1. Regenerated plants grew vigorously without undesirable basal branching or distorted leaves. This newly established regeneration method can provide the foliage plant industry with a means for rapidly propagating ‘Fire Flash’ liners in a year-round fashion.

Optimization in Agrobacterium-mediated transformation of Anthurium andraeanum using GFP as a reporter

Although Agrobacterium -mediated transformation protocols for many economically important plant species have been well established, protocol for a number of flowering plants including Anthurium andraeanum remains challenging. In this study, we report success in generating transgenic Anthurium andraeanum cv Arizona using Agrobacterium GV3101 strain harboring a binary vector carrying gfp as a reporter gene. The possibility of facilitating the screening process for transgenic plants expressing functional proteins using gfp marker was explored. In order to realize high transformation efficiency, different explant sources including undifferentiated callus pieces and petioles were compared for their regeneration efficiency and susceptibility to Agrobacterium -mediated transformation. We also optimized the concentration of AS added to co-cultivation media. Genomic PCR revealed that 11 of the 22 resistant plantlets regenerated on selective medium were successfully transformed. Green fluorescence was observed using a fluorescence microscope in 7 of the 11 PCR-positive plants, indicating GFP was expressed stably in the transformed Anthurium andraeanum . The highest transformation efficiency obtained in this study was 1.71% (percentage of explants with transgenic shoots in total explants) when callus explants were used as starting material and 125 μmol l -1 AS was added during the co-cultivation process.

Callus induction and plant regeneration in Alternanthera philoxeroides

To establish a successful in vitro plant regeneration system in Alternanthera philoxeroides (Mart.) Griseb, an orthogonal design was used to investigate the effects of three factors (plant growth regulators, explant types and dark treatment in initial-stage), each having three levels. The effects of these factors and levels on callus induction and shoot regeneration were quantitatively evaluated by analysis of variance. The experimental results showed that the callus induction was significantly affected by 2, 4-dichlorophenoxyacetic acid (2, 4-D), and shoot differentiation from subcultured pieces of callus was enhanced mostly by dark treatment in initial-stage. The optimal conditions for callus induction are obtained from the stem explants cultured on semi-solid Murashige and Skoog (MS) medium plus 2.2 μM BA and 2.2 μM 2, 4-D, with 20 days dark treatment in initial-stage. The highest frequency of shoot regeneration is obtained from the calli cultured on semi-solid MS medium plus 8.8 μM BA, without dark treatment in initial-stage.

Establishment of a novel plant regeneration system from suspension-derived callus in the halophytic Leymus chinensis (Trin.)

The establishment of cell suspension culture and plant regeneration of the halophytic Leymus chinensis (Trin.) are described in this study for the first time. Callus induction solid medium containing Murashige and Shoog (MS) basic salt, 2.0 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D), and 5.0 mg l -1 L-glutamic acid with 30.0 g l -1 sucrose and 4.0 g l -1 gelrite for solidification induced the highest rate of cell division in Type 1 callus among calli of various types. Liquid medium with the same hormone distribution was therefore, used for cell suspension culture from Type 1 callus. Over a 30 d suspension culture at 100 rpm, great amounts of biomass were accumulated, with 71.07% average daily increment and 22.32-fold total fresh weight increment. Comparison of before and after suspension culture, the distribution of different size callus pieces and the maintenance of callus type were basically unaltered, but a slight increase in relative water contents was observed. To induce the potential of plant regeneration, the directly transferring on plant regeneration solid medium containing MS basic salt, 0.2 mg l -1 α-naphthalene acetic acid (NAA), 2.0 mg l -1 kinetin (Kn), and 2.0 g l -1 casamino acid and indirectly transferring were simultaneously performed. Even now growth rates of suspension-derived callus on solid medium were approximately half of those of Type 1 callus, but faster somatic embryogenesis was observed. Rooting of all regenerated shoots was successfully performed on half- strength MS medium. All plants appeared phenotypically normal.