tTCL Explants Research Articles

Efficient somatic embryogenesis and regeneration from leaf main vein and petiole of Actinidia chinensis planch. via thin cell layer culture technology

Somatic embryogenesis (SEMs) is an effective micropropagation method that has been successfully applied to many different plant species; however, little is known about the SE in Actinidia chinensis Planch. - A species with high economic value. In this study, the efficiency of SEMs from leaf main vein and petiole of A. chinensis (seedlings germinated from seeds sterilized with 200 ppm silver nanoparticles for 7 min) via thin cell layer (TCL) technique was performed and compared with that of whole leaf main vein and whole petiole explants. The leaf main vein (mv - 1 mm × 10 mm in size) and leaf petiole (p - 1 mm × 10 mm in size) explants of in vitro A. chinensis were cut through transverse and longitudinal TCL (mv-tTCL and mv-lTCL for mv explants or p-tTCL and p-lTCL for p explants) with different sizes (½, ¼, 1/6, 1/8 of mv-lTCL or p-lTCL and ½, 1/3, ¼ of mv-tTCL or p-tTCL, respectively). Somatic embryo (SE) was recorded in the wounds of mv and p explants after 8 weeks of culture. For mv explants which cultured on MS medium supplemented with 0.02 mg/L NAA, 0.5 mg/L TDZ, 30 g/L sucrose and 8 g/L agar – MSE medium, the ½ mv-tTCL explant (mv was cut into 2 tTCL explants) gave the highest SEMs (98.67%) and number of somatic embryos per explant (10.66 embryos); meanwhile, p explants cultured on MSE medium, the ½ p-tTCL explant (p was cut into 2 tTCL explants) obtained the highest number of somatic embryos per explant (8.66 embryos) after 8 weeks of culture. In addition, the growth correction factor of SEMs was optimal at ½ mv-tTCL explant (21.04) or 1/6 p-lTCL (p was cut into 6 lTCL explants) and 1/8 p-lTCL (p was cut into 8 lTCL explants) (31.36 and 32.00). The current study has shown that the procedure of leaf main vein and petiole TCL derived- SEMs of A. chinensis is the optimal one. Large amounts of somatic embryo induction were observed from mv-TCL and p-TCL with full shapes of the globular, heart, torpedo and cotyledon. The shoot regeneration from somatic embryo with the cotyledon shape was optimal on medium containing 1.0 mg/L BA in terms of total number of shoots, shoot height and number of shoots. In addition, plantlets derived from shoot cultured on the MS medium supplemented with 0.4 mg/L NAA and planted in plastic pots with the combination of humus, coconut fiber and biofertilizers substrates (with ratio of 2:3:1 in weight) showed higher acclimatization and growth than those from other treatments in the greenhouse.

Genotype Screening of Recipient Resources with High Regeneration and Transformation Efficiency in Chrysanthemum

Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum, which relies on an optimal regeneration and transformation system. However, the regeneration system of different chrysanthemum cultivars varies, and the regeneration time of most cultivars is long. To screen cultivars with highly efficient regeneration, leaves and shoot tip thin cell layers (tTCL) from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media (MS) supplemented with 1.0–5.0 mg L−1 6-benzylaminopurine (6-BA) and 0.1–1.0 mg L−1 α-naphthaleneacetic (NAA). The results showed that the most efficient regeneration media were MS + 6-BA 1.0 mg L−1 + NAA 0.5 mg L−1 for leaf explants and MS + 6-BA 5.0 mg L−1 + NAA 0.1 mg L−1 for tTCL explants. Subsequently, another 13 chrysanthemum cultivars were screened by using the media, and finally, three cultivars with high regeneration efficiency were obtained from 21 cultivars. Among these, C1 had the highest regeneration efficiency: the regeneration rate of leaf explants reached 80.0% after 42 days of culture, and the regeneration rate of tTCL explants reached 100% after 31 days of culture. Furthermore, we also established the transformation system for C1 as follows: preculturing for one day, infecting with Agrobacterium suspension (OD600 = 0.6) for 10 min, and cultivating in the regeneration medium with 350 mg L−1 carbenicillin and 10 mg L−1 kanamycin, thus ultimately achieving a transformation rate of 4.0%. In this study, a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened, which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes.

Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shoot-derived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6- benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 µM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the β glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Somatic embryogenesis from leaf transverse thin cell layer derived-callus of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.)

No report on plant regeneration via somatic embryogenesis of P. vietnamensis has been previously published. In the present study, somatic embryogenesis via callus formation from cultures of leaf transverse thin cell layers (tTCLs) of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) was investigated. α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) and thidiazuron (TDZ) were added separately and in combination into the culture media. Explant necrosis or low callogenesis rates were observed when 1-mm wide leaf tTCLs were cultured on media with TDZ, BA, 2,4-D or NAA. On the other hand, calli were successfully induced from the tTCL explants cultured on medium supplemented with either 2,4-D and BA or 2,4-D and TDZ. Callogenesis was observed under both light and dark conditions. The highest callogenesis rate (100%) was obtained on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg l-1 2,4-D in combination with 0.1 mg l-1 TDZ in darkness after eight weeks of culture. White calli were cut into small pieces (1.0 x 1.0 cm dimension) and placed on MS media containing 1.0 mg l-1 2,4-D, 0.5 mg l-1 NAA and TDZ at various concentrations (0.01; 0.1; 0.2; and 0.5 mg l-1), and the best callus proliferation was recorded on medium containing 1.0 mg l-1 2,4-D and 0.2 mg l-1 TDZ. Somatic embryogenesis, with a success rate of 53.3% and 35 embryos per explant, was achieved when calli were subcultured onto MS medium supplemented with 1.0 mg l-1 2,4-D, 0.5 mg l-1 NAA and 0.2 mg l-1 TDZ.

Thin Cell Layer (TCL) Culture System for Herbal Biomass Production and Genetic Transformation of <i>Bacopa monnieri</i> L. Wettst.

Bacopa monnieri (L.) Wettst. (Scrophulariaceae) is a highly sought after medicinal plant with therapeutic properties as cognition enhancer as well as for other brain and body functions. Research was conducted to optimize a thin cell layer explant based micropropagation system to assist mass propagation. Thin cell layers (TCL) derived from leaf and internode segments were used as explants. Murashige and Skoog medium was used to formulate shoot induction, elongation, and rooting media. Shoot induction media were prepared by supplementing three concentrations (0.1, 1.0, and 10.0 μM) of four cytokinins 6-benzylaminopurine, 2-isopentenyl-adenine, 6-3-Hydroxybenzylaminopurine, and thidiazuron to study adventitious shoot bud induction response. An optimum shoot bud induction response was observed on MS medium supplemented with 10.0 μM 6-benzylaminopurine for both leaf and stem transverse thin cell layer (tTCL) explants. The average number of shoot buds from leaf tTCL explants was 59, whereas, on an average, 33 shoot buds were regenerated from internode tTCL explants. Elongation of adventitious shoot buds was achieved best in a liquid medium using Liquid Lab Rocker® system. Elongated shoots recorded 100% rooting in MS medium supplemented with 5 μM indole butyric acid. Bacopa micropropagation employing tTCL explants for initial shoot bud induction and using LLR® boxes in subsequent elongation step can achieve cost effective way to regenerate high volume of plantlets and biomass required for herbal industry. Leaf and stem tTCL explants both were suitable for Agrobacterium tumefaciens (EHA105) mediated genetic transformation. Successful transformation was scored within three days of co-cultivation with Agrobacterium suspension on the basis of Enhanced Green Fluorescent Protein (EGFP) expression as an early and non-destructible screening device. Transformation frequencies of 83% and 76% were accomplished for leaf and stem tTCL explants, respectively. Greenhouse grown Bacopa plants were analyzed as fresh and dry methanolic extracts for total polyphenol content (811.93 ± 7.98 and 814 ± 17.64 GAE mg g-1) and the Trolox Equivalent Antioxidant Capacity values were 1918.25 ± 173.12 and 3163.14 ± 403.25 μmol/g, respectively.

Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors

An efficient regeneration protocol for the medicinal plant, Paris polyphylla Sm. was developed through the formation of mini-rhizomes (MRs) using transverse thin cell layer (tTCL) culture technique. MRs were induced from tTCL explants derived from the basal and middle stem portions while apical portion failed to show any kind of response. Highest response percentage (86.6 %) of MRs formation with a maximum fresh weight (1.05 ± 0.08 g) was achieved from basal sections cultured on ½ MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (BAP). MRs transferred to plant growth regulator free medium gave rise to shoot buds that eventually regenerated into plantlets and were successfully acclimatized with a survival percentage of more than 95 % under greenhouse conditions. Quantification through reverse-phase HPLC showed 1.41-fold higher content of total steroidal saponins in MRs cultured on medium supplemented with 0.5 mg/l BAP as compared to the field-grown rhizome. Elicitation of MRs liquid culture with chitosan, salicyclic acid (SA) and yeast extract enhanced the production of steroidal saponins but resulted in reduced growth rate. Highest total steroidal saponins content (87.66 ± 1.66 mg/g DW) was achieved in cultures treated with SA at 50 mg/l after 30 days of elicitation which is 3.6 times higher than the in vivo rhizome. The developed protocol would facilitate the conservation of this valuable medicinal plant and could be used as a ready stock to meet the demands of the pharmaceutical industry for steroidal saponins productions.

In Vitro Propagation Using Transverse Thin Cell Layer Culture and Homogeneity Assessment in Ceropegia bulbosa Roxb.

Ceropegia bulbosa is an endangered medicinal plant used traditionally in the treatment of various diseases. Our aim is to develop a rapid and a competent procedure for direct and indirect organogenesis from transverse thin cell layer (tTCL) explants of C. bulbosa. Optimum response to direct adventitious shoot bud induction from tTCLs was observed on medium augmented with 8.8 µM 6-benzyladenine (BA) producing 15.6 ± 0.31 shoots per responsive explant. Best callusing response (95 %) was observed with tTCL explants in medium containing 4.5 µM 2,4-dichlorophenoxyacetic acid and 2.2 µM BA. High frequency shoot regeneration (75 %) was observed from tTCL derived calli. Medium containing 8.8 µM BA and 0.27 µM α-naphthalene acetic acid produced 22.2 ± 0.64 shoots with shoots acquiring an average length of 4.6 ± 0.12 cm. In vitro rooting was recorded on ½ strength Murashige and Skoog medium, producing 10.9 ± 0.23 roots with a length of 4.24 ± 0.16 cm. Plants were successfully transferred to the field with a survival rate of 89 %. The clonal nature of the regenerants was assessed using Inter-simple sequence repeat markers.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.

An improved and efficient micropropagation of Eclipta alba through transverse thin cell layer culture and assessment of clonal fidelity using RAPD analysis

An improved and efficient in vitro regeneration system has been developed for Eclipta alba, a medicinally important plant, through transverse thin cell layer culture (tTCL). The transverse section of the nodal segment of field grown plants was used as tTCL explants for plant regeneration. Shoot multiplication from tTCL nodal explants was influenced by BAP and their interaction with Kin or NAA. MS medium containing 13.2μM BAP and 4.6μM Kin was most effective for shoot multiplication from tTCL nodal explants. Upon this medium, percent response for shoot proliferation was 100% with an average of 32.6 shoot buds per tTCL nodal explant. Regenerated shoots from tTCL nodal explants were rooted on the growth regulator free MS medium. The rooted plantlets were successfully acclimatized and established in soil with a survival frequency of 90–100%. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic fidelity of the micropropagated plants. RAPD profile analysis indicated that micropropagated plants were genetically similar to mother plant.

Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m−2 s−1, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.

High frequency plant regeneration from transverse thin cell layers in Indian mustard (Brassica juncea L.)

An efficient and reproducible plant regeneration system was established using transverse thin cell layers (tTCLs) in five cultivars of Brassjca juncea L. The effects of medium conditions, explant types (tTCLs of hypcotyl and cotyledonary petiole) on shoot regeneration were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog (MS) medium supplemented with 4 mg/L 6-benzylaminopurine (BA) and 0.2 mg/L 1-naphthaleneacetic acid (NAA). The hypocotyls derived tTCL explants had more shoot regeneration frequency (52%) than the cotyledonary petiole derived tTCL explants. Shoot induction was further improved by the addition of silver nitrate (<TEX>$AgNO_3$</TEX>) in the regeneration medium. A significant genotypic effect was also observed between the five cultivars; Rai-5 displayed higher capacities to produce shoots than other cultivars. Regenerated shoots were rooted on MS basal medium without PGRs which induced 90% of roots. The plantlets established in greenhouse conditions with 99% survival, flowered normally and set seeds. The regenerated plants were fertile and identical to source plants.

Rapid shoot regeneration from thin cell layer explants excised from petioles and hypocotyls in four cultivars of Brassica napus L.

The present work describes a procedure that allows for the easy and rapid induction of caulogenesis in four cultivars of Brassica napus L. from transversal Thin Cell Layers (tTCLs). In order to investigate the regeneration ability of this crop, the effects of genotype, explant source and culture medium were examined on shoot regeneration. The tTCL explants were excised from hypocotyl and petiole of 2-week-old seedlings and cultured on a solid basal MS medium supplemented with α-naphthaleneacetic acid (NAA: 0.1–0.4 mg l−1), 6-benzylamino-purine (BAP: 1–4 mg l−1) and sucrose (20–40 g l−1). A significant genotypic effect was observed between the four cvs; Jumbo and Drakkar displayed higher capacities to produce shoots than Pactol and Cossair. Regeneration commenced earlier and the percentage of shoot-producing explants as well as the number of shoots per regenerating explant was greater. The comparison between the regeneration ability of different explants showed that the hypocotyls exhibited a high rate of shoot organogenesis when they were cultured on MS medium supplemented with 3 mg l−1 BAP, 0.3 mg l−1 NAA and 30 g l−1 sucrose. Adventitious shoot buds developed from 46% of the tTCLs, with a mean of 7.5 buds. Furthermore, the method was fast with shoot formation occurring by 7 days culture. Plantlets regenerated from all shoots and developed normally. The regenerated plants were fertile and identical to source plants.

오리엔탈 백합의 인편 및 줄기의 박판 세포층 절편으로부터 고빈도 자구형성

An efficient system for in vitro bulblet formation of Lilium oriental hybrids(cvs). Casa Blanca and Siberia is described. Transverse thin cell layer(tTCL)(1mm thick) explants of 'Casa Blanca' formed bulblets at a frequency of 97.7％ when cultured on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4 dichlorophenoxyacetic acid(2,4-D) (On average 15.6 bulblets were formed per explant). The frequency of bulblet formation was drastically reduced when the explant ghickness was thinner than 1 mm. Explants from the outermost layer of bulb scale produced greater frequency of bulblet formation than middle or innermost layer. Among auxins supplemented to culture medium at 1 mg/L, 2,4-D led to greater frequency of bulblet formation on explants than dicamba, picamba, or phenylacetic acid(PAA). tTCL explants from the middle region of the outermost layer bulb scale yielded greater frequency of bulblet formation than the upper or lower region. tTCL stem explants of 'Siberia' formed bulblets at a frequency of 95.3% when cultured on MS medium with 1 mg/L 2,4-D(On average 9.1 bulblets were formed per explant). The system estabilished in this study will be useful for in vitro rapid propagation and genetic transformation of Lilium Oriental hybrids.

Somatic embryogenesis and direct shoot regeneration of rice ( Oryza sativa L.) using thin cell layer culture of apical meristematic tissue

Summary A new and simple method was designed to directly produce shoot regeneration of rice ( Oryza sativa L.), within a short time-span, with a high yield, and without the need for subculture. Transverse thin cell layers (tTCLs) excised from the shoot apical meristems of two-week old seedlings, which received pretreatment in darkness with 2,4-D from 1–20 μmol/L, were placed on Murashige and Skoog. (1962) medium supplemented with 3% (w/v) sucrose and a combination of 2,4-D varying from 1–20 μmol/L and BAP varying from 1–10 μmol/L. Somatic embryos and the embryo-like structures which developed into shoots were kept in the dark for 2 weeks after the culture of tTCL explants, and produced a maximum of 6 somatic embryos and 16 shoots per tTCL explant. Also important for shoot regeneration was the effect of the 2-week period of exposure to darkness followed by light exposure. The regenerated shoots were transferred to a medium with no hormone for rooting. More than 95% of plantlets developed successfully when they were transferred to the greenhouse.