Thin Cell Layer Explants Research Articles

Effects of Thin Cell Layer Technique and Nutrient Media Contents of Regeneration and Browning Characteristics of the Laurus nobilis L. Explants

The aim of this study is in order to investigate the effects of explant source, explant type and MS media composition containing different concentrations of sucrose, activated carbon and Coconut milk for regeneration and browning of Laurus nobilis L. using thin cell layer (TCL) culture system. A higher rate of callus (57.15%) and shoot (2%) regeneration and a lower rate of blackening were determined in transversely cut stem TCL explants compared to leaf explants. While 1.33% callus regeneration was achieved in leaf explants; shoot regeneration could not be achieved. While more callus regeneration (35.17%) was found in explants taken from the field, more shoot regeneration (1.5%) and lower rate of browning were obtained in explants taken from in vitro. In the sugar trials, the highest callus regeneration (40.83%) was defined in MS medium containing 30 g/L sucrose supplemented with 1 mg/L BAP, and the highest shoot regeneration (2.5%) was determined in MS medium containing 45 g/L and 60 g/L sucrose supplemented with 1 mg/L BAP. When explant type, explant source and nutrient media composition are considered together; the highest callus regeneration (100%) was obtained in field-sourced stem TCL explants cultured in medium containing 25 mL/L coconut milk and 1 mg/L BAP. The highest shoot regeneration (6.6%) was determined in in vitro stem TCL explants cultured in MS media containing 30, 45, 60 g/L sucrose and 1 mg/L BAP. The lowest percentage of browning (50%) was obtained from in vitro stem TCL explants cultured in MS medium containing 2 g/L activated carbon and 1 mg/L BAP.

Enhancing drought resistance in Pogostemon cablin (Blanco) Benth. through overexpression of ACC deaminase gene using thin cell layer regeneration system.

Pogostemon cablin cultivation faces massive constraints because of its susceptability to drought stress that reduces patchouli propagation and oil yield. The present study has achieved an efficient and rapid direct regeneration system for the transgenic production of P. cablin using Agrobacterium-mediated genetic transformation. To establish an efficient regeneration protocol for fast in-vitro multiplication of patchouli plants, leaf, petiole, and transverse thin cell layer (tTCL) explants were used and inoculated on an MS medium supplemented with different combinations of phytohormones. A comparative study showed a maximum regeneration frequency of 93.30 ± 0.56% per explant was obtained from leaf segments on optimal MS medium fortified with 0.2mg/L BAP and 0.1mg/L NAA. Leaf and petiole explants took 25-35 days to regenerate while tTCL section showed regeneration in just 15-20 days on the same medium. Subsequently, productive genetic transformation protocol OD600 0.6, AS 200µM, 30mg/L kanamycin, and infection time 5 min. was standardized and best-suited explants were infected at optimum conditions from the Agrobacterium tumefaciens (LBA 4404) strain harboring ACC deaminase to generate transgenic P. cablin Benth. (CIM-Samarth) plants. The investigation suggested that the optimized protocol provides a maximum transformation frequency of 42 ± 1.9% in 15-20 days from tTCL. The transgenic plants were shifted to the greenhouse with a 52.0 ± 0.8% survival frequency. A molecular docking study confirmed significant binding affinity of ligand ACC with ACC deaminase at the catalytic site, and ligand interactions showed four H-bonds at the binding pocket with amino acids Cys-196, Val-198, Thr-199, and Gly-200 that validate gene relative expression in transgenic plants. Among all transgenic acclimatized greenhouse-grown patchouli plants, line PT4 showed improved drought resistance under severe water stress as its RWC was 71.7 ± 2.3% to 75.7 ± 2.1% which is greater than the RWC of the control plant, 58.30 ± 0.21%. Analysis of the other physiological indicators, H2O2, chlorophyll content, and ROS result support drought resistance ability. Our study concluded that the first report on P. cablin, tTCL direct regeneration, and standardized transformation protocol created a new opportunity for genetic manipulation to achieve drought-resistant patchouli plants for cultivation in all seasons at the commercial level.

The Application of Plant Growth Regulators in Propagation of Dendrobium Orchid with Thin Cell Layer (TCL) Technique: A Review

Plant growth regulators is one of the success factors for in vitro propagation. Plant growth regulators are organic compounds that are not nutrients and, in small amounts, promote, inhibit, or regulate physiological processes in plants. The function of growth regulators is to stimulate the growth of morphogenesis in cell, tissue and organ cultures. The most used plant growth regulators in tissue culture are auxins, cytokinins and gibberellins. One of technique for tissue culture is the thin cell layer technique. Thin Cell Layer (TCL) is a technique using small thin sections produced from organ pieces. TCL explants more effective than large conventional explants. Thin explants facilitate contact and diffusion of media into the tissue, better than thick explant slices. This literature review aims to gather information related to the application of plant growth regulators in propagation of Dendrobium orchid with thin cell layer (tcl) technique. A systematic Literature Review (SLR) uses in this paper. The growth regulators commonly used in Dendrobium orchid propagation using the TCL technique are auxins and cytokinins and the type of media that is often used in Dendrobium orchid propagation is Murashige and Skoog (MS). Protocorm Like Bodies (PLB) is the most potential explant in Dendrobium orchid propagation. The TCL technique both transverse and longitudinal has its own specificity with each type of explant used. TCL explants proved to be more effective than large conventional explants.

An Overview: Effect of Plant Growth Regulatory on Orchid Propagation through The Thin Cell Layer technique

Thin cell layer (TCL) is a micropropagation method using thin-sized explants. A TCL can be prepared from any explant source and the thichness of explant less than 5 mm. TCL is more efficient in producing total plantlet output than conventional in vitro methods. TCL have been applied to the in vitro culture of orchids, field, vegetable crops and medicinal plants The successful in vitro orchid propagation is influenced by many factors, such as plant genotype and media composition. Additions of plant growth regulator (PGR) in media culture is essential factor. The formation of complete plant depent on concentration and type of plant growth regulator. TCL explants require growth regulators to form an embryonic callus and zygotic embryos. Explant in medium without supplemented of growth regulators resulted in browning and failed to grow. The most commonly plant growth regulator in tissue culture are thidiazuron (TDZ), 6-benzylaminopurine (BAP) 2,4 dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and meta-Topolin. Recently, microalgae can also be used as an alternative source of hormones to increase plant growth through in vitro culture techniques

Somatic embryogenesis and plant regeneration from transverse thin cell layers of adult peach palm (Bactris gasipaes) lateral offshoots.

In this work, we report a successful protocol to obtain in vitro peach palm (Bactris gasipaes Kunth) “Diamantes 10” plants through somatic embryogenesis from transverse thin cell layer (TCL) explants, dissected from three sections (basal, medial, and apical) of lateral offshoots of adult plants cultured on different concentrations of 4-amino-3,5,6-trichloropicolonic acid (picloram). After swelling and development of primary callus in all treatments, without any strong effect of explant origin or picloram concentration, it was possible to observe the formation of embryogenic structures and the exact point from where they developed. Browning was also observed and correlated to the induction treatments, although it was not an impairment for the production of embryogenic structures. Subsequent maturation and conversion of somatic embryos into plantlets allowed their acclimatization 17 months after culture initiation (ACI), which was quicker than previous reports with juvenile tissues (from embryos or seed-germinated plantlets). To the best of our knowledge, this is the first report on peach palm regeneration through somatic embryogenesis from TCL explants from adult plants and could constitute, after fine-tuning the acclimatization stage, a tool for mass clonal propagation of elite genotypes of this open-pollinated crop, as well as for the establishment of conservation strategies of in situ gene bank plant accessions endangered due to aging and other threats.

Effect of microalgae Messastrum gracile and Chlorella vulgaris on the in vitro propagation of orchid Cattleya labiata

The aim of this study was to evaluate the effect of two green microalgae (Messastrum gracile and Chlorella vulgaris) in comparison with plant growth regulators (6-benzylaminopurine, BAP; thidiazuron, TDZ; zeatin, ZEA) on the in vitro propagation of Cattleya labiata, an endangered orchid, using the thin cell layer (TCL) technique from protocorms. TCL sections were cultivated in MS/2 medium containing M. gracile extract (EM) and biomass (BM); C. vulgaris extract (EC) and biomass (BC); and BAP, TDZ, and ZEA in different concentrations. Subsequently, the explants were grown in MS/2 medium, with 2 g L−1 of activated charcoal, to induce elongation and roots formation. For acclimatization, plants were transplanted in trays using Sphagnum sp. as substrate. TCL explants showed a higher formation of protocorm-like bodies (PLBs) than entire protocorms. Explants cultivated in media supplemented with BM showed a high rate of PLB regeneration (59%) and high mean number of PLBs (4 per explant) and 85% survival after acclimatization of the plants. Supplementation with BAP stimulated similar morphogenic responses to those observed with BM and superior results obtained with ZEA and TDZ. Mass propagation of C. labiata plants was successfully achieved using TCL, and it is recommended to supplement the MS/2 medium with 4 g L−1 of BM or 4 mg L−1 of BAP. Microalgae extracts and biomasses are effective alternatives for in vitro propagation of C. labiata that can replace plant growth regulators, as they favored the formation of PLBs and plants.

Transverse thin cell layer (t-TCL)-mediated improvised micropropagation protocol for endangered medicinal orchid Dendrobium aphyllum Roxb: an integrated phytomolecular approach

Clonal propagation is an important biotechnological tool for the conservation of rare, endangered and threatened as well as commercially important plant species of which orchids are an important representative. In the present research, an attempt was made to develop a fast, clonally stable as well as phytochemically enriched regeneration protocol for propagating Dendrobium aphyllum, an important medicinal orchid using transverse thin cell layer (t-TCL) explants. Murashige and Skoog (MS) medium supplemented with 15 µM meta-topolin along with 10 µM TDZ and 10 µM AgNO3 was found to be most conducive for shoot proliferation. After 8 weeks of culture, an average of 39.41 shoots/explant proliferated; till date this is the best shooting frequency for D. aphyllum and one of the best amongst the orchids. The highest rooting frequency of 82.34% was accomplished in half-strength MS medium supplemented with 15 µM IBA. The plantlets were successfully acclimatized in greenhouse exhibiting normal developmental patterns and flowering occurred after 6 months of acclimatization with 3–4 flowers/stalk irrespective of the flowering season. The genetic homogeneity of the micropropagated plants was ascertained using IRAP and ISSR markers. A comparative assessment of the antioxidant activity (mother and micropropagated plant) was performed using DPPH and FRAP assays which was recorded to be higher amongst the in vitro-regenerated plants. A rapid, genetically stable regeneration protocol coupled with comprehensively higher phytochemical yield is reported in the present study on D. aphyllum. The results of this study confirm the use of in vitro propagation protocol for sustainable commercial utilization as well as conservation of medicinal orchid bio-resources within a limited time period.

An improved thin cell layer culture system for efficient clonal propagation and in vitro withanolide production in a medicinal plant Withania coagulans Dunal

In vitro micropropagation based on transverse thin cell layer (tTCL), which utilizes a very small number of cells or tissues, has now emerged as a powerful tool in plant tissue differentiation and regeneration. In this study, intense, fast rate shoot multiplication has been achieved utilizing thin cell layer explants for endangered pharmaceutical plant Withania coagulans Dunal (family Solanaceae) having high hypoglycemic potential. To develop an efficient protocol for rapid in vitro propagation of W. coagulans stem node, shoot apical meristem (SAM), and tTCL were used as explants and inoculated in different strength of MS medium. The tTCL explant showed high frequency of shoot regeneration and was also affected by the concentration of plant growth regulators. The full strength solid MS medium was optimal for shoot regeneration (31.94 ± 1.39%) and the highest percent of shoots (93.05 ± 2.77) were observed in MS medium fortified with 2.0 mg l−1 BAP and 0.5 mg l−1 NAA. In contrast to tTCL, SAM cultured on the same medium showed significantly lower multiplication rate with only 1.16 ± 0.08 average shoots per responsive explants. The best rooting of the regenerated shoots was achieved in half strength liquid MS medium supplemented with 2 mg l−1 IBA. Plants were successfully transferred to the field after acclimatization with a survival rate of 90.6%. Monomorphic nature of ISSR and RAPD markers in this study confirmed the genetic fidelity of the in vitro raised tTCL clones. Quantification of withanolides content through high performance liquid chromatography (HPLC) showed 1.4 fold increases in withaferin A content and 1.6 fold withanolide A content in acclimatized field grown plants compared to field grown (wild type) plants. In acclimatized field grown plants the over-expression of SQS gene further attested the improved withanolide production in tTCL micropropagated plants having valuable anti-diabetic potential. The current study reveals the fast rate, cost effective micropropagation and conservation of W. coagulans through tTCL technique for its use as anti-diabetic agent at commercial level.

English

Oil palm hybrid BRS Manicore is important for plantations in the north of Brazil, as it is resistant to fatal yellowing and is compact. Seed germination is slow and reduced, so somatic embryogenesis is a promising alternative for its propagation. Two kinds of starting explants were used: Zygotic embryos (ZE) and thin cell layers (TCL) from the base of seedling aerial parts. Two culture medium formulae were compared (Y3 and modified MS), and several growth regulators (2,4-D, Picloram, BA, 2iP), putrescine and activated charcoal (AC) were used during the different stages. Somatic embryos (SE) were obtained from ZE-derived embryogenic masses cultured on Y3 medium with or without 2,4-D (9 µM) + 1000 µM putrescine and 40% were converted into seedlings in the Y3 medium supplemented with 2 g.L-¹ AC and without growth regulators, under light. SE developed in 49 to 53% TCL-derived calli and 50% of them were converted into seedlings in the same conditions described for ZE calli. The yield of both processes was compared, showing the superiority of TCL explants for SE production. Key words: Y3 culture medium, clonal propagation, putrescine, thin cell layer.

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.

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.

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.

The effect of ethylene inhibitors (AgNO3, AVG), an ethylene-liberating compound (CEPA) and aeration on the formation of protocorm-like bodies of hybrid Cymbidium (Orchidaceae)

Protocorm-like bodies (PLBs) or thin cell layers (TCLs) derived from PLBs of hybrid Cymbidium Twilight Moon ‘Day Light’ can induce new or neo-PLBs on Teixeira Cymbidium (TC) medium, which contains 0.1 mg/L α-naphthaleneacetic acid, 0.1 mg/L kinetin, 2 g/L tryptone and 20 g/L sucrose, and is solidified with 8 g/L Bacto agar. This study aimed to assess the response of neo-PLB formation to an ethylene-liberating compound (2-chloroethylphosphonic acid (CEPA)), to two ethylene inhibitors (silver nitrate (AgNO3) and aminoethoxyvinylglycine (AVG)), and to aeration (made possible by using Milliseal® or autoclaved filter paper). AgNO3 at 1 or 2 mg/L in TC medium significantly increased the fresh weight of PLBs while 1 mg/L of AgNO3 also showed a significant increase in the number of neo-PLB from both half-PLBs and from tTCLs. In contrast, AVG and CEPA inhibited neo-PLB formation. Neo-PLB formation from half-PLB or TCL explants in the presence of aeration resulted in significantly lower neo-PLB weight. The use of AgNO3 and aeration are alternative means to mass produce neo-PLBs for micropropagation purposes.

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and AimsAdventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.MethodsExpression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.Key ResultsThe accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.ConclusionsIn ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.

Rapid Shoot Regeneration from Thin Cell Layer Explants of an Endangered Medicinal Asclepiad Ceropegia spiralis L.

The thin cell layers of nodes and internodes of Ceropegia spiralis L. were cultured on MS supplemented with BAP 13.32 µM/l + NAA 0.537 μM/l induced 17.34 ± 0.55 shoots showing extensive growth. Later on the organogenesis was also induced on MS containing BAP 13.32 µM/l + 2, 4-D 1.130 µM/l, whereas the medium with BAP 13.32 µM/l + 2, 4-D 4.52 µM/l has the highest callus producing ability in recalcitrants as well as in normal explants. Shoots developed were rooted best on 0.5 MS with NAA 10.74 μM/l. Optimum shoot and root multiplication was obtained within eight weeks. In vitro plantlets were successfully weaned and transferred to soil with about 90 per cent survival rate. So far, more than 650 weanlings have been produced successfully and reintroduced into nature for their recovery. Key words: Ceropegia spiralis, Thin cell layer explants, Recalcitrants, Regeneration D. O. I. 10.3329/ptcb.v21i1.9564 Plant Tissue Cult. & Biotech. 21(1): 63-73, 2011 (June)

English

  This study highlights procedures for embryogenic calluses induction from immature female inflorescences of oil palm using thin-cell-layers explants (TCL). In three experiments, the ability of calluses induction were examined and identified through different types of basal media, position of the TCL explants in the rachillae and concentrations of 2,4-D and types of antioxidants added into the medium. Samples of embryogenic calluses obtained were isolated and transversal and longitudinal cross sections were obtained and stained for observations in light microscopy. The results achieved suggest that immature female inflorescences of oil palm can be reverted from the floral state to the embryogenic vegetative state and are excellent alternative sources of explants for the induction of somatic embryogenesis. In general, 225 to 450 µM of 2,4-D are required to induce embryogenic callus in explants composed of immature oil palm inflorescences and the composition formed by salts and vitamins of MS medium provides superior results than Y3 medium. The activated charcoal at concentration of 3.0 g l-1 is the most indicated antioxidant for preventing the oxidation of floral oil palm explants and its presence can be considered essential for the formation of embryogenic callus.   Key words: Elaeis guineensis, somatic embryogenesis, micropropagation, floral explants, morphogenesis, agroenergy.

MICROPROPAGATION OF A RARE ORCHID DENDROBIUM GRATIOSISSIMUM USING THIN CELL LAYERS

Dendrobium gratiosissimum Rchb. f. is a very graceful wild orchid of Thailand and is a rare species. Establishment of an efficient method for micropropagation is urgently needed because of its conservation and commercial requirements. An efficient method of rapid and mass propagation of D. gratiosissimum using thin cell layers (TCLs) had been developed. Four-week-old protocorms were segmented into two halves and each half was considered as a TCLs explant. The shoots, each with 1 to 2 nodes were cut into approximately 0.3 to 0.5 mm thickness from the base to the shoot tip. The TCLs explants from both protocorms and young stems were cultured on MS (Murashige and Skoog, 1962) medium supplemented with different combinations of N 6 -benzyladenine (BA), kinetin (Kn), 1-naphthaleneacetic acid (NAA) and 20 mg/L sucrose. MS medium without growth regulators failed to induce protocorm-like bodies (PLBs) formation from stem explants. The TCLs explants from both protocorm and stem developed PLBs within 3 to 4 weeks on the MS containing growth regulators. Of the two different TCLs explants types used, the highest percentage of PLBs formation (83%) and highest number of PLBs (18) per explants were observed on the protocorm sections, whereas only 66% of stem sections were able to produce PLBs with an average 9 PLBs per explants. MS containing 2 mg/L Kn was found to be the optimum concentration for PLBs development from thin protocorm section. In contrast, the highest PLBs production of stem sections was obtained from explants on 5 mg/L Kn and 1.0 mg/L NAA. Plantlet conversion from PLBs was successfully achieved on regulator-free growth medium.

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 µM picloram and 2,4-D with 3.0% sucrose, 500 mg L−1 glutamine, and 0.3 g L−1 activated charcoal and gelled with 2.5 g L−1 Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 µM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 µM NAA and 12.30 µM 2iP, 0.3 g L−1 activated charcoal, and 500 mg L−1 glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L−1 activated charcoal and gelled with 2.5 g L−1 Phytagel.

STUDY ON MORPHOGENESIS OF BLACK PEPPER (PIPER NIGRUM L.) IN LEAF THIN CELL LAYER CULTURE

A histological study of callus regeneration of black pepper using leaf transverse thin cell layer (TCL) explants was accomplished. The undifferentiated cells culture of callus were found to differentiate into vascular nodules called meristemoids, which then develop into xylem elements, especially tracheids. Culturing in the shooting medium, these nodules differentiated into shoot apical meristem.

Somatic embryogenesis and plant regeneration from pistil transverse thin cell layers of lemon (Citrus limon)

Callus induction, somatic embryogenesis and plant regeneration were obtained in Citrus limon (L.) Burm. (cv. Femminello) from cultures of pistil transverse thin cell layer explants [(t)TCLs]. Explants were cultured on two different media, based on Murashige and Skoog salts and vitamins, supplemented with 500 mg l−1 malt extract (MSI), or 500 mg l−1 malt extract and 13.3 μM 6-benzylaminopurine (MSII). Sucrose (146 mM) was used as carbon source. Somatic embryos appeared 3 months after culture initiation from stigma and style (t)TCLs; they were observed at the surface of the (t)TCL-derived callus. Although ovary (t)TCLs showed the highest callus formation, they never differentiated somatic embryos. Percentages of embryo formation from (t)TCLs incubated on MSI (13% and 2% for stigma and style, respectively) were lower than those from (t)TCLs incubated on MSII (36% and 7% for stigma and style, respectively). The embryogenic response of stigma (t)TCLs was usually higher than that of style (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies (57% and 62% for stigma- and style-derived somatic embryos, respectively).