Search

The efficiency of direct somatic embryogenesis (SE) induced on internode explants of the tree fern Cyathea delgadii treated with different concentrations of sucrose at various periods after their excision from donor sporophytes was investigated. Internode explants, about 1.5 mm in length, were dissected from etiolated plantlets grown in vitro and cultured on half-strength hormone-free MS medium supplemented with 10 g l−1 sucrose, under continuous darkness. The impact of four concentrations of sucrose (0.6, 0.8, 1.0, 1.2 M) applied every second day between days 0 and 10 was studied. The highest number of somatic embryos was obtained when explants were treated with 1.0 M sucrose on the 4th day (59.8 on average) or 0.8 M sucrose on the 8th day (67.6 on average) after excision. Light and scanning electron microscopy observations revealed that a nearly 22-fold increase in SE efficiency induced by sucrose treatment was associated with a change in the origin of somatic embryos from multicellular to unicellular. Application of high sucrose concentrations on the 4th day after explants excision stimulated the accumulation of starch, a valuable energy source for intensively dividing cells and developing embryos. The study's results may be useful for the in vitro mass production of plantlets of the valuable tree fern C. delgadii and helpful in enriching the knowledge of the mechanisms governing the initial phases of SE.Key message: Sucrose-induced osmotic stress changes the way of somatic embryo origin and delaying its application stimulates somatic embryogenesis of Cyathea delgadii which may be useful for in vitro mass production of plantlets. [ABSTRACT FROM AUTHOR]

The thin cell layers (TCLs) of zygotic embryos of Aesculus hippocastanum were cultured on Murashige and Skoog (MS) medium with and without 2,4-D. Somatic embryos developed directly on the TCLs of zygotic embryos at the highest frequency on hormone-free medium. The frequency of direct somatic embryo formation was suppressed by 2,4-D treatment. Microscopic observation revealed that compact and nodular structures formed on epidermal surfaces of TCLs, and these structures further developed into somatic embryos without the intervention of the callus stage, indicating direct somatic embryogenesis. Somatic embryos were not formed on the internal excised portion of zygotic embryos. Similar to zygotic embryo culture, direct somatic embryogenesis occurred from the culture of TCLs excised from cotyledonary somatic embryos on hormone-free MS medium. A similar suppression of direct somatic embryo development was observed in the presence of 2,4-D. Instead, friable embryogenic callus formation from TCL culture of somatic embryos was stimulated by 2,4-D treatment. Well-developed somatic embryos formed from the culture of embryogenic calluses. Dormancy of somatic embryos was observed because somatic embryos were not germinated on hormone-free medium without special treatment. Both cold treatment and GA3 treatment resulted in the germination of somatic embryos. [ABSTRACT FROM AUTHOR]

Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others. [ABSTRACT FROM AUTHOR]

Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L− 1) + BAP (0.5 mg L− 1) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L− 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L− 1) + NAA (0.2 mg L− 1) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L− 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants. [ABSTRACT FROM AUTHOR]

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures.

Abstract Background Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Results Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l−1 in darkness. TDZ at 3 mg l−1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95–100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. Conclusions In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore ‘Elsa’, was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

Wild populations of Paphiopedilum niveum (Rchb.f.) Stein, an endangered orchid species, have been threatened with extinction due to climate change and overcollection. Conventional methods of propagation are unable to meet market demands. The current study reports the production of genetically stable, protocorm-derived protocorm-like bodies (PLBs) of P. niveum that can be used for commercial production. These PLBs are referred to hereafter as somatic embryos (SEs). Factors affecting SE induction and SE proliferation were examined. The highest percentage of SE formation (68.33 ± 11.77%) and the maximum number of SEs per explant (5.19 ± 0.67) were obtained on modified Vacin and Went (MVW) medium containing 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The highest increase in fresh weight (FW) of proliferated SEs (183.33 ± 28.93 mg per 100 mg initial FW) was gained on hormone-free MVW. These SEs eventually developed into vigorous plantlets after culturing on MVW supplemented with 0.2% (w/v) activated charcoal and 50 g L−1 banana homogenate for 12 wk. A histological study revealed that SEs originated from both the epidermal and the sub-epidermal layers of the original protocorm via direct somatic embryogenesis. The genetic homogeneity between the mother generation (V1; the original protocorm) and subsequent generations (V2 and V3; the primary and secondary somatic embryos, respectively) was shown to be identical via random amplification of polymorphic DNA (RAPD) assessment. All regenerated plants grew well under greenhouse conditions. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU). A medium containing 0.1 mgL−1 2,4-D and 0.25 mgL−1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL−1 2,4-D and 0.5 mgL−1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury—an emerging model in developmental biology.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures. [ABSTRACT FROM AUTHOR]

Background: Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Results: Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l in darkness. TDZ at 3 mg l gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. Conclusions: In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry. [ABSTRACT FROM AUTHOR]

The development of protoplast derived from somatic embryos and some of their characteristics were compared with embryos from suspension and in vivo in the same B line. Embryos formed in a protoplast culture differed from others that their younger stages contained vacuolated cells, and older ones had altered morphological and histological structure. Somatic embryogenesis is more regular from suspension then from protoplasts. No distinct differences were observed in the rate of embryo development in vivo and in vitro, and in vitro embryos show a larger variation in size at the same stage. Embryos in vitro with fluorescence are generally larger than zygotic ones at each stage. The use of fluorescence is suggested for the selection of heterokariocytes after protoplast fusion.

We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N6-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm-3 resulted in highest number of secondary embryos that were derived from guard cells. The present communication provides a model system for studying factors and mechanism affecting totipotency or embryogenetic capacity of guard cells.

We present here a method for the in vitro propagation of Anthurium andraeanum cv. Fantasia through direct somatic embryogenesis. The embryos were directly formed at the cut end of most of the leaf explants when cultured on MS basal medium supplemented with N-benzyladenine (BA) plus α-naphthalene acetic acid (NAA). MS basal media supplemented with BA (0.27 μM) and NAA (2.68 μM) induced maximum number of somatic embryos per leaf explant (15.33 ± 1.45) after 28 d of continuous culture. The highest numbers of embryos (12.33 ± 0.88) were matured into plantlets in MS basal medium augmented with 0.27 μM BA and 58.4 mM sucrose. Histology showed the presence of scutellum, coleoptile, well-developed root, and shoot initials at different stages of somatic embryo (SE) development directly on leaf explants. About 85% of the plantlets were successfully acclimatized, and of these, 80.3% plants survived after secondary hardening under greenhouse conditions. The embryo-derived plants successfully flowered. The presence of monomorphic DNA bands in random amplified polymorphic DNA (RAPD) marker analysis confirmed the genetic homogeneity of direct somatic embryo-derived plantlets in respect to their mother plant. [ABSTRACT FROM AUTHOR]

Key message: This paper provides an important direct proembryogenic mass initiation and plant regeneration method that might be supportive and stable for gene transformation and commercial production of Torreya grandis. Abstract: Torreya grandis is an important and commercially valuable tree that is utilized as a source of essential oil and nuts. Propagation researches of T. grandis have been focused on micropropagation and somatic embryogenesis which derived from callus but limited data regarding direct somatic embryogenesis and plant regeneration are available in the literature. This study investigated the effects of plant growth regulators combinations on direct proembryogenic masses initiation and plant regeneration. Proembryogenic masses were initiated from cotyledons and hypocotyls of immature zygotic embryos directly. The frequency of direct proembryogenic masses initiation (52.6 %) was significantly greater than other treatments when the explants were cultured on Schenk and Hildebrandt medium which supplemented with 0.1 mg l N6-benzylaminopurine, 0.1 mg l Kinetin and 0.1 mg l 2,4-dichlorophenoxyacetic acid. Combined application of Abscisic acid and Polyethylene glycol 6000 stimulated cotyledons formation and resulted in morphologically superior somatic embryos. Schenk and Hildebrandt medium supplemented with 10 mg l Abscisic acid and 20 mg l Polyethylene glycol 6000 was optimum (88 %) for somatic embryos maturation. Highest germinated somatic embryos percentage (35.6 %) was obtained on Schenk and Hildebrandt medium supplemented with 2000 mg l activated charcoal, with root and shoot lengths of 2.3 and 1.9 cm, respectively. As proembryogenic masses were initiated without the formation of callus, this study will aid to establish stable genetic transformation and will also be useful in improving understanding of the somatic embryogenesis of T. grandis. [ABSTRACT FROM AUTHOR]

This study describes for the first time a simple and quick direct somatic embryogenesis and plant regeneration protocol for turmeric ( Curcuma longa L.) using leaf base explants. Preincubation of explants was carried out on solid Murashige and Skoog (MS) medium containing 4.49 μM 2,4-dichlorophenoxy acetic acid for 20 days under continuous dark condition. Subsequently transfer of the explants for 2 weeks to MS liquid medium containing 1.32 μM 6-benzyladenine (BA) under illuminated condition resulted in the highest percentage of somatic embryo (91.1 %) induction. Secondary somatic embryos which originated from primary somatic embryos were also observed in the same medium supplemented with 2.20 and 2.64 μM BA. Thereafter, mature somatic embryos germinated readily and the maximal plantlet development (87 %) was achieved on 1/2-strength MS medium containing gibberellic acid (GA) under dark condition. Histological analysis revealed that the proembryogenic masses were initiated directly from the area near the vascular tissues of the leaf base explants. Presence of clear protoderm in the globular structure and procambial strands in the matured stage of somatic embryos confirmed that these structures were true somatic embryos. Somatic embryos were successfully acclimatized ex vitro at a survival rate of 72.9 % and they were normal phenotypes. [ABSTRACT FROM AUTHOR]

Epidermal cell wall biogenesis with emphasis on cuticular layer formation during direct somatic embryogenesis in rice was examined by transmission electron microscopy. The first phenomenon noticed in the early globular stage of somatic embryos was a high activity of cell wall formation. In this stage, many endoplasmic reticula, Golgi bodies and their vesicles were present and epidermal cells were covered by fibrillar material. Cutin was initially deposited on the epidermal cell wall of late globular or early elongate stage embryos. In the late elongate stage, a lamellated cuticular layer formed, and in the late scutellar stage, a more homogenous continuous cuticular layer formed. The cuticular layer disappeared gradually from the scutellum cell wall as the somatic embryo germinated. The disappearance of the cuticular layer was similar to that of the zygotic embryo. There is no cuticular layer on the scutellar epithelial cell wall of the immature zygotic embryo 5 days after germination. Our observations lead to the conclusion that one of the structural and physiological markers of direct somatic embryogenesis is the transformation of fibrillar material into the cuticular layer.

Direct somatic embryogenesis in rice scutella was studied by scanning electron microscopy paying attention to the morphological change of surface structure. Somatic embryos were formed when the scutella of immature embryos of cv. Nipponbare were cultured on MS media containing 2 mg L−1 2,4-D. Embryos were then transferred to the media containing 1 mg L−1 2,4-D and germinated on hormone-free media. The developmental stages of typical monocotyledonous somatic embryo were classified into proembryo, globular, scutellar and coleoptilar stages by referring to the terminology used for zygotic embryos. Each stage had a characteristic surface structure. The surface of proembryo was smooth. Fibrillar material was observed during the transition from proembryo to globular stage. Fibrillar material was then gradually replaced by a mesh-like structure at the globular stage. With subsequent development of globular embryo, the apical scutellum region became flattened and the ridge of mesh-like structure became lower. Development of scutellum followed by the emergence of coleoptile from the scutellar notch was observed at the scutellar stage. The coleoptile surface was covered with elongating cells. The coleoptilar stage was characterized by the growth of coleoptile and the development of root. However, aberrant somatic embryos which had leafy scutellum and multiple shoot meristem, were also observed.

An efficient and simple procedure was systematically developed for inducing direct somatic embryogenesis and plantlet regeneration from leaf sheath explants of Curcuma amada Roxb. A two-step culture system was used to induce somatic embryogenesis. The optimized procedure resulted in direct somatic embryogenesis from 93.3% explants after 3-wk culture. Leaf sheath explants were incubated for 2 wk on medium containing 2.24 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine to initiate direct somatic embryogenesis. Thereafter, these explants were transferred to a medium containing 9.10 μM thidiazuron and 1.33 μM α-naphthaleneacetic acid. Elongated somatic embryos obtained from these cultures germinated readily, and the optimal frequency of plantlet development (86.7%) was achieved when embryos were cultured in darkness on 1/2 strength Murashige and Skoog medium containing 1.44 μM gibberellic acid. Histological and scanning electron microscopic studies showed that the initial cell divisions that led to embryo formation occurred in epidermal and subepidermal cells, followed by the development of globular and elongated structures that appeared to be somatic embryos. The presence of a clear protoderm in the globular structures and procambial strands in the elongated structures confirmed that these structures were true somatic embryos. Plantlets derived from somatic embryos were acclimatized successfully to ex vitro conditions at a survival rate of 87.43% and developed with normal phenotypes. [ABSTRACT FROM AUTHOR]

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants. [ABSTRACT FROM AUTHOR]

Highlights: [•] Direct SE was induced from the hypocotyl and leaf explants of gherkin. [•] The sucrose (5%), 2,4-D, BAP and l-glutamine to induce somatic embryogenesis. [•] Activated charcoal (AC) had a positive effect on embryo development. [•] Plantlet formation of somatic embryos was positively influenced by AC and GA3. [ABSTRACT FROM AUTHOR]

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4- D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4- D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4- D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide. [ABSTRACT FROM AUTHOR]

Abstract: This study describes, for the first time, an in vitro protocol for the direct development of somatic embryos and subsequent plant regeneration from hypocotyl segments excised from 21-days-old in vitro-germinated seedlings of Digitalis trojana Ivan (Helen of troy foxglove). Two sets of experiments were carried out, the first compared different concentrations of four cytokinins: N6-benzyladenine [BAP], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-(4-hydroxy-3-methylbut-2-enylamino) purine [zeatin] alone, while the second set tested TDZ or BAP combinations with IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA (α-naphthalene acetic acid). In the first set of experiments, TDZ was found the most effective at 1.0mg/l concentration, producing a mean of 10.7 somatic embryos per explant. In the second set, a combination of 1.0mg/l TDZ with 0.5mg/l IAA produced significantly more somatic embryos per explant (13.8 embryos) than with BAP (8.8 embryos). During subculture on growth regulator-free half-strength MS medium, somatic embryos gradually developed into plantlets. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large-scale production of cardenolides and genetic transformation of this valuable medicinal plant for its further improvement. [Copyright &y& Elsevier]

The comparison of direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic plants with DSE (12 weeks from explant to plants in soil). Microprojectiles of 0.3 μm diameter produced 5 times more transgenic lines than 1 μm microprojectiles when used at the same weight basis per shot. A significant reduction of the number of hybridization signals in the Southern blot was also observed with 0.3 μm microprojectiles when compared to 1.0 μm microprojectiles. This suggests that the lower DNA carrying capacity and greater number of the smaller microprojectiles contributes to more transgenic lines with less complex transgene integration. When geneticin sulfate, was used for selection following DSE, significantly more (4.8 times) transgenic plants were produced than with paromomycin sulfate and an equal number of non-expressing plants were produced with both selection agents. In conclusion, optimization of two alternative morphogenic routes for regeneration (DSE and ISE), biolistic and selection parameters generated rooted, transgenic plants of a commercially important sugarcane cultivar with simple transgene integration and within 12 or 19 weeks of culture initiation, respectively. Reducing the complexity of the transgene integration and minimizing the time in tissue culture will likely enhance the performance of the transgenic events. [ABSTRACT FROM AUTHOR]

We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm resulted in highest number of secondary embryos that were derived from guard cells. The present communication provides a model system for studying factors and mechanism affecting totipotency or embryogenetic capacity of guard cells. [ABSTRACT FROM AUTHOR]

A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 mo from culture initiation. Biolistic gene transfer into cross-sections of immature leaf whorl explants followed by direct somatic embryogenesis resulted in the stable genetic transformation of the commercially important sugarcane cultivar CP 88-1762. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Southern blot analysis revealed simple transgene integration patterns ranging from one to five hybridization products. NPTII-ELISA confirmed that most of the transgenic plants expressed the transgene stably in vegetative progeny. Using a minimal, linear expression cassette (MC) without vector backbone sequences for the biolistic gene transfer and reducing the amount of MC to 10 ng per shot may have led to simple transgene integration and stable transgene expression. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events. [ABSTRACT FROM AUTHOR]

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l benzyl aminopurine (BAP) and 0.1 mg l naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60-148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis. [ABSTRACT FROM AUTHOR]

n improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35-45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L GA respectively. The 2,4-D alone at 1.0 or 1.5 mg L was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L 2,4-D in combination with 0.1 mg L BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants. [ABSTRACT FROM AUTHOR]

Induction, generation mid development of embryo genic somatic phenomena are matters that can be modeled by monitoring the interplay between exogenous phytohorniones and explants. As a result, this work presents experimental studies that illustrate the influence that exogenous cytokithns (BAP and TDZ), and auxins (NAA or IBA) have, added in the composition of the artificial nutritive medium, for modeling the in vitro propagation through direct somatic embryogenesis in two cultivars of basil (Dc/mum ba'silicum L. var. Marseille and Dc/mum basilicrem L. var. Red Rub'in), starting from different types of explants (leaf; cotyledon, epicotyl, hipocotyl and radicle). The best embryogenic response was recorded for experimental variant consisting in TDZ cytokiniu and IBA auxin in quantities of 1 mg/l respectively 0.5 mg/l, and cotyledon type explant. [ABSTRACT FROM AUTHOR]

Abstract: The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limonium sinensis var. Golden Diamond in vitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N6-benzyladenine (BA) (0.44–2.2μM) or thidiazuron (TDZ) (4.54μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2μM) and NAA (1.07μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44μM BA and 4.5μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition. [ABSTRACT FROM AUTHOR]

Abstract: Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat. [Copyright &y& Elsevier]

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected. [ABSTRACT FROM AUTHOR]

To induce somatic embryogenesis in habanero pepper (Capsicum chinense Jacq.), the cultivar BVII-03, belonging to the red type, was used. Different explants were evaluated, as were different culture media, the composition of which varied in the content of plant growth regulators. Results showed the formation of somatic embryos from cotyledons, zygotic embryos, germinated zygotic embryos, hypocotyls, and cotyledonary leaves. Explants were cultured on Murashige and Skoog medium supplemented with 2,4-D (9.05 µM). The somatic embryos always formed directly from the explant, without callus formation, and the greatest efficiency was obtained when segments of hypocotyls were cultured, obtaining 175 ± 20 somatic embryos per explant. Only the somatic embryos obtained on Murashige and Skoog medium containing 2,4-D (9.05 µM) and treated with abscisic acid (ABA) (1.89 µM) before their transfer to the germination media (Murashige and Skoog + 1.1 µM GA3) emitted their radicule and expanded their cotyledonary leaves (60%), whereas the remaining embryos did not achieve germination because of different causes (abnormalities, delayed development). Not only is this protocol of somatic embryogenesis the first to be reported for this species (C. chinense Jacq.), but it is also the most efficient reported so far, within the Capsicum genus. [ABSTRACT FROM AUTHOR]

Sugarcane (Saccharum officinarum L.) plantlets derived through direct somatic embryogenesis were evaluated using random amplified polymorphic DNA (RAPD) markers to assess genetic fidelity. Thirty-five arbitrary 10-mer oligonucleotide primers produced amplification products and seventeen were used to screen for variation among 100 regenerants. In general, 4384 bands were scored of which only 125 bands were polymorphic. With all the primers used, extent of polymorphism ranged from 0.86 to 8.45%. All the RAPD profiles were monomorphic and similar with very low variation among the plantlets. The results suggest that direct somatic embryogenesis as a method can generate plants with maximum uniformity and that RAPD can be useful as a tool to check genetic variation among sugarcane regenerants. [ABSTRACT FROM AUTHOR]

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, α-naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l-1 casein hydrolysate and 10 mg l-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 m M CaCl2. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata. [ABSTRACT FROM AUTHOR]

Somatic embryos differentiated directly on the rachis of immature inflorescences of Paspalum scrobiculatum L. cv. PSC 1 on culture to MS or N6 medium supplemented with different concentrations (4.5–22.5 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). Direct embryogenesis on the rachis of inflorescence explants forms the first instance in graminaceous plants. Highest frequency of direct embryogenesis (34%and 30% cultures, respectively) was possible on N6 medium supplemented with 4.5 μM of 2,4-D and MS medium fortified with9.0 μM of 2,4-D. Other tissues of the explant, floral-primordia, only after an initial phase of callusing differentiated into somatic embryos; indirect embryogenesis. Somatic embryogenesis, direct as well as indirect, was resolved by scanning electron microscopy. The somatic embryos germinated and developed into plantlets on regeneration medium. Interestingly, one week incubation of somatic embryos on activated charcoal (0.5%) fortified basal medium, supported high potential for ‘germination’ on transfer to charcoal-free basal medium. This beneficial effect of activated charcoal on regeneration of somatic embryos into plantlets is the first record in the Gramineae. [ABSTRACT FROM AUTHOR]

In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU). A medium containing 0.1 mgL−1 2,4-D and 0.25 mgL−1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL−1 2,4-D and 0.5 mgL−1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury—an emerging model in developmental biology. [ABSTRACT FROM AUTHOR]

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm ( Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop. [ABSTRACT FROM AUTHOR]

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation. [ABSTRACT FROM AUTHOR]