Surface Of Callus Research Articles

Mechanisms for introducing 250 kDa fluorescent molecules and Cas9/sgRNA into plant cells by plasma treatment

Plant cell walls prevent molecules with high molecular weights from reaching the cell membrane, challenging genome editing in plants. To overcome this challenge, the microplasma method, established as a gene and molecule transfection technology in animal cells, was investigated in tobacco plants. We found that plasma treatment of tobacco leaves and calluses introduced fluorescent molecules into epidermal and callus cells. Scanning electron microscopy revealed that plasma treatment decomposed the cuticula layer on the surface of tobacco leaves and that plasma treatment decomposed the extracellular matrix and caused cracks on the cell wall surface of tobacco callus. These results suggest that when external molecules are introduced into plant cells by plasma treatment, the external molecules’ transport pathway reaches the cell membrane by degradation of the cuticula layer and extracellular matrix. Additionally, the introduction of molecules by plasma treatment was inhibited by an endocytosis inhibitor, indicating that plasma stimulation induces endocytosis. In summary, plasma treatment decomposes the cuticula layer and cellular interstitium, allowing molecules to reach the cell membrane, after which they are introduced into the cell via endocytosis.

Induksi dan Multiplikasi Kalus Eucalyptus sp. pada Berbagai Media Callus Induction Medium (CIM) Secara In Vitro

Callus Induction and Multiplication of Eucalyptus sp. on Various Media Callus Induction Medium (CIM) In Vitro. Callus induction in vitro can be done by planting specific part of plant such as leaf, root, stem, etc in aseptic condition by using medium that contains 2,4-D, a plat regulator for promoting callus formation. One of the objectives of callus production in vitro is to form secondary metabolites, because eucalyptus is known as a producer of essential oils. This research is an initial study for in vitro production of secondary metabolites for further research. The aim of this study was to determine the most suitable callus induction medium for callus formation and callus multiplication for Eucalyptus grown in vitro. This research was designed as a Completely Randomized Design (CRD), five treatments, repeated five times. Woody Plant Medium (WPM) was used as the basic media. Several Callus induction Medium (CIM) were used as treatments, i.e. CIM1 = 1 ppm 2,4-D; CIM2 = 1,5 ppm 2,4-D; CIM3 = 2 ppm 2,4-D; CIM4 = 1 ppm NAA+1 ppm BAP; and CIM5 = 2 ppm NAA+ 2 ppm BAP. Variables observed were the time of curly leaf formation, time of the emerging callus, colour and texture of the callus, callus’s weight, callus surface area, the weight of callus after multiplication. The result showed that CIM4 and CIM5 had the fastest emerging callus (7 days after planting), CIM1 and CIM2 formed green and crumb texture of callus, CIM1 produced the highest of callus’s weight, and CIM2 resulted in the highest of callus’s surface area. In conclusion, the most suitable CIM for callus induction was CIM1, while for callus multiplication was CM2.

In vitro regeneration and its histological characteristics of Dioscorea nipponica Makino

Dioscorea nipponica Makino is an optimal candidate to develop the diosgenin industry in North China. Due to its increasing demand in the medicine industry, it is urgent to apply new biotechnological tools to foster breeds with desirable traits and enhanced secondary metabolite production. The production of useful metabolites by the in vitro cultured rhizomes can be explored successfully for utilization by various food and drug industries. In this study, we reported callus formation and plantlet regeneration of the medicinal plant D. nipponica. Explants of leaves, stem segments and rhizomes of aseptic seedlings were cultured on Murashige and Skoog (MS) medium containing various combinations of auxin and cytokinin to find the optimal PGRs of each type of explant for callus induction and shoot regeneration of D. nipponica. The paraffin section technique was also used to observe of the morphogenesis of callus and adventitious bud. Explants of seeds and rhizomes formed calli at high frequency in all lines we examined. However, the explant of leaves rarely formed callus. Three kinds of callus were detected during the induction phase. Here, we describe three types of callus (Callus I–III) with different structure characteristics. Greenish in color and a nodule-like protrusion surface (Callus type III) were arranged more closely of cells with less interstitial substance, cell differentiation ability stronger than other callus types. The optimum combination was the maximum shoot differentiation frequency of 90% in callus derived from seeds cultured on MS medium with 2.0 mg L−16-BA + 0.2 mg L−1NAA. The shoot differentiation frequency (88.57%) of rhizome-induced callus was obtained by the combination of MS medium supplemented with 3.0 mg L−16-BA + 2.0 mg L−1NAA. 1/2 MS medium plus 0.5 mg L−1NAA resulted in a higher root regeneration frequency of 86.70%. In vitro propagated plantlets with healthy roots were domesticated and transplanted into small plastic pots containing sterile soil rite under greenhouse conditions with 80% survivability. Bud differentiation is mostly of exogenous origin, mostly occurring on the near callus surface. Therefore, it may be surmised that in vitro morphogenesis of D. nipponica is mainly caused by indirect organogenesis (adventitious bud).

Androgenic haploid plant development via embryogenesis with simultaneous determination of bioactive metabolites in Cambod tea (Camellia assamica ssp. lasiocalyx)

This pioneering work reports successful androgenic plant development via embryogenesis from microspore calluses in anther cultures and estimation of bioactive metabolites in in vitro regenerants and parent plant (control) of Cambod tea, Camellia assamica ssp. lasiocalyx (Planch MS) cultivar TV19. Anthers bearing microspores at early-to-late uni-nucleate stage were selected to initiate androgenesis. A pre-treatment of 5 °C for 5 days in the dark was most effective to initiate profusely growing white callusing from microspores within 10 weeks of culture on MS medium (6% sucrose) supplemented with high cytokinin/auxin ratio maintained by 6-benzylaminopurine (BAP) and 2,4-dichlorophenxoyacetic acid. Nodular structures on the callus surface differentiated into embryos. Further developement of the embryos occurred on embryogenesis medium but, with ten times reduced concentration of growth regulators and additives. Germination of embryos into complete plantlets was achieved when major salts in medium were reduced to half MS (½ MS) and augmented with BAP, GA3 and IBA along with glutamine and serine. Cytological examination of the root-tip cells revealed that regenerated plantlets were haploids (2n = x = 15), which was further confirmed through flow cytometry. The hot-water extracts from in vitro haploid calluses, embryos and field-grown donor plant were utilized for quantification of (+)-catechin, (−)-epicatechin, (−)-epigallocatechin gallate, caffeine and theophylline. Our findings revealed that the metabolite profile of in vitro regenerated haploid cultures is comparable to that of the mother plant, thereby presenting them as potential source for genome duplication and development of genetically stable homozygous pure breeding lines. This is first report on haploids in out-breeding tree, Cambod tea. It’s a significant achievement towards generating homozygous lines, which is impossible using conventional methods. Haploids showed consistent metabolite production.

Guided Bone Regeneration with Ammoniomethacrylate-Based Barrier Membranes in a Radial Defect Model.

Large bone defects pose an unsolved challenge for orthopedic surgeons. Our group has previously reported the construction of a barrier membrane made of ammoniomethacrylate copolymer USP (AMCA), which supports the adhesion, proliferation, and osteoblastic differentiation of human mesenchymal stem cells (hMSCs). In this study, we report the use of AMCA membranes to seclude critical segmental defect (~1.0 cm) created in the middle third of rabbit radius and test the efficiency of bone regeneration. Bone regeneration was assessed by radiography, biweekly for 8 weeks. The results were verified by histology and micro-CT at the end of the follow-up. The AMCA membranes were found superior to no treatment in terms of new bone formation in the defect, bone volume, callus surface area normalized to total volume, and the number of bone trabeculae, after eight weeks. Additional factors were then assessed, and these included the addition of simvastatin to the membrane, coating the membrane with human MSC, and a combination of those. The addition of simvastatin to the membranes demonstrated a stronger effect at a similar radiological follow-up. We conclude that AMCA barrier membranes per se and simvastatin delivered in a controlled manner improve bone regeneration outcome.

Phenylethanoids, phenylpropanoids, and phenolic acids quantification vis-à-vis gene expression profiling in leaf and root derived callus lines of Rhodiola imbricata (Edgew.)

Salidroside and rosavins have been found as the most potent ingredients used in Rhodiola based herbal formulations. Rhodiola imbricata is a medicinal herb of the trans-Himalayan Ladakh region of India. Since, the natural supply of this herb is rapidly decreasing, due to its over-exploitation, high altitude region, and traditional usage in the Amchi system of medicine. In the present study, friable callus culture was developed for the first time from leaf and root explants of R. imbricata. Furthermore, callus cell lines were also evaluated for rapid growth rate and high metabolite accumulation. The results revealed that callus induction frequency observed in juvenile leaves (100 %) and roots (87.50 %) in MS medium enriched with 0.5 mg/L TDZ and 1 mg/L NAA. Scanning electron microscopy (SEM) analysis was done for histological observations of the callus surface. A selective and efficient ultra-performance liquid chromatography with PDA detector (UPLC-PDA) method was developed and validated for the quantification of phenylethanoids, phenylpropanoids, and phenolic acids. The maximum salidroside was detected in leaf derived friable green calli (3.59 mg/g DW) subsequently followed by leaf derived friable white calli (2.31 mg/g DW). While rosavin and rosarin were detected maximum in root derived compact green calli (0.15 mg/g DW) and root derived friable green calli (0.07 mg/g DW). Genes encoding enzymes involved in salidroside and rosavins biosynthesis were also investigated for transcript abundance in wild as well as in vitro cultures using the RT-qPCR approach. The present study explained the friable callus culture as a potential alternative approach to obtain higher metabolite yield in R. imbricata. Further, it can be used for sequential scale-up of metabolite at bioreactor level to meet the industrial demand.

Callus Induction of Mangosteen (Garcinia mangostana L.) In Vitro with Addition of Growth Regulators

This research was conducted to determine the best combination of plant growth regulators on callus induction of shoot explants mangosteen (Garcinia mangostana L.). The research used complete randomized design (CRD) which is consisting of 12 treatments with 3 times repetitions. The parameters observed were time of callus formation, callus texture, callus color, callus pile height, callus surface area, callus weight, and callus performance. Data was analyzed by using ANOVA, and DMRT as follow up. The result showed that addition of plant growth regulators have effect on callus induction of shoot explant mangosteen (Garcinia mangostana L.). The best concentration and composition of media for induction of mangosteen callus is by giving sucrose 1,5 g / L + 2,4D 3 ppm because it was able to produce callus with the fastest time with a compact texture and color that is identical to green and white. The callus has 0.67a cm in height, 1.70a cm2 in width, and 0.51a g in weight. The callus performance is growing all over the surface.

Treatment of closed calcaneal fractures by open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision

Objective To report the treatment effects of open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision on closed calcaneal fractures. Methods A retrospective study was done of the 20 patients (25 feet) who had been treated at Ward One, Department of Orthopaedics, People's Hospital of Yunfu from February 2016 to February of 2019 for closed calcaneal fractures by open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision. They were 16 males and 4 females, aged from 16 to 60 years. According to the Sanders classification, there were 3 cases of type Ⅱ, 15 cases of type Ⅲ and 2 cases of type Ⅳ. Their fractures were reduced by traction, extruding, prying and direct visualization through the tarsal canal window; the bone plates were inserted through a small incision at the back of the heel and fixated by screws. Postoperative observation was done to address fracture healing, and length, width, height, Bohler angle and Gissane angle of the affected calcaneus, as well as functional recovery of the ankle-hindfoot by the American Orthopaedic Foot and Ankle Society (AOFAS) evaluation. Results The operation time for a single foot ranged from 45 min to 70 min, averaging 64.5 min; the intraoperative fluoroscopy for a single foot ranged from 3 times to 6 times, averaging 4.5 times. Local skin necrosis of about 0.5 cm×0.3 cm appeared in one foot after operation but responded to dressing change. No other wound complications occurred. Their follow up was carried out for 6 to 36 months (average, 17.3 months). The fractures healed well with well-shaped bony callus and flat articular surface after 4 to 6 months. The length (80.5 mm±4.2 mm), width (44.8 mm±5.2 mm), height (44.4 mm±3.0 mm), Bohler angle (25.0°±5.1°) and Gissane angle (113.8°±8.6°) of the calcaneus at the last follow up were significantly improved than the preoperative values (79.4 mm ± 4.5 mm, 50.5 mm ± 6.3 mm, 40.0 mm±4.4 mm, 12.0°±13.8° and 107.0°±13.3°) (all P<0.05). By the AOFAS ankle-hindfoot scale, functional recovery of the foot was excellent in 20, good in 3 and fair in 2 cases, giving an excellent to good rate of 92%. Conclusion In the treatment of closed calcaneal fractures, open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision may lead to fine outcomes due to its advantages of small incision and fine fracture reduction. Key words: Calcaneus; Fractures, bone; Fracture fixation, internal; Tarsal Canal; Small incision

In vitro Callus Induction of Sipahutar Pineapple (Ananas comosus L.) from North Sumatra Indonesia.

Sipahutar pineapple (Ananas comosus L.) is a indigenous of pineapple grown in Sipahutar district, North Sumatra, Indonesia. Propagation of Sipahutar pineapple that being done traditionally is less effective, because the number of seeds that produced is very limited and requires a long time. Propagation through in vitro culture is an alternative solution to solve this problem. It is necessary to add plant growth regulator (PGR) to stimulate callus formation in Sipahutar pineapple explants (Ananas comosus L.). Callus induction of pineapple from Sipahutar was carried out by PGR treatment on MS medium. The purpose of this study was to determine the effect MS medium treatment with added dichlorophenoxyacetic acid (2,4-D) and benzyl amino purin (BAP) PGR on Sipahutar pineapple callus formation (Ananas comosus L.) with light and dark treatment. This callus induction research used a completely randomized design (CRD) with 2 factors, the first factor was treatment 2,4-D (0, 1, 2) ppm. The second factor is BAP (0, 0.5, 1) ppm. Nine combinations of treatments are obtained. Each combination of treatments is treated in both light and dark conditions. The parameters of this study were the percentage (%) of explants that formed callus, the time of callus formed, callus texture, callus biomass, callus surface height and callus surface area. Data were analyzed with two-way ANOVA, followed by Duncan Multiple Rate Test (DMRT). The study showed that the interaction between 2,4-D and BAP significantly affected the time of callus formed but 2,4-D and BAP did not significantly affect callus biomass, callus surface height and callus surface area. All explants can form callus, except explants without the addition of 2,4-D and BAP. The callus formed on 10 days after induction (DAI) and 12 DAI with the treatment of light and dark. The color of the produced callus were white, yellowish white, greenish white, brown, brownish yellow, brownish white, brownish green, yellowish green and greenish white. The callus formed is generally compact textures, except for explants which by giving 1 ppm 2,4-D produce friable callus.

Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations

Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells.

Functional differentiation in pollination processes among floral traits in Serapias species (Orchidaceae).

Floral displays, influencing attractiveness to insects, increase the number of pollinator visits and the efficiency of each visit in terms of pollen exchange and thus affect the plant reproductive success. Here, we conducted an in situ manipulation experiment to investigate whether the floral modifications affect reproductive success in natural orchid populations of Serapias lingua and Serapias vomeracea. We estimated male and female reproductive success of three treatment groups, disassembly of floral tube, cutting of lip, and painting of the callus surface, in terms of pollinaria removed/deposited and fruit production. Results revealed that phenotypic modification had opposite effects on reproductive success of two examine species. Indeed, reproductive success was significantly increased by the detached of the petals and sepals, and decreased, due to callus painting and lip removal, in S. lingua. On the contrary, unmanipulated plants of S. vomeracea showed significantly higher value of pollinaria removed and deposited and fruit set than manipulated ones. The differences between S. lingua and S. vomeracea agree to the different pollination strategy of examined species. S. vomeracea shows shelter imitation strategy, and thus, the disassembly of tunnel‐like corolla does not allow the insects to use the flower as a refuge, while S. lingua is a sexually deceptive orchid and therefore the opening of the flower made more visible callus (visible at a greater distance) increasing the pollinators attraction. This study provides evidence that pollinators were largely sensitive to the experimental modification of the flower phenotype, which is consistent with the presence of significant selection on individual floral characters. Our experimental investigations of the effects of variation in display on pollinator visitation provide insights into the evolution of floral morphology in orchid with shelter imitation strategy.

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda).

Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue.

Below the Callus Surface: Applying Paleohistological Techniques to Understand the Biology of Bone Healing in Skeletonized Human Remains

Objectives: Bone trauma is a common occurrence in human skeletal remains. Macroscopic and imaging scrutiny is the approach most currently used to analyze and describe trauma. Nevertheless, this line of inquiry may not be sufficient to accurately identify the type of traumatic lesion and the associated degree of bone healing. To test the usefulness of histology in the examination of bone healing biology, we used an integrative approach that combines gross inspection and microscopy. Materials and Methods: Six bone samples belonging to 5 adult individuals with signs of bone trauma were collected from the Human Identified Skeletal Collection from the Museu Bocage (Lisbon, Portugal). Previous to sampling, the lesions were described according to their location, morphology, and healing status. After sampling, the bone specimens were prepared for plane light and polarized light analysis. Results: The histological analysis was pivotal: (1) to differentiate between types of traumatic lesions; (2) to ascertain the posttraumatic interval, and (3) to diagnose other associated pathological conditions. Conclusion: The outer surface of a bone lesion may not give a complete picture of the biology of the tissue's response. Accordingly, microscopic analysis is essential to differentiate, characterize, and classify trauma signs.

Effect of collection season, type and length of explant on callus induction and its survival in peach

It takes 5–6 weeks to complete callus formation over callus surface in peach. During the course of investigations effect of explants collection season, type and length of explants for callusing in peach were standardized. Among various combinations best response in terms of maximum callus induction (97.78%) was noted in spring x internodal segments x 0.5 cm length of explants closely followed by (95.55%) in spring x leaf disc x 0.5 cm length of explants however maximum callus culture survival (100%) was noted in spring x leaf disc as well as in internodal segments x 0.5 cm length of explants. Observations on callus morphology indicate that the leaf disc explants collected during winter season formed white, watery type callus with poor callus growth while no callus formation was noticed in internodal segments in winter season. However, yellowish green, friable type callus with excellent growth was observed during spring season in leaf disc and green colour callus along with compact callus type were observed with internodal segment. During summer season brown colour callus with friable type were noticed in leaf disc explants with good callus growth and whitish green colour callus with semi-compact type were observed in internodal segment explants along with excellent callus growth.

Effect of plant growth regulators on in vitro plant regeneration of sandalwood (Santalum album L.) via organogenesis

Santalum album L. is an economically important tropical tree species. Sandalwood trees are the source of highly priced and fragrant heartwood, the ‘East Indian Sandalwood’ that on steam distillation yields on an average 5–7 % oil of high perfumery value. Owing to extensive logging, changes in land-use patterns and poor natural regeneration, the natural sandal populations are rapidly dwindling. An efficient protocol of plant regeneration from node explant has been developed for S. album. Optimal callus was developed from nodal segments on woody plant medium (WPM) supplemented with 0.6 mg 1−1 TDZ and 1.5 mg 1−1 2,4-D. Fresh weight (132.3 mg) and dry weight (41.0 mg) of node derived callus were highest in medium supplemented with 0.6 mg l−1 TDZ. Shoot bud initiation was achieved from the surface of callus when transferred on shoot induction medium supplemented with BA and NAA in combination. Highest number of shoot buds (16.0) per callus was observed in medium containing 2.5 mg l−1BA + 0.4 mg 1−1 NAA. Shoot proliferation (number of shoots/callus) (21.5) and shoot length (4.7) was highest in media containing 3.0 mg 1−1 Kn. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). Highest rooting percentage (82.37) and survival was achieved on WPM media with 1.5 mg 1−1 IBA. The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics.

Studies on floral nectary, tepals' structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae).

The lip of Epipactis palustris consists of two movably joined parts: the basal part (hypochile) with central broad isthmus and epichile with callus. The analysis of flowers provides strong evidence to conclude that the whole surface of lip callus and abaxial side of isthmus are secretory. The exudation at first appears on callus, at early stages, later on isthmus. It could be a strategy to prolong the emission of volatile substances and nectar, and this means to prolong luring pollinators. The results from transmission electron microscopy (TEM) support this conclusion. The plastids noted in callus were without starch, whereas the isthmus’ cells contained partly hydrolyzed starch. Some plastids, noted in callus, had polymorphic shapes, which were often related to a starch reduction. During the depletion of starch in callus cells, the number of plastoglobuli within the plastids increased, and also lipid bodies appeared in the cytoplasm whereas, in isthmus cells, proplastids with phytoferritin were noted. The endoplasmic reticulum was in contact with plasmalemma, and the vesicles were fusing with plasmalemma in secretory cells of callus and isthmus, which is a way of granulocrine secretion. The cross-sections of sepals revealed that abaxial epidermis was tomentose, with stomata at the top of substomatal cavities. The pollen grains adhering to the rostellum-viscidium prove previous ecological observations that the rostellum-viscidium is not a barrier preventing self-pollination.

Extracellular matrix surface network is associated with non-morphogenic calli of Helianthus tuberosus cv. Albik produced from various explants

&lt;em&gt;Helianthus tuberosus&lt;/em&gt; is economically important species. To improve characters of this energetic plant via genetic modification, production of callus tissue and plant regeneration are the first steps. A new, potentially energetic cultivar Albik was used in this study to test callus induction and regeneration. Callus was produced on leaves, petioles, apical meristems and stems from field-harvested plants but was totally non-morphogenic. Its induction started in the cortex and vascular bundles as confirmed by histological analysis. The surface of heterogeneous callus was partially covered with a membranous extracellular matrix surface network visible in scanning and transmission electron microscopies. The results clearly indicate that: (&lt;strong&gt;&lt;em&gt;i&lt;/em&gt;&lt;/strong&gt;) the morphogenic capacity of callus in topinambur is genotype dependent, (&lt;strong&gt;&lt;em&gt;ii&lt;/em&gt;&lt;/strong&gt;) cv. Albik of &lt;em&gt;H. tuberosus&lt;/em&gt; proved recalcitrant in in vitro regeneration, and (&lt;strong&gt;&lt;em&gt;iii&lt;/em&gt;&lt;/strong&gt;) extracellular matrix surface network is not a morphogenic marker in this cultivar.

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.

Anatomy of the floral nectary of ornithophilousElleanthus brasiliensis(Orchidaceae: Sobralieae)

Orchidaceae show enormous floral diversity. However, anatomical studies of nectary tissues relative to nectar composition and pollinators are scarce. This work aims to present a detailed anatomical study of the labellar nectary of Elleanthus brasiliensis, analyse the chemical composition of its nectar and relate these findings to pollination biology. Basally, the labellum bears a pair of fleshy, whitish, ovoid calli on its adaxial surface. Nectariferous callus tissue consists of a papillate epidermis and enlarged subepidermal parenchyma cells with thin walls, large nuclei and dense cytoplasm which stained positively for hydrophilic substances, interpreted as pre-nectar. The paired calli lack vascular tissues, but at the point of callus insertion, the diameters of vascular bundles supplying the lip are larger. Nectar is secreted as droplets on the adaxial callus surface. It is produced in small quantities, c. 4 μL per flower. Callus cell contents tested negative for polysaccharides, lipids and phenolic compounds. The nectar is sucrose-dominant, as in other hummingbird-pollinated species. It is suggested that other ornithophilous species of Sobralieae have anatomically similar nectaries.

Somatic embryogenesis and bulblet regeneration in snakehead fritillary (Fritillaria meleagris L.)

Induction of in vitro morphogenesis of mature zygotic embryos of Fritillaria meleagrisL. was investigated. Somatic embryogenesis and whole plant regeneration were achieved. Isolated zygotic embryos were cultured on MS medium that contained 3% sucrose, 0.7% agar, 250.0 mg/l casein hydrolysate, 250.0 mg/l L-proline, 80 mg/l adenine sulfate and 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ). Embryogenic callus was derived from mature zygotic embryos after 4 weeks on TDZ containing medium. Somatic embryos at the early stages of development arose from the surface of the embryogenic callus. Multiplication of somatic embryos, formation of bulblets and shoot development were observed on the same medium. Scale sections prepared from the formed bulblets were cultured on MS media supplemented with 2,4-D (0.1 to 10.0 mg/l) and TDZ (0.05 to 2.0 mg/l). Somatic embryos and bulblets were frequently produced from the scale sections. This is a successful report of plant regeneration through somatic embryogenesis for this very important medicinal and horticultural plant. Histological observation revealed that plants of F. meleagris L. were regenerated via somatic embryogenesis. Key words: Bulbous plant, medicinal plant, snakehead fritillary, somatic embryos.