Weeks Of Subculture Research Articles

Ex-situ Conservation of Bulbophyllum leopardinum, A Threatened Medicinal Orchid of Nepal

A successful micropropagation method was developed via the in-vitro seed germination and seedling growth of the epiphytic and/or lithophytic orchid Bulbophyllum leopardinum, a species having horticultural and therapeutic significance. To enhance seed germination, several quantities and combinations of naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), indole acetic acid (IAA), gibberellic acid (GA3), and coconut water (CW) were added to 0.8% (w/v) agar-solidified MS medium. Half-strength MS medium has been experimented with alone and in combination with BAP, Kinetin (Kn), and GA3 to promote shoot development. In-vitro-developed healthy shoots were chosen to establish roots in a half-strength MS (HMS) medium supplemented with various auxins. The best and earliest seed germination with the greenest protocorms (96.3±0.5% in 7 weeks) was achieved on HMS medium fortified with 15% CW (H15C). Further tests for the shoot as well as root development were continued with an H15C medium. H15C with 1 mg/l BAP and 1.5 mg/l Kinetin was most effective for early in vitro development and differentiation into seedlings with the many long shoots (9.3±0.1 shoots and 2.4±0.1 cm per culture) within 12 weeks of sub-culture. The most suitable rooting hormone was 1 mg/l NAA (4.2±0.26 roots per culture). This medium also produced the longest roots (1.9±0.09 cm per culture). By successfully developing a protocol for the mass propagation of B. leopardinum, this research has enhanced both the cultivation and the commercialization potential of this species.

Optimal condition for Propagation and Growing of Dendrobium thyrsiflorum

Dendrobium thyrsiflorum is a medicinal orchid that is being gradually destroyed due to over-exploitation. This research focuses on in vitro production of D. thyrsiflorum plantlet under optimal acclimatization conditions. The results of the study showed that the best medium for seed germination was MS basal medium (full-strength MS nutrient plus 100 ml L-1 coconut water; 7 g L-1 agar, 30 g L-1 sucrose, and 0.5 g L-1 active charcoal) supplemented with 60 g L-1 mashed sweet potatoes, which induces 97.8% of seed germination. The shoot was well developed in MS basal medium supplemented with 60 g L-1 mashed sweet potatoes and 0.4 mg L-1 6-Benzylaminopurine (BA) and, corpulent and green in shoot morphology. The shoot multiplication rate was greatest on MS basal medium supplemented with 0.4 mg L-1 BA and 0.4 mg L-1 Kinetin with 4.53 times, and the shoot height was reported at 3.45 cm after 8 weeks of subculture. Further, The shoot was 100% rooting, with an average of 4.51 roots/shoot and 5.34 cm per root in length when the shoot was implanted on MS basal medium plus 60 g L-1 mashed sweet potatoes and 0.75 g L-1 active charcoal. Especially, plantlets after transplanting to the orchid net house reached a 94.8% survival rate on tree fern after 24 weeks. Hence, the results of the study suggested a successful production of D. thyrsiflorum plantlet on the selected media compositions.

Indoleamine 2,3-Dioxygenase-1 Expression is Changed During Bladder Cancer Cell Invasion.

The severity of the bladder carcinoma (BC) is directly linked to cell invasion and metastasis. Indoleamine 2,3-dioxygenase-1 (IDO-1) is an INF-γ-induced immunomodulating enzyme that has been linked to the cancer cell invasiveness. Because IDO1 is variable among the tumors, we analyzed its expression in the BC invasion using BC mice models and cell culture. MB49 cells were orthotopically or ectopically inoculated in C57Bl6 mice to evaluate IDO1 by immunohistochemistry. For in vitro experiments, expression of IDO1 and INF-γ was evaluated in grade-1 (RT4) and in grade-3 (T24) BC cell lines. Invading and non-invading T24 cells were separated using the Matrigel/Transwell system, of which total RNA was extracted immediately or after 2 weeks of subculture. Finally, IDO1 was silenced in T24 cells to verify its role on cell invasiveness. In both animal models, IDO1 was differentially expressed between non-invading and invading cells. In cell culture, T24 cells expressed more IDO1 than RT4 cells, independently of the INF-γ expression. IDO1 was differentially expressed between non-invading and invading T24 cells, a difference that was lost by long-time subculture. IDO1 silencing resulted in diminished cell invasiveness. In conclusion, IDO1 expression is changed during bladder carcinoma invasion, playing an important role in this process.

First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

Organogenesis from Leaf Tissue of Spondias pinnata (L. f.) Kurz, SEM study and Genetic Fidelity Assessment by ISSR and ScoT

In vitro raised plantlets were obtained from nodal tissue through direct organogenesis and they served as donor plants for the collection of leaf explants. Leaf explants were inoculated on Murashige and Skoog’s medium with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Hundred percent callogenesis was observed on medium supplemented with 5 mg l−1 2,4-D. For the multiplication of cells (proliferation), calli were transferred to either basal medium or media containing different types and concentrations of cytokinins. Proliferation was observed maximum on media containing 0.5 mg l−1 BAP or 1.0 mg l−1 BAP. Shoot differentiation from calli took place on media supplemented with BAP in combinations with TDZ or ZN. Initiation of organogenesis was observed in calli within two weeks of their subculture on differentiation medium. Shoot differentiation was maximum, when calli proliferated on medium having 1 mg l−1 BAP were transferred to the medium containing 1 mg l−1 BAP and 0.5 mg l−1 TDZ. Organogenic responses after four weeks of subculture on above differentiation medium were as such: number of shoots per explants (25.33 ± 0.88), number of shoots per calli replicate (3.67 ± 0.33) and maximum shoot length (3.43 ± 0.20 cm). Medium supplemented with 2.5 mg l−1 NAA was most responsive for rooting of shoots (54.16 ± 1.39%). About 62.5% plantlets survived after hardening and 54.17% plantlets got acclimatized. All acclimatized plants were transferred to field condition successfully. Formation of unipolar shoots and their multicellular attachment with callus were observed by scanning electron microscopy. To confirm the genetic fidelity of micropropagated plants, five micropropagated plants derived from different leaf explants and two mother plants (randomly selected from micropropagated plants raised from nodal explants) were subjected to molecular analysis. The genetic fidelity of in vitro regenerated plants was assessed by using SCoT and ISSR molecular markers. 12.5% polymorphism was reported in both studies, which may be due to callus mediated regeneration of shoots. Indirect organogenesis from leaf tissues of Spondias pinnata can be used both for conservation as well as the improvement of plants.

Expression of SSEA-4 and Oct-4 from somatic cells in primary mouse gastric cell culture induced by brief strong acid.

Environmental changes can stress and alter biology at the molecular and cellular level. For example, metal-protein interaction is a classic physic and biological property of nature, which is fundamentally influenced by acidity. Here, we report a unique cellular reprogramming phenomenon in that a brief strong acid treatment induced the expression of pluripotent stem cell (PSC) markers. We used strong acid to briefly challenge mix-cultured gastric cells, and then subcultured survived cells in anormal cell culture medium. We found that survival acid-treated cells expressed PSC markers detected by commonly used pluripotent antibodies such as SSEA-4 and Oct4. In addition, we observed that the survived cells from theacid challenge grew faster during the second and third weeks of subculture and hada relative short doubling time (DT) than the controls. PSC marker-labeled 'older' cells also presented immature cell-like morphology with some having marker Oct4 in the nucleus. Finally, the expression of the markers appeared to be sensitive to metal ion chelation. Removal of the metals during a brief acid treatment reduced pluripotent marker-positive cells, suggesting the dissociation of metals from metal-binding proteins may be a factor involved in the induction of stem cell markers. Our findings reveal that somatic cells appear to possess a plasticity feature to express pluripotent marker proteins or to select cell subpopulations that express pluripotent marker proteins when cells are transiently exposed to strong acid. It opens new directions for understanding conserved regulatory mechanisms involved in cellular survival under stressful stimulation.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12weeks of subculture following cryopreservation, similar efficiencies were obtained after 16weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

The Effect of Organic Nutrient and Growth Regulators on Seed Germination, Embryo and Shoots Development of Dendrobium antennatum Lindl. Orchid by In Vitro

<p><em>Dendrobium antennatum</em> has high economic value as cut flowers and flowerpots. Like orchid seeds in general, <em>D. antennatum</em> is difficult to germinate under natural conditions. This study aimed to determine the effect of coconut water on seed germination and embryo development, as well as the effect of NAA on shoots development of <em>D. antennatum</em>. This study consisted of two stages. In the first stage, the 12 weeks-old seeds after pollination were sown on MS medium containing 2 g/L peptone + 0%; 5%; 10%; and 20% coconut water. After 8 weeks of culture, the seeds germinated and the shoot formed were recorded. The highest in seed germination (92.2%) and the formation of shoots (51.4%) were obtained when seeds were cultured on MS medium containing 2 g/L peptone + 20% coconut water. In the second stage, the shoots were sub-cultured on MS medium containing 1 mg/L thidiazuron + 0 mg/L; 1 mg/L; 2 mg/L; and 3 mg/L NAA. After 16 weeks of sub-culture, the height of <em>plantlets</em>, the length of the roots and leaves, number of leaves and roots formed were recorded. MS medium containing 1 mg/L thidiazuron + 1 mg/L NAA was the most suitable for the shoots development of <em>D.antennatum</em>. The embryo development of<em> D.antennatum in vitro</em> begins with the enlargement of embryo, with further it emerges from the seed coat (germinated) followed by the formation of the apical meristems to form the shoots and the roots.</p><p> </p>

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl−1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA3), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO4·7H2O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.

In Vitro Regeneration Potential of Seven Commercial Soybean Cultivars (Glycine max L.) for Use in Biotechnology

Abstract This work is aimed to evaluate in vitro regeneration potential of seven commercial soybean varieties Bohemians, Cardiff, Gallec, Merlin, Moravians, Naya and Silensia (Glycine max L.) cultivated in Central Europe. Our results showed the half-seeds could be effectively used as an explant source for all tested cultivars. The regeneration was initiated on the media containing growth regulators 1.67 mg.l-1 BAP and 0.25 mg.l-1 GA3. Within the first five days culture, green chlorophyll-containing explants were observed with frequency from 18.3% to 55.9%. Two weeks later, the explants responded by production of calli with the efficiency up to 83.0%. First shoots appeared after 2–3 weeks of subculture on the media. The soybean regeneration showed to be genotype-dependent with variable efficiencies from 5.7% (cv. Naya) to 37.7% (cv. Gallec). The cultivars Cardiff, Merlin and Gallec appear to be the most promising candidates for further biotechnological use. Application of antioxidants such as L-cysteine, dithiothreitol and sodium thiosulfate does not have effect on the explant regeneration for the first five days.

Increased biofilm formation ability in Klebsiella pneumoniae after short-term exposure to a simulated microgravity environment.

Biofilm formation is closely related to the pathogenetic processes of Klebsiella pneumoniae, which frequently causes infections in immunocompromised individuals. The immune system of astronauts is compromised in spaceflight. Accordingly, K. pneumoniae, which used to be isolated from orbiting spacecraft and astronauts, poses potential threats to the health of astronauts and mission security. Microgravity is a key environmental cue during spaceflight. Therefore, determining its effects on bacterial biofilm formation is necessary. In this study, K. pneumoniae ATCC BAA‐1705 was exposed to a simulated microgravity (SMG) environment. K. pneumoniae grown under SMG formed thicker biofilms compared with those under normal gravity (NG) control after 2 weeks of subculture. Two indicative dyes (i.e., Congo red and calcofluor) specifically binding to cellulose fibers and/or fimbriae were utilized to reconfirm the enhanced biofilm formation ability of K. pneumoniae grown under SMG. Further analysis showed that the biofilms formed by SMG‐treated K. pneumoniae were susceptible to cellulase digestion. Yeast cells mannose‐resistant agglutination by K. pneumoniae type 3 fimbriae was more obvious in the SMG group, which suggests that cellulose production and type 3 fimbriae expression in K. pneumoniae were both enhanced under the SMG condition. Transcriptomic analysis showed that 171 genes belonging to 15 functional categories were dysregulated in this organism exposed to the SMG conditions compared with those in the NG group, where the genes responsible for the type 3 fimbriae (mrkABCDF) and its regulator (mrkH) were upregulated.

Study of in vitro selection and plant regeneration of Indica rice tolerant to iron

Iron tolerant callus lines of Indica rice Vars. Khandagiri and Jagannath has been achieved on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l Kinetin, 3.0 mg/l 2,4-D, 12 mM Fe. Somatic embryos derived from both tolerant non-tolerant calluses are germinated on MS medium supplemented with 1.0 mg/l 6-benzylaminopurine (BA), 0.25 mg/l 1-napthalen-eacetic acid (NAA) and 12 mM Fe within four weeks of subculture. The growth parameters like fresh and dry weight of callus, growth tolerance index, enzyme activity (Catalase and Peroxidase) and biochemical markers were used as indicators of iron tolerance. Measurements as early as two weeks after the beginning of the treatments did not yield consistent results. However, the growth tolerance index in the four weeks after the beginning of treatments yielded significant differences among non-tolerant and tolerant calluses. The enzyme activity is enhanced in the tolerant calluses than non-tolerant one. The in vitro raised plantlets derived from tolerant callus lines are grown in vitro in the presence of BA, NAA and 12 mM iron. The tolerant plantlets are established in the pots with iron-rich soil. This study will help to develop iron tolerant rice plants for breeding program.

Standardization of in Vitro Regeneration Protocol for Conservation of Shirui Lily (Lilium Mackliniae)-An Endangered Heritage Flower under Changing Climatic Conditions

Shirui lily (Lilium mackliniae), an endangered Asiatic lily species, hails from the Shirui peak of Siroi hill range in Ukhrul district of Manipur, India. This rare species is now at the verge of its extinction due to uncontrolled trampling and plucking by the human intrusions and extensive slash and burning cultivation in this region, as a result of which, it has been recorded in the ‘Red Data Book’ of ‘Indian Plants: Volume I’. Conservation of this endangered species under changing climatic conditions is a great concern for the researchers. An attempt has been made in the present investigation to establish an efficient in vitro regeneration protocol in L. mackliniae for its conservation. Callus cultures were established in MS medium containing different concentrations of 2,4-D (1-2mg/l). The regeneration ability in friable callus was 12 fold higher as compared to the compact callus. Direct regeneration and somatic embryogenesis was achieved in friable callus at 6-8 weeks of subculture in MS with benzyladenine (BA, 1.0mg/l). Multiple shoot proliferation and rooting were achieved in MS+0.5mg/l BA and 0.5mg/l napthalene acetic acid (NAA), respectively. Hardening under mist house resulted in higher survivability (85%) over poly house (62.2%) and shed net (55.5%). The in vitro plantlets were evaluated under different temperature and moisture regimes. The first time report of in vitro regeneration protocol will be helpful to augment breeding strategies in this rare endangered species for wide adaptation under changing climate conditions.

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers.

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog's (MS) medium supplemented with 2.0mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25-0.5mg/l NAA (1-napthalene acetic acid) and 3% (w/v) sucrose within 4weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0mg/l BA, 0.5mg/l NAA, 40mg/l Ads (adenine sulphate) within 4-6weeks of subculture. Inclusion of 0.25-0.50mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25-1.0mg/l NAA or indole-3-butyric acid (IBA) and 2% (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5mg/l NAA with 3% (w/v) sucrose. About 80% of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.

Rapid In Vitro Plant Regeneration of Black Gram (Vigna mungo L. Hepper) Var. Sarala, an Important Legume Crop

Efficient in vitro regeneration of black gram (Vigna mungo L. Hepper) var. Sarala was achieved through organogenesis using cotyledonary explants excised from 4 days old seedlings on MS medium supplemented with 2.0 mg/l BAP. Organogenic calli were developed from cotyledonary tissues within 4–6 weeks of culture on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BAP) along with 2.0 mg/l 1-napthaleneacetic acid (NAA). Shoot bud regeneration was achieved on MS medium supplemented with 2.0 mg/l BAP within 3–4 weeks of subculture. The number of shoots per culture varied from 1.12 to 8.75 in different growth media. The cultures incubated initially on dark photoperiod for 2 weeks and subsequently transferred to 16 h photoperiod showed higher number of shoot bud regeneration. The proliferated shoots were further sub-cultured on similar medium for higher rate of shoot bud regeneration. The elongated shoots were rooted on ½ strength MS medium fortified with 0.1–0.5 mg/l NAA or indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) with 2 % (w/v) sucrose within 2–3 weeks of culture. The higher percentage of rooting was obtained on 0.1–0.25 mg/l NAA as compared with IBA or IAA. The rooted plantlets were transferred to soil mixture (soil: sand: vermi-compost, 1:1:1 ratio) and kept in the greenhouse with 85 % humidity. The regenerated plantlets were successfully grown with 75 % survival rate. This protocol can be used for genetic improvement of black gram.

Free radical scavenging activity in in vitro-derived tissues of Eruca sativa

Feasible regeneration protocol for economically important plant Eruca sativa was established and 1, 1-diphenyl-2-picrylhydrazyl scavenging activity of regenerated tissues was evaluated and compared with plant material collected from the wild. Leaf portions inoculated onto Murashige and Skoog (MS) medium responded to all plant growth regulators exploited. Optimum callus production was achieved on a combination of 2.0 mg l(-1) 6-benzyladenine (BA) + 1.0 mg l(-1) α-naphthalene acetic acid (NAA) and the lowest response was recorded for 0.5 mg l(-1) gibberellic acid (GA3) + 1.0 mg l(-1) NAA. The callus was subcultured on similar composition/concentrations of plant growth regulators after 4 weeks of culture time. A 5.0 mg l(-1) 6-BA + 1.0 mg l(-1) NAA produced optimum percentage shoot organogenesis after 4 weeks of subculturing. However, optimum number of shoots per explant was recorded for moderate concentrations (1.0 and 2.0 mg l(-1)) of kinetin. Incorporation of NAA into MS medium-containing GA3 also produced a feasible number of shoots/explant. Similar mean shoot length was recorded for 2.0 mg l(-1) kinetin + 1.0 mg l(-1) NAA and optimum concentrations (2.0, 5.0, and 10.0 mg l(-1)) of GA3 + 1.0 mg l(-1) NAA. In vitro generated shoots were shifted to MS medium augmented with indole acetic acid (IAA) for rooting after 4 weeks of subculturing. Moderate concentrations (5.0 mg l(-1)) of IAA produced feasible rooting. Investigation of radical scavenging activity showed that callus possesses higher levels of radical scavengers than other plant tissues tested. Phenolics and glucosides are reported to be active components of Eruca sativa phytochemistry.

Spinal fusion using tissue engineered bone. A prospective, randomized clinical pilot trial

Spinal fusion is performed using bone harvested from the ilium. However, graft harvest is associated with frequent complications and pain. If a tissue engineered bone (TEB) was available, spinal fusion could be performed without damaging normal tissues. In 8 patients, 10-20 mL of marrow fluid was collected from the iliac crest to fabricate. After primary culture in the standard medium, marrow mesenchymal cells were combined with porous tricalcium phosphate block and were cultured in osteogenic medium containing dexamethasone, b-glycerophosphate, vitamin C phosphate, and estriol. After 3 weeks of subculture, spinal fusion was performed using TEB. Nine patients who had undergone spinal fusion using iliac autografts served as the controls (AG group). In all patients, significant improvement in JOA score was seen in both TEB and AG groups. The radiographic fusion rate was 87.5% (7/8) in TEB group and 77.8% (2/9) in AG group at 6 months after surgery. The mean operating time in TEB group was shorter than in the AG group. Compared with the AG group, the patients receiving TEB graft had significantly less total blood loss. In the AG group, all of the patients complained of graft site pain for 2 to 4 weeks after the operation. Two patients (22.2%) still had graft site pain at 6 months postoperatively. Bone regeneration therapy using the TEB graft introduced in this report makes it possible to perform spinal fusion as is done using autogenous bone grafts, but with the minimally invasive procedure of bone marrow aspiration.

IN VITRO CULTURE OF Pogostemon cablin BENTH. (NILAM PLANT): THE EFFECT OF NAA AND BAP ON EMBRYOGENIC CALLUS PROLIFERATION AND SUBSEQUENT SOMATIC EMBRYOGENESIS

An experiment to investigate the somatic embryogenesis from shoot-derived callus of Pogostemon cablin (nilam plant) has been conducted at the Plant Biotechnology Laboratory, Agricultural Faculty, University of Jambi from January through to July 2004. Callus proliferation was induced on explants taken from young shoots cultured on solid MS medium supplemented with phytohormones NAA (0.8, 1.1, 1.4, and 1.7 ppm) and BAP (1.1, 1.4, 1.7, and 2.0 ppm) under in vitro conditions. Cultures were maintained at 25 􀂱 1 oC, light intensity 50 􀁭mol m-2 s-1, and 16 hours photoperiod. The results indicated that all cultured explants showed positive responses on callus proliferation on all treatments within two weeks of culture initiation. The effect of phytohormones, however, was unspecific as all callus showed similar properties, from non-embryogenic to embryogenic. The addition of NAA and/or BAP to the culture medium was not significantly affected the number of days to callus proliferation. Callus fresh weight was significantly affected by NAA (P = 0.01) or BAP (P = 0.05), but the interaction of these phytohormones resulted in a non-significant effect on callus fresh weight (P = 0.18). Also, BAP significantly affected callus dry weight (P =0.03). However, neither NAA nor its interaction with BAP significantly affected callus dry weight (P = 0.07 and 0.16, subsequently). Embryogenic and non-embryogenic callus were subcultured separately onto new fresh media with the same composition as for callus induction. Following this subculture, embryogenic callus regenerated somatic embryos within ten days, whereas non-embryogenic callus did not show any symptom of embryogenesis, and lost their proliferative capacity after six weeks of subculture. The regenerated somatic embryos continued to grow to form profuse mass of young plantlets ready for in vivo acclimatization. Keywords: Pogostemon cablin, nilam, patchouli oil, tissue culture, in vitro culture, NAA, BAP

Gibberellic acid promotes in vitro regeneration and shoot multiplication in Lotus corniculatus L.

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA3)-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA3 for 3 weeks, and then returned to a hormone-free medium for a further 3–5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2–13 shoots during the first 3 weeks of subculture in GA3-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA3 was not the consequence of transformation events. The results point to a possible involvement of GA3 in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.

Mycobacterium sherrisii sp. nov., a slow-growing non-chromogenic species

'Mycobacterium sherrisii' is an undescribed species that appears to be emerging, in particular, among HIV-positive patients originating from Africa. To describe 'M. sherrisii', to ensure that the species name is validly published and to define its phylogenetic position, we collected 11 of these strains reported in five previous studies, and subjected them to biochemical identification, cell-wall mycolic acid analysis and sequencing of multiple housekeeping genes. The bacteria formed smooth and generally non-chromogenic colonies after 2-3 weeks of subculture at 24-37 °C; photochromogenic and scotochromogenic pigmentation were exhibited by three and two strains, respectively. The strains were positive for the heat-stable catalase test, but negative in tests for hydrolysis of Tween 80, nitrate reduction, β-glucosidase and 3-day arylsulfatase. Mycolic acid patterns, obtained by HPLC, resembled a trimodal profile similar to those of type strains of Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium triplex and Mycobacterium genavense. The 16S rRNA gene sequences of the 11 strains differed by 4 bp (99.7 % similarity) from that of the type strain of the closest related species, M. simiae ATCC 25275(T). Levels of internal transcribed spacer (ITS) and partial hsp65 and rpoB gene sequence similarity between the two taxa were 95.8 % (271/283 bp), 97.5 % (391/401 bp) and 95.2 % (700/735 bp), respectively. On the basis of these results, we propose the formal recognition of Mycobacterium sherrisii sp. nov. The type strain is 4773(T) ( = ATCC BAA-832(T) = DSM 45441(T)).