High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.     

High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell layer method

A method for a high frequency and direct in vitro bud regeneration of a woody species, the trifoliate orange (Poncirus trifoliata L. Raf), was designed. Transverse thin cell layer (tTCL) explants excised from the stem internodes of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 μM) and N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1–10 μM). The optimal concentrations for bud induction were 25 μM BAP and 1 μM TDZ leading to 87 and 72 % of responsive tTCLs and 24 and 15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher frequency of bud regeneration were obtained with BAP and TDZ combined. With a combination of 10 μM BAP and 1 μM TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium containing 1 μM GA₃. Rooting of individual shoot was induced using 5 μM NAA. One hundred per cent of rooted shoots developed normally after transfer to the greenhouse; no phenotype variation was observed. High numbers of regenerated viable plants can be produced directly without callus formation from tTCL after 9 weeks of culture.     

Rapid Multiplication and Induction of Early In Vitro Flowering in Dendrobium Primulinum Lindl

Dendrobium primulinum is an important epiphytic orchid. A successful protocol for mass multiplication and early in vitro flowering was developed. Immature embryos of 4 week after pollination exhibited about 96% germination within 30 days of culture on MS medium containing sucrose (3%) (w/v), NAA and BA (6 and 9 μM) in combination. Protocorm-like bodies (PLBs) formed from the germinating seeds on the germination medium. Rooted plantlets were formed within 2-3 wk on MS medium containing sucrose (3%), NAA and BA (3 and 12 μM in combination) where about 29 shoot/buds produced per cycle of 4 wk interval. The well rooted plantlets produced 4-5 floral buds per spike when they were maintained on MS medium containing sucrose (3%), fresh apple juice (10%) (v/v) for four wk followed by on MS medium freed of apple juice but enriched with NAA and BA (3 and 12 μM respectively). The hardened plantlets were transferred to community potting mix where the about 80% transplants survived after two months of transfer.     

High-frequency shoot regeneration through transverse thin cell layer culture in <Emphasis Type="Italic">Dendrobium Candidum</Emphasis> Wall Ex Lindl.

An efficient in vitro propagation protocol for Dendrobium candidum Wall ex Lindl. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration and the number of adventitious buds produced from the regenerated shoots significantly relied on the concentration of plant growth regulators, and the position and orientation of the explant. Murashige and Skoog (MS) medium with half-strength macronutrients and 2% sucrose, supplemented with 1.2 mg l⁻¹ naphthaleneacetic acid (NAA) and 1.2 mg l⁻¹ 6-benzyladenine (6-BA), was optimal for shoot regeneration. Upon this medium, the youngest explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (92%), and the highest number of adventitious buds (an average of 24.5) per explant. Rooting of shoots and adventitious buds was achieved on MS medium with half-strength macronutrients and 2% sucrose with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ indole-3-acetic acid (IAA). Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Ontogenetic studies revealed that the shoots originated from the stem vascular bundles.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

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

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

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.     

正常人卵巢绒毛膜促性腺激素(hCG)受体的研究

23例正常卵巢制备之细胞膜,可与绒毛膜促性腺激素(hCG)发生特异性结合,最大结合率为13.2±2.24％,Scatchard作图分析,得一直线。23例正常卵巢之受体量为O.66±0.142×10~(-10)M/μg膜蛋白,Kd值为10.16±5.5×10~(-9)M。     

Sex expression in monoecious cucumbers micropropagated in vitro

The effects of plant growth regulators (PRGs) on the induction of flowering and sex expression in micropropagated cucumbers are presented. The highest number of male flowers (6.0 ± 0.7 per plant) was produced by cv. Kmicic F1 on the Murashige and Skoog (MS) medium supplemented with 4.0 μM kinetin. The highest number of female flowers (3.1 ± 0.3) was also observed in cv. Kmicic F1 on either control (PRG-free) medium or medium supplemented with 6.4 μM indole-3-acetic acid (IAA). The MS medium supplemented with 4.4 μM benzyladenine inhibited flower formation. The highest percentage of flowering plantlets (67.5 ± 7.5) was observed on the control MS medium after 16 weeks of culture. Female-to-male flower ratio was influenced by the culture media and changed during cultivation. The highest pollen viability (60–70 %) was observed in anthers of plants cultured on the control medium and the medium with IAA.     

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

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

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

The effect of sugars on niger embryogenesis and plant regeneration in anther culture

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05–0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg’s B5 medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 μM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 μM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 μM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.     

Sucrose effects on in vitro fruiting and seed production of Centaurium pulchellum

The effect of sucrose on fruiting, seed production, and seed germination of lesser centaury [Centaurium pulchellum (Sw.) Druce] was examined using explants of flowers and flower buds. Sucrose concentrations in the culture medium ranged from 0.003 to 0.3 M. It has been shown that the number of auxiliary buds, capsules dimension, number of viable seeds per capsule and seed dimensions increased with the increase of sucrose concentrations. The highest values were recorded at sucrose concentrations higher than 0.03 M, except for seeds size, which were larger at sucrose concentration ranging from 0.003 to 0.1 M. The germination of in vitro produced seeds was affected by previous culture history: a higher germination percentage was obtained in seeds that were raised from explants originally grown on medium with sucrose concentrations higher than 0.003 M.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

In vitro shoot proliferation of 5-leaf aralia explants from field grown plants and forced dormant stems

A. Sieboldianus (5-leaf aralia) is recalcitrant for micropropagation, but has very good landscaping potential. This research was conducted with the following objectives: (1) to study effects of BA, TDZ, CPPU, 2iP, kinetin and zeatin in woody plant medium on the performance of softwood shoot nodal explants produced by field grown 5-leaf aralia plants; (2) to investigate influences of BA or TDZ in the forcing solution on subsequentin vitro shoot initiation of nodal explants taken from forced softwood growth. Shoot initiation of softwood nodal explants from field-grown plants was promoted by adding BA, TDZ or CPPU to the culture medium. Kinetin, zeatin and 2iP were ineffective for micropropagation ofA. Sieboldianus. The forced softwood growth for use as explants was “primed” by forcing dormant stems in solution containing 200 mg 8-HQC per liter plus 2% sucrose, 44.4, 222, or 444 μM BA, or 45.4, 227, or 454 μM TDZ. BA and TDZ in the forcing solution enhanced subsequentin vitro axillary shoot initiation of nodal explants taken from forced stems by doubling the number of shoots produced per explant to 3.3 from 1.65 shoots per explant taken from field grown plants. This forcing solution technique also reduced the time needed from culture initiation to potted plants to half of the time needed for the conventional micropropagation method (12 to 14 vs. 25 to 27 weeks), thus expediting the micropropagation ofA. Sieboldianus.     

In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential

Present study describes rapid in vitro propagation of Caralluma tuberculata, a traditional medicinal plant, and antioxidant potential of calli and plants extracts. The highest callus induction rate (93.3%) with maximum weight of calli 5.2 g was achieved from shoot tip explants on MS medium supplemented with 9.04 μM 2,4-D and 4.44 μM BA. The maximum shoot induction rate (71.1%) with mean number of shoots 3.66 ± 1.53 and 4.6 cm average shoot length was observed on 13.32 μM BA, 4.52 μM 2,4-D and 2.89 μM GA3 appended in MS medium. The developed shoots were best rooted in the presence of 5.07 μM IAA with 3.0 ± 0.15 roots per plantlet. The plants were successfully acclimatized under in vivo conditions. The plants and calli extracts exhibited good antioxidant activities, however, plant extract activities were more pronounced. The phenolic compounds in plant and calli extracts were 0.16% and 0.057%, respectively. While the flavonoids were 0.092% in plant and 0.039% in calli extract. Total Phenolics, flavonoids; DPPH radical scavenging activity and reducing power potential distributed among different fractions depending upon polarity of the solvent. The highest DPPH scavenging activity and reducing power was exhibited by water fractions; 4.95 mg/mL and 0.729 OD at 10 mg/mL, respectively. The micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant. Furthermore, antioxidant value describes importance of this valuable plant as food and medicine.     

Effects of thidiazuron and N6-benzylaminopurine on shoot regeneration of Phalaenopsis

Adventitious bud formation from the vegetative buds of the flower stalks of Phalaenopsis occurred on Vacin and Went medium with 15% coconut water and 5 to 40 μM thidiazuron (TDZ) or 40 μM N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 μM TDZ. Adventitious buds developed into shoots on VWC medium. TDZ was more effective than BAP in stimulating the axillary buds of intact shoots to develop. Regenerated shoots rooted after about two months of culture on VWC medium with 1% sucrose. Shoot tips excised from the regenerated shoots initiated protocorm-like bodies after two months of culture on VWC medium.     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.