An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.

An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.     

Investigations on influencing plant-associated bacteria in tissue cultures of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.)

During tissue culture of black locust (Robinia pseudoacacia L.), serious problems with plant-associated bacteria led to a reduction of propagation potential in several clones. Four dominant strains of plant-associated bacteria could be isolated and were assigned to the genera Acidovorax, Dyella, Microbacterium and Sphingomonas. Out of five essential oils tested, thyme and lemongrass oil at a concentration of 0.03% each and 0.015% of both oils in combination clearly inhibited the growth of these bacteria strains on bacteriologic medium. There were no significant differences in total bacterial population density when penicillin, thyme and lemongrass oil or thyme plus lemongrass oil were added to the plant propagation media. The use of lemongrass oil changed the proportion of dominant bacterial strains.     

Plant development and synthesis of essential oils in micropropagated and mycorrhiza inoculated plants of Origanum vulgare L. ssp. hirtum (Link) Ietswaart

Biomass production of micropropagated oregano was induced by inoculation with the fungus Glomus viscosum. The effects of arbuscular mycorrhizal (AM) symbiosis on morphological and metabolic variations of regenerated oregano plants were investigated at different growth stages. AM greatly increased parameters such as plant leaf area, fresh and dry weight, number of spicasters and verticillasters in infected plants. An increase of the gland density, especially on the upper leaf epidermis, was also observed following the physiological ageing of the tissues. The in vitro plants of O. vulgare ssp. hirtum described in this study provided a qualitatively and quantitatively good source of essential oils that have a chemical profile comparable to that of the control mother plants with carvacrol as the main compound.     

Efficiency of neural networks for prediction of in vitro culture conditions and inoculum properties for optimum productivity

This study represents an ANN based computational scheming of physical, chemical and biological parameters at flask level for mass multiplication of plants through micropropagation using bioreactors of larger volumes. The optimal culture environment at small scale for Glycyrrhiza plant was predicted by using neural network approach in terms of pH and volume of growth medium per culture flask, incubation room temperature and month of inoculation along with inoculum properties in terms of inoculum size, fresh weight and number of explant per flask. This kind of study could be a model system in commercial propagation of various economically important plants in bioreactors using tissue culture technique. In present course of study the ANN was trained by implementing MATLAB neural network. A feed-forward back propagation type network was created for input vector (seven input elements), with single hidden layer (seven nodes) and one output unit in output layer. The ‘tansig’ and ‘purelin’ transfer functions were adapted for hidden and output layers respectively. The four training functions viz. traingda, trainrp, traincgf, traincgb were randomly selected to train four networks which further examined with available dataset. The efficiency of neural networks was concluded by the comparison of results obtained from this study with that of empirical data obtained from the detailed tissue culture experiments and designated as Target set (mean fresh weight biomass per culture flask after 40 days of in vitro culture duration). Efficiency of networks for better training initialization was judged on the basis of comparative analysis of ‘Mean Square Error at zero epoch’ for each network trained in which the least error at initial point was observed with trainrp followed by traincgb and traincgf. A comparative assessment between experimental target data range obtained from wet lab practice and all trained network output range for the efficiency of trained networks for least deviation from target range revealed the output range of network ‘trainrp’ was closest to the empirical target range while least comparison was worked out from network ‘traincgb’ which had output range more than the target decided and ultimately showed meaningless result.     

Ethylene and rooting of mung bean cuttings. The role of auxin induced ethylene synthesis and phase-dependent effects

We have re-examined the role of ethylene during rooting of mung bean cuttings. Cuttings were treated for 5 days with a low or a high concentration of NAA (naphthaleneacetic acid). During this 5 days period, we also applied STS (silverthiosulfate, an inhibitor of ethylene action) or ACC (1-aminocyclo-propane-l-carboxylic acid, a direct precursor of ethylene). At high NAA concentration, STS promoted and ACC inhibited rooting, respectively. At low NAA concentration, the effects were opposite, STS being inhibitory and ACC promotive. AVG (aminoethoxyvinylglycine, an inhibitor of ethylene synthesis) gave similar results as STS. Together, these data suggest supraoptimal and suboptimal ethylene levels in the tissue at high and low NAA concentration, respectively. We also examined whether the effect of ethylene varied during the successive phases of the rooting process. Thus, we gave 24 h pulses with either STS or ACC during the rooting treatment. During the first two days (0–48 h), ACC-pulses were promotive and STS-pulses inhibitory. Later on (48–168 h), the ACC-pulses were inhibitory and the STS-pulses promotive. Whether this effect was observed or not was dependent on the NAA concentration. These data indicate that ethylene promotes or inhibits rooting depending on the stage in the rooting process. When ACC was added only during the initial period, rooting was increased at all NAA concentrations in a NAA dose-response curve and the optimal NAA concentration remained the same. This suggests that ethylene renders more cells responsive to NAA.     

Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap (Dionaea muscipula)

In vitro culture of Venus fly-trap (Dionaea muscipula) was initiated using flower stalk explants. Activated charcoal was required for bud initiation, but omitted in the subculture of regenerated plantlets. Regenerated plants were subsequently used as explant source for investigations concerning effects of source of tissue, etiolation, orientation and illumination of leaf explants on plant regeneration. Etiolation of source plantlets increased the rate of regeneration from explants and decreased explant failure. Generally, adventitious buds developed at the adaxial side and proximal end of an explant. However, when explants were incubated in the dark, 20–30% of bud initiation occurred at the distal end. The site of shoot regeneration on a leaf explant was affected by both illumination and orientation of explants. Placing an explant adaxial side up resulted in the highest rate of regeneration. The most effective condition for plantlet regeneration was found with etiolated petioles incubated with the adaxial side facing the light. Received: 18 March 1998 / Revision received: 12 August 1998 / Accepted: 7 September 1998     

In nature dormant buds and in vitro dormant-like buds ofFraxinus excelsior L.

Summary Microcuttings ofFraxinus excelsior, sampled from adult trees during the period of cell cycle blockage of bud in G_0–1, developed long rejuvenated sprouts on the Woody Plant Medium (WPM) with benzylaminopurine (BAP) (4.0 mg/l) and indolebutyric acid (IBA) (0.03 mg/l). These sprouts had the ability to enter a resting period, building dormant-like buds when maintained on the original WPM. Sprouts developed from subcultures also entered a resting period without any transfer. Comparison of in nature buds in active growth and dormancy with buds of growing sprouts and in vitro dormant-like buds revealed similarity in behaviour at the shoot apical level. In particular, in dormant-like buds in a constant environment, shoot apical functioning was suppressed while the cell cycle of the shoot apex was blocked at the G_0–1 phase, like in nature dormant buds.Abbreviations a.u. arbitrary unit - BAP benzylaminopurine - CF cumulative frequency - d₁, d₂ diameters of the shoot apex - IBA indolebutyric acid - P_n last opposite primordia of range n - WPM woody plant medium     

RAPD,ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.- an important endangered orchid

Dendrobium fimbriatum is an ornamental and medicinal orchid listed in the Red data book of IUCN. Phytohormones’ effect on the in vitro regeneration of the orchid was studied using Mitra medium supplemented with different growth regulators. KN produced effective shoot formation when present alone or in combination with IBA or NAA. The shooting was gradually increased when KN concentration was increased from 0.8 to 4.8 mg L⁻¹, but the opposite response was observed with BAP at higher concentration (4.8 mg L⁻¹). IBA either in combination with BAP or KN promoted effective root development and multiplication. Micropropagated orchids grown in the basal medium devoid of any phytohormone showed 100% monomorphism, while low genetic polymorphism of 1.52% (RAPD—Random Amplification of Polymorphic DNA), 1.19% (ISSR-Inter Simple Sequence Repeat) and 3.97% (SCoT—Start Codon Targeted) was exhibited among the regenerants propagated in the hormone enriched medium. UPGMA (Unweighted pair group method using arithmetic averages) dendrograms showed the grouping of mother plant (MP) with the in vitro regenerants. The principal coordinate analysis (PCoA) further confirmed the clustering patterns as determined by the cluster analysis. The study reported for the first time the successful in vitro propagation of Dendrobium fimbriatum and their genetic stability assessment using molecular markers.     

In vitro propagation of <Emphasis Type="Italic">Huernia hystrix</Emphasis>: an endangered medicinal and ornamental succulent

An efficient and rapid method for in vitro clonal propagation of Huernia hystrix was developed, resulting in shoot regeneration within 3 weeks of culture. This endangered medicinal and ornamental succulent is in high demand. Multiple shoots were regenerated from stem explants (10 mm length) cultured on Murashige and Skoog (MS) medium containing 3% sucrose and supplemented with a range of NAA (0.00–8.06 μM) and BA (4.44–22.19 μM) concentrations. A 100% shoot response with a multiplication rate of four shoots per explant was obtained on MS medium containing 5.37 μM NAA and 22.19 μM BA. Callus produced at the base of the explant on the same medium showed root organogenic potential. The in vitro regenerated shoots produced roots when transferred to half strength MS medium with or without auxin. The micropropagated plants were easily acclimatized within 2 months under greenhouse conditions when potted in a soil and sand mixture (1:1; v/v) treated with a fungicide (Benlate, 0.01%). More than 95% survival with no observable morphological variations was obtained. The developed protocol provides a simple, cost-effective means for the conservation of endangered H. hystrix by clonal propagation within a short time.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.