Rapid in vitro propagation of Eclipta alba (L.) Hassk. through high frequency axillary shoot proliferation

An efficient protocol has been developed for rapid in vitro propagation of Eclipta alba L. (Asteraceae) through axillary bud multiplication. Murashige and Skoog (MS) medium supplemented with BA (10 M) was found to be most effective in breaking bud dormancy. An average number of 23 ± 0.57 shoots per explant was recorded after 30 days. Culture of node segments on fresh medium with lower concentration of BA (2 M) enhanced the multiplication rate. A maximum of 79 ± 1.90 mean number of shoots were obtained after three subcultures without any decline in multiplication rate. The regenerated microshoots showed the most efficient rooting on half strength MS medium augmented with 0.5 M IBA. Plantlets went through a hardening phase prior to ex vitro transfer and established in earthen pots containing garden soil; survival of about 90%. The established plants were uniform and exhibited morphological characters identical to mother plants.     

Micropropagation of Eclipta alba and Eupatorium adenophorum using a single-step nodal cutting technique

Protocols for the micropropagation of two traditional medicinal plants Eclipta alba (L.) and Eupatorium adenophorum (L.) from nodal segments were developed. Proliferated microshoots of Eclipta alba and Eupatorium adenophorum were obtained through axillary branching by culturing nodal segments in modified MS medium and half strength of MS, respectively, with minimal strength of nutritional support. Simultaneous rooting could also be induced in the same medium. Regenerated rooted plantlets were successfully acclimatized in soil where they grew normally without showing any morphological variation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Reducing bushiness in micropropagated Gerbera

Bushiness in Gerbera is a major problem causing more than 30% loss of production during micropropagation and greenhouse production. We studied two Gerbera cultivars `Rosabella' (bushy) and `Sunset' (non-bushy). Three different types of inocula (main, adventitious and axillary shoots) were investigated. The choice of the inoculum had an important influence on bushiness, the main axis being the most sensitive. Higher KIN concentrations enhanced bushiness. Addition to the fungicide imazalil to the propagation medium reduced bushiness. The appropriate concentration of imazalil depended on the degree of bushiness and the inoculum type.     

One-step acclimatization of plantlets using a mist reactor

A mist reactor was used to grow and acclimatize carnation plants in vitro without using ex vitro acclimatization techniques. The acclimatization protocol in the reactor consisted of altering the mist-on period during the course of the culture period and a stepwise reduction in the relative humidity surrounding the plants from 98% to 70% relative humidity (RH) during the final week of in vitro growth. After transfer and further growth in a greenhouse for 5 weeks, survival was 91% for plants grown in reactors, 81% from vented boxes, and 50% from unvented boxes. Ex vitro survival directly correlated with increased in vitro rooting and decreased hyperhydration. In vitro rooting also correlated with high-quality plants, but did not significantly correlate with low hyperhydration, as normal plants often lacked roots. After 5 weeks in the greenhouse, the quantity of mid- and high-quality plants obtained from reactors and ventilated boxes was similar. Conditions in the mist reactor can be manipulated to produce plants that are readily acclimatized and are equal or better in quality and yield than plants produced using conventional methods.     

Arbuscular mycorrhizal fungi influence water relations, gas exchange, abscisic acid and growth of micropropagated chile ancho pepper (Capsicum annuum) plantlets during acclimatization and post-acclimatization

Little is known about the role of arbuscular mycorrhiza fungi (AMF) on physiological changes of micropropagated plantlets during acclimatization and post-acclimatization. Using chile ancho pepper (Capsicum annuum L. cv. San Luis), measurements were made of water relations, gas exchange, abscisic acid (ABA), plantlet growth and AMF development. Plantlets had low photosynthetic rates (A) and poor initial growth during acclimatization. Relative water content (RWC) decreased during the first days after transfer from tissue culture containers to ex vitro conditions. Consequently, transpiration rates (E) and stomatal conductance (gs) declined, confirming that in vitro formed stomata were functional and able to respond ex vitro to partial desiccation--thus avoiding excessive leaf dehydration and plant death. Colonization by AMF occurred within 3 days after inoculation. Colonized plantlets had lower leaf ABA and higher RWC than noncolonized (NonAMF) plantlets during peak plant dehydration (6 days after plant transfer)--and a higher A and gs as early as days 5 and 7. During post-acclimatization [after day 8, when RWC increased and stabilized], A increased in all plantlets; however, more dramatic changes occurred with AMF plantlets. Within 48 days, 45% of the roots sampled of inoculated plantlets were colonized and had extensive arbuscule development. At this time, AMF plantlets also had greater E, A, leaf chlorophyll, leaf elemental N, P and K, leaf dry biomass and leaf area, fruit production and differences in carbon partitioning [lower root/shoot ratio and higher leaf area ratio] compared with NonAMF plantlets. Rapid AMF colonization enhanced physiological adjustments, which helped plantlets recover rapidly during acclimatization and obtain greater growth during post-acclimatization.     

Optimization of wedelolactone accumulation in shoot cultures of Eclipta alba

Influence of different environmental factors on accumulation of wedelolactone, a potential anti-hepatotoxic principle of E. alba in shoot cultures was investigated. A significant increase in the content of wedelolactone due to kinetin treatment, temperature change and photoperiod alteration in shoot cultures was observed. Incorporation of phenylalanine in the medium also increased content of wedelolactone significantly in a dose-dependent manner.     

Field performance of micropropagated forestry species

Summary Micropropagated plants both from angiosperms (bamboo, birch, eucalyptus, tamarind, teak, willow) and gymnosperms (Douglas-fir, loblolly pine, Monterey pine, and redwood) have been established in the field. Plantlets were regenerated from juvenile explants (via adventitious or axillary buds) as well as explants from mature trees [apical and axillary buds (nodal segments)]. Plantlets regenerated from adventitious buds tend to show early maturation traits (Douglas fir, loblolly pine, Monterey pine). A population of selected clones showed superior performance and yield over seedlings derived from the same trees. Increased biomass production was obtained with plantlets derived from tissue culture ofEucalyptus spp. when compared to seedlings. No morphologic variation was observed in micropropagated plants. Plantlets derived from tissue culture grew very uniformly. Early flowering was observed with plantlets derived from tissue culture (tamarind, teak). Based on a presentation at an International Training Course on the Application of Biotechnology in Forest Trees held in Caracas, Venezuela, May 1991.     

Cold-induced genetic instability in micropropagated Pistacia lentiscus L. plantlets

Genetic stability of plants during in vitro propagation and conservation is one of the important aspects of plant biotechnology. In the present study, micropropagated P. lentiscus L. shoot cultures, which are cultivated for the mastic resin, have been cold stored up to 12 months at 4 °C in the dark for different durations (2, 4, 6, 8, 10 and 12 months) and genetic alterations in cold storage conditions were evaluated. Growth parameters such as proliferation rate, shoot numbers per explant, shoot lengths and shoot forming capacity were also calculated. Since the highest proliferation rate (100 %) was obtained in 6?month-stored shoot cultures without any severe influence of cold stress on proliferation ability, amplified fragment length polymorphism (AFLP) and inter-retrotransposon amplified polymorphism (IRAP) marker systems were used to determine genetic stability in the plantlets after this storage period. Totally, 702 scorable bands were produced by 10 AFLP primer pairs. Genetic similarity value of the non-stored (control) plant and cold-stored clones ranged from 0.66 to 0.84 with a mean of 0.74. In the case of IRAP, 159 bands were produced by 8 IRAP primers. Genetic similarity value of the non-stored plant and cold-stored clones varied from 0.65 to 0.83 and the average genetic similarity value was determined as 0.72. The genetic similarity indices revealed that genetic variability was similar in both techniques. Our results showed that tissue culture and especially cold storage of P. lentiscus L. may result transposons activation, thus could cause genetic instability.     

Regeneration of plantlets from hypocotyl-derived callus of Coptis teeta

Callus cultures of Coptis teeta were established from hypocotyl segments (excised from aseptically germinating seeds) on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Microshoots were produced within 6–7 weeks of subculturing this callus in 1/2 MS nutrient medium supplemented with kinetin alone. Excised microshoots were rooted in 1/2 MS nutrient medium containing indolebutyric acid (IBA). The complete plantlets were hardened and established.     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Application of rapd in the determination of genetic fidelity in micropropagated Drosera plantlets

Summary Random amplified polymorphic DNA (RAPD) markers were used to verify the clonal fidelity of two micropropagated Drosera species, D. anglica and D. binata, which were regenerated by adventitious budding from leaf explants and shoot tips, respectively. Twenty arbitrary decamers were used to screen 15 randomly selected plantlets of each species. No genetic variation was detected among D. binata regenerants, whereas a 0.08% polymorphism frequency was estimated for D. anglica plantlets. These results indicate that the regeneration of plants through shoot-tip culture is a low-risk method for generating genetic variability, whereas material regenerated through leaf explants requires further verification.     

Field performance and biochemical evaluation of micropropagated mulberry plants

Microclones of different mulberry genotypes were successfully transferred to the field. The same genotypes were raised through conventional methods (cuttings). A comparative study using morphological and biochemical tests of field established micropropagated and cutting derived plants of mulberry genotypes was conducted. Micropropagated mulberry plants showed significant morphogenic vigour when compared to plants raised through cuttings. Biochemical tests of leaves revealed that, there was no significant nutritional difference between micropropagated plants and those originated from cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

Control of hyperhydricity in micropropagated apricot cultivars

Summary The effects of different factors on the control and reversion of hyperhydricity during the in vitro propagation of Prunus armeniaca were studied. Treatments that decreased the hyperhydricity but did not affect micropropagation rates were the use of the bottom cooling system for 1 or 2 wk and agargel as gelling agent in ‘Helena’, whereas the best results were obtained with the bottom cooling system for 2 wk and the use of 0.8% agar as gelling agent in ‘Lorna’. Hyperhydric shoots reverted to normal after keeping them for 3 wk in the bottom cooling system.     

Physiology of effects of temporary immersion bioreactors on micropropagated pineapple plantlets

Summary Temporary immersion bioreactors are an efficient tool for plant mass propagation because they increase multiplication rate and plant quality. Little knowledge is available on the ecosystem and physiological behavior of plantlets when using this new culture technique. In order to evaluate the effects of the conditions on physiological change of pineapple plantlets, a factorial experiment was conducted, where axillary clusters were cultured under two levels of photosynthetic photon flux (PPF): 30 μmol m⁻²s⁻¹ (low) and 225 μmol m⁻²s⁻¹ (high), using two culture methods (conventional micropropagation in liquid medium and a temporary immersion bioreactor) during the elongation phase. CO₂ concentration in the headspace volume container was measured during a whole cycle of temporary immersion (3h). At the time before the next immersion period, the levels of CO₂ increased significantly to 14171 μmol mol⁻¹ at high PPF. The maximal photosynthetic rate as well as the maximum quantum yield of photosystem II were low for plantlets cultivated in the femporary immersion bioreactor at high PPF. However, these plantlets showed large increases in sugar and nitrogen uptake and also increases in dry weight and foliar area. These results indicate that shoot growth did not totally depend on the photosynthesis process. In vitro pineapple plantlets appeared to use more nutrients in the culture medium than those from photosynthesis. In summary, temporary immersion bioreactor-derived plantlets showed remarkable nutrient uptake, indicating a higher photo-mixotrophic metabolism.     

Antihyperglycemic activity of Eclipta alba leaf on alloxan-induced diabetic rats

Eclipta alba, an indigenous medicinal plant, has a folk (Siddha and Ayurvedha) reputation in rural southern India as a hypoglycemic agent. In order to confirm this claim, the present study was carried out to evaluate the antihyperglycemic effect of E. alba and to study the activities of liver hexokinase and gluconeogenic enzymes such as glucose-6-phosphatase and fructose 1,6-bisphosphatase in the liver of control and alloxan-diabetic rats. Oral administration of leaf suspension of E. alba (2 and 4 g/kg body weight) for 60 days resulted in significant reduction in blood glucose (from 372.0 +/- 33.2 to 117.0 +/- 22.8), glycosylated hemoglobin HbA(1)c, a decrease in the activities of glucose-6 phosphatase and fructose 1,6-bisphosphatase, and an increase in the activity of liver hexokinase. E. alba at dose of 2 g/kg body weight exhibited better sugar reduction than 4 g/kg body weight. Thus, the present study clearly shows that the oral administration of E. alba possess potent antihyperglycemic activity.     

Vesicular arbuscular mycorrhizal fungi improves establishment of micropropagated Leucaena leucocephala plantlets

Investigations were carried out to achieve cent per cent transplantation success of micropropagated Leucaena leucocephala (a fast growing multipurpose leguminous tree species) plantlets using two vesicular arbuscular mycorrhizal fungi, Glomus fasciculatum and Glomus macrocarpum. Plantlets were obtained by rooting the shoots [obtained through; hypocotyl callus in presence of 10^-5M BAP + 10^-6M NAA; and axillary bud sprouting from cotyledonary and other nodes in presence of 10^-5M BAP, on Gamborg's B5 medium], on half strength B5 medium supplemented with 5×10^-6M IBA. Subsequent to the nodulation of their roots with Rhizobium (strain PRGL 001)in soilrite, these plantlets were tranferred to sterilized garden soil by laying inoculum of either Glomus fasciculatum or Glomus macrocarpum around their roots. Only 20% of the plantlets survived in soils lacking VAM fungus. In contrast, cent per cent of the plantlets of Leucaena leucocephala established very well and showed good growth in VAM inoculated soil. Roots of the later plantlets showed presence of both external and internal hyphae with well formed arbuscules and vesicles confirming the establishment of good mycorrhizal association. These studies convincingly demonstrate that the mycorrhizal association help in successful establishment of tissue culture raised plantlets of Leucaena leucocephala in the field conditions by alleviating the transplantation shock. This revised version was published online in June 2006 with corrections to the Cover Date.     

The transmission of banana bunchy-top virus in micropropagated bananas

Plants were established in vitro from banana bunchy0top virus (BBTV) infeeted plants. Explants containing either vegetative shoot apices or terminal floral apices were used to initiate cultures. Plants multiplied in culture were indistinguishable from non-infected (control) plants and lacked characteristic symptoms of BBTV infection. After 16 months in culture plants were established in the glasshouse and after 1 month in pots some plants started to show symptoms of the disease. After a further 5 months, 73% of the plants showed characteristic symptoms of the disease while 27% were symptomless and similar in appearance to control plants. These plants have been grown to maturity in the field without showing recognizable symptoms. This study demonstrates that BBTV can be transmitted in an apparently symptomless condition in culture and has important consequences for the dissemination of banana germplasm within Australia and internationally.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Effect of combinations of antibiotics on micropropagated Clematis,Delphinium, Hosta,Iris and Photinia

The phytotoxic effects of antibiotic treatment on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia were determined by assessing multiplication and rooting rates of plants in vitro and weaning success and flowering in vivo.Abbreviations BA 6-benzyladenine - IAA 3-indole-acetic acid - NAA 1-naphthyl-acetic acid     

ISSR marker based analysis of micropropagated plantlets of Nothapodytes foetida

A DNA-based, inter simple sequence repeat (ISSR) markers were used to monitor genetic stability in micropropagated plantlets of Nothapodytes foetida. A total of 146 clear and distinct bands were produced using 26 primers resulting in 3 212 fragments. Out of 146, 135 bands (92.4 %) were monomorphic and 11 bands (7.53 %) were polymorphic which ranged from 200 to 21 226 bp in size. The number of bands per each primer varied from 1 to 11 with an average of 5.6 bands per primer. The banding pattern for each primer was uniform and comparable to mother plant from which the cultures had been established. The dendrogram based on the unweighted pair-group method with arithmetic averaging (UPGMA) depicted about 97 % homology between the mother plant and micropropagated plants. An attempt was made to reintroduce the micropropagated plants in the natural habitat and over 500 plants were successfully established.