In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill

Aloe species are highly-prized for their ornamental value and have been utilized for centuries in traditional medicine. Due to their habitat loss and exploitation for medicinal and ornamental plant trade, many species in this genus have become threatened. One of the most important globally rated medicinal species in Aloe genus is A. arborescens. The current study evaluated the roles of different aromatic cytokinin types and concentrations on direct organogenesis, in vitro bioactive secondary metabolite production and antioxidant activity of regenerated shoots of A. arborescens. There was an increase in the number of adventitious shoots produced per explant with an increase in concentration in cultures treated with meta-topolin (mT), meta-topolin riboside (mTR), meta-methoxytopolin (MemT) and benzyladenine riboside (BAR), reaching an optimum at either 5.0 or 7.5???M. Overall, the treatment with 5.0???M mT gave the largest number of transplantable shoots (regenerated shoots with length greater than 10?mm). Rooted shoots were successfully acclimatized after 8?weeks with a survival frequency above 90?% and no observable morphological abnormalities. Variable amounts of total iridoids, phenolics, flavonoids and condensed tannins were detected in regenerated shoots from all the cytokinin treatments. An increased free-radical scavenging activity with an increase in concentration was recorded in regenerated shoots from mT and mTR treatments, reaching an optimum at 7.5???M concentration. The present study shows that the choice of cytokinin type and concentration exogenously supplied during tissue culture markedly influences not only shoot proliferation but also the in vitro production of bioactive secondary metabolites.     

Responses of the photosynthetic system and peroxidase activity to the rooting conditions of oak micropropagation

The effects of indole butyric acid (IBA) concentration and the duration of the plant growth regulator (PGR) treatment on the rooting ability, peroxidase level and photosynthetic activity of young Quercus robur L. plants were studied. Four-week-old oak shoots were transplanted to rooting media supplemented with 10 or 20 μM IBA. After 2, 3, 4, 5 or 7 days the shoots were transplanted again to a fresh, PGR-free medium. On the tenth day after transfer to rooting medium, the CO₂ fixation capacity, pigment content and peroxidase activity were measured. The photosynthetic parameters varied as a function of the time spent on medium containing PGR, showing maximum values in plants transplanted on the third to fourth day to PGR-free medium. The rooting percentage of these plants reached its maximum within two weeks. However, peroxidase activity was the highest in plants transferred later to PGR-free medium. The most pronounced stimulating effect on rooting was achieved with the higher initial IBA concentration followed by a transfer to PGR-free medium on the third to fourth day. These plants showed the highest vitality and the best rooting ability. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of <Emphasis Type="Italic">meta</Emphasis>-topolins in alleviating micropropagation problems

Low shoot multiplication, morphological abnormalities, poor rooting frequency and high cost of production are among the factors challenging the micropropagation of ornamental perennials and garden plants. Most of these problems can be alleviated by using the appropriate type and concentration of plant growth regulator(s) (especially cytokinins) in developing efficient micropropagation protocols. In this study, we investigated the effects of five different aromatic cytokinins (BA, Kin, mT, mTR and MemTR) on adventitious shoot production from shoot-tip explants of B. greenii, a critically endangered plant with horticultural potential. Of all the cytokinin concentrations evaluated, the highest adventitious shoot production (5.88 ± 0.73 shoots/explant) was observed in cultures containing 7 μM MemTR. Low adventitious shoot production, which was not significantly different from that of the control, was observed at all the concentrations of kinetin (Kin), suggesting that it is a weak cytokinin for shoot production in this species. All the treatments with BA alone showed higher adventitious shoot production when compared to the BA treatments supplemented with NAA concentrations. At equimolar concentrations, however, all the BA concentrations had a higher abnormality index than the other cytokinins. It is noteworthy that the abnormality index in all the topolin treatments was much lower than that recorded at the lowest BA concentration. Almost all the abnormality indices recorded with mTR and MemTR concentrations were lower than that of the control. Given that the explants used were from BA-containing cultures, it is likely that the abnormalities recorded using mTR and MemTR were carry-over effects of BA. Culturing under 16 h light/8 h dark conditions resulted in a higher production of adventitious shoots with lengths greater than 10 mm compared to culturing under continuous light. This measure could help reduce the cost of production. Regenerated shoots were successfully rooted and acclimatized with a 65% survival frequency and no observable morphological variation. The developed micropropagation protocol has the potential for producing more than 60,000 transplantable shoots per year from a single shoot-tip explant of this critically endangered species.     

Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana

The effects of five topolins (meta-Topolin, mT; meta-Topolin riboside, mTR; meta-Methoxy topolin, MemT; meta-Methoxy topolin riboside, MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl, MemTTHP) on the phenolic content and subsequent acclimatization potential of micropropagated ??Williams?? bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30 ??M of the above aromatic cytokinins (CKs) for 42?days. Phenolic contents were quantified spectrophotometrically. Growth parameters and photosynthetic pigments of the greenhouse-acclimatized plants were determined after 5?months. Total phenolic levels were highest in 10 ??M mT-treated plantlets within the aerial parts and 30 ??M MemTTHP for the underground parts. In the underground parts, 10 ??M mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. Furthermore, 10 ??M MemTTHP-treated plantlets had significantly higher total flavonoids (30.1?±?0.24 mg CE/g DW) within the aerial parts. Plantlets regenerated using MemT, MemTR and MemTTHP had significantly longer roots and better shoot/root ratios compared to the control and BA-treated plants. In terms of root fresh weight, it was significantly higher in MemT-treated plantlets than in the control and BA treatments. Chlorophyll a/b ratio was significantly improved with the use of MemT, mTR and mT compared to control. Current findings indicate the potential of topolins in stimulating the accumulation of phenolic compounds in micropropagated plantlets. In view of the importance of plant secondary metabolites, their substantial accumulation probably enhanced the acclimatization and subsequent ex vitro survival of the micropropagated plantlets. Topolins, particularly, the new derivative MemTTHP could be an alternative CK for the micropropagation of plant species based on their stimulatory effect on ex vitro rooting that inevitably enhances acclimatization competence. Furthermore, topolins are demonstrated as potential elicitors in micropropagation.     

Physiological effects of a novel aromatic cytokinin analogue in micropropagated Aloe arborescens and Harpagophytum procumbens

Globally, there is a continuous effort geared toward improving micropropagation protocols with much emphasis on the type of cytokinins (CKs). We investigated the effect of meta-topolin tetrahydropyran-2-yl [mTTHP—a novel derivative of the aromatic cytokinin meta-topolin (mT)] on shoot proliferation, photosynthetic pigment content, phytochemicals and antioxidant activity of two widely used medicinal plants, Aloe arborescens and Harpagophytum procumbens. In terms of shoot proliferation, besides mTTHP and mT (at equimolar level) showing comparable effects, both topolins were significantly better than the control and benzyladenine riboside (BAR) in micropropagated A. arborescens. In H. procumbens, mT-treated cultures were the most responsive treatment at 2.5 μM when compared to the control. At 5.0 μM concentration, mTTHP and meta-topolin riboside (mTR) demonstrated a comparable effect on shoot proliferation. To a certain extent (particularly at low concentrations), mTTHP had a better rooting stimulatory effect when compared to the other CKs in both investigated species. In micropropagated A. arborescens, 2.5 μM mTTHP showed the best antioxidant activity (ferric reducing power) which was significantly higher than the control and all BAR-treated plants. Although 2.5 μM mTTHP exhibited a significant antioxidant activity (in β-carotene based assay) when compared to mTR at equimolar level in H. procumbens, it was not significantly different from the treatment with 2.5 μM mT and the control. As evidenced in the current study, the efficacy of the novel cytokinin may be species-specific and beneficial for few physiological parameters. It is conceivable that mTTHP is another viable alternative topolin with the extra advantage of inducing rooting at a low concentration.     

Shoot and root proliferation in ‘Williams’ banana: are the topolins better cytokinins?

The effects of five topolins (meta-Topolin?=?mT; meta-Topolin riboside?=?mTR; meta-Methoxy topolin?=?MemT; meta-Methoxy topolin riboside?=?MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl?=?MemTTHP) on shoot and root regeneration of micropropagated ??Williams?? bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30???M of the above cytokinins (CKs) for 42 days. Using shoot regenerated from optimum CK concentration, rooting experiments involving the use of indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid, smoke?Cwater (SW) and karrikinolide (KAR₁) were conducted. Apart from 10???M BA and 30???M MemTTHP treatments, the number of shoots produced in all the CK treatments were significantly higher than the control. In addition, 30???M mT resulted in the highest number of shoots (7.3?±?1.0). Unlike other CK treatments requiring higher concentrations, optimum mean shoot number per explant rate was attained at the lowest concentration in MemT and MemTTHP (10???M) treatments. The use of 10???M MemTTHP had the least root inhibitory effect during the shoot proliferation phase. In terms of abnormality index, mTR-regenerated plantlets were of the best quality across all the CKs tested. In mT- and BA-derived shoots, SW and KAR₁ significantly increased the number and length of roots compared to the control. Overall, when compared to BA, the use of topolin demonstrated higher mean shoot number per explant (MemT and MemTTHP) at lowest CK concentrations and the ease of rooting during the shoot proliferation phase (MemTTHP). During the rooting phase, topolin treatments produced more off-shoots than BA-treated ones. The current finding is a further demonstration of the increasing importance of topolins in micropropagation.     

Optimizing the micropropagation protocol for the endangered <Emphasis Type="Italic">Aloe polyphylla</Emphasis>: can <Emphasis Type="Italic">meta</Emphasis>-topolin and its derivatives serve as replacement for benzyladenine and zeatin?

Benzyladenine (BA) is the most widely used cytokinin in the micropropagation industry due to its effectiveness and affordability. It, however, has disadvantages such as genetic alteration and abnormal growth in some plants. Naturally occurring zeatin on the other hand is not as widely used as BA and is far more expensive. The use of meta-topolin and its derivatives as alternatives to BA and zeatin, both of which frequently have negative effects in tissue culture was investigated. In vitro grown Aloe polyphylla (an endangered medicinal and ornamental aloe) were cultured on full strength Murashige and Skoog basal medium with different concentrations of cytokinins and solidified with 1% Bacteriological Agar (Oxoid No. 1). mT was the preferred cytokinin both in terms of multiplication rate and rooting. The optimum concentration that induced regeneration and rooting is 5.0 μM. The problem of hyperhydricity was totally controlled. Plants rooted spontaneously in multiplication medium, thus avoiding the extra rooting step of the protocol. More than 91% of the plants transferred to ex vitro conditions were successfully acclimatized.     

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-ß-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

Effect of meta-Topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks

The effect of meta-Topolin (mT), an aromatic natural cytokinin, on micropropagation and adventitious shoot regeneration was evaluated on Prunus rootstocks, Torinel (Prunus domestica L.) and Ferdor (Prunus insititia  ×  domestica). In vitro shoots were grown for three subcultures on a multiplication medium containing 2.1, 4.2 or 6.3 μM of mT or 2.1 µM N ⁶-benzyladenine (BA). Then, apical leaves were excised and transferred on a medium supplied with BA, thidiazuron (TDZ) or zeatin for adventitious regeneration. Shoots multiplied on 2.1 μM mT or BA, were also induced to root with α-naphthalene acetic acid and acclimatized. meta-Topolin did not improve shoot proliferation, respect to BA, however, positively influences growth and quality of shoots. Ferdor from mT showed higher rooting percentage (92 %), root number and length, respect to the control, while a similar response was observed in Torinel with both cytokinins. Acclimatisation was higher than 90 % for both genotypes and, after 5 months, the highest length of roots was found in plants from mT. Adventitious regeneration was obtained only in leaves from shoots previously grown on mT. The highest regeneration responses, 65 and 42 %, respectively for Ferdor and Torinel, were obtained in the regeneration medium supplied with TDZ.     

Recalcitrant effects associated with the development of basal callus-like tissue on caulogenesis and rhizogenesis in <Emphasis Type="Italic">Sclerocarya birrea</Emphasis>

In the micropropagation of woody plant species, adventitious root and shoot formation remain some of the major bottlenecks due to their recalcitrance to in vitro manipulation. Some plant growth regulators may ameliorate these recalcitrant effects and improve in vitro caulogenic and rhizogenic processes. Shoot induction on shoot meristems, hypocotyls and epicotyls was evaluated using equimolar concentrations of benzyladenine (BA), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxytopolin riboside (MemTR). Three auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) were used in the induction of adventitious roots. Moderately high shoot formation (62.7%) was achieved at a concentration of 8.0 μM mT after 8 weeks in culture. The highest number of adventitious shoots per explant (2.4 ± 0.3) and the longest shoots (23.5 ± 3.16 mm) were recorded on 8.0 μM mT, though not significantly different from BA treatments. Most shoots progressively produced brown basal callus, which is a potential sink for cytokinin conjugates that are inhibitory to further proliferation of adventitious shoots. Good adventitious shoot formation occurred in 55% of hypocotyl explants on 8.0 μM mT. The highest rooting (91.6%) was achieved with IBA-treated shoots at a concentration of 4.0 μM. The use of mT and IBA provide an efficient micropropagation method for S. birrea, though further research is required especially in overcoming ex vitro plantlet survival challenges.     

Antioxidant and phenolic acid profiles of tissue cultured and acclimatized Merwilla plumbea plantlets in relation to the applied cytokinins

Merwilla plumbea (Lindl.) Speta is an important medicinal plant widely used in traditional medicine. We evaluated the effect of five cytokinins [benzyladenine (BA), 2-isopentenyladenine (2iP), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxy-9-tetrahydropyran-2-yl-topolin (MemTTHP)] on the level of phenolic acids and antioxidant activity of M. plumbea during the tissue culture and acclimatization stages. Two cytokinins (mT and mTR) significantly improved the antioxidant activity of tissue culture plantlets while the control plantlets were better after acclimatization. Using UPLC–MS/MS, the levels of hydroxybenzoic and hydroxycinnamic acid derivatives (phenolic acids) varied significantly during tissue culture and acclimatization, depending on the cytokinin and plant part analyzed. Vanillic acid (24.9 μg g⁻¹) detected in underground parts of tissue culture plants supplemented with BA was the most abundant phenolic acid detected. The current findings indicate that the phytochemicals together with the bioactivity during in vitro propagation of M. plumbea is influenced by the cytokinin type used and the stage of plant material collection.     

Factors affecting micropropagation and acclimatization of an elite clone of Eucalyptus tereticornis Sm.

Several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm. were investigated. Amongst different cytokinins tested, 6-benzyleadenine proved to be the most effective cytokinin for shoot multiplication and elongation. The initial size of the shoot clump (inoculum) also influenced shoot multiplication and elongation. The number of shoots proliferated per culture vessel were significantly higher (342 shoots per culture vessel) when larger shoot clumps (15?C20 shoots) were inoculated, compared to smaller shoot clumps (4?C5 shoots), which resulted in a reduced shoot proliferation rates (245 shoots per culture vessel). However, the number of elongated shoots (65 per culture vessel) and shoot length (5.23?cm) were higher in cultures which were inoculated with smaller shoot clumps in comparison to those cultures which were inoculated with larger shoot clumps (54 shoots per culture vessel with shoot length of 4.17?cm). The maximum number of rooted shoots (80.7?%) was obtained on one fourth-strength MS medium supplemented with 5.0???M indolebutyric acid. The number of shoots proliferated, elongated, rooting frequency, and subsequent survival of plants after acclimatization were higher in cultures incubated under photosynthetically active radiation (PAR) compared to those incubated under cool fluorescent lights (CFL). Osmotic potential of the sap and chlorophyll content of cultures incubated under PAR were also higher than those incubated under CFL. Following transfer of plants to soil, inoculation with a suspension of Bacillus subtilis (plant growth-promoting bacterium) increased the survival rate of plants by 10?%, yielding successful transfer of 84?% of plants. Random amplified polymorphic DNA and inter simple sequence repeat analyses indicated a high level of clonal uniformity amongst regenerated plants and also with that of the mother plant.     

Plant growth regulator induced phytochemical and antioxidant variations in micropropagated and acclimatized Eucomis autumnalis subspecies autumnalis (Asparagaceae)

Eucomis species is a valuable plant with both medicinal and horticultural potential. The current study evaluated the role of plant growth regulator (PGR) on growth, phytochemicals, and antioxidant activity in Eucomis autumnalis subspecies autumnalis. Five cytokinins including topolins and benzyladenine (BA) at 2 µM in combination with varying (0–15 µM) concentrations of naphthalene acetic acid (NAA) were tested. In vitro regenerants were acclimatized in the greenhouse for 4 months. Highest number of shoots (9 shoots/explant) was observed with 15 µM NAA alone or when combined with BA. Acclimatized plants derived from the 15 µM NAA treatment had the highest number of roots, largest leaf area and biggest bulbs. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. Among the in vitro regenerants, 5 µM NAA and 2 µM BA treatments produced the best antioxidant activity in the DPPH (55 %) and beta-carotene (88 %) test systems, respectively. A remarkable carry-over effect of the PGR was conspicuous in the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the optimized micropropagation protocols, the current findings present a promising potential for manipulating the type and concentration of applied PGRs to improve phytochemical production and hence medicinal value in E. autumnalis subspecies autumnalis.     

In vitro plant regeneration of caper (<Emphasis Type="Italic">Capparis spinosa</Emphasis> L.) from floral explants and genetic stability of regenerants

A new technique to regenerate caper plants (Capparis spinosa L. subsp. rupestris) starting from flower explant is reported. In vitro plant regeneration was attempted using stigma, anthers and unfertilized ovules of unopened flowers collected in the field. Plant regeneration was achieved from unfertilized ovules on MS medium supplemented with 88 mM sucrose and 13 μM 6-benzyladenine (BA). New individuals obtained from unfertilized ovules were used as source material for micropropagation and multiple shoots were obtained on MS medium supplemented with the adeninic cytokinin BA and the auxin indole-3-butyric acid (IBA). Explants obtained in micropropagation step were used for rooting step under several treatments. The best results (100% of rooted explants) were obtained when explants were dipped for 10 min in 50 μM IBA solution and successively maintained in growth regulator free medium. New plants were vigorous, of good quality and presented phenotypic characters similar to mother plants. Furthermore genetic stability of regenerants was verified through flow cytometric analysis and two different DNA-based techniques.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

Micropropagation of Gagnepainia godefroyi K Schum and Gagnepainia thoreliana (Baill) K Schum — Rare Medicinal Plants of Thailand

A micropropagation protocol for Gagnepainia godefroyi K Schum and G thoreliana (Baill) K Schum, two rare medicinal plants of Thailand, has been developed using terminal buds. After a total of twenty weeks of culture (4 weeks on MS added with 36.33 μM TDZ and 16 weeks on PGR-free MS), 28.77 and 13.50 shoots/explant were obtained, respectively. Rooting was spontaneous and regenerated plants were successfully transplanted to soil.     

The role of cytokinins during micropropagation of wych elm

We have compared the influence of two aromatic cytokinin derivatives, N ⁶-benzyladenine (BA) and meta-topolin (mT), on the in vitro multiplication and senescence of wych elm (Ulmus glabra Huds.). After 3 months of cultivation, the micropropagation rate was higher (approx. six times more shoots developed) on Murashige and Skoog (MS) medium supplemented with mT than on MS supplemented with BA. Quantification of 50 endogenous cytokinins, using a recently developed UPLC-ESI(+)-MS/MS method, showed significant differences in the cytokinin metabolites (especially different glucosides) in explants cultivated on media supplemented with BA or mT. Differences in efficiency of photosystem II was also detected.     

The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (<Emphasis Type="Italic">Musa</Emphasis> spp. AAA)

The effect of the cytokinins mT (meta-topolin), mTR (meta-topolin riboside), MemT (meta-methoxy topolin) and MemTR (meta-methoxy topolin riboside) on micropropagation of banana cultivars ‘Williams’ and ‘Grand Naine’ was studied and compared to BA (6-benzylaminopurine). In vitro cultures, at the third sub-culture level, were purchased from African Biotechnologies (Pty) Ltd., South Africa. These were then sub-cultured on MS media containing 7.5, 15 and 30 μM of all the cytokinins tested. Results recorded after 6 weeks of growth demonstrated that there were statistically significant differences between the parameters analyzed for the treatments. Superior multiplication rates were recorded for mT and mTR treatments. This result was consistent when compared to BA at 22.2 μM (previously published standard concentration). Contrary to previous findings with other species, these cytokinins inhibited rooting. The effect on somaclonal variation was not significantly different when BA, mT and mTR were tested at the seventh multiplication cycle for ‘Williams’ banana. These results support the possible use of topolins as an alternative to BA for Cavendish banana tissue culture. The role of these cytokinins on somaclonal variation however, requires a more stringent investigation as the results obtained in this investigation could have been influenced by carry-over effects from the initial cultures.     

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.