Effect of Auxins on in vitro Rooting of Plumbago Zeylanica: Peroxidase Activity as a Marker for Root Induction

Induction of rooting in the microshoots of Plumbago zeylanica was achieved on halfstrength basal Murashige and Skoog's medium supplemented with 0.25 mg dm^–3 indole-3-butyric acid. Rooting was totally inhibited when the microshoots were cultured in vitro under continuous light, however, maximum percentage of microshoots rooted when incubated in continuous light for 4 weeks before transfer to the rooting media. Peroxidase activity increased markedly during root induction indicating a key role of peroxidase in rooting of microshoots of Plumbago zeylanica in vitro.     

In vitro rooting of juvenile and mature<Emphasis Type="Italic"> Acacia mangium</Emphasis> microcuttings with reference to leaf morphology as a phase change marker

Rooting capacity of microshoots derived from a mature clone of Acacia mangium Willd. and from its juvenile progeny was compared in different in vitro conditions and in relation to leaf morphology as a phase change marker. Rooting capacity of the mature clone appeared to depend a lot on rooting medium composition, in contrast to the juvenile plant material. On a SH/3 derived medium with 4 µM IAA, the rooting rates varied significantly according to microshoot morphology and light regime starting from day 14. Maintaining the microshoots for 3 weeks in total darkness prior to transfer to the standard 16-h photoperiod increased the rooting rates overall. Mature-like microshoots of juvenile origin rooted in much lower proportions than the juvenile-like ones under the 16-h photoperiod, whereas no difference in rooting rates between the two morphological types was observed after 3 weeks in total darkness. The results were totally opposite for the mature clone for which 3 weeks in darkness, in contrast to the 16-h photoperiod, induced morphology-related differences in rooting rates with higher scores for the juvenile type. This study shows that under certain conditions, A. mangium microshoots from mature origin but with juvenile morphology can root in vitro with similar success rates (70%) to microshoots from juvenile plant material. However, the inconsistencies observed between the two age classes of microshoots in relation to photoperiod refute the use of foliar morphology as a reliable marker of rooting ability in A. mangium, although these two traits are classically referred to as phase change indicators.     

Micropropagation of juvenile and adult Annona squamosa

A micropropagation system for Annona squamosa L. (Sugar Apple) using hypocotyls of seedlings and nodal cuttings from 3-year-old plants was developed. Shoot proliferation was achieved with Woody Plant Medium supplemented with BA. Silver thiosulphate was added at 0.5 mg l^–1 to control leaf abscission. Rooting was obtained when subcultured shoots were preconditioned for 2 weeks in medium with 10 g l^–1 activated charcoal before treatment with 43 µm NAA or 39 µm IBA. Rooting was improved when galactose was used instead of sucrose in the rooting medium. The rooted plantlets were acclimatised successfully.Abbreviations NAA naphthaleneacetic acid - IBA indolebutyric acid - MS Murashige & Skoog Medium - WPM Woody Plant Medium - NN Nitsch Medium - Juv juvenile explant - Adu adult explant     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

In Vitro Root Formation in Anacardium Occidentale Microshoots

Cultural conditions affecting the induction of rhizogenesis in vitro were evaluated in cashew (Anacardium occidentale L.) shoot-node-derived microshoots. The application of auxins was essential for the formation of adventitious roots. A 5-d indole-3-butyric acid (IBA) induction period was more suitable than continuous IBA treatment or a shorter induction period. N⁶-[2-Isopentenyl]adenine in low concentrations (0.3 – 1 µM) in the root induction medium supported root formation. Precultivation of microshoots with gibberellic acid (GA₃) suppressed the subsequent rhizogenesis. Activated charcoal did not affect rooting. No significant differences in rooting abilities of cashew shoots were observed between 25, 29 and 35 °C and roots did not develop at 19 °C. Salts of low osmotic composition were more suitable than richer media. Microshoots originated from cotyledonary nodes showed higher rooting when compared to standard microshoots.     

Onset of in vitro rhizogenesis response and peroxidase activity in Zingiber officinale (Zingiberaceae)

The induction of rooting in microshoots of Zingiber officinale cvs. Suprava, Turia local, Suruchi and V3S18 was achieved on half-strength basal Murashige and Skoog's medium supplemented with 0.5-1.0 mg/l either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) and 2% (w/v) sucrose within 7-9 days of culture. Rooting was inhibited when the microshoots were cultured under higher concentration of auxins. The microshoots cultured on medium supplemented with NAA induced large number of thin root hairs with friable calluses within 6-7 days. Peroxidase activity was determined during root induction (0-day to the 10th day at every 2 day interval) from microshoots derived in vitro. The activity was minimum in the inductive phase (primary) and at the maximum level during the root initiative phase. These finding may be useful in monitoring the rooting behaviour in microshoots derived from different subculture and peroxidase activity as a marker for root initiation.     

Micropropagation of Pistachio from mature trees

Multiple shoots were induced from nodal segments of mature trees of Pistacia vera L. on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BA). Maximum shoot production was obtained from shoot tips taken from in vitro proliferated shoots when cultured on solidified MS medium containing 8.8 μM BA. The multiplication rate was 20 microshoots per explant on the 30th day. Rooting of microshoots was achieved in MS medium supplemented with indole butyric acid (IBA). Rooted plantlets reassumed independent growth after a short period of acclimatisation. Stable regenerated plants were established in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of thidiazuron on micropropagation of rose

Summary Rose (Rose hybrida L.) plants were micropropagated by axillary shoot proliferation method. Maximum number of microshoots per shoot tip explant were obtained on MS medium supplemented with 5 to 10&#x00B5;M thidiazuron (TDZ). The microshoots formed rooted plants on MS hormone-free medium. No difference in the rooting of microshoots produced on medium containing TDZ or N⁶-benzyladenine was observed. The regenerated plants were successfully transplanted to the field and appeared similar to the parent plant in morphologic features.     

Role of Amino Acids in Evolution of Ethylene and Methane, and Development of Microshoots in Cajanus Cajan

In pigeonpea (Cajanus cajan L.) shoot buds were induced when cotyledonary node explants were supplemented with benzylaminopurine (BAP; 2 mg dm^–3). When 0.1 mg dm^-3 BAP and 0.01 mg dm^-3 naphtahalene acetic acid were supplemented to the medium, the 34 – 35 % of induced buds developed to microshoots. By supplementing amino acids like proline, glutamine, asparagine and L-cysteine, shoot bud development to microshoots was enhanced at least by two fold. Amongst the amino acids proline gave maximum number of microshoots per explant. With increase in concentration of amino acids, fresh mass increased but microshoot number decreased. Also methane evolution was increased by addition of amino acids, and also in medium containing more of its nitrogen in the form of ammonia. Increased evolution of methane was accompanied by reduction in evolution of ethylene, and enhancement of efficiency of microshoot development.     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Influence of auxins and darkness on in vitro rooting of micropropagated shoots from mature and juvenile Acacia mangium

The influence of total darkness versus a 16/8 photoperiod and of auxins added to the culture medium on the in vitro root formation capacity of Acacia mangium microshoots of juvenile and mature origin was examined. Rooting of the mature clone was significantly increased by exposing the microshoots to auxins (4 and 6 μM IAA or IBA) in darkness, while the promoting effect of darkness combined with 4 μM IAA was more time-restricted for the juvenile-origin microshoots. Overall, the latter rooted in greater proportions than those from the mature source. Maintaining the microshoots of both origins on auxin supplemented medium in darkness resulted in a greater number of adventitious roots formed than under the standard 16/8 lighting conditions. On the other hand, light stimulated root elongation. These results are discussed mainly from the viewpoint of auxin metabolism in relation to adventitious root formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation of Ulmus pumila L. from mature trees

Summary Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l^–1 BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l^–1 naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.     

Ex Vitro Rooting of Micropropagated Shoots of Stackhousia Tryonii

Micropropagated shoots of Stackhousia tryonii were exposed (individually or in combination) to indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthalene acetic acid (NAA) at concentrations 1, 2 or 4 g dm^–3 with the view to induce rooting under ex vitro conditions. The treated microshoots were grown in a mist room for four weeks and assessed for survival, rooting percentage, number of roots and root length. The results showed that IBA at 2 g dm^–3 was most effective in inducing roots. Mixing of two or more auxins markedly reduced rooting percentage indicating antagonistic effects. The results demonstrated the potential of combining ex vitro rooting and hardening in one step, with view to reducing costs of multiplying plants via micropropagation.     

Direct shoot organogenesis from hypocotyl explants of Jatropha curcas L. an important bioenergy feedstock

Multiple shoots were induced on hypocotyl segments reared from in vitro germinated seedlings of Jatropha curcas L., using Murashige and Skoog (MS) medium supplemented with N⁶‐benzyl adenine (BA) and kinetin (kin) either alone or in combination with indole‐3‐acetic acid (IAA). The combined treatment of 7.05 μm kin and 1.425 μm IAA resulted in maximum shoot production with an average shoot bud initiation of 12.1 per explant. The regenerated microshoots were transferred to root induction medium containing half‐strength MS salts supplemented with either indole‐3‐butyric acid (IBA) or IAA. Rooting of microshoots was best achieved on half‐strength MS medium supplemented with IBA (9.8 μm ). Rooted plantlets were subsequently acclimatized under green house condition and the plantlets showed 70% survival.     

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88 μM BAP with 108.6 μM adenine sulphate and 11.84 μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84 μM IBA, with 81.1 % rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.     

Sequential rooting media and rooting capacity of Sequoiadendron giganteum in vitro. Peroxidase activity as a marker

The rooting capacities of tips of seedling, juvenile and mature shoots of Sequoiadendron giganteum were compared on different rooting media (inductive and expressive media) after passage on an elongating medium. None of the cuttings rooted when continuously kept on medium containing the auxin NAA and vitamin D2. Peroxidase activity of all those cuttings on NAA+D2 first increased during the 7–9 first days and decreased in the days after. Rooting was obtained by transfer of the cuttings after periods longer than 7–9 days from the NAA+D2 inductive medium to a basal medium supplemented or not with rutin (expressive medium). The rooting capacity was emphasized by rutin treatment and was in correlation with the peroxidase peak reached on the NAA+D2 medium. Seedlings, characterised by the highest peroxidase activity, were most performing in rooting.Abbreviations BM basal medium - D2 ergocalciferol - NAA naphtaleneacetic acid     

Micropropagation of Psiadia arguta through cotyledonary axillary bud culture

A micropropagation protocol for Psiadia arguta, an endangered endemic plant from Mauritius is described using 15-day old in vitro seedling explants without the radicle. MS basal medium supplemented with TDZ (0.5–1 mg/l) proved to be the most effective medium for the induction of cotyledonary axillary buds as compared to MS medium containing NAA (0.5 mg/l) or both NAA (0.5 mg/l) and TDZ (0.5–1 mg/l). In fact, after transfer to hormone free MS medium, microshoots were obtained only from seedling explants cultured on media containing only TDZ. Regenerated shoots elongated and rooted when cultured on MS8900 containing IBA (0–1 mg/l). Hormone-free MS8900 was the best medium for rooting and development of plantlets for acclimatization.     

Micropropagation of Mexican redbud,Cercis canadensis var. mexicana

Mexican redbud (Cercis canadensis var. mexicana) shoot cultures were initiated from explants taken from both mature and juvenile stock plants. Culture conditions affecting shoot growth and proliferation and rooting of three clones were investigated. Shoot growth was best on media supplemented with 0.25% activated charcoal and solidified with 0.2% Gelrite. Four commercially available salt formulations (Anderson's rhododendron medium, WPM, MS, DKW) were tested for growth of shoot cultures, and Anderson's rhododendron basal salt mixture was superior. Axillary shoots grew from explants cultured media supplemented with a wide range of concentrations of benzyladenine and thidiazuron. Benzyladenine at 5.6–22.2 M supported the best combination of shoot quality and number. Rooting of microshoots in vitro was best on half-strength WPM containing 6.71 M naphthaleneacetic acid and 0.1% activated charcoal.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - 2iP 6-(, -dimethylallylamino)purine - DKW Driver & Kuniyuki Walnut - kinetin 6-furfurlaminopurine - MS Murashige & Skoog - NAA -naphthaleneacetic acid - WPM Woody Plant Medium - TDZ thidiazuron - 1-phenyl-3 (1,2,3-thidiazol-5-yl)urea     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture

An efficient clonal multiplication system was developed for in vitro propagation of crocin — producing Gardenia jasminoides Ellis plants. Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP 1 mg l^–1) and indole-3-butyric acid (IBA 1 mg l^–1) resulted in multiple shoot initiation at the rate of 21 shoots per explant in 60 d of culture. Transfer of the microshoots into liquid MS medium supplemented with BAP (5 mg l^–1) with two subcultures of 15 d duration in the same medium resulted in 400 ± 25 shoots per explant. Efficient rooting was achieved in MS medium supplemented with -naphthaleneacetic acid (5 mg l^–1). The in vitro raised plants were hardened in a greenhouse and transplanted to the field successfully. The method described will be useful for rapid multiplication of Gardenia for commercial exploitation.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - Kn kinetin - 2ip 6-(,-dimethylallylamino)purine - NAA -naphthalene- acetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid