Modified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots

ABSTRACT

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the D8 wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurred at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in the basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed.     

Adventitious root formation in woody plant tissue: The influence of light and indole-3-butyric acid (IBA) on adventitious root induction in <Emphasis Type="Italic">Betula Pendula</Emphasis>

Summary The effects of auxin concentration and photoperiod on rooting were examined with a view to establishing a rooting regime for Betula pendula shoots cultured in vitro. Optimum concentrations of indole-3-butyric acid (IBA) were determined: the effects of a 16-h photoperiod and a pretreatment of 8d total darkness were examined. Maximum rooting rates and rooting densities (root number) were achieved using relatively low levels of IBA (0.39–0.74 μM). Both the dark and the light regimes produced roots, higher yields occurring with the latter. Maximum rooting percentage was reached after 30 d growth. in the light-treated cultures.     

Control of plant growth resides in the shoot,and not in the root,in reciprocal grafts of flacca and wild-type tomato (Lysopersicon esculentum), in the presence and absence of salinity stress

Grafted plants of flacca, an ABA-deficient mutant of tomato (Lycopersicon esculentum), and the wild-type variety Rheinlands Ruhm were grown with and without salinity stress to test the roles of roots and shoots in the regulation of plant growth. Fourteen days after exposure to 200 mM NaCl, shoot and root fresh weight, endogenous ABA concentrations, nitrate concentration, activities of selected enzymes related to nitrogen assimilation, and cation accumulation were determined. Rootstock genotype had little influence on the growth of the grafted plants, whereas grafted plants having wild-type shoots (Ws) produced more biomass than those having flacca shoots (Fs), irrespective of the salinity level. Growth of flacca shoots grafted onto wild-type rootstock (Fs/Wr) was superior to that of flacca shoots grafted onto flacca rootstock (Fs/Fr). The improved growth correlated with enhanced levels of ABA in the flaccashoots of Fs/Wr. In all the graft combinations, ABA content was higher in wild-type shoots than in flacca shoots, with or without salinity. There were no significant differences in root ABA concentrations among the different grafted types. Enhanced growth correlated with higher nitrate levels and higher nitrate reductase activity in the roots and shoots of plants with wild-type shoots and with higher shoot concentrations of ABA in plants with wild-type shoots. There were no significant differences in glutamine synthetase and phosphoenol pyruvate carboxylase activities in the shoots and roots of all the grafted plants, regardless of the salinity level. While shoot genotype determined the accumulation of K⁺ and Na⁺ in grafted plants regardless of salinity, it had no influence on Ca²⁺ concentrations. Regardless of the salinity, the total concentration of cations was the same in all the plants, while salinity decreased Mg²⁺ concentration in roots and shoots of all grafts, with the exception of flacca grafted shoots. The scion genotype – and its ABA level – thus played the major role in the growth of grafted plants, regardless of the rootstock genotype and the salinity of the growth medium.     

Changes in peroxidase activity,auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloïdes) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA naphthaleneacetic acid - IAA indole-3-acetic acid - 2-iP 2-isopentenyladenine - IAAsp indole-3-acetylaspartic acid - IBA indole-3-butyric acid - GC gas-chromatography     

The responses of wild-type and ABA mutant Arabidopsis thaliana plants to phosphorus starvation

The growth patterns of plants subjected to phosphorus starvation resemble those caused by treatment with ABA, suggesting that ABA could mediate the response of the plant to phosphorus starvation. We examined the role of ABA in phosphorus stress by comparing growth and biochemical responses of Arabidopsis thaliana ABA mutants aba-1 and abi2-1 to those of wild-type plants. We first characterized acid phosphatase production of wild-type Arabidopsis in response to phosphorus starvation. We found that several acid phosphatase isozymes are present in roots and shoots, but only a subset of these isozymes are induced by phosphorus stress, and they are induced in both organs. Production of acid phosphatase in response to phosphorus stress was not affected by the aba-1 or abi2-1 mutations. Low phosphorus also resulted in decreased growth of both wild-type and ABA mutant plants, and the root-to-shoot ratio was increased in both wild type and mutants. Anthocyanins accumulated in response to phosphorus stress in both wild-type and mutant plants, but the increase was reduced in the aba-1 mutant. Thus, two different ABA mutants responded normally in most respects to phosphorus stress. Our data do not support a major role for ABA in coordinating the phosphorus-stress response.     

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACT

Pinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.     

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.     

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.     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

插穗因素对闽楠扦插苗生根、生长及相关酶活性的影响

选择40年生无病虫害闽楠植株半木质化枝条为插穗,采用L_8(2~7)正交试验方法,设置插穗来源(枝条顶部、枝条中部)、枝条长度(10cm、15cm)和留叶数(保留1片和2片全叶)3个插穗因素,研究插穗因素对闽楠扦插苗生根、生长及理化性质等方面影响,筛选适宜插穗处理方式,揭示插穗调控扦插苗生根和生长机理。结果表明:(1)闽楠插穗来源、长度及留叶数显著影响扦插苗的生根、生长及理化特性。(2)闽楠扦插最适合的插穗处理是取长度为10cm的中部枝、保留2片叶。(3)影响闽楠扦插生根率的最主要因素是插穗来源,关键生理指标是根系PPO和POD活性;影响闽楠扦插苗新梢生长最主要的因素是插穗的留叶数,关键生理指标是根系IAAO活性和MDA含量。研究表明,各插穗因素显著影响着闽楠扦插苗的生根及生长,并以长度10cm、保留2片的中部枝为插穗最佳;该研究结果为闽楠扦插繁殖技术制定和推广应用提供理论与技术指导。     

Metabolic changes during rooting in stem cuttings of Casuarina equisetifolia L.: effects of auxin, the sex and the type of cutting on rooting

Rooting in terminal shoot and lateral shoot cuttings from 10-year-old elite trees of Casuarina equisetifolia L. in different sex groups was achieved after 20 days when the basal ends of the cuttings were dipped for 3 h in 20 ppm indole-3-butyric acid (IBA). Shoots derived from male plants rooted better than their female and monoecious counterparts, and the lateral shoots were more responsive to rooting than the terminal shoots. During rooting, the metabolic activities varied in both lateral shoot and terminal shoot cuttings derived from plants under different sex groups. Peroxidase and polyphenoloxidase activities were high during root initiation and showed a sharp decline thereafter. The polyphenoloxidase activity was higher in the lateral shoot than the terminal shoot cuttings. The rooted plantlets survived and established well in the field.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

Stock-plant etiolation causes drifts in total soluble sugars and anthraquinones, and promotes adventitious root formation in teak (Tectona grandis L. f.) coppice shoots

The effects of stock-plant etiolation on coppice-shoot growth, drifts in total soluble sugars and anthraquinones (AQs; C₁₄H₈O₂), and rooting potentiality of shoot cuttings were examined in Tectona grandis L. f. (clone FG1). When seedlings were one-year-old, they were coppiced and maintained in the dark for etiolation, with a parallel set kept under natural light in an open environment. Coppice shoots were made into single-node leafy cuttings (SNCs), which were cultured under intermittent mist for rooting. These SNCs were treated with different concentrations of NAA (0, 2000 and 3000 mg l⁻¹). Etiolation significantly increased the coppice-shoot length, internode length, number of coppice shoots, number of leaves, number of nodes and total soluble sugars. The HPTLC analysis showed qualitative and quantitative differences in AQs in coppice shoots obtained from etiolated and non-etiolated stock plants. The study showed that AQs could be used as a marker for maturity and juvenility in teak. Stock-plant etiolation caused a significant increase in percent rooting and sprouting, shoot length, number of shoots and number of leaves per SNC, but a decrease in callusing at the base of the SNC. NAA at 2000 and 3000 mg l⁻¹ had inhibitory effects on rooting and sprouting of SNCs. The result showed that stock-plant etiolation fostered rooting by rejuvenating the coppice shoots.     

Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation

The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vegetative Propagation of Pinus sylvestris

Methods for the vegetative propagation of Pinus sylvestris L. from interfascicular shoots are described. Using 5-year-old plants the outgrowth of interfascicular shoots was promoted by removal of terminal and lateral buds; this response was augmented substantially by application of cytokinin, tri-iodobenzoic acid, alar and morphactin alone and especially in combination. The rooting capacity of shoot cuttings from interfascicular shoots appeared to be largely determined by the state of growth of the stock plant. Cuttings from dormant stock plants subjected to short-day treatment followed by a period of low temperature gave the best rooting, especially when the cuttings themselves had been cold-stored prior to planting. Rooting was optimal when such cuttings were treated with a mixture of 25 mg/l of indolebutyric acid and 25 mg/l of napthaleneacetic acid as a 48 h basal soak, and were planted on a heated mistbench under extended illumination; over 90% of such cuttings could be rooted. These results are discussed in relation to bud activity, endogenous hormone levels and promoting tissue extracts also tested.     

继代培养次数对杜梨生根能力和叶形态及其激素水平的影响

为了探索杜梨组培复幼变化规律,对10年生杜梨进行连续继代培养,统计不同继代次数杜梨丛生芽繁殖系数和生根率,观察记录叶片形态变化并测定内源激素含量。结果表明:(1)通过连续继代培养,杜梨丛生芽生根率由0提升到66.70%,繁殖系数由第1代的2.13提升到第10代的4.20。(2)叶片在继代第3次时出现裂刻且随后裂刻程度逐代加深;在继代过程中,丛生芽叶面积和叶脉数显著降低,叶周长和叶形指数呈先下降后上升的变化趋势。(3)丛生芽叶片内源IAA含量在继代第6次时达到46.39 ng·g-1,且显著高于初代丛生芽;随着继代次数的增加,叶片内源ZR呈先上升后下降的变化趋势,内源GA3含量没有发生显著性变化,而内源ABA含量逐渐降低;叶片IAA/ABA和IAA/ZR的值随着继代次数的增加而增加。(4)丛生芽叶片ABA含量和IAA/ZR与其生根率分别呈显著负相关和显著正相关关系,叶片裂刻数和IAA/ABA与生根率均呈现极显著正相关关系,而叶脉数与生根率则呈现极显著负相关关系。研究认为,连续继代培养可显著提高杜梨丛生芽的生根能力,并且与丛生芽叶形和激素含量及其比值有密切的关系,该研究结果为难生根植物无性繁殖以及树木复幼提供了重要技术借鉴。     

Rapid Axillary Bud Proliferation and ex vitro Rooting of Eupatorium triplinerve

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 M benzylaminopurine (BAP) and 2.46 M indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 M IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.     

Rooting blockage in the tobacco rac mutant occurs at the initiation phase,and induces diversion to xylem differentiation

ABSTRACT

The rac mutant of Nicotiana tabacum L. cv. Xanthii is impaired in adventitious root formation. The objective of the present study was to determine whether or not the root induction phase occurs in the rac mutant, and if so, to determine what causes the induced cells to become incapable of organising root primordia. To this end, rac and wild-type shoots were cultured in vitro for 7 days under conditions suitable for obtaining roots in the wild-type (i.e., exposure to 5 µM indole-3-butyric acid for 4 h, and then transfer to hormone-free medium), and then histologically and biochemically analysed during culture. The variations in peroxidase activity, and in cellular levels of auxins and polyamines revealed that the induction phase occurs in rac shoots, although it lasts longer than in the wild-type ones. Furthermore, both auxin and polyamines were consistently higher in rac shoots compared to the wild-type. After induction, auxin and putrescine levels abruptly decreased in the wild-type shoots, whereas they decreased much more slowly in the rac mutant. The histological analysis of the wild-type shoots showed that the abrupt decrease in auxin and polyamine levels were correlated with a normal initiation phase. In fact, wild-type shoots showed cell divisions in the procambium already at day 2, resulting in the formation of root primordia at day 4, and in root emergence between days 5 and 7. In rac shoots, despite the fact that the procambium cells were activated to undergo cell division, the initiation phase was highly perturbed, and the procambial cells developed tracheary elements instead of adventitious roots. The different morphogenic responses of the two genotypes are discussed in the light of the differences in auxin content after the induction phase.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.