Genetic instability in calamondin (<Emphasis Type="Italic">Citrus madurensis </Emphasis>Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives

Somatic embryos were regenerated in vitro from calamondin style–stigma explants cultured in the presence of N ⁶-benzylaminopurine (BAP) cytokinin and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), N-phenyl-N′-benzothiazol-6-ylurea (PBU) and N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at micromolar level (12 μM) were able to induce a percentage of responsive explants significantly higher from that obtained with BAP and hormone-free (HF) conditions. In order to verify the genetic stability of the regenerants, 27 plants coming from different embryogenic events were randomly selected from each different culture condition and evaluated for somaclonal variations using inter-simple sequence repeat and random amplified polymorphic DNA analyses. We observed that 2,3-MDPU and PBU gave 3.7% of somaclonal mutants, whereas 4-CPPU gave 7.4% of mutants. No somaclonal variability was observed when plantlets were regenerated in BAP or HF medium. Although diphenylurea derivatives show a higher embryogenic potential as compared to BAP, they induce higher levels of somaclonal variability. This finding should be taken in consideration when new protocols for clonal propagation are being developed.     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.     

Diphenylurea Derivatives Induce Somatic Embryogenesis in Citrus

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N′-benzothiazol-6-ylurea (PBU), N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somatic embryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently as regards the concentrations tested and the citrus species. PBU was able to induce somatic embryogenesis at all the concentrations tested and in all the three species with percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-MDPU were completely unable to induce the production of somatic embryos in C. myrtifolia while both the compounds at the higher concentration (12 μM) acted positively in both C. madurensis and C. limon (68% of embryogenic explants). The phenylurea derivatives, used for the first time in this study to induce somatic embryogenesis in plant, showed a higher embriogenic performance when compared with 6-benzylaminopurine (BAP), a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a classical DPU derivative.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

N,N′-bis-(2,3-methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea interact with auxin in enhancing root formation of M26 apple (Malus pumila Mill.) stem slices

Stem slices cut from micropropagated cuttings of apple rootstock M26 were cultured in the presence of indole-3-butyric acid (IBA) plus N,N-bis-(2,3-methylenedioxyphenyl)urea or N,N-bis-(3,4-methylenedioxyphenyl)urea, to verify if there was an interaction between them in enhancing root formation. The N,N-bis-(methylenedioxyphenyl)ureas were supplemented after, before and in the simultaneous presence of auxin. Our data demonstrate that only the simultaneous presence of auxin and N,N-bis-(methylenedioxyphenyl)ureas in the culture medium enhanced root formation on M26 stem slices. The percentage of rooted slices obtained in the presence of the mixtures was significantly different from that obtained in the presence of low auxin concentration alone (1µM). Moreover both the percentage of rooted slices and the number of roots per slice obtained in these culture conditions was not significantly different to that of the optimal auxinic treatment in which the auxin concentration was threefold higher.     

A three-stage micropropagation system for a novel color morph of the wildflower, <Emphasis Type="Italic">Porteranthus trifoliatus</Emphasis> (L.) Britt

A three-stage micropropagation system was devised for Porteranthus trifoliatus ‘Pink Profusion’, a cultivar distinguished from the wild type by its pink flowers, purple stems and darker reddish leaves. Nodes of young shoots were used as explants, disinfested, and placed on several different Murashige and Skoog’s (MS) media formulations containing two levels of indole-3-butyric acid (IBA) and four levels of benzylaminopurine (BAP) in a factorial combination. An optimal number of commercially-usable shoots was achieved with BAP concentrations between 10 μM and 30 μM, while the addition of IBA made no significant difference in the number of shoots produced. Proliferated shoots could be rooted ex vitro without auxin treatment.     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.     

Micropropagation of gum karaya (Sterculia urens) by adventitious shoot formation and somatic embryogenesis

Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 μm N⁶-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 μm indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 μm 2,4-dichlorophenoxyacetic acid and 8.90 μm BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 μm thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field. Received: 26 November 1997 / Revision received: 14 April 1998 / Accepted: 11 May 1998     

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS⁺ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR⁺ plants.     

Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for short-term preservation and distribution of the medicinal and timber plantation tree, Khaya senegalensis, using alginate-encapsulated shoot tips. The study assessed the effects of culture medium, storage temperature, auxin concentration and planting substrate on shoot regrowth or conversion into plantlets of four different clones. Optimal shoot growth was obtained, with high frequencies (92–100%) of shoot emergence, on Murashige and Skoog (MS) culture media containing 4.4 μM benzyladenine (BA). Encapsulated shoot tips survived longer at 25°C than at 4°C, with viability of 73–88% after 8 weeks. Conversion into plantlets was achieved on half-strength MS medium by pre-culture treatment of shoot tips with 49–490 μM indole-3-butyric acid (IBA) before encapsulation. Treatment with 245 μM IBA provided 52–98% conversion, and 90–95% of plantlets survived acclimatisation under nursery conditions. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile substrates. Highest frequencies of plantlet formation from pre-converted synthetic seeds (42–86%) were obtained by transferring synthetic seeds to organic compost, and these plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion is a novel and convenient method for producing synthetic seeds that are suitable for distribution to commercial nurseries.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Micropropagation of <Emphasis Type="BoldItalic">Bambusa balcooa</Emphasis> Roxb. through axillary shoot proliferation

Bambusa balcooa is one of the most commercially important bamboo species. Regeneration of this species by sexual means is impossible because no seeds are set after flowering. Vegetative propagation is hindered due to bulky propagules, low rooting ability of the culm and branch cuttings, and seasonal specificity. This makes in vitro-based methods of regeneration important. This paper describes an efficient micropropagation protocol for multiplication of B. balcooa from nodal explants. Nodal segments were surface sterilized with 0.1% mercuric chloride for 10 min, and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP), 2.32 μM kinetin (Kn), and gelled with 0.2% w/v gelrite. Eighty-five percent of explants could be established in vitro with 90% of these achieving bud break. In vitro-formed shoots were successfully multiplied in MS liquid medium supplemented with 6.6 μM BAP, 2.32 μM Kn, 2.5% v/v coconut water, and 100 mg l⁻¹ myo-inositol. Subculturing shoots every 3 wk yielded a consistent proliferation rate of 4.11-fold without decline in vigor. Shoot clusters, containing 5 to 8 shoots, were rooted with 87.5% success in 1/2 MS supplemented with 5.71 μM indole-3-acetic acid (IAA), 4.9 μM indole-3-butyric acid (IBA), and 5.37 μM naphthaleneacetic acid (NAA) within 3 wk. Plants regenerated in this manner were acclimatized in the greenhouse and under a shade net with 88% success.     

Propagation and Conservation of Picrorhiza kurrooa Royle ex Benth.: An Endangered Himalayan Medicinal Herb of High Commercial Value

Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or α-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 μM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA₃), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 μM GA₃. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 μM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations.     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants

Summary Multiple shoots were grown from seedling explants of Alnus cremastogyne Burk by a two-stage culture procedure: initiation on WP medium supplemented with 2–8 M benzylammopurine(BAP) for 6 weeks, thereafter 3 weeks of subculture(shoot multiplication) on the same medium with 1 M BAP. A 5–9 fold multiplication rate was achieved. Type and concentration of sugar used in the multiplication medium were shown to be critical factors for both multiple shoot induction and bud elongation, the optima being 87.5mM glucose and 87.5mM sucrose respectively. After transfer to half-strength WP media either containing indolebutyric acid (IBA) or lacking plant growth regulator, almost all the shoots rooted. However, high rhizogenesis could be achieved only with shoots cultured in rooting medium containing 87.5mM sucrose or 175mM glucose, and shoots from multiplication media containing 87.5mM sucrose. Survival of the plantlets following transfer to vermiculite was 100%.Abbreviations BAP 6-benzylaminopurine - 2iP N⁶-(²-isopentenyl)adenine - kinetin 6-furfurylaminopurine - zeatin trans-6-(4-hydroxy-3-methylbut-2-enyl)aminopurine - IBA indol-3-butyric acid - WPM Woody plant medium (Lloyd and McCown, 1981)     

Rapid micropropagation of mature wild cherry

Explants taken from the mature vigorous tree of wild cherry (Prunus avium L.) were assayed for their organogenic capacity under various phytohormonal treatments. The highest rate of adventitious shoot multiplication was recorded at a combination of 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ thidiazuron (6.83 shoots per explant). No differences in multiplication rates were found among media supplemented with BAP, BAP + α-naphthaleneacetic acid (NAA) or BAP + indole-3-butyric acid (IBA). Shoot elongation was significantly affected by the concentration of BAP, regardless of auxin addition to medium. Up to 73 % of microshoots rooted after using 0.3 mg dm⁻³ IBA, otherwise the adventitious rooting occurred at reasonable frequencies in all auxin treatments. Regenerated plantlets were successfully hardened ex vitro and continued to grow after the transfer to soil. No morphological aberrations were observed in the regenerates.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium