Effects of pH Changes on Ion and 2,4-D Uptake of Wheat Roots

The quantitative relationships between pH-dependent ion and 2,4-D uptake in winter wheat seedlings (Triticum aestivum L. cv. Yubileynaya 50) have been investigated. The movement of various ions (potassium, phosphate, nitrate and ammonium) and 2,4-D across the root membranes was monitored with radioactive and stable isotope tracer methods. It was found that the H₊ ion concentration of the absorption solution strongly influences the 2,4-D uptake of the roots. Simultaneously, the 2,4-D uptake stimulates secretion of H⁺ into the absorption solution, that is, a H⁺ efflux can accompany the uptake of 2,4-D. This finding is consistent with the acid secretion theory of auxin and fusicoccin action. At pH 4 the 2,4-D uptake was much higher than at pH 6, thereby inhibiting the ion uptake and increasing the phytotoxicity in the plant. The results indicate that 2,4-D enters the root cells rapidly at the lower pH, mostly as undissociated molecules. With reference to the 2,4-D concentration in the roots at pH 4, a possible transport mechanism of the auxin herbicide is briefly discussed.     

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

Colonisation of seedling roots by 2,4-D degrading bacteria: A plant-microbial model

The development of model plant-microbial associations between Gram negative soil microbes capable of degrading phenoxyacetate herbicides, such as 2,4-D and 2,4-D methyl ester, and the crops canola and wheat was described. Both an Acinetobacter baumannii pJP4 transconjugant and Alcaligenes eutrophus JMP 134 colonised non-parasitically on the roots of sterilised seedlings in a hydroponic system. Laser scanning confocal microscopy has shown that colonisation occurred both on the root surface and deeper inside the mucilage layer or inside some surface root cells. When 2,4-D was added to the hydroponic medium supporting the growth of those seedlings colonised by 2,4-D degrading bacteria, the gas chromatographic analysis showed a rapid decrease in the concentration of this herbicide. These bacteria colonising the root system were shown to be responsible for the degradation of 2,4-D. Plants inoculated with the 2,4-D degrading microbes were subsequently found to be less susceptible to damage by the herbicide in such hydroponic systems.     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of microshoots of <Emphasis Type="Italic">Cotinus coggygria</Emphasis> Mill,a woody ornamental plant

Studies on rooting of microshoots of smokebush (Cotinus coggygria Mill, var. Royal Purple), a woody ornamental, were carried out in vitro. Microshoots were rooted in a mixed-auxin regime (indole 3-acetic acid, indole butyric acid [IBA], and naphthalene acetic acid) or singly in the above auxins and the 2,4 dichlorophenoxyacetic acid (2,4-D) over a wide concentration range. Indole butyric acid at 10 μM proved to be the most suitable treatment, producing less basal callus, 100% rooting, and earlier root emergence than the other treatments. No roots were formed with 2,4-D. A 6-day root induction period was obtained with 10 μM of IBA. Histological studies revealed increased mitotic activity after 3 d in culture in the medullary ray cells, which led to root primordium formation, several of which were formed simultaneously around the base of the explant. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 10. Thus, direct rhizogenesis occurred with the IBA treatment, as opposed to the roots that were formed in the basal callus under the mixed-auxin regime.     

Auxin effects on rooting in pea cuttings

Light-grown stem cuttingss of Pisum sativum L. cv. Weibull's Marma were rooted in a nutrient solution. The presence of 10 μ M indolylacetic acid (IAA) in the solution for 24 h or longer periods decreased the number of roots subsequently formed to about 50% of control, provided IAA was present in the solution during any of the 4 first 24 h periods. Treatment for 6 h or shorter periods caused no or small response. IAA did not appreciably change the time needed for root formation, the time course of root appearance or the pattern of root distribution along the basal internode. IAA at 100 μ M usually increased the number of roots although variable results were obtained with this IAA concentration.
The number of roots was strongly increased by treatment with indolylbutyric acid (IBA) or 2,4-dichlorophenoxyacetic acid (2,4-D). None of these or other synthetic auxins decreased the number of roots in suboptimal concentrations. Experiments with 10 μ M IBA showed that stimulation of rooting was obtained only if the auxin was present in the rooting solution for several days. Simultaneous treatment with IAA decreased the stimulating effect of IBA to some extent, whereas no such response was obtained if IAA was combined with 2,4-D.
IAA applied in lanolin to the stem of intact cuttings decreased the number of roots formed. Decapitation and debudding of the cuttings decreased the number of roots formed. If at least 2 leaves were left this decrease was efficiently counteracted by an optimal IAA dose applied to the upper part of the stem. A five times higher dose was less effective, indicating a negative effect on rooting also by IAA applied to the shoots.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

Interaction between two auxin-resistant mutants and their effects on lateral root formation in rice (Oryza sativa L.)

Since root elongation is very sensitive to auxin, screening for reduced inhibition in root elongation has been an important method for the detection of auxin-resistant mutants. Two recessive auxin-resistant lines of rice (Oryza sativa L. ssp. indica cv. IR8), arm1 and arm2, have been isolated by screening for resistance to 2,4-dichlorophenoxyacetic acid (2,4-D). arm1 displays a variety of morphological defects including reduced lateral root formation, increased seminal root elongation, reduced root diameter, and impaired xylem development in roots, while the arm2 phenotype is almost similar to wild-type IR8 except for a slightly reduced lateral root formation, impaired xylem development in roots and an enhanced plant height. Although the growth of arm2 roots exhibited a resistance to 2,4-D, it was sensitive to 1-naphthaleneacetic acid (NAA) as the wild type. At the same time, the arm2 roots showed a reduced [14C]2,4-D uptake while uptake of [3H]NAA was normal, suggesting that the resistance to 2,4-D of arm2 roots is due to a defect in 2,4-D uptake. To investigate the possible interaction between arm1 and arm2 genes, a double mutant has been constructed. The roots of arm1 arm2 double mutant were more resistant to 2,4-D and formed fewer lateral roots than those of either single mutant, suggesting that the two genes show synergistic effects with respect to both auxin response and lateral root formation. By contrast, all these mutants displayed the normal gravitropic response in roots, as did the wild-type plants. Taken together, Arm1 and Arm2 genes seem to function in different processes in the auxin-response pathways leading to lateral root formation.     

Mutants of Arabidopsis thaliana with altered responses to auxins and gravity

Auxin-resistant mutants of Arabidopsis have been induced and isolated by screening for survivors on a medium containing the herbicide 2,4-D. Thirty independently arisen mutants have been isolated in this way and one of them, P 83, has been investigated in detail. When wild type and P 83 are compared in concentration/response curves, where the response is the inhibition of root growth, the ED₅₀ values of the auxins, 2,4-D and IAA, are 14-fold higher for the mutant. The mutant also responds differently to gravity: its roots do not show positive geotropism, but tend to grow with a clockwise curvature on agar surfaces. The seedling roots of the mutant also grow more rapidly than those of the wild type in the absence of 2,4-D, following faster germination. The F₁ between P 83 and wild type is similar to the latter, but has a slightly increased resistance to 2,4-D. Results obtained from the F₂, F₃ and backcross generations suggest monofactorial inheritance. Most of the other 29 mutants have the P 83 phenotype, but at least five are different. Four have lower levels of resistance to 2,4-D and P 83, and their roots appear to respond normally to gravity. One mutant has an abnormal georesponse and a much higher level of resistance to 2,4-D than P 83.     

Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al. [1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.Abbreviations Ri-root Agrobacterium rhizogenes transformed root - BAP benzylaminopurine - IAA indoleacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid - 2,4-D 2,4 dichlorophenoxyacetic acid     

The ambiguous role of 2,4-dichlorophenoxyacetic acid in wheat tissue culture

The very basal, highly immature regions of dissected young leaves of Triticum aestivum L. cv. Kite formed adventitious roots on a nutrient medium supplemented with comparatively low concentrations (0.16 to 0.63 μ M ) of 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentrations (up to 640 μ M ) had to be applied to stimulate growth from more mature regions higher up the leaf. Yet, already at 2.5 μ M roots were less distinct and more callus-like, and eventually (at 10 to 640 μ M ) only a subculturable callus of apparently suppressed, slowly proliferating root primordia developed. Furthermore, at the most basal, highly immature regions growth was significantly retarded when the auxin concentration was raised. The leaf culture system appears to reflect the dual action of 2,4-D known from herbicide research, namely growth stimulation from differentiating (or differentiated) cells, but growth suppression at or in the vicinity of apical meristems. Correspondingly, when the callus of apparently suppressed, slowly proliferating root primordia was transferred to media without 2,4-D or with low concentrations (0.16–2.5 μ M ) rapid proliferation commenced, leading to profuse root outgrowth. The system demonstrates the ambiguous role which this auxin appears to have, at least in wheat tissue culture.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Effect of 2,4-D on Seedling Physiology and Cytogenetical Studies in Triticum aestivum and Phalaris minor (Gramineae)

Effects of 2, 4 -D on seedling growth and chromosomal abnormalities were studied in Triticum aestivum and Phalaris minor. Seeds were soaked at different concentrations of 2,4 -D （0.01%, 0.1%, 1.0% ） for 4, 8, 12 and 16 hours. 2,4-D suppressed the germination more severely in P. minor than in T. aestivum. Shoot and root length was retarded with the increase of concentration and time of treatment in both species. Generally radical was more negatively affected than coleoptile and emergence of radical was not observed at 1.0% concentration at 8, 12, and 16 hours of treatment in T. aestivum while in P. minor there was a total lack of radical emergence at 1.0% concentration for all durations of treatment. Stiff and curled roots and undifferentiated callus like scutellar tissues were observed in T. aestivum, while in P. minor the coleoptile obtained was lean, pale green in colour and was lying flat on filter paper. Mitotic index decreased, while chromosomal abnormalities, bridges and laggards were increased with the increase of concentration and soaking time however, laggards were not observed in T. aestivum. Clumping and chain formation of chromosomes at metaphase was also noticed in P. minor.     

Colonization of wheat para-nodules by the N2-fixing cyanobacterium Nostoc sp. strain 2S9B

The treatment of wheat roots with 2,4-dichlorophenoxyacetic acid (2,4-D) led to the formation of tumour-like structures, para -nodules, which were readily colonized by cyanobacteria. The amount of cyanobacteria found on 2,4-D treated roots was 3.6 times higher than that found on untreated roots. Cyanobacteria penetrated the para - nodules by migrating in between loosely arranged cells that covered their surfaces or by penetrating the space at the junction of root and para -nodule. In plants treated with 2,4-D and co-cultivated with cyanobacteria in medium without combined nitrogen, the rate of acetylene reduction was three times that seen in untreated but colonized roots. Addition of 2,4-D itself did not change the rate of acetylene reduction in free-living culture of Nostoc sp. strain 2S9B. In plants treated with 2,4-D and co-cultivated with Nostoc sp. strain 2S9B in medium without combined nitrogen the nitrogen content of roots but not shoots was significantly increased.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.