Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

Growth at high CO2 affects the chloroplast number but not the photosynthetic efficiency of photoautotrophicMarchantia polymorpha culture cells

Shoot tips from accessions of wild cherry (Prunus avium L.) selected from British woodland, and also theP. avium rootstock cvs. F12/1 and Charger, were successfully establishedin vitro, and most were easily micropropagated on Murashige and Skoog (MS)-based media. In one accession, adventitious shoots occasionally developed from the extrafloral nectaries positioned at the base of leaf petioles of the initial explants. Micropropagation of cv. Charger was improved by culture on a Quoirin and Lepoivre and Woody Plant-based medium, and by supplementing the medium with 1-phenyl-3-(1, 2, 3-thiadiazol-5yl)urea (thidiazuron). Poor shoot production by F12/1 on 1, 3, 5-trihydroxybenzene (phloroglucinol)-free media was not improved by up to 18 months of regular subculture. Study of cv. F12/1 showed that shoots produced on a MS-based medium with 1 mM phloroglucinol, 0.49 μM indolebutyric acid, 4.4 μM benzyladenine (BA) and 0.29 μM gibberellic acid (GA₃), were easier to root over several subcultures than shoots produced on a similar medium with no GA₃ and only 2.2 μM BA, but only in a rooting medium supplemented with 1 mM phloroglucinol.     

Influence of gibberellic acid and different salt concentrations on germination percentage and physiological parameters of oat cultivars

Gibberellic acid (GA₃) is one of the plant growth regulators which improve salt tolerance and mitigate the salt stress impact on plants. The extant analysis was carried out to study the effect of GA₃ and different salt concentrations on seed germination and physiological parameters of oat cultivars. Oats is substantially less tolerant to salt than wheat and barley. Experimentation was conducted as factorial with Completely Randomized Block Design with three replicates. Different concentration of NaCl salt ((25, 50, 75 and 100 mM) were used in test control group and 100 and 150 ppm of GA₃ were used in two group by pre-treated (after 24 h of the seed soaking) and plants were analyzed on 15th day. Results indicate that increasing salinity would decrease the germination percentage and growth parameter in three oat cultivars. Quotes data indicating a 13%, 19.9% and 32.48% in cultivars NDO-2, UPO-212 and UPO-94 germination reduction when soil salinity reaches 50 mM. A 36.02%, 47.33% and 56.365 reduction in germination is likely when soil salinity reaches 100 mM respectively same cultivars. Seeds treated with GA₃ significantly promoted the percentage of germination, shoot and root length, total fresh and dry weight of seedling, tissue water content and seedling vigor index by NDO-2 and UPO-212 under different saline concentration. The maximum average of germination and growth parameters were observed from 150 ppm GA₃ treated seeds. But this concentration was significantly inhibited root length in sensitive cultivar UPO-94 at 75 and 100 mM salt as compared to 100 ppm. We observed that, the high concentration of GA₃ was not suitable for sensitive oat cultivars. Because the plant root are the real workforce behind any plants success. Thus, it may be concluding that, GA₃ treatment could curtail the toxic effect of salinity by increasing germination percentage and shoot and root length, total fresh and dry weight, tissue water content and seedling vigor index in tolerant cultivar.     

Stimulation of putrescine biosynthesis via the ornithine decarboxylase pathway by gibberellic acid in the in vitro rooting of globe artichoke (Cynara scolymus)

Rooting and growth of globe artichoke (Cynarascolymus) cuttings were improved by supplementing the rooting mediumwith 10 M gibberellic acid(GA₃). Exogenous putrescine (Put) (0.5mM) raised the percentage of rooting to thesame level as GA₃, but decreased the number of roots per plant.Addition of Put to GA₃ produced the same effects on growthparametersand rooting percentage as Put alone. Changes in endogenous contents of freepolyamines (PAs) were determined in plants cultured 4 weeks in a rootingmedium.Whatever the treatments, rooting caused a large decrease in Put, the largelypredominant PA, in the leaves. GA₃ increased the free Put content ofthe leaves and roots. DL--difluoromethylarginine (DFMA), which inhibitsarginine decarboxylase (ADC), did not affect rooting while Put content wasdecreased by 12–15%. DFMA changed neither GA₃ effectsnor those from DFMO when used in combination. An inhibitor of ornithinedecarboxylase (ODC), DL--difluoromethylornithine (DFMO), inhibited leafgrowth as well as rooting and decreased the Put content; these effects werepartially reversed by addition of Put. GA₃ combined with DFMO didnotalleviate the inhibitory effects of DFMO. The GA₃ treatment caused aclear stimulation of ODC activity in the leaves; it also stimulated¹⁴C-putrescine uptake (2.8-fold) by leaves and its translocationtowards roots. These results indicate that the ODC pathway mediates thehormone-induced rooting response in globe artichoke. Moreover, a release of Putfrom its conjugates could contribute to the increase of free-Put levels in theroots under GA₃ treatment. Thus, Put can be considered to be a goodmarker of rhizogenesis.     

Gibberellin structure-activity effects on flower initiation in mature trees and on shoot growth in mature and juvenile Prunus avium

When applied to spurs of mature Prunus avium before floral initiation, gibberellins GA₁, GA₄ and GA₃ inhibited floral initiation by 9–17%, GA₇ by 43%, GA₃ by 65–71% and 2,2-dimethyl GA₄ by 78%. GA₉ and GA₂₀ were inactive. Thus activity only of the GAs with a C-3 hydroxyl was increased markedly by a double bond in the C-1,2 or C-2,3 position, and activity increased with increasing hydroxylation. None of the GAs affected the total number of buds (vegetative and floral) surviving in the spur. Measured by the threshold dose required for activity, seedling shoot growth responses to GA₃, GA₇, GA₁ or GA₄ resembled those of floral initiation, but di-methylation of GA₄ at C-2 had no effect, and GA₉ was as active as GA₇. Mature shoots, including those on rooted cuttings, were less responsive to GA treatment than were juvenile shoots, with terminal shoots on mature trees more responsive than spur shoots. Spur shoot growth on mature trees responded to GA₃ and to a lesser extent GA₇, but not to GA₁ or GA₄. However, all these GAs promoted the growth of terminal shoots on mature trees to similar extents, whereas 2,2-dimethyl GA₄ was less active than GA₄ The differences between juvenile and mature shoot growth in sensitivity to a C-1,2 or C-2,3 double bond, and between mature shoot growth and floral initiation in GA-structure requirements, indicate that phase change alters the GA complement and/or GA receptor/transduction mechanisms of P. avium. The difference in sensitivity to 2,2-dimethyl GA₄ indicates that floral initiation and growth have different requirements for GA transport and/or action.     

Adventitious Root Formation Induced by Gibberellic Acid and Regulated by the Irradiance to the Stock Plants

Pea plants (Pisum sativum L. cv. Alaska) were grown from seeds for eleven days at different irradiances. Cuttings were then excised and rooted at 16 W × m^?2. Gibberellic acid (GA₃, 10^?11 to 10^?3M) was applied to the cuttings immediately after excision. Cuttings from stock plants grown at the highest level of irradiance (38 W × m^?2) formed the lowest number of roots. An increasing number of roots per cutting was obtained by decreasing the irradiance to the stock plants. In cuttings from stock plants grown at low irradiances, low concentrations of GA₃ (10^?8 and 10^?7M) promoted root formation further. No effect on rooting by these GA₃ concentrations was observed when applied to cuttings originating from stock plants grown at the high irradiances. Root formation in all cuttings was inhibited by GA₃ at concentrations higher than 10^?6M. The degree of inhibition by GA₃ was influenced by the irradiance pretreatment and was increased with an increase in the irradiance during the stock plant growth. Seeds from different years produced cuttings with different response patterns regarding the irradiance and GA₃ effects on rooting.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Growth Performance of Cuttings Raised from in vitro and in vivo Propagated Stock Plants of Rosa damascena Mill.

Comparative studies on rooting and growth performance of cuttings raised from in vitro and in vivo grown plants of Rosa damascena are described. Cuttings were treated with different auxins and upon transfer to soil their growth performance was recorded. Overall, the auxin treated cuttings of in vitro raised plants responded better than the cuttings of in vivo raised plants. Optimal response for percentage of rooting, root number, root length and bottom breaks was observed at 100 mg dm^–3 IBA. The cuttings derived from in vitro raised plants showed a significantly better response for percent rooting, root number, root length and bottom buds in control treatments.     

Leaf number and position effects on the survival and performance of grape microcuttings in vitro,and the sensitivity of the cut nodal region to the medium

The presence of leaf in microcuttings of grape cvs. Arka Neelamani and Thompson Seedless promoted rooting in vitro (MS, 1 μM IAA, 0.1 μM GA₃, 3% sucrose) but the effect varied depending on the number of leaves and position of the leaf on the cutting. Single node cuttings with a full-length lower internode and a lamina at top (LAT) showed earlier rooting and more root and shoot growth than cuttings with lamina positioned at the middle (LAM), while cuttings with a leaf at the base (LAB) of the cutting and full-length upper internode exhibited a lower percent rooting and sprouting, poor root and shoot growth, and low survival. Partial or complete removal of the upper internodal segment in LAB cuttings improved rooting and sprouting suggesting the possible operation of an inhibitory effect by the upper internode. Retaining an upper leaf in LAB cuttings (LAB+UL) resulted in necrosis of the upper leaf often followed by the lower one. The extent of necrotic damage was influenced by the leaf area and position or age of the cutting on the stock shoot. Retaining the lower internode in LAB and LAB+UL cuttings which held the node–leaf junction away from the medium, or reducing the concentration of MS medium helped significantly in improving the survival and performance of these cuttings. The difference in reaction between LAB and LAT cuttings was attributable mainly to the difference in the sensitivity of the stem part that came in contact with the medium. Removal of the leaf in LAB cuttings reduced this sensitivity. The majority of the LAB and LAB+UL cuttings, as well as non-rooting or delayed rooting LAT and LAM cuttings, exhibited high purple pigmentation of leaf, petiole and stem. Two-leafed cuttings in vitro showed poor survival, less rooting and low plantlet output compared to single-leafed cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

外源赤霉素对紫斑牡丹种子萌发的影响

以紫斑牡丹种子为试验材料,研究不同浓度的赤霉素(GA_3)处理对种子生根以及生根过程中营养物质、酶活性和内源激素水平变化的影响,为探讨紫斑牡丹种子萌发机制提供依据。结果表明:(1)GA_3处理能够促进种子生根,并以300 mg/L GA_3处理对种子生根效果最好,与对照相比可提前14.67 d生根,生根率可达71.00%。(2)与对照相比,GA_3处理可以在0～15 d时促进种子淀粉水解和可溶性糖的积累,并加速可溶性蛋白的消耗,在0～30 d促进过氧化物酶(POD)活性的提高,从而促进种子萌发生根。(3)在种子沙藏生根过程中,种子脱落酸(ABA)含量呈下降趋势,赤霉素(GA)、玉米素核苷(ZR)和吲哚乙酸(IAA)含量均表现出先上升后下降的趋势,与对照相比,GA_3处理可使种子GA、ZR和IAA的含量在沙藏前期明显上升,以解除种子休眠。研究发现,外源GA_3处理可以调控紫斑牡丹种子内源激素含量和POD活性的变化,促进营养物质转化,从而提前解除种子休眠使其萌发。     

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.     

Interaction of gibberellic and indole-3-acetic acid on root formation in pea (Pisum sativum L.) epicotyl cuttings

Indole-3-acetic acid (IAA) strongly enhanced rooting of etiolated pea epicotyl cuttings while gibberellic acid (GA₃) enhanced rooting only slightly. The promoting effects of the hormones appeared not until 14 d after the onset of treatment. When GA₃ and IAA were applied together, the initiation of rooting started already after 6 d after onset of treatment. It is suggested that gibberellin plays an important role, in combination with auxin, in the initiation of root formation in Pisum cuttings.Abbreviations IAA Indole-3-acetic acid - GA₃ Gibberellic acid     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

A Relationship between Length of Basis and Adventitious Root Formation in Pea Cuttings

A positive correlation between the length of the basis and the ability of the cuttings to form adventitious roots was observed in pea cuttings. Plants with a different basis length (the third internode) were obtained in different ways: Regulation by the level of irradiance, dark treatment or gibberellic acid. The length of the basis was also regulated by excision of the cuttings at different places on the stock plants. With increasing basis length an increase was found in the number of roots subsequently formed. The results were similar in cuttings from plants grown at different levels of irradiance or from dark treated plants. Optimal rooting was obtained by cutting the plants just above the second scale leaf. Cuttings from plants treated with 10^?3M GA₃ showed the same correlation between the length of the third internode and root formation as found in the other experiments, but the number of roots were at a lower level.     

Virus elimination and pathogen-free plantlets regeneration in Cucurbita pepo L.

An efficient in vitro protocol was established for developing pathogen-free plantlets in Cucurbita pepo through meristem culture. Meristems of about 0.3–0.5 mm in size were isolated from shoot tips of 25–30 day old in vitro grown plants. For primary establishment of isolated apical meristem, MS liquid medium supplemented with 2.0 mgl KIN and 0.5 mg/l GA₃ was found to be most effective in both cultivars. MS semisolid medium containing 2.0 mg/l BAP were found to be most effective for shoot development from primarily established meristem in both cultivars. A good number of shoots were not concomitant with good rooting. The best root induction was found in media having 1.0 mg/l IBA in cv. Bulum. It was found that cv. Bulum was better than cv. Rumbo in all stages of meristem culture. The presence of virus in plantlets was achieved by DAS-ELISA test, where 68–81% plantlets have been proved to be virus free among the studied viruses. Healthy growth and vigour was observed in meristem derived plants over their source plants after cultivation under natural conditions.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Distribution of calcium in tomato plants in response to heat stress and plant growth regulators

Two cultivars of tomato with contrasting response to elevated temperature were compared: sensitive-Roma and tolerant-Robin. Experiments were done on fruit explants and on rooted cuttings with small fruits. In both cases⁴⁵Ca was poorly transported to the fruits. Nevertheless in fruit explants elevated temperature (40°C) increased⁴⁵Ca import into the fruits in both cultivars. In the compared cuttings, treated or not treated with growth regulators and at various temperatures, the greatest differences were observed in the amount of⁴⁵Ca transported to the fruits. Sensitive Roma cuttings scantily supplied their fruits with⁴⁵Ca both under optimal temperature and heat stress. In plants previously treated with NOA+GA₃ high temperature increased⁴⁵Ca transport to the fruits. Robin cuttings inversely responded to heat stress by transporting a much higher portion of⁴⁵Ca to the fruits, both in control and NOA+GA₃ cuttings.The diversity of⁴⁵Ca distribution during elevated temperature in cuttings, but not in fruit explants of both cultivars seems to be connected with an ability to control calcium supply to fruit or at least to prevent its decrease; this mechanism is perhaps located outside the cluster.     

The influence of branches and leaf area on rooting and development of Cotinus coggygria cv. Royal Purple cuttings

The influence of branches and mature leaves on the rooting and subsequent development of cuttings was examined, using Cotinus coggygria cv. Royal Purple. A model system was developed, whereby branched cuttings could be harvested from stock hedges and manipulated to alter leaf area, the number of actively‐growing, lateral branches and thus the source: sink ratio for photoassimilates. Highest percentage rooting ((80%) was promoted by retention of branches and a full leaf area. Reducing leaf area resulted in a lower rooting percentage (44%); however, greatest reductions in rooting were associated with the removal of lateral branches ((22%). Applying exogenous auxin (indole‐3‐butyric acid) at the excision point where branches had been removed significantly improved rooting potential, but did not fully substitute for the presence of branches with active shoot tips. Negative effects associated with removing a proportion of mature leaves appeared to relate to alterations in carbon balance rather than an influence on the supply of endogenous auxin to the potential rooting zone. The use of branched cuttings accelerated root and shoot development and resulted in a finished plant being produced more rapidly than is achieved from conventional, non‐branched cuttings. The results presented indicate a means for improving the efficiency of production of Cotinus coggygria, which may be applicable to a wider range of ornamental plants.     

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.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.