Rapid Micropropagation of Edible Bamboo Dendrocalamus asper

Abstract

Micropropagation protocols for Dendrocalamus asper using nodal shoots and seeds culture are described. Multiple shoots were induced through forced axillary branching. Ninety-five percent of the nodal shoot explants taken from juvenile primary and lateral branches, produced multiple shoots through axillary buds activation within 2 to 8 weeks on Murashige & Skoog's (MS) medium supplemented with 0.1-15 mg/l benzyladenine (BA). The cultured seeds also produced multiple shoots (1-20) within 6 weeks on this medium. The multiple shoot differentiation was influenced by the concentration of BA in the medium. The in vitro generated shoots were excised and subculture on MS + 3.0 mg/l BAP for further shoot multiplication. Fifteen to 20 fold rate of shoot multiplication was achieved by regular subculturing. These shoots were multiplied for more than 3 years without loss of vigor. Ninety-five percent of the shoots were rooted, when propagules (each consisting of cluster of 3 shoots) were transferred on to MS medium with 3.0 mg/l NAA or 10 mg/l indole-3-butyric acid (IBA).

To date, 18,000 plants (through axillary bud initiated from nodal ex-plants) and 6,000 plants from seed culture have been hardened and acclimatized. 12,000 plants have been field transferred.     

An Efficient Micropropagation Protocol for High-Frequency Plantlet Regeneration from Liquid Culture of Nodal Tissues in a Medicinal Plant,Turnera ulmifolia L.

A highly efficient, stable, and cost-effective micropropagation protocol for the conservation of a medicinal plant Turnera ulmifolia L. was established from nodal tissues via multiple axillary shoot proliferation on using Murashige and Skoog’s (MS) liquid nutrient medium. To begin with, nodal explants were placed on agar gelled medium amended with 2.0 mg L^?1 6-benzylaminopurine (BAP) and 0.1 mg L^?1 indole-3 acetic acid (IAA) for shoot induction. Subsequently, elongation of regenerated shoots could be possible on liquid MS medium supplemented with 0.5 mg L^?1 BAP and Kin (kinetin) each along with 0.1 mg L^?1 IAA where high frequency of regeneration in terms of number of shoots (47.2 shoots/explant) was achieved. Furthermore, long and healthy shoots (4?5 cm in length) were rooted on agar gelled half-strength of MS medium supplemented with 2.0 mg L^?1 indole-3 butyric acid (IBA). Finally, in vitro regenerated plantlets were gradually acclimatized in the greenhouse and transferred to the field successfully.     

Clonal propagation ofGmelina arborea Roxb. byIn vitro culture

In vitro propagation technique ofGmelina arborea multipurpose and a fast growing tree species was studied. Nodal segment including axillary bud was used as a explant. They were cultured on MS media containing various concentrations (0–10 mg/l) of BAP alone or in combination with 0.002 mg/l of IBA. Nodal segments showed axillary bud proliferation in almost all media tested. MS media containing 0.22 mg/l of BAP alone and 2 mg/l of BAP in combination with 0.002 mg/l of IBA were effective for inducing multiple shoots and shoot elongation. MS medium supplemented with 0.02 mg/l of NAA and 1 mg/l of IBA gave the best result for rooting. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the green house. Adventitious shoots production from stem explants that were taken from regenerated plantletin vitro was also discussed. Stem segments were tested for their morphogenetic potential on MS media with various combinations and concentrations of BAP, zeatin and TDZ. Successfull result was obtained on MS media supplemented with 2 mg/l of BAP and 1 mg/l of zeatin or supplemented with 0.5 mg/l of BAP and 0.5 mg/l of TDZ. The shoots obtained on MS media containing 2 mg/l of BAP and 1 mg/l of zeatin rooted on MS media containing 0.02 mg/l of NAA and 1 mg/l of IBA, and plantlets were successfully obtained. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forest Society (1998).     

Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

Micropropagation of Anogeissus rotundifolia Blatt,and Hallb.-An Endemic and Rare Tree of the Thar Desert

Abstract

Multiple shoots and plantlets were developed in vitro from cotyledonary nodal segments of in vitro raised seedlings of Anogeissus rotundifolia (syn. A. sericea var. nummularia)-a rare and endemic tree species of the Thar Desert. About 15-20 shoots differentiated from a single cotyledonary node within four weeks on Mu-rashige and Skoog's (MS) basal medium containing 0.1 mg l^?1 indole-3-acetic acid (IAA) + 2.0 mg l^?1 6-benzylaminopurine (BAP) + additives (25 mg l^?1 each of adenine sulphate, L-arginine, and citric acid, and 50 mg l^?1 of ascorbic acid) at 26 ± 2°C temperature and 36 μmol m^?2 s^?1 photon flux density with a 12 h/day photoperiod. The shoots produced in vitro were further multiplied by subculturing on fresh medium. The original cotyledonary nodal segment was repeatedly transferred (5 to 6 times) onto fresh medium containing 1.0 mg l ^?1 BAP + 0.1 mg I ^?1 IAA + additives to yield fresh crops of multiple shoots. These shoots were rooted on half-strength MS medium supplemented with 0.1 mg l^?1 indole-3-butyric acid (IBA). Plantlets were transferred to pots containing sand-dune soil and ver-miculite it the ratio of 4:1 (v/v) and hardened in a growth chamber for two weeks and finally transferred to a greenhouse. From a single cotyledonary node about 1500 plantlets could be developed within four months. The method developed is useful for mass multiplication and for the conservation of germplasm of Anogeissus rotundifolia.     

爬行卫矛下胚轴高频离体再生体系的建立(英文)

In this paper,a protocol for efficient shoot regeneration was successfully developed from hypocotyl explants of Euonymus fortunei var.radicans.Some factors that influenced shoot regeneration such as different combinations of plant growth regulators,types of medium and inoculation ways were studied in order to establish an efficient plant regeneration for transformation.The results showed that hypocotyl explants wero horizontally cultured on a basic medium composed of MS medium supplemented with 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA for induction and development of adventidous shoots.Ninety-four percent of regeneration frequency and 5.1 shoots per explants were obtmned after 30 days of culture.Regenerated shootsproliferated efficiently on a shoot multiplication medium consisting of MS medium containing 1.0 mg·L-1 BAP and 0.1 mg·L-1 NAA.Microshoots were rooted on a rooting medium made up of MS medium enriched with O.5 mg·L-1 IBA and O.5 mg·L-1IAA.After hardening,90% of plants were successfully established under greenhouse conditions.Histological observation revealed that shoot primordium originated from subepidermal cells of hypocotyl explants and directly developed into adventitious shoots without caHus formation.     

Micropropagation of Selected Genotype of Aloe vera L.—An Ancient Plant for Modern Industry

Aloe vera Linn. (Syn. Aloe barbadensis Mill; Gwar-patha in Hindi) belongs to family Liliaceae. The plant, for its medicinal properties, has commercial value. Some of the genotypes of Aloe vera are consumed as a vegetable and processed to make curry and other edible products. We report here on the development of an efficient method for rapid clonal propagation by shoot proliferation from axillary meristem(s) of selected germplasm of Aloe vera. Explants were pretreated with 0.1% aqueous solution of both streptomycin and bavistin separately, each for 15 min. These were surface sterilized with 0.1% aqueous solution of mercuric chloride (HgCl₂) for 4–5 min and washed several times with autoclaved water. These were kept in a chilled, sterile antioxidant (200.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 of citric acid, and 25.0 mg L^?1 of polyvinylpyrrolidone; PVP) solution and cultured on semi-solid Murashige and Skoog's (MS) medium. The bud explants produced multiple (10.3 ± 0.675/explant) shoots on MS medium containing 13.32 μM of 6-Benzylaminopurine (BAP) and 100.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 each of citric acid and PVP, with 25.0 mg L^?1 each of arginine and adenine sulphate as additives. The shoots were further multiplied by (a) repeated transfer to fresh MS medium with additives + 13.32 μM BAP, and (b) subculturing on MS medium with a lower (4.44 μM) concentration of BAP. On MS medium containing 4.44 μM of BAP and additives, a maximum number (27.8 ± 0.63) of shoots were produced. In liquid MS medium with 4.44 μM of BAP, the rate of shoot multiplication increased and the vigor of the shoots improved. One hundred percent of the cloned shoots rooted under in vitro conditions on hormone-free half-strength MS salts containing 200.0 mg L^?1 of activated charcoal at 32 ± 2°C. The cloned shoots treated with 2.46 mM of indole-3-butyric acid (IBA) or 2.473 mM of β-naphthoxyacetic acid (NOA) for 5 min rooted under ex vitro conditions in the greenhouse. The rooted plants were hardened in the greenhouse and stored under an agro-net house. The cloned plants were transferred under different field conditions at various sites in Western Rajasthan. These plants grew normally. The higher rate of shoot multiplication and easier approach of direct rooting and hardening make this method superior to the methods previously reported on cloning/tissue culture of Aloe species. From a single shoot bud, approximately 5000 plants can be produced within 180 days.     

Micropropagation of Anogeissus latifolia (Roxb. Ex DC.) Wall,ex Guill. & Perr.- A Tree of Fragile Ecosystems

Abstract

A micropropagation process was developed for Anogeissus latifolia-a tree of fragile ecosystems. Multiple shoots were regenerated from cotyledonary node and epicotyl explants on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole-3-acetic acid (IAA) + 1.5 mgl ^-l 6-benzylaminopurine (BAP) + additives (25 mgl ^-l each of adenine sulphate, L-arginine, ascorbic-, citric acids and 1.0 mM L-asparagine) + 200 μM Fe-EDTA (ferric-ethylenediaminetetraacetic acid) salt. The shoots differentiated in vitro were subcultured and repeatedly transferred onto fresh medium but with 1.0 mgl^-1 of BAP to achieve 4-5 fold rate of shoot multiplication. After every 4th week of culture, from each culture bottle 8-10 shoots could be harvested for rooting. The shoots produced in culture were rooted in vitro on half strength MS medium with 1.0 mgl ^-l either of IBA (indolebutyric acid) or NAA (naphthaleneacetic acid). The shoots were pulse treated with combination (100 mgl ^-l each) of IBA and NOA (2-naphthoxy acetic acid) rooted ex vitro. The ex vitro root induction method is highly efficient and plantlets so generated could be acclimatized and pot transferred. The process developed can be used for large scale production of plants of A.     

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA₃). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.This revised version was published online in November 2005 with corrections to the Cover Date.     

Regeneration and multiplication of Albizia procera Benth. through organogenesis

Vegetative propagation techniques are recognized as indispensable tools for mass multiplication of important multipurpose trees adopted in different agroforestry systems. Albizia procera, one among important species, is difficult to propagate commercially either by stem / root cuttings or layering. A study was undertaken to develop procedure for its in vitro regeneration through organogenesis. Explants collected from 15±2 yr-old mature plus trees and from 15 days old juvenile seedlings were regenerated with exogenous application of different hormones. Epicotyl and hypocotyl explants excised from juvenile seedlings showed higher callusing than axillary bud and shoot tip explants derived from mature trees. Benzylaminopurine (BA) at 3 μg/l was most effective, which induced hundred percent callusing in epicotyl and hypocotyl explants in 1/2 Murashige and Skoog (MS) medium. Callus originated from axillary buds and apical shoot tips of mature trees failed to form organs, however callus derived from epicotyl and hypocotyl explants proliferated and formed de novo shoots and leaflets. A concentration of 3 μg/l of BA was found effective for shoot proliferation. Shoots grew vigorously in 2 μg/l gibberellic acid (GA₃) treatment and rooted in 1/2 MS medium supplemented with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Rooting was most successful on medium supplemented with 6 μg/l IBA alone on which 93.3% of the shoots formed roots. Sand or vermiculite supplemented with 4 ml of yoshida solution proved as best hardening media, which recorded 70-80% survival of plantlets. One year old tissue culture raised plants had comparatively more height, collar diameter, biomass, and root shoot ratio than plants raised from cuttings and seeds of the same age. The procedures enumerated provide a basis for the development of in vitro techniques for rapid multiplication of A. procera. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue culture ofRosa Chinensis var.Floribunda

The tissue culture of Schloss Mannheim(Rosa Chinensis var.Flaribunda) with full and unsprouting bud of stem segments as the explants was experimented. The result shows that the buds sprouted best on MS medium with the addition of 6-BA 1.0 mg/L, and differentiation was best on MS medium with addition of 6-BA 1.5 + NAA 0.05 + ZT 0.1 mg/L or KT 1.0 + NAA0.05 + ZT 0.1 mg/L. The MS medium with addition of 6-BA 0.3 + NAA 0.0 5+ ZT 0.1 mg/L or KT 0.3 + NAA0.05 + ZT 0.1 mg/L showed a good result for developing strong shoots. 1/2 MS medium with the addition of IBA 0.1 mg/L or IBA 0.1 + NAA 0.02 mg/L had best result for rooting. The plantlets should be transplanted from test-tube to soil when they grew to 2.5 ∼ 4.0 cm high and have 3 ∼ 5 strips short roots. A higher survival rate was obtained under the conditions of controlling humidity and temperature.     

Establishment of in vitro culture of Populus euphratica Olivier

The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine (BAP) and gibberellic acid (GA) in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingjiang, west China, on selected media with MP2 0.5 mg·L-1 BA 0.1 mg·L-1 NAA. The shoots were elongated on a medium with 0.25 mg·L-1 BAP, 0.1 mg·L-1 NAA and 2 mg·L-1 GA and were then rooted on a medium with 0.2-0.5 mg·L-1 IBA. All the media were incorporated with 30 g·L-1 sucrose and an adjusted pH at 6.3.     

罗扶木瓜组培快繁技术

以罗扶木瓜茎尖为试材进行离体组培快繁技术的研究结果表明：最佳诱导产生不定芽的培养基为MS 6-BA1．0mg／L NAA0．2mg／L，增殖培养基为MS 6-BA2．5mg／L NAA0．1mg／L，生根培养基1／2MS IBA0．2mg／L ABT1．0mg／L。试管苗经适宜条件练苗驯化的移植大田，成活率可达82％以上，而且生长良好。     

金冠白蜡快繁体系建立的研究

以金冠白蜡带腋芽的茎段作为外植体,进行组织培养快繁体系的建立。结果表明:使用消毒剂0.1%HgCl2+吐温消毒3min为最佳外植体消毒处理,存活率可达88.3%;启动培养基采用MS+6-BA 1.0mg/L+IBA 0.2mg/L为宜;继代增殖培养选择MS+6-BA 2.0mg/L+KT 1.0mg/L+IBA 0.5mg/L的培养基,其增殖系数为4.617;生根培养以1/4MS+IBA 1.0mg/L的培养基进行培养,生根率达75%。     

Micropropagation of an endangered Indian sandalwood (Santalum album L.)

Santalum album is known as East Indian sandalwood. It is the most economically important tree harvested for heartwood oil, and India is among the chief exporters of sandalwood and its products. Multiple shoots were induced from nodal shoot segments derived from a 50- to 60-year-old candidate plus tree (CPT) on Murashige and Skoog (MS) medium supplemented with 0.53 μM α-naphthaleneacetic acid (NAA) and 11.09 μM 6-benzylaminopurine (BA). In vitro differentiated shoots were multiplied on MS medium with 0.53 μM NAA, 4.44 μM BA, and additives: 283.93 μM ascorbic acid, 118.10 μM citric acid, 104.04 μM cystine, 342.24 μM glutamine, and 10% (v/v) coconut milk. New shoots were harvested repeatedly for up to three subculture passages on fresh medium at 4-week intervals. Microshoots treated with 98.4 μM indole-3-butyric acid (IBA) for 48 h produced roots on growth-regulator-free, quarter-strength MS basal salts medium with vitamin B₅ and 2% sucrose. In vitro root induction was achieved from microshoots pulsed with 1230 μM IBA for 30 min in soilrite rooting medium. The percentage of rooting in soilrite was higher than that for agar medium, and in vitro raised plants were established in the field and showed normal growth.     

Micropropagation of Salvadora oleoides—An Oil Yielding Tree of Arid Forests

Salvadora oleoides is an ecologically important multipurpose tree of the arid forest that occurs in saline areas of northwest India. The seed of this plant yields non-edible commercially usable oil. Poor seed germination, low seed viability, and increasing industrialization are some of the constant factors which significantly affect the status of the natural population of this plant. Therefore, there is a great need to develop an efficient propagation system using the tissue culture technique. In the present communication, we demonstrate the development of an in vitro propagation system for S. oleoides. Multiple shoots were induced from nodal segments harvested from about 25- to 30- yr-old lopped trees of S. oleoides on MS medium + 0.1 mg L^?1 NAA (Naphthalene acetic acid) + 2.5 mg L^?1 BA (6-Benzylaminopurine) + additives. The shoots were multiplied by (a) repeated transfer of the mother explants on MS medium + 1.0 mg L^?1 BA + 0.1 mg L^?1 NAA + additives and (b) subculturing of shoot on MS + 1.0 mg L^?1 BA + additives. About 84% shoots rooted ex vitro on soilrite within 3–4 weeks when base (4–5 mm) of shoots was treated with 100 mg L^?1 of IBA (Indole-3-butyric acid) for 5 min. The plantlets were hardened successfully in the greenhouse and transferred to the pots and field. To the best of our knowledge, this is the first report of a regeneration protocol for S. oleoides from explants obtained from mature trees. Use of the ex vitro rooting technique for plant production serves as a more economical option as it reduces labor, cost, and time. We suggest that the methods developed and described in this article can be used for large-scale plant production and conservation of germplasm of this tree species.     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Preservation of Juniperus thurifera L.:a rare endangered species in Algeria through in vitro regeneration

Juniperus thurifera L.is an endemic Cupres saceae from the Aure`s Mountains of north eastern Algeria and endangered,in part,due to the scarcity of viable seeds It is threatened by other abiotic factors and the lack of an effective management strategy will increase its risk o extinction.The dearth of information on its in vitro regeneration impedes its application in forest managemen programs.We therefore developed a micropropagation protocol using microcuttings with auxiliary buds.Cuttings were grown on different combinations of media supplemented with plant growth regulators at different concentrations.The highest number of shoots and branches regenerated from original shoots was obtained on Woody Plant Medium(WPM)supplemented with 6-benzylaminopurine(BAP)(0.5 mg L^-1)and 2,4-dichlorophe noxyacetic acid(2,4-D)(0.25 mg L^-1).The best elongation of shoots was achieved with WPM supplemented with0.5 mg L^-1of BAP and 0.25 or 1 mg L^-1 of 2,4-D.On the second subculture,shoots had a higher number of branches than those of the first.The highest rooting rate,38.8%,was obtained with shoots cultured in 1/2 Murashige and Skoog(MS)medium supplemented with 5.0 mg L^-1each of indol-3-butyric(IBA)and naphthalene acetic acid(NAA).Similarly,the highest root numbers and lengths were produced on 1/2 MS medium supplemented with IBA and NAA(5.0 mg L^-1each).During transfer to acclimatization,rates of plant losses of 50% occurred.The second part of the experiment showed that the best shoot callusing was on WPM supplemented with BAP and 2,4-D,with either the combination 0.5+0.25 or 0.25+0.25 mg L^-1.The results of this research provide a starting point for further studies on in vitro regeneration of J.thurifera for the sustainable management of its unique ecosystem in the Mediterranean basin.     

油茶优良无性系组织培养与植株再生

以油茶优良无性系的腋芽为外植体,分别采用附加不同种类激素的MS培养基对其进行组织培养试验,结果表明:不定芽的最佳继代增殖培养基为MS+6-BA0.8 mg.L-1+NAA0.1 mg.L-1,诱导生根最适培养基为1/2MS+IBA0.5 mg.L-1+NAA0.2 mg.L-1,生根率达87.5%。     

An efficient method for clonal propagation and in vitro establishment of softwood shoots from epicormic buds of teak (Tectona grandis L.)

Softwood shoots were produced from 40 cm long stem segments placed horizontally in flat trays containing sterilized sand under natural light or shade conditions for subsequent rooting and micropropagation studies in teak (Tectona grandis L.). Higher number of shoots (6.17) per log was produced under natural light as compared to shade conditions. Forcing was also better in natural light as compared to shade in terms of shoot length, number of nodes or leaves. For rooting, 2–4 cm long softwood shoots were excised and treated with either indole-3-butyric acid (IBA) or α-naphthyl acetic acid (NAA) at 0, 1000, 2000 or 3000 μmol·L^–1 each or with combinations (1000 + 1000, 2000 + 2000 or 3000 + 3000 μmol·L^–1) and then placed in flat trays containing autoclaved sand at 25 ± 2ºC in 16 h photoperiod at 35 µmol·m^–2·s^–1. After 28 days, softwood cuttings treated with IBA + NAA (3000 + 3000 μmol·L–1) had highest rooting percentage (89.3%) with 5.5 mean roots. Shoot apex and nodal explants of softwood cuttings were pretreated with 0.1% (w/v) ascorbic acid, boric acid, activated charcoal, citric acid, glutamine or polyvinylpolypyrollidone (PVP) for 24 h to remove phenolic compounds before surface disinfestation. Glutamine (Gl) and PVP were equally effective resulting in 60% establishment of shoot apices on MS medium supplemented with 10 μmol·L–1 6-benzylaminopurine (BAP) + 5 μmol·L–1 NAA. Using shoot apices, highest (42.80) number of multiple shoots with 54.33 mm shoot length were obtained on MS + BAP (8.8 μmol·L–1) + IBA (2 μmol·L–1) after 45 days. Shoots were successfully rooted and acclimatized to greenhouse