Changes in the concentrations of auxins and polyamines during rooting of in-vitro-propagated walnut shoots

Rooting was induced in in-vitro-propagated walnut (Juglans regia L.) shoots by subculturing the shoots on rooting medium containing agar and 3 mg l(-1) indole-3-butyric acid (IBA) for 7 days in darkness. Changes in the concentrations of endogenous free indole-3-acetic acid (IAA), indole-3-acetylaspartic acid (IAAsp) and free polyamines were determined during culture on root-inducing medium. In extracts of whole shoots, the concentration of free IAA showed a transient peak at 60 h (around 48 h in extracts from basal shoot portions) and then remained at a relatively low concentration for the remainder of the 7-day culture period. The concentration of IAAsp in extracts of whole shoots peaked at about the same time as the concentration of free IAA, whereas the IAAsp concentration in extracts from basal shoot portions peaked earlier, at around 12 h. The concentrations of free polyamines in extracts of whole shoots increased soon after the shoots were transferred to root-inducing medium. The concentrations of IAA and IAAsp remained stable when the rooted shoots were transferred to a vermiculite/gelrite mixture (without auxin) and grown in light.     

Effects of cutting types and hormonal concentration on vegetative propagation of Zanthoxylum armatum in Garhwal Himalaya,India

Experiments were carried out to define the effects of hormonal concentrations on semi-hard wood(SHW) and hard-wood(HW) branch cuttings of the Z.armatum. SHW and HW cuttings were collected in the month of March. The SHW and HW cuttings were treated with different concentration of indole-3-acetic acid and indole-3-butyric acid(IAA and IBA) and placed in vermiculite rooting medium for 90 days under 1-min misting after 10 min. Sprouting, rooting percentage, sprout number, sprout length, root number, and length were measured.The highest rooting and sprouting rate, 64.0 %, was obtained at the 0.3 % IBA treatment in the SHW cuttings.Similarly sprout length and number of roots per cutting were also higher at the 0.3 % IBA treatment in the SHW cuttings. The number of shoots per cutting was higher at the 0.3 % IAA treatment in the SHW cuttings. Root length per cutting was higher in 0.4 % IBA treatment in the SHW cuttings. The results indicated that 0.3 and 0.4 % IBA treatment produce higher rooting percentages as well as the number of roots and their length in SHW cuttings. The HW cuttings produced maximum rate of 18.0 % rooting in0.5 % IBA treatment. The control set and lower concentrations of IBA and IAA completely failed to root in the mist chamber.     

金叶银杏硬枝扦插繁殖生根过程及其生根机制研究

【目的】研究不同生长调节剂处理对金叶银杏硬枝扦插生根率和成活率的影响,探讨金叶银杏的生根机制,为金叶银杏苗木扦插快繁技术体系的建立提供技术支持和理论指导。【方法】通过NAA和IBA处理金叶银杏硬枝插穗,检测它们对插穗的生根状态、生根指标及IBA对扦插过程中插穗皮部可溶性糖、抗氧化酶、激素含量的影响,探究金叶银杏硬枝扦插繁殖技术和生根机理。【结果】1)金叶银杏插穗愈伤组织诱导期和形成期、不定根发生期和形成期分别在插后的20~26、26~42、42~53、53~79 d。2)800 mg·L^-1 IBA和400 mg·L^-1 NAA+400 mg·L^-1IBA(混合)处理的插穗愈伤组织和不定根出现期及生根率与根数都较其他相应处理的效果好,其中生根率分别达64.4%和51.1%;不定根数分别达8.6条和9.3条。3)插穗生根过程中(500 mg·L^-1 IBA处理)基部韧皮部的SOD、PPO活性在插后40、60 d达到高峰,均比对照提前20 d达到峰值;插穗的POD活性出现上升-下降-升高的趋势,插穗可溶性糖也出现升高-下降-升高的趋势,但时间点不一致;而淀粉的含量出现下降-上升的趋势。4)生根期间插穗基部韧皮部的IAA含量出现了2个峰值和一个谷值;而ZR含量在第40天达到峰值后下降;GA含量先下降后上升;ABA含量在插后的前期高后期低,且值都低于同期对照。IAA/ABA比值在插后的第40天(愈伤组织形成期)达到谷底,后又上升(不定根形成期)。IAA/GA在插后的40~60 d内快速上升,80 d后又开始大幅下降;IAA/ZR值在扦插愈伤组织形成期(40 d)呈下降趋势,到60 d时(不定根发生期)又急剧上升到峰值。【结论】高活性的SOD和POD有利于插穗初期愈伤组织的形成(40 d),高活性的POD与PPO有利于不定根的形成(60 d)。较高的IAA含量是保证愈伤组织形成(20~40 d)的必要条件,不定根形成期(60 d)需要较高的IAA/ZR、IAA/ABA、IAA/GA比值。本研究为金叶银杏扦插繁殖技术和生根机理的研究及金叶银杏苗木的快速繁殖生产提供技术支持。     

Effects of IBA on the rooting of branch cuttings of Chinese jujube (<Emphasis Type="Italic">Zizyphus jujuba</Emphasis> Mill.) and changes to nutrients and endogenous hormones

‘Zhongqiusucui’ jujube secondary shoots were treated with 3-indolebutyric acid (IBA) at three concentrations, 500, 1000 and 1500 mg/L. Results show that IBA could significantly enhance rooting and root characteristics of cuttings and were best with IBA at 1500 mg/L. In the rooting process, the formation of adventitious roots was related to the consumption and accumulation of nutrients (soluble sugars and proteins) and the changes in endogenous hormones in phloem, leaf tips and leaf bases. The rooting of cuttings had a positive correlation with the consumption of soluble sugars during the period of callus formation and with the accumulation of soluble sugars during adventitious root formation and growth. Rooting was positively related to the breakdown of soluble proteins in the phloem when the callus formed, and had a positive correlation with its accumulation during adventitious root formation and growth. Leaf tips and leaf bases showed a reverse trend in changes of soluble protein. However, together with the phloem, leaf tips and leaf bases regulated and controlled the formation and development of adventitious roots. The main activities of soluble proteins exist in the leaf tips as this was the main source of soluble proteins. The relation between rooting and IAA (indole-3-acetic acid) content in phloem was positive and thus a high concentration of IAA could benefit the induction and formation of adventitious roots. However, rooting was negatively related with ABA (abscisic acid) and GA (gibberellic acid) and a high concentration of both could inhibit the induction and formation of adventitious roots. Rooting had a positive correlation with phloem IAA/ABA ratios, and higher ratios could improve rooting. Low concentrations of ZR (zeatin riboside) triggered the induction of adventitious roots, while higher concentrations promoted root growth. Endogenous hormones in leaf tips and bases had an impact on rooting. The activities of endogenous hormones mainly existed in leaf tips because they play a major role in the production and consumption of IAA and its ABA content increased during rooting. The ZR in leaf tips influenced the rooting of cuttings, especially in the callus formation and rooting stage. Leaf tips were the main source of GA.     

Vegetative propagation of Colutea arborescens L., a multipurpose leguminous shrub of semiarid climates

The vegetative propagation of the multipurpose leguminous shrub Colutea arborescens from leafless hardwood cuttings was examined. Specific attention was paid to the factors: 1) cutting origin (apical, medial or basal); 2) indole-3-butyric acid (IBA) treatment (0, 50 or 200 ppm); and 3) relative humidity of the propagation environment (high or low humidity). After 120 days, 67.6% of the C. arborescens cuttings had rooted. The rooting ability of the cuttings was significantly influenced by the cutting origin and hormonal treatment. No effects on rooting percentage were shown by the relative humidity of the propagation environment. The best rooting response (95%) was shown by basal cuttings treated with 200 ppm IBA. Cutting origin, IBA treatment and relative humidity were significantly related to the root number, longest root length and the fresh/dry biomass of roots and leaves. Under conditions of high humidity, basal cuttings treated with 200 ppm IBA gave rise to the most highly developed plants. The positive relationship between sprouting and rooting suggests that successful propagation is related to the shoot formation potential of the cutting's food reserves. These findings were used to propose an efficient method for the vegetative propagation of C. arborescens plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Requirements for in vitro rooting of Quercus robur and Q. rubra shoots derived from mature trees

Stabilized shoot cultures initiated from crown material of six adult Quercus robur L. trees and from basal epicormic shoots of a Quercus rubra L. tree showed good in vitro rooting capacity. An initial five-day dark period generally improved the rooting response but was detrimental to plantlet quality. There were clonal differences in rooting capacity. The concentration and exposure time of the indolebutyric acid (IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium containing 25 mg l(-1) IBA for 24 h and subsequent transfer to an auxin-free medium containing 1% activated charcoal. For all clones tested, the charcoal benefited both shoot quality and root system development, the latter being enhanced by the formation of many lateral roots. Total root system area and length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium lacking charcoal. Because darkening the basal part of the shoots with aluminum foil during the rooting phase only caused a small increase in rooting, we conclude that the large effect of charcoal on rooting was the result of adsorption of inhibitory compounds from the medium or the explant or both, rather than of basal darkening. Other factors affecting the rooting response of Q. robur were: (a) the position on the tree of the material from which cultures were initiated (the topophysical effect); and (b) shoot quality. Recycling the same horizontally placed explant on multiplication medium allowed three successive crops of shoots to be obtained, and rootability was typically maintained from crop to crop.     

Ectomycorrhizal fungi and exogenous auxins influence root and mycorrhiza formation of Scots pine hypocotyl cuttings in vitro

We studied the ability of the ectomycorrhizal (ECM) fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr. (Strain H), to produce indole-3-acetic acid (IAA) and to affect the formation and growth of roots on Scots pine (Pinus sylvestris L.) hypocotyl cuttings in vitro. Effects of indole-3-butyric acid (IBA) and the auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), on rooting and the cutting-fungus interaction were also studied. Both fungi produced IAA in the absence of exogenous tryptophan, but the mycelium and culture filtrate of Pisolithus tinctorius contained higher concentrations of free and conjugated IAA than the mycelium and culture filtrate of Paxillus involutus. Inoculation with either fungus or short-term application of culture filtrate of either fungus to the base of hypocotyl cuttings enhanced root formation. Inoculation with either fungus was even more effective in enhancing root formation than treatment of the hypocotyl bases with IBA. Fungal IAA production was not directly correlated with root formation, because rooting was enhanced more by Paxillus involutus than by Pisolithus tinctorius. This suggests that, in addition to IAA, other fungal components play an important role in root formation. Treatment with 5 microM TIBA increased the rooting percentage of non-inoculated cuttings, as well as of cuttings inoculated with Pisolithus tinctorius, perhaps as a result of accumulation of IAA at the cutting base. However, the marked reduction in growth of Pisolithus tinctorius in the presence of TIBA suggests that the effects of TIBA on rooting are complicated and not solely related to IAA metabolism. The high IAA-producer, Pisolithus tinctorius, formed mycorrhizas, and the IBA treatment increased mycorrhizal frequency in this species, whereas TIBA decreased it. Paxillus involutus did not form mycorrhizas, indicating that a low concentration of IAA together with other fungal components were sufficient to stimulate formation and growth of the roots, but not the formation of ECM symbiosis.     

Influence of growth hormones on adventitious root formation in semi-hardwood cuttings of <Emphasis Type="Italic">Celasturs paniculatus</Emphasis> Willd.: a contribution for rapid multiplication and conservation management

Celastrus paniculatus Willd. (Celastraceae) is an important medicinal tree valued for its seeds. Indiscriminate collection of this plant from the habitat zones has posed a serious threat to its existence in the wild, results in globally a threatened plant species. Ex situ conservation is a prioritized agenda of research for redlisted plant species. Semi-hardwood stem cuttings collected from 10 to 15 year old trees were treated with different concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 g l⁻¹) of growth hormones; indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) for positive effect on root induction. After 90 days of treatment, results were analyzed for percentage of rooting, root number and average root length per rooted cuttings. The highest rooting (57%) response with highest number of roots (~77.2) was obtained in indole-3-acetic acid at 3.0 g l⁻¹. Overall, high significant (P ≤ 0.05) rooting response was noticed in IAA treatment comparative to hormones IBA and NAA. Rooted cuttings exhibited 100% survival in the experimental field. This study reveals the propagation of semi-hardwood cuttings derived from partially matured branches is possible and it provides a basis for conservation of this threatened plant species by reducing pressure on its natural populations.     

Genetic variation in rooting ability of loblolly pine cuttings: effects of auxin and family on rooting by hypocotyl cuttings

After about 20 days, hypocotyl cuttings from 20-day-old loblolly pine (Pinus taeda L.) seedlings rooted easily in the presence of the auxin indole-3-butyric acid (IBA), with roots forming directly from xylem parenchyma. In contrast, woody cuttings from 1-2-year-old hedged seedlings formed roots indirectly from callus tissue in 60-90 days, but IBA had little effect on rooting. Variation in rooting among hypocotyls from both half- and full-sib families was highly significant in response to IBA, and rooting did not occur within 20 days unless IBA was applied. Hypocotyls from poor rooting families tended to produce fewer roots per cutting than hypocotyls from good rooting families. Rooting by woody cuttings and hypocotyl cuttings from the same nine full-sib families was weakly correlated, raising the possibility that at least some common genetically controlled processes were affecting rooting by both types of cutting. The phytotropin N-1-naphthylphthalamic acid (NPA), supplied at 1 micro M with 10 micro M IBA, significantly inhibited rooting by hypocotyl cuttings from both good and poor rooting families, but there was no significant family x treatment interaction. Family variation in rooting ability may be a function of the frequency of occurrence of auxin-responsive cells in the hypocotyls.     

青钱柳子叶不定根的发生机制

本文以青钱柳子叶为材料,WPM为基本培养基,研究不同质量浓度的外源ABA和IBA对子叶不定根发生的影响,并对不定根发生与内源激素的变化关系进行探讨.结果表明:添加0.5～5 mg·L~(-1) IBA,对子叶不定根发生均有不同程度的促进作用,以1.5 mg·L~(-1)效果最佳,诱导生根率、平均生根条数和根长均达最大,生根率可达到100%;而添加0.01～0.5 mg·L~(-1) ABA和不加激素的对照没有不定根的发生.内源激素的动态测定表明IAA对不定根的发生起关键性作用,根原基的启动需要较高的内源IAA,而不定根的形成与根的伸长需要相对较低的内源IAA水平.较高的IAA/ABA,IAA/GA_3比值有利于不定根的发生,能较好反映出生根过程中内源激素间的平衡需求,GA_3可能不是影响不定根发生的关键因素.研究结果为青钱柳生根诱导及其机制的研究提供依据.     

Low temperature, IBA concentrations and optimal time for adventitious rooting of Eucalyptus benthamii mini-cuttings

Eucalyptus benthamii is a forest species of economic interest that has difficulty with seed production and also is considered to have difficulty with adventitious rooting using propagation techniques,such as cutting or mini-cutting.We aimed to assess the adventitious rooting percentage under different storage times in low temperatures and at various IBA(indole-3-butyric acid) concentrations to determine the optimal time of permanence for rooting Eucalyptus benthamii minicuttings in a greenhouse.Shoots collected from mini-stumps cultivated in a semi-hydroponic system were used to obtain the mini-cuttings.For the first experiment,the mini-cuttings were stored at 4℃ for 0(immediate planting),24,48,72,96 and 120 h.The second experiment evaluated the rooting dynamic to determine the optimal time of permanence for minicuttings in a greenhouse.The basal region of the mini-cutting was treated with various IBA solutions:0(free of IBA),1,000,2,000,3,000 and 4,000 mg·L-1.Every seven days(0(immediate planting),7,14,21 and 28 days),destructive sampling of the mini-cuttings was performed to evaluate the histology of the adventitious rooting.Eucalyptus benthamii minicuttings should be rooted immediately after the collection of the shoots.The 2,000 mg·L-1 IBA concentration induced a greater speed and percentage of adventitious rooting,and an interval of 35 to 42 days was indicated for permanence of the mini-cuttings in the greenhouse.Exposure to low temperature induced adventitious root formation with diffuse vascular connections.     

Vegetative propagation of African Blackwood (<Emphasis Type="Italic">Dalbergia melanoxylon</Emphasis> Guill. &; Perr.): effects of age of donor plant,IBA treatment and cutting position on rooting ability of stem cuttings

A study to determine the best vegetative propagation protocol was carried out for Dalbergia melanoxylon. Natural regeneration of D. melanoxylon is limited due to poor seed viability and germination. The effects of age of donor plant, indole-3-butyric acid (IBA) treatment and cutting position on rooting of stem cuttings were investigated. The effect of age of donor plant was significant (P < 0.001) only for percent rooting, position effect was significant for all studied parameters, and IBA treatment at 300 ppm was insignificant only for percent callusing. Interactive effects of cutting position × age of donor and cutting position × IBA treatment were significant for percent rooting and root number per cuttings while interactive effect of age × IBA was significant only for percent rooting. Observed difference in rooting performance could be due to uneven distribution of root promoting and inhibiting substances with respect to age of donor plant and IBA which intensified root formation. Cuttings from juvenile donor plant performed better in all rooting parameters than mature donor plant. The highest percentages in rooting were (71.11%) and (24.42%) for cuttings from juvenile and mature donor plants, respectively. IBA treated cuttings produced higher percentage rooting, number of roots and root length than untreated cuttings which revealed a strong IBA influence on rooting ability of stem cuttings in D. melanoxylon. It was concluded that stem cuttings should be taken from juvenile donor plants and basal cutting position should be used to produce high quality planting stock material for the afforestation and conservation programme of D. melanoxylon.     

3种植物生长调节剂对紫叶稠李扦插生根的影响

试验研究不同质量浓度（50、100、200 mg/L）的植物生长调节剂吲哚乙酸（IAA）和吲哚丁酸（IBA）、萘乙酸（NAA）和不同处理时间（30 min、60 min）对紫叶稠李扦插生根的影响。结果表明：30 min、60 min不同质量浓度IAA和NAA均未显著促进紫叶稠李扦插生根,而与CK相比,30 min、60 min IBA处理50、200 mg/L显著促进了紫叶稠李的生根率、根长及根数,且60 min IBA处理50 mg/L效果显著。表明植物生长调节剂IBA对紫叶稠李扦插生根的效果优于IAA和NAA,且60 min IBA处理50 mg/L是最佳组合。     

Softwood cuttings for propagation of red alder

Tests were conducted to assess rootability of red alder softwood cuttings from shoots of young trees and epicormic sprouts of mature individuals. Ortets were 3 to 34 years old, and treatments with and without indole-3-butyric acid (IBA) were compared. The extent of rooting and root vigor on the cuttings varied greatly among ortets and treatments. Among nonterminal cuttings, the best overall rooting success, with and without IBA, was obtained with cuttings of the youngest ortet. Average rooting of terminal and nonterminal cuttings of one clone were 100% and 64%, respectively. Although cuttings of some trees rooted well without IBA, the IBA treatment appeared to be generally beneficial. For all nonterminal cuttings of all clones, best results (80.0%/x) were obtained with a 10-sec dip in 2,000 or 4,000 ppm IBA. Vigorous planting stock was obtained from the rooted cuttings of all clones. Using epicormic sprouts from mature trees of proven performance will presumably allow propagation of superior trees and establishment of new plantations with improved stock.     

Regulation of phytohormones on root primordium initiation and adventitious root formation in the etiolated shoots of Paeonia suffruticosa''''Yinfen Jinlin''''

Tree peony is well known and sought after for its large, colorful flowers. Its propagation is via vegetative methods. Mech-nisms of the adventitious rooting and the regulation of rooting processes are the principles and techniques of plant propagation and improvement. Microstructures and fluctuations of phytohormones in the adventitious rooting were studied with the etiolated softwood shoots of Paeonia suffruticosa 'Yinfen Jinlin'. There are no pre-primordia in the shoots of the cultivar. Adventitious roots are produced in five stages: shoot selection, primordium initiation, primordium growth, conducting tissue differentiation and root protrusion. Primordia initiated in the cortex. The contents of the endogenous hormones, IAA, ABA and GA, were 5.842, 0.873 and 1.043 nmol·g-1 FW on the bases of shoots, respectively. CTKs which included isopentenyl adenine (iPA), zeatin riboside (ZR) and dihy-drozeatin riboside (DHZR) were 0.949, 0.695 and 2.034 nmol·g-1 FW, respectively. DHZR is active among CTKs. The ratio of IAA to GA, CTK and ABA clearly increased at the stage of primordium initiation, while they showed low levels at the stages of primordium growth. The ratios were restored at the shoot levels at the stage of root protrusion. IBA provoked primordia initiation in the cortex, the vascular cambium, the pith and even in the callus induced on the base of shoots. IAA levels in the treated shoots increased gradually to its highest level (three times of control) at the stage of conducting tissue differentiation. The ratios of IAA to GA, CTK and ABA clearly decreased at the stage of primordium initiation. The ratio of IAA to ABA is regulated at 10:1.     

国槐组织培养生根的研究

通过不同基本培养基、植物生长调节剂IAA、NAA、IBA和活性炭等对国槐试管苗不定根的诱导进行了系统研究。筛选出国槐最佳生根培养基是1/2MS+IAA0.5mg/L+蔗糖2%+琼脂0.65%,生根率达86%以上;并对不同苗龄试管苗不定根的发育过程进行了研究,结果表明,苗龄为20～25天的试管苗生根率最高可达90%,而且生根周期较短。     

植物生长素对东北红豆杉嫩枝扦插生根的响

以ABT-1^#、2．4-D、IAA、IBA、NAA5种植物生长素处理东北红豆杉当年生半木质化嫩枝，进行扦插生根试验，结果表明：嫩枝插穗扦插40-60d后在形态下端形成愈合瘤，并逐渐长出不定根，属于愈合组织生根型；在激素配比均为100×10^-6的条件下，经2．4-D处理的插穗生根率达到68％以上，经ABT—1^#和IBA处理的达到52％以上；采自幼龄植株的嫩枝插穗生根率高于老龄植株。     

Vegetative propagation of Colutea istria Mill. from leafy stem cuttings

This study investigates the optimal conditions for the vegetative propagation of the multipurpose leguminous shrub Colutea istria from leafy stem cuttings. The conditions examined included 1) the origin of the cutting (apical, medial or basal), 2) indole-3-butyric acid (IBA) treatment (0, 50, 200 or 400 mg/l), 3) the relative humidity of the propagation environment (high or low), 4) wounding base of cutting, and 5) the season in which the cutting was taken. The rooting ability of the cuttings was significantly influenced by all these factors. Winter cuttings obtained from the middle and base of the branch showed a greater rooting capacity than those harvested from the apical area, while autumn cuttings harvested from the middle and apical areas of the branch showed a greater rooting capacity than those harvested from the base. Winter cuttings showed the best rooting capacity than autumn cuttings. IBA treatment (200 mg/l), high humidity (98-100%), and wounding increased the rooting capacity of the cuttings. The sprout of new leaves was higher in basal cuttings, while IBA and wounding also significantly enhanced the root number of rooted cuttings. A method for vegetatively propagating C. istria plants is given.     

不同处理对红雾水葛插穗生根的影响

试验表明，对红雾水葛扦插生根影响最主要的因素是插穗部位，其次是激素种类厦浓度水平。红雾水葛以嫩枝扦插效果最好，在激素中以ABT生根粉和吲哚丁酸的效果较佳，而蔡乙酸的效果最差。生根效果最好的是A1B1C3和A1B3C12个处理，扦插后5d，分别有96．67％和86，67％的插穗形成愈伤组织，6d生根率分别迭80％和93．33％，12d全部生根，每株分别有新根90．6根和97，2根；而对照扦插后5d，有66．67％的插穗形成愈伤组织，6d生根率达83．33％，12d全部生根，每株仅有新根27根。     

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L^?1 L-ascorbic acid, 25 mg L^?1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L^?1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.