Light quality influences the polyamine content of lettuce (Lactuca sativa L.) cotyledon explants during shoot production <Emphasis Type="Italic">in vitro</Emphasis>

Light quality has previously been shown to influence morphogenesis in lettuce cotyledon explants, with white or red light promoting adventitious shoot production, and blue light inhibiting it. Endogenous polyamine (PA) concentrations were compared between explants cultured under different light qualities. Explants cultured under white or red light accumulated PAs during shoot primordia production, with a 5.6-fold increase compared to initial concentrations under white light, and 6.7-fold increase under red light. These results suggest polyamines are involved in the formation of shoot primordia. After 18 days in culture PA concentrations decreased under white light, and to a lesser extent under red light, signaling a shift in polyamine metabolism that correlates with shoot expansion, which occurs more readily under white light. Explants cultured under blue light accumulated polyamines for the first 7 days, to a level 1.3 times greater than initial values, followed by a gradual decline during the remainder of the culture period. Explants cultured under blue light also contained a greater proportion of PCA-insoluble conjugated PAs, compared to explants under white or red light, which contained greater proportions of free or PCA-soluble conjugated polyamines. The ratio of putrescine to spermidine was also different with a lower Put:Spd ratio being associated with shoot production under white or red light, and higher Put:Spd ratio being associated with culture under blue light.     

Adventitious shoot regeneration from Begonia × erythrophylla petiole sections is developmentally sensitive to light quality

The influence of light quality on organogenesis in vitro was investigated using Begonia  ×  erythrophylla petiole explants. Pre-treatment of in vitro donor plants by growth in the dark or under far-red or blue light reduced their competence for shoot formation when compared with those grown under red or white light. Culture of competent petiole explants under far-red, blue light or in the dark reduced the number of shoots produced per explant compared to those cultured under red or white light. Explants were found to be developmentally sensitive to both far-red and blue light, because meristem, but not primordia development was inhibited. In addition, blue light inhibition of shoot formation is not mediated directly through phytochrome, as few shoots formed on explants cultured under a mixture of red and blue light which resulted in a high P _fr/ P _tot (0.82) and would allow shoot formation in the absence of blue light. Unlike the inhibitory influence of far-red light, which is reversible, exposure to blue light permanently reduces an explant's competence for shoot formation. Our results suggest that phytochrome and an independent blue light photoreceptor, possibly a cryptochrome, can regulate shoot production from B. erythrophylla petiole explants.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon

The influence of light incubation during embryo germination on shoot organogenesis from cotyledons of four diploid watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai cultivars was examined. Germinating embryos in darkness significantly improved the number of explants that produced harvestable shoots during the 6 week incubation period on shoot regeneration medium under a 16-h photoperiod. The percentage of explants with shoots more than doubled for `Crimson Sweet' and was about 1.5-fold greater for `Sweet Gem' and `Yellow Doll' when embryos were germinated in darkness. The percentage of explants with shoots was not significantly improved for `Minilee' by pretreating seedlings in darkness. This study demonstrates that optimal shoot regeneration can be obtained by germinating embryos in darkness before preparing cotyledon explants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Effects of light quality on growth and development of protocorm-like bodies of <Emphasis Type="Italic">Dendrobium officinale</Emphasis> in vitro

The effect of light quality on protocorm-like bodies (PLBs) of Dendrobium officinale was investigated. PLBs of D. officinale were incubated under a number of different light conditions in vitro, namely: dark conditions; fluorescent white light (Fw); red light-emitting diodes (LEDs); blue LEDs; half red plus half blue [RB (1:1)] LEDs; 67% red plus 33% blue [RB (2:1)] LEDs; and 33% red plus 67% blue [RB (1:2)] LEDs. Growth parameters, number of shoots produced per PLB, chlorophyll concentration and carotenoid concentration were measured after 90 days culture. The percentage of PLBs producing shoots was 85% under blue LEDs. In contrast, the percentage of PLBs producing shoots was less than 60% under dark conditions, fluorescent white light and red LEDs. The number of shoots produced per PLB was more than 1.5 times greater under blue LEDs, RB (1:1) LEDs and RB (1:2) LEDs than those cultured under other light treatments [dark, Fw, red LEDs and RB (2:1)]. Chlorophyll and carotenoid concentrations were significantly higher under blue LEDs and different red plus blue LED ratios, compared to other light treatments (dark, Fw and red LEDs). Blue LEDs, Fw, and RB (1:2) LEDs produced higher dry matter accumulations of PLBs and shoots. This study suggests that blue LEDs or RB (1:2) LEDs could significantly promote the production of shoots by protocorm-like bodies of D. officinale and increase the dry matter of PLBs and the accumulation of shoot dry matter in vitro.     

毛地黄(Digitalis purpurea)叶离体培养过程中光质与培养基对器官发生的交互作用

培养基成分影响毛地黄叶外值体的生长,不含BA的培养基中芽不能发生,无NAA的培养基中无根发生。光质的效应与培养基成分有关,黄、蓝、绿光在未加有机成分(NAA为0.ling·L~(-1))的培养基中能促进芽的生长,当NAA为0.5 mg·L~(-1)时则抑制芽的生长。红光、黑暗处理与培养基成分关系不大,一般均抑制发芽;根的发生不需要光。光质和培养基之间有交互作用。     

The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein peas

Summary The production of whole plants from explants of protein pea (Pisum sativum L.) using an efficient, reliable and rapid strategy, while maintaining trueness to type, will be required before regeneration can be exploited for genetic transformation. Seeds of the pea genotypes Terese, Solara, Frisson and P64 (a hypernodulating mutant line of Frisson) were surface-sterilized and imbibed overnight, whereafter embryo axes were dissected and germinated on hormone-free medium for 7–10 d. Hypocotyl sections lacking pre-existing meristems were harvested and cultured on a range of media with various concentrations and combinations of growth regulators in order to induce either caulogenesis or somatic embryogenesis. Differences in responsiveness were apparent between genotypes, but regeneration via caulogenesis was consistently more reliable than via the induction of somatic embryos. Few explants underwent somatic embryo production and their conversion into plants has remained elusive so far, irrespective of the genotype studied. Conversely, large numbers of buds were produced within 10 d by organogenesis, and healthy, rootable shoots were obtained. A clear relationship was observed between the growth regulators employed for bud regeneration and shoot rooting phases and the subsequent competence of the regenerated plants for flowering, pod formation and viable seed production.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.     

光质及种子大小对普洱地区14种植物种子萌发的影响

以普洱地区14种常见植物种子为材料,在实验室条件下研究了其在白光、黑暗、红光和蓝光条件下的萌发特性,并分析了种子大小与萌发率、萌发速率、萌发开始时间的关系。结果表明:光质对四方蒿、沙针、尖子木、藿香蓟种子萌发率和萌发速率均有显著影响(P0.05)。光质对大叶斑鸠菊、云南山枇花、臭灵丹、车桑子、光萼猪屎豆、葫芦茶、云南地桃花、西南宿苞豆、岗柃、中国宿苞豆10个物种的种子萌发率和萌发速率均没有显著影响(P0.05),以上物种中除中国宿苞豆外,其他物种种子萌发率均在20%以下,处于休眠状态。四方蒿种子在白光(89.9%)和红光(84.7%)下萌发率最高,红光下种子萌发最快(4.93),蓝光下种子萌发开始时间最晚(11.3 d);沙针种子在白光下萌发率最高(80.4%)、萌发速率最快(2.71),在黑暗和蓝光下萌发率较低(43.9%和38%)、萌发速率最慢(0.73和0.85),白光、红光下萌发开始最早(11 d),黑暗条件下萌发开始最晚(21.7 d);尖子木种子萌发率在白光、黑暗、蓝光下均在86%以上,而红光下仅32%且萌发速率最慢(1.29),在蓝光下萌发开始时间最晚(13 d);藿香蓟种子萌发率和萌发速率在红光下最高(分别为71.3%和6.46),黑暗条件下最低(分别为42.5%和2.62);大叶斑鸠菊萌发开始时间在黑暗条件下最早(6 d),其次是白光下(7 d),蓝光和红光下较晚,分别为8 d和7.7 d。14个物种种子的萌发率与种子大小间均有显著负相关关系;种子萌发速率、萌发开始时间与种子大小间也有负相关关系,但不显著;种子大小与萌发率、萌发速率和萌发开始时间的关系不会随着光质的变化而发生变化。     

Shoot regeneration and the relationship between organogenic capacity and endogenous hormonal contents in pumpkin

To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after in vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0 mg l⁻¹). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5 mg l⁻¹ BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1 mg l⁻¹ BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1 mg l⁻¹ NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.