High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

Adventitious Shoot Induction from Internode and Root Explants in a Semiparasitic Herb Monochasma savatieri Franch ex Maxim

A highly efficient protocol for the induction of adventitious shoots from young internode and root explants of a semiparasitic medicinal herb Monochasma savatieri Franch ex Maxim was developed. MS basal medium supplemented with 5 µM thidiazuron (TDZ) induced 32 adventitious shoots/explant, which was double the number obtained using the same concentration of 6-benzyladenine (BA). Hyperhydric shoots were observed when 10 µM of any cytokinin was added to MS media. Use of any cytokinin at 2.5 µM produced an average of 14–21 adventitious shoots/root explant. Shoots formed roots in vitro more effectively than α-naphthaleneacetic acid when indole-3-butyric acid and indole-3-acetic acid were used at 1.0 µM. Two-month-old rooted plantlets were transplanted to vermiculite and 70% survived after 4 months.

     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

Increased Copper Content of the Medium Improves Plant Regeneration from Immature Embryo Derived Callus of Wheat (Triticum aestivum)

The effect of various concentrations of CuSO₄ on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO₄. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO₄ was significantly higher.     

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4?±?1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Regeneration of Indian ginseng plantlets from stem callus

The development of stem callus mediated plant regeneration system for Withania somnifera is described. Maximum callus proliferation was obtained on Murashige and Skoog medium supplemented with 2.26 μM 2,4-D. Three-week-old, white, friable callus was used for shoot regeneration. The maximum shoot regeneration (6.2 ± 0.34 shoots/explant) was achieved in four weeks when callus was cultured on MS medium fortified with 4.44 μM BA and 0.57 μM IAA. Regenerated shoots were excised and multiplied (8.4 ± 0.43 shoots/explant) on MS medium supplemented with 4.44 μM of BA. Multiple shoots were divided into single shoots and were rooted (5.1 ± 0.49 rootlets/shoot) on half strength MS medium supplemented with 9.84 μM of IBA. After a hardening phase of 3 weeks the plantlets were transferred to the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity

An efficient in vitro regeneration protocol was developed for medicinally important aromatic plant Anethum graveolens. Nodal segments were cultured onto Murashige and Skoog (MS) basal medium supplemented with different auxins and cytokinins singly as well as in combinations. The optimum callus induction (93.33 %) was obtained on medium fortified with 2.2 μM N⁶-benzyladenine (BA) and 0.21 μM α-naphthaleneacetic acid. The best shoot regeneration (85.7 %) with 12.86 shoots per explant was achieved in two weeks when callus was subcultured on MS medium amended with 2.2 μM BA and 1.85 μM kinetin. The average length of regenerated shoots varied from 3.15 to 4.8 cm. The rooting of regenerated shoots was nearly 100 % on ? MS augmented with 4.9 μM indolebutyric acid with a maximum root length of 5.1 cm. Plantlets were successfully acclimatized with 60 % survival rate. During organogenesis, catalase and ascorbate peroxidase activity increased while superoxid dismutase activity decreased. Clonal fidelity of in vitro raised plants has been checked by random amplified polymorphic DNA using 10 selected decamer primers. It has been found that regenerated plants are true to type plants.     

In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

In vitro propagation of Mussaenda erythrophylla Schum and Thom cv. scarlet through multiple shoot regeneration

Axillary bud explants were induced to form shoots on Murashige and Skoog's (MS)' basal medium. Best yield (9 shoots per explant) was obtained when the medium was supplemented with adenine sulphate (40 mg/L) and 6-benzylaminopurine (2.25 mg/L). The shoots were rooted on half strength MS basal medium supplemented with indole butyric acid (0.5 mg/L) and having thiamin-HCl (800 mg/L). Regenerated plantlets were successfully acclimatized. This is the first report of micropropagation in the genus Mussaenda without callus intervention.     

In vitro propagation and the acclimatization effect on the synthesis of 2-hydroxy-4-methoxy benzaldehyde in Decalepis hamiltonii Wight and Arn.

The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N₁) from the basal cut end similar to cytokinin–auxin combination (N₂). Between two types of nodular tissues, N₂ was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA₃). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation

A reliable plant regeneration system is described for the production of adventitious shoots from root explants of spinach. Explants from roots of axenic shoots and roots induced on cultured hypocotyl explants were used for adventitious shoot induction. Explants from apical, middle and basal root regions were incubated on Nitsch and Nitsch medium supplemented with α-naphthaleneacetic acid, gibberellic acid and kinetin. Optimum shoot regeneration was from explants of apical and middle root regions on medium with 20 μm α-naphthaleneacetic acid and 5.0 μm gibberellic acid. Shoots originated directly from root tissues without an intervening callus phase. Adventitious shoots were rooted and were grown to maturity in the glasshouse. This plant regeneration procedure has been exploited in preliminary studies of Agrobacterium-mediated transformation. Received: 27 February 1996 / Revision received: 22 August 1996 / Accepted: 30 September 1996     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

In Vitro Propagation of the Elephant Yam, Amorphophallus campanulatus var. hortensis Backer (Araceae)

Lateral buds from the corms of field-grown Amorphophallus campanulatusvar. hortensis Backer were cultured on Murashige and Skoog (MS)medium in the presence of various combinations of naphthaleneaceticacid (NAA) and kinetin (KN) and on MS medium lacking hormoneswith and without the addition of coconut water. Callus and plantletdevelopment was optimal at 0.5 µM NAA and 0.05 µMKN. Plantlets transferred to soil after 24–36 weeks ofculture grew normally. Amorphophallus campanulatus, elephant yam, callus, in vitro propagation, micropropagation plantlets, tissue culture