High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

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 Regeneration of Mat Sedge (Cyperus pangorei Rottb)

An in vitro protocol was developed for regeneration of Cyperus pangorei that may supplement enough raw materials for the mat weaving community. Callus was initiated from inflorescence explants on Murashige and Skoog’s (MS) medium supplemented with 5 and 10 μM each of 2, 4-D, 2, 4, 5-T and CPA. Development of numerous de novo spikelets from immature inflorescence explants grown in (10 μM) 2, 4, 5-T was observed. MS with 5 μM Kn and 100 ml l^?1 Coconut milk (CM) promoted shoot regeneration from calli. Calli from 2,4-D and CPA medium sub-cultured on medium containing 5 μM BAP, 5 μM Kn, 1 μM IAA and 100 ml l^?1 CM produced extensive and rapid rhizogenesis with wiry and scaly roots. Micropropagation using rhizome buds on MS medium with BAP, Kn and Zeatin at 10 μM concentrations resulted in shoot release and multiplication by breaking the bud dormancy. An average of 10 shoots per explant was produced in 10 μM BAP, whereas (10 μM) Kn and (10 μM) Zeatin induced only single shoot formation. The shoots were transferred to rooting media comprising 10 μM IAA with 1 μM BAP or Kn and then acclimatized. The results accomplished were found to be useful in developing a complete in vitro regeneration protocol towards the mass production of Cyperus species, which may provide a basis for further genetic improvements that may prove its use as an alternative natural fibre resource in commercial applications.     

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun')

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and β-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 μM brassin, benzylaminopurine and β-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'. Received: 9 April 1997 / Revision received: 27 August 1997 / Accepted: 20 September 1997     

Somatic embryogenesis and shoot organogenesis from leaf explants of <Emphasis Type="Italic">Primulina tabacum</Emphasis>

Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced. This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite).     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 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 with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

Efficient In vitro Plant Regeneration Protocol from Leaf Explant of Jatropha curcas L — A Promising Biofuel Plant

An efficient in vitro plant regeneration from leaf-disc culture of Jatropha curcas L has been established. Adventitious shoot buds along with callus were induced from leaves of 2-year-old J. curcas plants cultured on Murashige and Skoog’s (MS) medium supplemented with TDZ (2 μM) BAP (2 μM) and IBA (1 μM), wherein 63.3% leaf explants responded. The multiplication of shoots was achieved from the adventitious shoot buds after transferring them to shoot induction medium. The highest number of shoots (9.7/explant) was achieved after 6 weeks of culture on MS medium containing 3 μM of BAR The welldeveloped shoots were rooted on MS medium supplemented with IBA (1.5 μM) with the rooting frequency of 53.3%. Addition of phloroglucinol (200 μM) to the medium enhanced the frequency of rooting to 76.7%. Regenerated plantlets were successfully transferred to field after initial acclimatization.     

A micropropagation protocol for <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl. from root explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.     

The influence of thidiazuron on shoot regeneration from leaf explants of fifteen cultivars of Rhododendron

The influence of cytokinin thidiazuron (TDZ) and auxin indole-3-acetic acid (IAA) on in vitro shoot organogenesis of fifteen Rhododendron genotypes was investigated and a protocol for high frequency adventitious shoot regeneration from leaf explants was developed. High genotypic variation was observed and regeneration frequencies ranged from 0 to 100 %. Genotype Ovation had the highest number of shoots (26.4 per explant) after 12 weeks on medium with 0.57 μM IAA and 1.20 μM TDZ, but only 65 % of explants regenerated. Catawbiense Grandiflorum had 17.7 shoots per explant and 75 % regeneration on medium with 5.70 μM IAA and 0.45 μM TDZ and Van Werden Poelman had 14.3 shoots per explant and 100 % regeneration on medium with 0 57 μM IAA and 0.45 μM TDZ.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid micropropagation of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium

An efficient protocol has been developed for rapid micropropagation of Ocimum basilicum. Multiple shoots were induced by culturing shoot tip explants excised from mature plants on a liquid Murashige and Skoog (MS) medium supplemented with 5–100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplementation to the culture medium was 50 μM for 8 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (78 %), mean number of shoots (11.6 ± 1.16) and shoot length (4.8 ± 0.43 cm) per explant. A culture period longer than 8 d with TDZ resulted in the formation of fasciated or distorted shoots. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA). The micropropagated shoots with well developed roots were successfully established in pots containing garden soil and grown in greenhouse with 95 % survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell layer method

A method for a high frequency and direct in vitro bud regeneration of a woody species, the trifoliate orange (Poncirus trifoliata L. Raf), was designed. Transverse thin cell layer (tTCL) explants excised from the stem internodes of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 μM) and N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1–10 μM). The optimal concentrations for bud induction were 25 μM BAP and 1 μM TDZ leading to 87 and 72 % of responsive tTCLs and 24 and 15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher frequency of bud regeneration were obtained with BAP and TDZ combined. With a combination of 10 μM BAP and 1 μM TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium containing 1 μM GA₃. Rooting of individual shoot was induced using 5 μM NAA. One hundred per cent of rooted shoots developed normally after transfer to the greenhouse; no phenotype variation was observed. High numbers of regenerated viable plants can be produced directly without callus formation from tTCL after 9 weeks of culture.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot (Rhodiola rosea L)

A procedure for in vitro propagation of roseroots (Rhodiola rosea L), a medicinal plant, was developed using a RITA bioreactor system containing liquid medium, combined with a gelled medium. Wild roseroot clones: ‘RCi’, ‘RC2’ and ‘RC3’ were established on a basal medium (BM) from in vitro-germinated seedlings on half-strength Murashige and Skoog (MS) salts. TDZ at 2–4 μM supported shoot proliferation but inhibited shoot elongation of ‘RCi’ shoots on gelled medium. Clones differed significantly with respect to multiplication rate with ‘RCi’ producing the most shoots per explant on gelled BM with 2 μM zeatin. In a bioreactor system, TDZ supported rapid shoot proliferation at lower concentration (0.5 μM) but induced hyperhydricity at more than 0.5 μM. Bioreactor-multiplied hyperhydric shoots of all clones when transferred to gelled medium containing 1–2 μM zeatin produced normal shoots within 4 wk of culture. Shoots were rooted in vitro on BM void of growth regulators. Almost all (9U to 95%) in vitro plantlets survived when transferred to potting medium.     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

Regeneration of <Emphasis Type="Italic">Aloe arborescens</Emphasis> via somatic organogenesis from young inflorescences

A system for in vitro regeneration of Aloe arborescens was developed using young inflorescences as explants. Different phytohormone combinations of N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ), benzyladenine (BA), 6-(γ,γ-dimethylallyl-amino)purine riboside (2iPR), zeatin ribozide (ZR), N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and kinetin (K), with or without ancymidol, were examined in order to induce plant regeneration. Efficient shoot regeneration was initiated on Murashige and Skoog (MS) medium supplemented with BA or TDZ. MS medium enriched with 19.6, 22.2 μM BA and 3.92 μM ancymidol (MSBA5/1 medium), promoted organogenesis enabling 87.3% of the explants to regenerate 6.04 ± 1.79 shoots/explant. Subsequent shoot elongation and plant regeneration were strongly affected by the medium composition used for shoot induction. Optimal elongation (three to four shoots per explant) was obtained when shoots, initiated on MSBA5/1 medium, were subsequently transferred onto MS containing only 4.4 μM BA. Rooting was performed on MS media lacking growth regulators. Histological analysis revealed that the initiated shoots originated from the receptacle tissue surrounding the residual vascular tissue of the flower buds.