Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

Enhancement of shoot regeneration potential by liquid medium culture from mature cotyledons of sunflower (Helianthus annuus L.)

We describe here a liquid culture system for the regeneration of shoots at high frequencies from mature cotyledon tissues of three genotypes of sunflower (Helianthus annuus L.) one of which had previously been found to be recalcitrant to regeneration when cotyledons were cultured on solid medium. Cotyledons were excised from 2-day-old seedlings and incubated in liquid Murashige and Skoog's modified medium supplemented with 5.4 M naphthaleneacetic acid (NAA) and 4.4 M benzylaminopurine (BAP). After two weeks in culture, the whole upper surface of regenerating explants was covered with green shootlets. The percentages of regenerating explants of three genotypes varied between 60 and 70%, and the number of shoots per regenerating explant was highly increased. The shootlets were transferred to solid Murashige and Skoog's medium allowing shoot development, then to rooting medium. Rooted plantlets were successfully acclimatized and gave fertile plants. The role of liquid medium culture in the induction of sunflower regeneration is discussed.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

In vitro propagation of the leguminous tree Swartzia madagascariensis

Shoot induction frequency for the leguminous tree Swartzia madagascariensis Desv. was higher on MS and WP media than on B5. Explants incubated on media solidified with agar produced more shoots with a lower tendency to hyperhydricity than explants on agarose or Gelrite media. Maximum shoot induction was obtained with an agar-solidified MS medium containing 2.2 M benzyladenine (37 shoots/explant). Shoots rooted after transfer to half-strength MS medium supplemented with 26.8 M naphthaleneacetic acid.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA naphthaleneacetic acid - WP(M) woody plant (medium)     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and -naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 M BA and 1.0 M NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 M BA and 1.0 M NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 M indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.     

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro

High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 M indole-3-acetic acid and 50 M benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.     

In vitro multiplication of Calophyllum apetalum (Clusiaceae), an endemic medicinal tree of the Western Ghats

Young shoots collected from mature trees of Calophyllum apetalum during the flush season (November–February) were red (1–2 weeks), pinkish-red (3–5 weeks), pale green (6–8 weeks) and dark green (9–10 weeks) coloured after different periods of growth. All the shoot tip and single node explants of the youngest 1–2-week-old shoots cultured in Murashige and Skoog's (1962) agar medium were lost due to excessive browning and necrosis; nodes of the 6–8-week-old shoots subjected to transfers twice a week in fresh medium containing 8.8 ;M BAP responded the most (68% of explants) with the formation of 3.2 shoots per explant in 7 weeks. The shoot tip was a relatively poor source of regeneration (2.3 shoots per explant; 39% of explants). Subculturing of explants from in vitro derived shoots for 5 weeks in medium containing 4.4 M BAP resulted in the formation of an increased number and percentage of shoots in the nodes (5.3 per explant; 74% of explants). The shoot cultures were transferred to 1/2 MS basal medium for 4 weeks to induce the elongation of shoots (;3.0 cm). Rooting of the microshoots (>2.0 cm) was achieved when cultured in quarter strength MS medium supplemented with 9.8 ;M IBA for 4 weeks followed by transfer to 1/4 MS basal medium for 4 weeks. The rooted plantlets transferred to clay pots filled with soil, sand and farmyard manure (1:1:1), maintained in a mist chamber at a relative humidity of 80–90%, acclimatised at a 56% rate after 6 weeks. Out of 345 plants restored to their native habitat in the forest at three locations of the institute campus, 293 plants survived and showed uniform growth free of morphological defects.     

Rapid clonal propagation of Vitex trifolia

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

A method for shoot regeneration from leaf explants in two cultivars of cranberry (Vaccinium macrocarpon Ait.) is described. Modified Anderson's medium supplemented with combinations of thidiazuron (TDZ) with or without 1 M NAA (-naphthaleneacetic acid) was used to optimize shoot regeneration. The effect of light or dark incubation was also determined. Maximum regeneration was obtained in the light in the presence of 10 M TDZ and 1 M NAA. While this medium was suitable for leaf explants obtained from shoot cultures, regeneration did not occur from leaves collected from greenhouse-grown plants. Elongation of the regenerated shoot tips did not occur until explants were transferred to growth regulator-free medium at which time only a minority of shoots elongated. Elongated shoots could be dissected away from leaf tissue, rooted easily, and acclimitized to ambient conditions.Abbreviations NAA -naphthaleneacetic acid - TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea     

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     

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.     

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 M TDZ and 2.85 M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 M benzyladenine and 5.77 M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 M IAA and 2.46 M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.     

Efficient Regeneration of Tetraploid Isatis indigotica Plants via Adventitious Organogenesis from Hypocotyl Explants

An efficient in vitro plant regeneration system via hypocotyl segments of tetraploid Isatis indigotica Fort. was established. Murashige and Skoog's (MS) and Gamborg's (GB₅) media were found to be superior to White medium for promoting shoot regeneration. The highest shoot regeneration (92 %) was achieved from hypocotyls cultured on MS medium containing 8.9 M benzyladenine (BA) and 2.7 M naphthaleneacetic acid (NAA), with an average of 4.2 shoots developed per explant. Plant regeneration was also improved when the explants were cultured in MS basal medium containing 3 % (m/v) sucrose and grown under a 12-h photoperiod. The developed shoots were well rooted in a half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA) and were morphologically normal after transfer to soil.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.