In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μm benzyladenine (BA)+0.54 μm naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N⁶-(Δ²-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μm kinetin+0.54 μm NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction. Received: 20 August 1997 / Revision received: 29 December 1997 / Accepted: 5 February 1998     

Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomile (Chamomilla recutita L.)

A protocol has been developed for the induction of somatic embryogenesis from flower explants of chamomile (Chamomilla recutita L.). The effects of several plant growth regulators [α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA) and kinetin (Kin), alone or in combination] and the flower type (disk or ray flower) were investigated. Both types of flowers responded to the callus and shoot induction treatments, but formation of globular somatic embryos took place only on disk-flower-derived explants after 2–4 weeks of culture on a Murashige and Skoog (MS) medium supplemented either with 8.87 μm BA and 1.07 μm NAA or with 26.8 μm NAA and 11.5 μm Kin. However, fully developed, cotyledonary-stage somatic embryos could be induced only on the NAA/Kin medium, 10 weeks after culture initiation. Germination of the embryos and plant regeneration took place after subculture for 4–5 weeks onto medium of the same composition. Plantlets regenerated from embryos flowered in vitro on a MS medium supplemented with 8.87 μm BA and 1.07 μm NAA. The significance of the results with respect to chamomile micropropagation and the utilization of wild populations in breeding programs is discussed. Received: 6 April 1998 / Revision received: 12 October 1998 / Accepted: 28 October 1998     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron

Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75–100 buds/explant) were achieved in the presence of 0.2 μm TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 μm IAA. Received: 1 March 1997 / Revision received: 29 October 1997 / Accepted: 15 November 1997     

Micropropagation of gum karaya (Sterculia urens) by adventitious shoot formation and somatic embryogenesis

Nodal explants from selected trees of gum karaya (Sterculia urens Roxb.) in the adult growth phase cultured on Murashige and Skoog (MS) medium supplemented with 6.62 μm N⁶-benzylaminopurine (BAP) produced an average of six adventitious shoots in 30 days. Shoots were rooted in vitro on 1/4-strength MS medium containing 9.82 μm indole-3-butyric acid. Nodulated callus was produced from hypocotyl explants cultured on MS medium supplemented with 4.52 μm 2,4-dichlorophenoxyacetic acid and 8.90 μm BAP. Somatic embryos developed when the nodulated callus was transferred to MS medium containing 0.45 μm thidiazuron (TDZ). TDZ treatment for 2 days gave the optimum response. Over 30% of the somatic embryos developed into plantlets when transferred to 1/4-strength MS basal medium without any growth regulators. Plantlets produced from adventitious shoots and somatic embryos were acclimatized to ex vitro conditions and established in the field. Received: 26 November 1997 / Revision received: 14 April 1998 / Accepted: 11 May 1998     

In vitro plant regeneration in Melia azedarach L.

Nodal explants of 3- 6-week-old seedlings cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) (17.75 μm) produced multiple shoots. Shoots were isolated and induced to root on 1/2-strength MS medium supplemented with indole-3-butyric acid (4.92 μm). In-vitro-rooted shoots resumed growth after a short period of acclimatization and resulted in plantlets which were successfully established in soil. In vitro flowering was observed in some of the nodal explants in the above medium, and also in cotyledonary leaves and internodal explants on MS medium supplemented with a combination of indole-3-acetic acid (IAA) (0.06 μm)+BA (4.44 μm) and IAA (0.06 μm)+kinetin (4.65 μm). Received: 25 October 1997 / Revision received: 1 May 1998 / Accepted: 15 May 1998     

Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb

A procedure was developed for plant regeneration of Hybanthus enneaspermus, a rare ethnobotanical herb from the Deccan peninsula in India, through seed-derived callus. Seeds demonstrated a high induction frequency (69.4±2.8%) and a high yield (364.4±2.5 mg) of light-yellow friable callus on Murashige and Skoog's (MS) medium containing 2.6 μm NAA and 2.2 μm BA within 4 weeks of incubation. After 1 year of subculture, yellow friable and light-green compact calli types were established from initial light-yellow friable callus. Shoot differentiation was achieved from light-green compact callus, but not from yellow friable callus. Shoot differentiation resulted when light-green compact callus was transferred to MS medium supplemented with 8.8 μm BA and 2.6 μm NAA; the highest percentage of calli forming shoots (66.6±4.8%) and the highest number of shoots (8.9±0.3) were achieved in this medium. Differentiated shoot buds elongated to 4–5 cm within 4 weeks. The addition of casein hydrolysate (500 mg/l) and more potassium phosphate (1.86 mm) to the culture medium enhanced shoot differentiation. Rooting was achieved on the shoots using half-strength MS medium containing 4.8 μm IBA. About 70% of the plants were established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. Received: 23 July 1998 / Revision received: 18 November 1998 / Accepted: 26 November 1998     

Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture

A protocol for micropropagation of Lilium longiflorum using pseudo-bulblets from in vitro shoot-tip-derived stem nodes was developed. Shoot tips from stems of dormant bulbs were cultured on one-half Murashige and Skoog (MS) medium. Stems from plantlets derived from the shoot tips were cut into nodal segments, which were then cultured on one-half MS medium supplemented with 1 μm 6-benzyladenine (BA). Pseudo-bulblets formed on each node after 1 month. An average of 32 pseudo-bulblets were formed on all nodes of the plantlet. Pseudo-bulblets gave rise to multiple shoots when cultured on one-half MS medium supplemented with 2.3 μm BA. Shoots were rooted on one-half MS medium containing 1.1 μm α-naphthaleneacetic acid. A continuous system of propagation by multiplication of pseudo-bulblets with no dormancy period was developed. The 80 flowering plants produced from shoot tip culture were acclimatized in the greenhouse for 3 months and then grown in the experimental garden for 8 months. Received: 29 October 1997 / Revision received: 28 April 1998 / Accepted: 3 May 1998     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Salvia nemorosa</Emphasis> L. from shoot tips and leaf explants

Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with 8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo.     

Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.)

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5–7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 μm 2,4-dichlorophenoxyacetic acid and 0.93 μm kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology. Received: 29 December 1997 / Revision received: 9 April 1998 / Accepted: 24 April 1998     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.     

Plant regeneration from cotyledonary explants of jute, Corchorus capsularis L.

A liquid culture protocol was developed to regenerate shoots from cotyledons of germinating seeds of jute (Corchorus capsularis L.). Reproducibility of the protocol was tested in three genotypes of jute. Highest bud differentiation rates into normal shoots (via shoot-forming explants) were obtained on modified Murashige and Skoog's liquid medium containing 2.7 μM α-naphthaleneacetic acid and 4.4 μM benzylaminopurine. Regenerated shoots were excised, and the best root formation could be induced in medium with 2.5 μM indole-3-butyric acid and 1.5% sucrose. Bud primordia were formed directly on the cut surface of the cotyledons. Scanning electron micrographs and histological studies confirmed the organogenic nature of the regenerated shoots. The physical condition of the culture medium and the age of the explants played crucial roles in the induction of shoot development using shoots; 2-day-old explants being optimal. Approximately 70% of the shoots were successfully established in soil after hardening. Received: 20 October 1997 / Revision received: 4 October 1998 / Accepted: 27 October 1998     

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×10⁶ were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation. Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998     

Micropropagation of Guazuma crinita mart. by root and petiole culture

Summary Shoot organogenesis of Guazuma crinita Mart. from root and petiole explants was obtained via adventitious bud formation. Root segments and petiole explants excised from in vitro generated plantlets were cultured on woody plant medium (WPM) supplemented with [trans-6-(4-hydroxy-3-methylbut-2- enyl)aminopurine] (zeatin) or with [6-benzyladenine] (BA). After 45 d of culturing, clumps of green bulbous structures containing small adventitious buds (clusters) were generated in all explants cultured with 10 μM zeatin under a photon flux density of 65 μmol m⁻² s⁻¹. For subsequent shoot differentiation, clusters were transferred onto medium containing 1 μM zeatin. After 60 d of culturing, 30% of clusters generated from petiole explants developed into plants. The regenerated plantlets were successfully acclimatized and all survived and grew well. No morphological abnormalities were observed.     

The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern.

In cotyledon cultures of Brassica juncea, shoots and roots invariable differentiate at the cut end of the petiole. Organogenesis occurred only if the proximal cut end of the petiole was in contact with the medium. In the absence of the petiole, differentiation from the lamina was rare. Hence investigations were carried out to study the influence of the cotyledonary lamina on regeneration of shoots and roots from the petiolar cut end. The lamina tissue was surgically removed from the cotyledon explants at different durations (0–10 days) after culturing them on either root-forming (basal medium) or shoot-forming (basal medium containing 5.0 M N⁶-benzyladenine) media. The differentiation of roots or shoots from the petioles was dependent on the presence of the lamina for at least 7 days of culture. Quantitative removal of the laminar tissue had a corresponding negative effect on shoot bud differentiation from the petiole. The nature of the lamina factor was found to be auxin-like.     

Continuous callus production and regeneration of garlic (Allium sativum L.) using root segments from shoot tip-derived plantlets

Root segments from shoot tip-derived plantlets of the garlic (Allium sativum L.) clones `DDR7099', `PI383819', and `Piacenza' were utilized as an explant source for continuous, friable callus production. The best callus production occurred on root segments initially cultured on medium with 4,5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 weeks, then subcultured to medium with 4.7 μm 4-amino-3,5,6-trichloropicolinic acid (picloram) +0.49 μm 6-(γ-γ-dimethylallylamino)purine (2iP) for 8 weeks. Embryogenic, friable callus was transferred to liquid medium for 1 month and then transferred to solid regeneration medium for 14 weeks. The best shoot and root regeneration (85.3% and 35.8%, respectively) occurred on 4-month-old calli from the clone `DDR7099'. In all clones, regeneration rate decreased as callus age increased. Received: 14 October 1997 / Revision received: 26 December 1997 / Accepted: 12 January 1998     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Comparative adventitious shoot induction in kentucky coffeetree root and petiole explants treated with thidiazuron and benzylaminopurine

Summary Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10μM BA and 1.0μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation in root explants, it promoted shoot initiation in petiole explants. In contrast, TDZ was equally effective at inducing shoots in root and petiole explants. This suggests that root and petiole explants of Kentucky coffeetree could be a useful model system for studying the differences, in apparent mode of action between TDZ and BA on adventitious shoot initiation.