Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro plant regeneration of Vismia guianensis through organogenesis

A protocol for in vitro plant regeneration through organogenesis was established for Vismia guianensis(Hypericaceae), a species that produces an anti-cancer compound. The highest mean number of shoots per gram of callus (57.33) was obtained on Murashige and Skoog medium supplemented with 4.44 μM 6-benzyl-aminopurine, 5.70 μM indole-3-acetic acid and 12.88 μM gibberellic acid. Rooting was favoured by the addition of 10 μM indole-3-butyric acid, and by sucrose concentrations higher than 1%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

Micropropagation of lemon balm

Traditional propagation of lemon balm (Melissa officinalis L.) is inefficient for establishing a good quality clonal population. Results of the presented experiments outline an effective method for micropropagation of this species. Following culture initiation from shoots of field-grown plants on growth regulator free Murashige–Skoog medium, rapid shoot multiplication with only rudimentary root formation could be achieved on media containing various concentrations of indole-3-acetic acid and 6-benzyladenine. The combination of 5.71 μM indole-3-acetic acid and 6.66 μM 6-benzyladenine resulted in the best multiplication. Transfer of propagules to media containing indole-3-acetic acid and kinetin did not result in shoot proliferation; however, single plantlets grown on media containing 5.71 μM indole-3-acetic acid and 13.9 μM kinetin developed more compact shoots and stronger roots than the control plants and were suitable for acclimatisation with an efficiency over 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

Effect of vessel type and growth regulators on micropropagation of Capsicum annuum

Leaves from 14-d-old Capsicum annuum L. cv. Anaheim seedlings were cultured on Murashige and Skoog (MS) medium containing different combinations of indole-3-acetic acid (IAA) and 6-benzyladenine (BA). After 3 months, cultures were transferred to new medium where BA was replaced with 9 μM isopentenyladenine (2iP) to enhance the growth of shoot buds. Developing shoots were elongated and rooted on MS medium enriched with 9 μM indole-3-butyric acid (IBA). All cultures were maintained in 250 cm³ baby jars covered with a clear polypropylene lid with or without microporous polypropylene membrane. Vessel type and plant growth regulators significantly affected callus morphogenic appearance, organogenesis and in vitro plantlet growth. Ventilated vessels supported photomixotrophic culture and improved regeneration and growth of plantlets. Higher plantlet dry mass and content of photosynthetic pigments, and lower stomatal density of plantlets grown in ventilated than in non-ventilated vessels facilitated ex vitro acclimation and growth.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Indirect shoot organogenesis from leaves of Dieffenbachia cv. Camouflage

A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N ⁶-(Δ²-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and morphology have been observed in regenerated plants.     

Leaf explant response in in vitro culture of St. John’s wort (Hypericum perforatum L.)

For centuries Hypericum perforatum has been used in natural medicine. In the last decades, it has also attracted the attention of pharmaceutical industry due to its promising anti-depressant properties. The important factor in pharmaceutical application of plant material is its stable content of active compounds. Such stability requires standardized conditions of growth, e.g. an in vitro culture. Our aim was to establish a medium allowing for an effective regeneration of shoots from the standardized leaf explants in in vitro conditions. Cultures of the leaf explants carried out in darkness, on Murashige and Skoog agar medium, supplemented with auxins (2,4-dichlorophenoxyacetic acid, 2-metoxy-3,6-dichlorobenzoic acid, α-naphtaleneacetic acid, indole-3-acetic acid) and cytokinins (kinetin, N⁶-(benzyl)adenine, thidiazuron) resulted in callus formation. The callus produced roots on media containing indole-3-acetic acid or α-naphtaleneacetic acid alone. On media supplemented with auxins and cytokinins, indirect shoot organogenesis was also observed. The most efficient shoot formation was observed with 2.85 μM of indole-3-acetic acid and 4.44 μM of benzyladenine. Regenerated shoots were rooted on Murashige and Skoog without plant growth regulators medium or on a medium supplemented with indole-3-acetic acid. From a single leaf explant (one fifth of the leaf) after a month of the culture, 35 regenerated shoots were obtained (allowing for the formation of about 180 vegetative shoots per leaf). Successful multiplication of shoots from a standardized explant makes it possible to obtain a great quantity of uniform plant material for biotechnological purposes.     

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.     

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, <Emphasis Type="Italic">Tylophora Indica</Emphasis> (Burm. F.) merrill

Summary Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.     

Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls

Seedling hypocotyls were used as explants to establish a regeneration protocol for Eucalyptus urophylla and N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), one kind of di-substituted urea, was found useful growth regulator. The hypocotyls incubated on a modified Murashige and Skoog medium (SPCa), supplemented with 6.6 μM PBU and 0.57 μM indole-3-acetic acid (IAA) dedifferentiated and form calli (100 % after 7 d). Compared with other growth regulator combinations, PBU stimulated more vigorous calli and restrained their darkening. In addition, the calli induced by PBU showed high frequency of adventitious buds formation (57%). Shoot proliferation and elongation was then stimulated on SPCa medium containing 0.44 μM 6-benzyladenine (BA), 0.54 μM naphthalene acetic acid (NAA) and 0.3 μM gibberellic acid (GA3). For rooting, shoots were cultivated on root induction medium containing 2.5 μM indole-3-butyric acid (IBA). Plantlets were then successfully transplanted to greenhouse.