In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall

Curcuma attenuata is a highly valued ornamental. This study provides the first report on C. attenuata shoot organogenesis and plant regeneration. Immature anthers derived from 5 to 7?cm long inflorescences were isolated and cultured on different variations of Murashige and Skoog (MS) media to induce callus and then shoot organogenesis. When the 2-mm long anthers in which microspores were at the uninucleate developmental stage were cultured in the dark on MS medium containing 13.6???M 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3???M kinetin (KT) for 15?days and then transferred to 40???mol?m^?2?s^?1 fluorescent light for 30?days, the percentage callus induction reached 33.3?%. After callus was transferred to various differentiation media and cultured in the light, 33.1?% of all callus cultures could differentiate into adventitious shoots on MS medium supplemented with 22.0???M 6-benzyladenine (BA), 0.53???M ??-naphthaleneacetic acid (NAA) and 1.4???M thidiazuron (TDZ) after culturing for 60?days. Over 95?% of plantlets survived after transplanting plantlets into trays with a mixture of sand and perlite (2: 1) for 20?days. Chromosome number, determined from the root tips of young plantlets, indicated that all plantlets were diploid (2n?=?84).     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg?l^?1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg?l^?1 TDZ. The WPM medium supplemented with 2.5 mg?l^?1 BA?+?0.4 mg?l^?1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg?l^?1 BA and 3.0 mg?1^?1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg?l^?1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.     

Influence of type of explant,plant growth regulators,salt composition of basal medium,and light on callogenesis and regeneration in <Emphasis Type="Italic">Passiflora suberosa</Emphasis> L. (Passifloraceae)

Passiflora suberosa is used in popular medicine, improvement programs, and as an ornamental plant. The goal of this study was to establish efficient protocols for plant regeneration and callus induction from nodal, internodal and leaf segments excised from in vitro-grown plants. The different morphogenetic responses were modulated by the type and concentration of plant growth regulators, according to the basal medium and light conditions. Shoot formation occurred through three pathways: (1) development of preexisting meristems, (2) direct organogenesis, and (3) indirect organogenesis. Development of preexisting meristems was observed from nodal segments (1 shoot/explant) in response to α-naphthaleneacetic acid (NAA), picloram (PIC), and 2,4-dichlorophenoxyacetic acid (2,4-D), using two basal media (MS and MSM). Direct organogenesis in this species was obtained for the first time in this work, through shoot development from internodal segments in the presence of 6-benzyladenine (BA). The highest regeneration rates were achieved on MSM medium, regardless of the BA concentration. Indirect organogenesis was achieved from all explant types on media supplemented with BA, used alone or in combination with NAA. The highest regeneration efficiency was obtained from internodal segments cultured on MSM medium plus 44.4 μM BA. Compact, friable, or mucilaginous non-morphogenic calluses were induced by thidiazuron, PIC, 2,4-D, and NAA. High-yielding friable calluses obtained on MSM medium supplemented with 28.9 μM PIC are being used for the establishment of suspension cultures and further analysis of the production of bioactive compounds.     

Biochemical and histological changes associated with <Emphasis Type="Italic">in vitro</Emphasis> responses in sunflower cotyledonary explants

In vitro shoot regeneration from sunflower cotyledonary explants can be obtained in the presence of kinetin and indole-3-acetic acid. In contrast, callus proliferation is obtained in the presence of 2,4-dichlorophenoxyacetic acid on culture medium. The purpose of this study was to investigate changes in protein profiles during callus and shoot development from cotyledonary explants and to correlate them with ontogenic stages during in vitro culture. Cotyledons cultured in the presence of 2,4-dichlorophenoxyacetic acid produced friable callus as a result of early division of parenchymatic cells associated with the vascular bundles of the explant. The callogenic ability was independent of the cotyledonary region used as starting explant. Direct shoot organogenesis was observed from the same type of cells growing in culture media supplemented with kinetin and indole-3-acetic acid. In this case, the regeneration potential varied among regions from which the explants were obtained. Protein profiles revealed differences associated with shoots or callus developmental programs. A 27-kDa polypeptide was uniquely detected in the explants undergoing shoot organogenesis. The amount of this polypeptide during the first 4 d of culture increased and was followed by the appearance of meristematic centers in histologically analyzed samples. This polypeptide could be used as a specific marker for in vitro shoot development in this species.     

Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]

A successful, efficient system for multiple soybean shoot induction of soybean [Glycine max (L.) Merr.] is reported. Multiple shoots were induced from cotyledonary nodes and hypocotyl segments cultured on media supplemented with 2 mg/l thidiazuron (TDZ) or 1.15 mg/l benzyladenine (BA). It was found that TDZ induced adventitious shoots more efficiently than BA and that hypocotyl segments promoted more adventitious shoots than cotyledonary nodes. The optimal TDZ concentrations for shoot organogenesis from hypocotyl segments were between 1 and 2 mg/l. Basal media also influenced the efficiency of shoot organogenesis. The frequency of adventitious shoot formation tended to increase when the salt concentration in the basal media supplemented with 2 mg/l TDZ was reduced. Two media (1/2B5 and 1/2L2) stimulated shoot organogenesis efficiently from hypocotyl segments. This method can thus be advantageously applied in the production of transgenic soybean plants. Received: 3 July 1996 / Accepted: 9 May 1997     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Artemisia abrotanum</Emphasis> L. by means of somatic organogenesis

The goal of this project was to regenerate Artemisia abrotanum L., Southern wormwood, by means of organogenesis from leaves. In vitro plant propagation may greatly support the molecular characterization of the medicinal qualities of A. abrotanum. Young, intact leaves were excised from mature plants and surface sterilized. Abundant callus growth, as well as shoot formation, was produced on an MS medium supplemented with 4.44 μM BA and 0.54 μM or 0.81 μM NAA. Shoots, with some residual callus, rooted equally well in MS media with 0.49 μM IBA, 0.54 μM NAA, or without hormones. Rooted plants were best acclimated in potting soil.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Organogenesis plant regeneration from leaf explants of Exacum Styer Group

Exacum Styer Group plantlets were regenerated through direct organogenesis from leaf explants. Four genotypes were evaluated on MS media supplemented with combinations of BA (0, 0.44, 2.22, 4.44, or 8.88 μM) and NAA (0, 0.05, 0.54, or 2.69 μM) for direct shoot organogenesis without an intervening callus phase. Regression analyses were used to analyze and interpret the data. There were significant genotype, media, and genotype × media interactions for several variables. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively) while the 4.44 μM BA plus 0.54 μM NAA treatment induced the greatest number of shoots among the genotypes evaluated.     

Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii

In this study, mature zygotic embryos, plant growth regulators, and various media were tested with the aim of developing an efficient regeneration system for plantlets of the bamboo species Dendrocalamus hamiltonii. Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 1.0–3.0 mg/l 2,4-dichlorophenoxyacetic acid. Optimal shoot differentiation and subsequent shoot growth were also obtained in MS medium supplemented with 2 mg/l benzyladenine, 1 mg/l kinetin, and 1 mg/l naphthaleneacetic acid. Root induction was enhanced by the addition of 5 mg/l indole-3-butyric acid to the culture medium. Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus initiation, shoot differentiation, and the development of plantlets from the mature zygotic embryos. Our data provide a useful basis for developing culture protocols for the regeneration of bamboo plants.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

Plant regeneration from isolated cells ofLavandula latifolia medicus

Summary Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were subcultured to media containing 2 mg/liter BA and 15% coconut milk. Under these conditions, up to 75% of calli formed shoots that subsequently were rooted and established in soil.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Regeneration of grape (Vitis labruscana cv. Kyoho) by shoot-tip culture

We investigated the optimal levels of growth regulators, culture media, and pH on callus growth and organogenesis of in-vitro cultured ‘Kyoho’ grapes. Calli were induced by culturing leaf blades on an MS basal medium supplemented with 1 mg/IL BA and 0.01 mg/L 2,4-D. In addition, calli originating from the exocarp and mesocarp of grape fruits devel-oped on MS media supplemented with 0.1 mg/L IAA, NAA, or 2,4-D, or with 0.2 mg/L BA. In testing the potential for plant regeneration from shoot tips on various media, we found that the Nitsch medium, with I mg/L BA, was optimal for caulogenesis. The type of shoot development depended on the pH of the medium, with vigorous multiple-shoot devel-opment occurring at pH 6.0, and single shoots forming at pH 5.0. Finally, we were able to obtain rooted seedlings from the regenerated shoots that had been cultured on 1/4-strength Nitsch medium supplemented with 0.03 mg/L NAA.