High frequency plant regeneration from anther-derived cell suspension cultures via somatic embryogenesis in Catharanthus roseus

Summary A system for high frequency plant regeneration from cell suspension cultures in Catharanthus roseus is described. Calli were obtained from anthers cultured on Murashige and Skoog's medium supplemented with 1 mgl^-1 -naphthaleneacetic acid and 0.1 mgl^-1 kinetin. After the second subculture on solid medium, embryogenic callus was identified and transferred to liquid medium to initiate suspension cultures. Cells dispersed finely in the medium were subcultured at 14-day intervals. Upon plating onto the basal medium, yellowish compact colonies proliferated from the cells and more than 80% of them gave rise to somatic embryos. Subsequently, plantlets developed from the embryos. Both the plantlets and the source plants showed the normal somatic chromosome number of 2n=2x=16.Abbreviations MS Murashige and Skoog - MSNK MS medium + 1 mgl^-1 NAA + 0.1 mgl^-1 kinetin - NAA -naphthaleneacetic acid     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br.

Callus cultures were established from immature embryos of Calotropis gigantea (Linn.) R. Br. on a modified basal medium of Murashige & Skoog supplemented with 1 mgl^-1 2,4-D. In addition to 0.1 mgl^-1 of NAA the optimal BAP concentration for promoting shoot bud formation and growth was 2 mgl^-1. Rooting was induced when shoots were transferred to auxin-supplemented Bonner's solution or half-strength MS basal salt solutions.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-butyric acid - BAP 6-benzylaminopurine - Kin kinetin     

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 M (0.5 mgl^–1) -naphthaleneacetic acid and 0.33 M (0.05 mgl^–1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 M (1.0 mgl^–1) 6-benzylaminopurine and 0.53 M (0.1 mgl^–1) -naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 M (1.5 mgl^–1) -naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.     

Callus initiation,growth and plant regeneration in Plantago ovata Forsk. cv. GI-2

The technique for callus initiation, growth and plant regeneration from cultured hypocotyl explants of Plantago ovata cv. GI-2 is described. Best initiation and growth of callus was achieved on Murashige & Skoog's medium containing 2,4-dichlorophenoxyacetic acid (1.0 mgl^-1) and kinetin (1.0 mgl^-1). The callus showed maximum shoot differentiation on medium containing kinetin (4.0 mgl^-1) and -naphthaleneacetic acid (0.01 mgl^-1). Root formation of shoots was best on half-strength medium supplemented with 3-indolebutyric acid. The regenerated plants were successfully transferred into pots.     

In vitro regeneration from hypocotyl and seedling cotyledons of tronchuda (Brassica oleracea var. tronchuda Bailey)

Four tronchuda (Brassica oleracea var. tronchuda Bailey) cultivars were tested for their ability to regenerate in vitro on Murashige & Skoog (MS) medium supplemented with 3 different combinations of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Explants were either axillary bud-free whole cotyledons or hypocotyls from 7-day-old darkgrown seedlings. The ability to regenerate varied by cultivars, explants and the concentration of growth regulators. Hypocotyl explants of all 4 cultivars, and cotyledon explants of 2 cultivars, developed plantlets within 4 weeks. Hypocotyl explants produced more shoots than cotyledons. Cotyledon explants produced more roots than hypocotyls. Best shoot regeneration was on MS medium supplemented with 2 mgl^-1 BAP and 0.1 mgl^-1 NAA. Portuguesa produced the most shoots. Some regenerants varied in leaf shape and phyllotaxy.Abbreviations BAP benzylaminopurine - NAA napththaleneacetic acid - IBA indolebutyric acid     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

In vitro culture of Foeniculum vulgare: callus characteristics in relation to morphogenesis

Callus induction and morphogenic response of several fennel populations were determined by genotype and hormonal treatment. 100% callus formation occurred only in the Francia Pernod population under the action of 2, 4-dichlorophenoxyacetic acid or -naphthaleneacetic acid and kinetin. Only this last hormonal treatment induced shoot regeneration. Plant regeneration was observed especially in Francia Pernod population. Ths calluses grown in presence of 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid plus kinetin, showed considerable differences at cytological and histological level which were correlated to their different morphogenic capability.Abbreviations 2, 4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid     

Somatic embryogenesis from mesophyll protoplasts of Trigonella corniculata (leguminosae)

Mesophyll protoplasts isolated enzymatically from Trigonella corniculata divided to form callus, with a plating efficiency of 49% in Kao (1977) medium. Protoplast-derived tissues formed somatic embryoids at high frequency on MS medium with 2.0 mg L^–7 NAA and 0.5 mg L^–7 BAP. Embryoids developed into plants on MS medium lacking hormones.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - MES 2-(N-morpholine) ethanesulphonic acid - NAA -naphthaleneacetic acid On leave from Genetics Institute, Academia Sinica, Beijing, China     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Characterization of recurrent somatic embryogenesis of alfalfa on auxin-free medium

Callus cultures from 300 genotypes of alfalfa (Medicago sativa L.) were initiated from leaf, petiole, and internode explants placed on Blaydes medium containing 10.74 M -naphthaleneacetic acid, 11.42 M indole-3-acetic acid, and 9.29 M kinetin. Five genotypes produced somatic embryos. Upon transfer of these embryos to growth regulator-free Murashige and Skoog medium with B₅ vitamins, new somatic embryos repeatedly formed directly on older somatic embryos without an intervening callus phase in a cycle lasting about 30 days. These cultures have been maintained for two years, during which time their embryogenic capacity has remained stable. New embryogenic cultures could be started repeatedly from these genotypes. The elimination of sugars from the medium could stop recurrent embryogenesis. Glucose, maltose, and fructose stimulated recurrent embryogenesis more effectively than sucrose. Sucrose was superior to lactose, while sorbitol and mannitol did not stimulate recurrent somatic embryogenesis. The absence of nicotinic acid in the medium, as long as sucrose was present, was lethal to embryos of three of the five tested genotypes. The ability of this system to propagate embryos exponentially offers potential for development of new gene transfer systems and application to artificial seed technology.Abbreviations NAA -naphthaleneacetic acid - RSE recurrent somatic embryogenesis     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine