Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL^–1 picloram + 0.01 mgL^–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL^–1 BAP plus either 0.2 mgL^–1 picloram or 0.1 mgL^–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - TDZ thidiazuron     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Solanum Nigrum is a Model System in Plant Tissue and Protoplast Cultures

Solanum nigrum is a model system especially for newcomer to the subject of plant tissue culture. Shoot culture has been easily established from shoot cutting of germinated seeds on Gamborg (B5), or Murashige and Skoog (MS) medium without phytohormones. Direct regeneration was possible using basal media B5, B5C (B5 supplemented with 5 % coconut endosperm milk), Schenk and Hildebrandt (SH), and MS, leaf, stem, shoot tip as explants, cytokinins benzylaminopurine (BAP) or kinetin (KIN) at concentrations from 0.25 to 2 mg dm^–3, and different light treatments (dark, dim and normal light). The best culture condition for shoot formation was the culture of stem internode segments on B5 medium supplemented with 0.5 mg dm^–3 BAP at 16-h photoperiod (irradiance of 100 µmol m^–2 s^–1). Also, root formation was possible under different culture conditions. The best culture condition was the culture of microshoot segments on half strength MS medium supplemented with 1 mg dm^–3 isobutyric acid. Induction of callus formation from young and mature tissues on MS medium supplemented with 0.5 mg dm^–3 BAP, 0.1 mg dm^–3 2,4-dichlorophenoxyacetic acid and 1 mg dm^–3 naphthalene acetic acid, and subsequent plant regeneration on B5C medium supplemented with 0.5 mg dm^–3 BAP was easy. Regeneration of protoplasts isolated from shoot tips and fully expanded leaves was also simple. Finally, the transfer of rooted plantlets to the soil was successful.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Effect of brassinolide on callus growth and regeneration in Spartina patens (Poaceae)

The effect of brassinolide (BL) on cultured calluses of Spartina patens (Ait.) Muhl. (Poaceae), a halophyte monocot was studied. BL at 0.03–0.04 mg l^–1 at fixed concentrations of IAA (0.2 mg l^–1) and BA (3.0 mg l^–1) added in MS medium increased the ratio for fresh weight (CIRFW) to dry weight (CIRDW) by 96–111% and 235–326%. Similarly, in callus regeneration capacity, BL at 0.03 mg l^–1 was most effective, increasing the shoot regeneration ratio (SRR) by 425%. BL at 0.04 mg l^–1 had not such an increasing effect as BL at 0.03 mg l^–1, which increased SRR by 79%. However, BL at 0.005 mg l^–1 promoted regenerated shoot growth most significantly, increasing the shoot height increasing ratio (SHIR) by 395% after a 40-day culture. BL at 0.05 mg l^–1 was least effective in the callus regeneration and regenerated shoot growth, decreasing SRR by 27% and SHIR by 52%. Present results suggest that BL at 0.03 mg l^–1 is suitable for the callus growth and shoot regeneration, while BL at 0.005 mg l^–1 effectively enhanced the regenerated shoot growth.     

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.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Efficient plant regeneration in vitro in buckwheat

An in vitro highly efficient plant regeneration system was established from hypocotyl segments in buckwheat (Fagopyrum esculentum Moench.). Calli were induced on Murashige–Skoog (MS) medium containing 1.0 mg l^–1 to 2.0 mg l^–1 2,4-dichlorophenoxyacetic acid and 1.5 mg l^–1 6-benzylaminopurine. Shoot buds were formed on subcultured pieces of callus. A high frequency (over 80%) of shoot differentiation was obtained on MS medium supplemented with 2.0 mg l^–1 6-benzylaminopurine and 1.0 mg l^–1 6-furfurylaminopurine. The regenerated shoots rooted readily on MS medium plus 0.2 mg l^–1naphthaleneacetic acid and 0.2 mg l^–1 indole butyric acid. The regenerated plantlets were acclimatized and successfully transferred to pots. Chromosome examination showed that the regenerated plants had normal chromosome number (2n=16).     

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.     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Picloram-induced somatic embryogenesis in pejibaye palm (Bactris gasipaes H.B.K.)

Somatic embryogenesis in pejibaye or peach palm (Bactris gasipaes H.B.K.) was induced from callus derived from in vitro cultured shoot tips of young field-grown plants using a modified Murashige and Skoog medium supplemented with 5 mg L^–1 of N⁶-benzyladenine (BA) and 0.06 mg L^–1 of picloram for three months in the dark; this was followed by an additional three months with the same medium and incubation conditions, but using 0.03 mg L^–1 of picloram. The cultures were then transferred to light on a medium without hormones. This led to the formation of morphogenic callus, in which somatic embryos, as well as shoot primordia, and finally complete plantlets were formed. These plantlets continued to grow on transfer to the greenhouse.     

Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

A rapid and efficient method for regeneration of plantlets from embryo explants of cumin (Cuminum cyminum)

A new, simple and efficient method was developed for multiple shoot regeneration of cumin from imbibed embryo cultures. This method yielded a large number of shoots within short period of time (30–50 days) without any subculturing. The effects of different media, different embryo explants and various combinations of plant growth regulators (PGRs) on callus formation and shoot regeneration in cumin were investigated. Simultaneous callus formation and shoot regeneration was obtained. The best response for multiple shoot regeneration was observed on B5 medium containing 1.0 mg l^–1 BAP, 0.2 mg l^–1 NAA and 0.4 mg l^–1 IAA, with an average of 140 shoots per explant.     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

Embryogenesis and Regeneration of Green Plantlets from Wheat (Triticum aestivum) Leaf Base

A short-term regeneration system from leaf-base-derived callus of wheat (Triticum aestivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of 3–4-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). MS medium with 2,4-D 4.5–9.0 mol l^–1 was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

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.     

In vitro regeneration from excised leaves of Flaveria trinervia (Sprengel) C. Mohr

Flaveria trinervia (Compositae) leaves are used for the treatment of jaundice and fever. From the leaf callus cultures regeneration of plantlets has been achieved. The results showed that BAP greatly stimulated the bud formation in concentrations ranging from 2–5 mg l^–1 than at very low concentrations (0.2–1.0 mg l^–1). Roots developed on the regenerated shoots, over a range of treatments, but were most prolific in the medium containing 1 mg l^–1 IAA. Histological observations revealed that cultured spongy cells of the mesophyll were greatly enlarged and underwent repeated cell divisions leading to the formation of hard nodular callus from which shoot buds differentiated. The shoots obtained were readily rooted and transplanted into glass houses. Cytological studies of the callus showed abnormalities such as bridges, endomitosis and multinucleolate conditions. Root tip squashes of the regenerated plants showed no variations and were diploid in chromosome number.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA napthalene acetic acid - IAA indole acetic acid - BAP 6-benzyl aminopurine - Kn kinetin