Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l⁻¹) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l⁻¹) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l⁻¹ TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

Comparison of callus induction and plant regeneration from different explants in triploid and tetraploid turf-type bermudagrasses

The turf-type bermudagrasses are genetically variable and do not respond uniformly to tissue culture and plant regeneration protocols. We evaluated the callus induction response of two explant types, young inflorescences and nodes, from multiple genotypes including triploid TifSport, TifEagle, and Tift97-4 and tetraploid Tift93-132, Tift93-135, Tift93-156 and Tift93-157 on MS medium supplemented with 1–1.5 mg l⁻¹ 2,4-D + 0.01 mg l⁻¹ BA + 1.16 g l⁻¹ proline. Four types of callus were observed. Type I was fluffy, soft, and white non-embryogenic callus, common to all cultures. Type II was globular, transparent, and hard, but sticky callus, which was pre-embryogenic and could be selected for subculture. Type III callus was transparent, compact, and embryogenic. Type IV callus was opaque white and compact. Both Type III and Type IV calluses were embryogenic and regenerative. A combination of gelling agents in the medium (2 g l⁻¹ Gelrite and 5 g l⁻¹ agar) improved callus quality and increased the rate of compact callus formation during subculture. Embryogenesis from compact callus was observed in TifEagle, TifSport and Tift93-132, and shoots were regenerated on MS medium with 0.1 mg l⁻¹ 2,4-D + 0.5–4.0 mg l⁻¹ BA. Low intensity light treatment (30 μmol m⁻2 s⁻¹ of cool white fluorescence) to callus before regeneration greatly enhanced regeneration frequency from 6.7% to 40% in recalcitrant TifSport.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

In vitro callus induction and regeneration studies in Withania somnifera

Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-D (2 mg l⁻¹) and KN (0.2 mg l⁻¹). Shoots differentiated best from axillary shoot base callus on MS medium containing BA (2 mg l⁻¹). Regenerated shoots rooted best on MS medium containing IBA (2 mg l⁻¹) alone, and IBA (2 mg l⁻¹) with IAA (2 mg l⁻¹). Plantlets were transferred to pots containing sand and soil mixture, acclimatized in a culture room and afterwards transferred to the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency plant regeneration from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture

An in vitro protocol for efficient plant regeneration has been developed from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture. Embryos with (endosperm-supported culture, ES) or without endosperm (non-endosperm-supported culture, NES) were excised from mature seeds and cultured on MS medium supplemented with various concentrations of 2,4-D (1–5 mg l⁻¹) for callus induction. The percentage of callus induction from ES explants was significantly (P < 0.05) lower than that from NES. The highest frequency (97.6%) of callus induction was obtained from NES explants on MS medium containing 3 mg l⁻¹ 2,4-D. When the primary calli were maintained at a reduced concentration of 2,4-D (0.5 mg l⁻¹) for 3 weeks, embryogenic calli were formed. The embryogenic calli were then transferred to MS medium supplemented with different concentrations of BA (1–5 mg l⁻¹) and 500 mg l⁻¹ casein hydrolysate (CH) for shoot regeneration. However, the capacity of plant regeneration from ES explant-derived calli was significantly (P < 0.05) higher than that from NES. The best response (81.3%) was observed from ES explant-derived calli on MS medium containing 2 mg l⁻¹ BA. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds. This method could be employed for genetic manipulation studies.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Plant regeneration and production of embelin from organogenic and embryogenic callus cultures of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm. f.—a vulnerable medicinal plant

Embelia ribes, an important vulnerable medicinal liana, was regenerated through organogenesis and embryogenesis using leaf explants. Leaf explants produced organogenic calluses on MS medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzylaminopurine. Shoot regeneration was obtained from organogenic calluses on MS medium containing different concentrations of thidiazuron (TDZ) and indole-3-acetic acid (IAA). The frequency of shoot bud organogenesis was highest (23.9 shoots/explant) in MS medium containing 0.5 mg l⁻¹ TDZ and 0.1 mg l⁻¹ IAA. The best result for induction of embryogenic callus was noticed in the combination of 2.0 mg l⁻¹ TDZ and 0.5 mg l⁻¹ 2,4-D. This callus, maintained in the same medium, showed the highest differentiation of embryos (56.5%) after 6 wk of culture. Embryos were transferred to MS medium supplemented with different concentrations of TDZ, and this facilitated conversion of embryos into plants. After 6 wk of subculture, MS medium with 0.05 mg l⁻¹ TDZ favored the highest percentage (52.2%) embryo conversion. As per the present protocol, 52.2% of the embryos underwent conversion, and a mean number of 29.5 shoots per culture was obtained. Shoots developed from both types of calluses were rooted on half-strength MS basal medium supplemented with 1.0 mg l⁻¹ indole-3-butyric acid. HPLC-UV assay demonstrated the highest embelin content (5.33% w/w) in the embryogenic callus cultures. Embelin was isolated from embryogenic callus and was identified using IR and ¹H NMR studies.     

Synergistic effect of DFMO and 2,4-D on regeneration of Tabernaemontana persicariaefolia jacq in vitro

Callus cultures of Tabernaemontana persicariaefolia, (Apocynaceae), an endangered species endemic to the Mascarene Islands, were established from leaf explants on MS medium containing either 5 mg·l⁻¹ 2,4-D and 0.5 mg·l⁻¹ BA or 5 mg·l⁻¹ 2,4-D, 0.5 mg·l⁻¹ BA and 200 mg·l⁻¹ DFMO. Histological studies showed regenerating nodules resembling globular embryos in calli after 4 weeks on the DFMO medium. Green shoot formation was achieved by sequential subculture of the induced calli on media with gradually decreasing 2,4-D concentrations (5→1→0 mg·l⁻¹). Regeneration was greatly stimulated in the presence of DFMO. The first emergence of shoots occured 3 weeks earlier than in untreated callus cultures.     

Direct somatic embryogenesis from cotyledon and cotyledonary node explants in bishop’s weed <Emphasis Type="Italic">Trachyspermum ammi</Emphasis> (L.) sprague

A three-stage procedure for embryogenesis in Trachyspermum ammi was developed from cotyledon and cotyledonary node explants cultured in Murashige and Skoog (MS) liquid medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Globular somatic embryos without intervening callus phase developed in 4 wk. The development of embryos to heart and torpedo stages required second-stage subculture of the explants (along with developing embryos) in liquid medium with lower concentrations of 2,4-D. Further development of embryos required a third-stage subculture in hormone-free liquid medium supplemented with 100 mg l⁻¹ casein hydrolysate. Regeneration of complete plantlets occurred after the fully developed somatic embryos were transferred to solidified half-strength MS medium supplemented with 1 mg l⁻¹ gibberellic acid.     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> Swartz)

Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical, and subtropical regions. A callus induction and plant regeneration protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg l⁻¹ 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.1 or 1.0 mg l⁻¹ benzylaminopurine (BA). A multifactorial experiment demonstrated the combination of 3 mg l⁻¹ dicamba and 1.0 mg l⁻¹ BA for induction of callus resulted in 12 times higher plant regeneration frequency compared to 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone or ten times higher plant regeneration frequency than the combination of 3 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ BA. These results are expected to support the development of a genetic transformation protocol for seashore paspalum.     

Somatic embryogenesis from rhizome explants of <Emphasis Type="Italic">Cymbopogon winterianus</Emphasis>

Plantlet regeneration through indirect somatic embryogenesis was attempted from rhizome derived callus of Cymbopogon winterianus Jowitt (cv. Jorlab2). Optimum callus was induced on Murashige and Skoog (MS) basal medium supplemented with 4 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D). Initially the callus was friable, shiny white and watery in nature. After subculturing on MS medium containing 2,4-D and kinetin (Kn), callus was transferred onto the MS medium supplemented with 2,4 -D, Kn and coconut water to induce somatic embryogenesis. Optimum somatic embryogenesis (78.33 %) was achieved on MS medium containing 3.0 mg dm⁻³ 2,4-D and 0.5 mg dm⁻³ Kn. High frequency (65 %) plantlet conversion from embryos was achieved in MS medium supplemented with 2 mg dm⁻³ N⁶-benzyladenine (BA), 0.5 mg dm⁻³ Kn, 0.2 mg dm⁻³ calcium pantothenate and 0.2 mg dm⁻³ biotin.     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.) Marechal]: a recalcitrant grain legume

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with 0.75 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ 6-benzylaminopurine (BA) and with various additives (50 mg l⁻¹ ascorbic acid and 25 mg l⁻¹ each of adenine sulphate, citric acid and l-arginine). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with 0.25 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing 0.2 mg l⁻¹ BA and 2.0 mg l⁻¹ gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.