Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize

Six media compositions and different transfer times were compared in order to improve the quality of microspore derived structures and their regeneration capacity. The production of regenerated plantlets was increased by a factor of 10 by an early transfer of anthers to medium with low sugar concentration and the use of kinetin-like growth regulators. The best transfer date was 3 weeks after initial plating, onto medium containing 25 gl^–1 sucrose and 1 mg l^–1 kinetin. This study demonstrates that anther transfer at an early developmental stage, before macroscopic appearance of embryo-like structures, improves embryo quality and further regeneration. This production of androgenetic structures showing normal morphological features is the first step to obtain an improvement of androgenetic plant regeneration yield.     

Regeneration of <Emphasis Type="Italic">Pelargonium × hederaefolium</Emphasis> ‘Bonete’ from petiole explants

The adventitious shoot regeneration from petiole explants of Pelargonium × hederaefolium ‘Bonete’ was achieved via a mixed pathway i.e. organogenesis and somatic embryogenesis. The histological study of regenerated structures revealed the presence of both shoot primordia and embryo-like structures. The initial growth in petiole explants occurred on media with BAP + auxin or TDZ alone. However, the most effective regeneration (24 structures/explant) was noted in the presence of TDZ (2 mg l⁻¹) and IBA (0.1 or 0.2 mg l⁻¹). Moreover, the application of TDZ in the induction phase reduced the time needed for the formation of adventitious structures and positively influenced the further shoot development on the medium containing m-topolin and IBA.     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Callus induction and shoot regeneration of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. cv. Wonder Bush and cv. Pole Sieva

We report the first protocol for callus induction and shoot regeneration for Phaseolus lunatus L. cv. Wonder Bush and cv. Pole Sieva. We used different explants viz., epicotyls, cotyledons and hypocotyls. The medium used was MS basal medium with thidiazuron (0.5 mg l⁻¹) and IAA (0.05 mg l⁻¹) for the induction of callus followed by BAP (1.0 mg l⁻¹) for the induction of shoots. Epicotyl explants showed the fastest response and the highest percentage of shoot regeneration. This protocol opens new biotechnological strategies to transfer economically important genes to this important crop species.     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Crown and leaf slices of in vitro plantlets of a non-flowering Vetiveria zizanioides from Java were used to induce compact calli and to regenerate plantlets. The influence of different growth regulators (2,4-dichlorophenoxy acetic acid, 6-benzylaminopurine), sucrose concentrations (10–100 g l⁻¹), cultivation in light or dark, and cultivation time on callus induction medium (6 or 12 weeks), on the induction of compact callus and the subsequent regeneration of plantlets was studied. Up to 75% of crown slices cultured on modified Murashige and Skoog medium supplemented with 2.26 μM 2,4-dichlorophenoxy acetic acid, 2.22 μM 6-benzylaminopurine and 75 g l⁻¹ sucrose developed compact callus. For subsequent regeneration of plantlets, callus induction in the light for 6 weeks on the callus induction medium containing 10 g l⁻¹sucrose, and subsequent transfer to the regeneration medium, was the best procedure, regenerating plantlets on around 60% of the crown or leaf slices, with up to 100 plantlets per slice. We have compared the efficiency of the above mentioned procedure with several other methods to regenerate plantlets. Our findings indicate that the procedure developed in this study was best in regenerating plantlets for the used vetiver variant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

Regeneration of sorghum from shoot tip cultures and field performance of the progeny1

A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l⁻¹) and kinetin (0.1 mg l ⁻¹) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about 5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l⁻¹) and indole-3-acetic acid (0.5 mg l ⁻¹) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation over other protocols using different explants are discussed. 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.     

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.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

Efficient and rapid in vitro plant regeneration system for Indian cultivars of chickpea (Cicer arietinum L.)

Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l⁻¹ 6-benzyladenine (BA) and 0.04 mg l⁻¹ α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l⁻¹ indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop.     

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.     

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.     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.