A comparison of Hordeum bulbosum-mediated haploid production efficiency in barley using in vitro floret and tiller culture

Summary A high efficiency of Hordeum bulbosum-mediated haploid production in barley has been achieved using a floret culture technique in which florets pollinated with Hordeum bulbosum are cultured on modified N₆ media containing 0.5 mg/l kinetin and 1.2 mg/l2,4-D. Cultures were maintained at 25 °C with a 16 h photoperiod for 9 days before embryo rescue. In a comparison of haploid production efficiency using five F₁ hybrids from winter x winter and winter x spring barley crosses, 41.6 haploid plants/100 florets pollinated were produced using floret culture. Using detached tiller culture, 13.5 haploid plants/100 florets pollinated were produced. Higher efficiencies achieved with floret culture are attributed to the formation of larger, differentiated embryos. Such embryos lead to higher frequencies of plant regeneration. The F₁ from a winter x winter cross was inferior in haploid production compared to F₁s from winter x spring crosses. No genotype x technique interaction was observed.Oregon Agricultural Experiment Station Technical Paper No. 8653     

Somatic embryogenesis of agriculturally important lupin species (Lupinus angustifolius,L. albus,L. mutabilis)

Somatic embryos were obtained from immature cotyledons of Lupinus angustifolius, L. albus and L. mutabilis but not from L. luteus. Different kinds of basal media and plant growth regulators in primary and secondary culture were tested. The best induction media were based on B5 and were supplemented with 5 mg I^-1 2,4-D alone or with 0.25 mg I^-1 kinetin. Mature stage somatic embryos were obtained on media containing ABA (0.1–0.5 mg I^-1) and a high NH₄/NO₃ ratio. Embryo germination and plantlet development occurred on MS media supplemented with glutamine or GA₃.     

Regulation of Medicago sativa L. somatic embryogenesis by gibberellins

The influence of exogenous gibberellic acid (GA₃) andpaclobutrazol, an inhibitor of gibberellin biosynthesis, on growth of callusandsomatic embryogenesis in petiole-derived tissue cultures of Medicagosativa L. has been investigated. GA₃ (0.5–500M) or paclobutrazol(5–100 M) were added to either an induction (with 2,4 Dand kinetin) or a differentiation medium (without plant growth regulators).Gibberellin A₃, applied during the induction as well as thedifferentiation stage, reduced the weight of callus and increased the number ofsomatic embryos in Medicago sativa L. tissue cultures.Somatic embryo production was increased more by the presence of exogenousGA₃ in the differentiation than induction medium. The inclusion ofpaclobutrazol in the induction or differentiation medium caused the inhibitionof callus growth and embryo production. Callus growth was much less affectedthan embryogenesis. These results indicate that gibberellins are beneficial forboth embryoinduction and formation. The level of endogenous gibberellins is presumablysufficient for callus induction and growth. However, it seems not optimal forthe induction and particularly for the differentiation of embryos.     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.)

Summary Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos developed from these masses following transfer to a medium containing 3 mg/l 2,4-D. The embryo morphology was quite variable. Following transfer to hormone-free medium, these embryos germinated. Shoot elongation was obtained in 25% of the embryos following transfer to a medium supplemented with 0.5 mg/l each of BAP and Kn. The plants grown in vitro by this method survived in sand:soil mixture and were grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzyl amino purine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kn kinetin - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - Z zeatin     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr.

The effects of sucrose on maturation and of plant growth regulators on germination of soybean somatic embryos were investigated for the purpose of developing an efficient culture method for plant recovery. Somatic embryos produced on medium with a low sucrose concentration (5 gl^-1), less than 1 mm in length, 0.6 mg in fresh weight, and green in color, were grown for 2 weeks on MS medium containing 5 gl^-1 or 30 gl^-1 sucrose and then for another 5 weeks on MS medium containing 5–90 gl^-1 sucrose. The highest increase in fresh weight of somatic embryos was obtained in the treatment of transferring from 30 gl^-1 sucrose (2 weeks) to 60 gl^-1 (5 weeks). With the increase in fresh weight, the somatic embryos gradually changed color from green to yellow, and finally to white, when they stopped growth. Soybean seed storage proteins (-conglycinin and glycinin) were accumulated in somatic embryos under tissue specific and stage specific control analogous to that in zygotic embryos. Exogenous gibberellic acid was effective in promoting precocious germination of premature soybean somatic embryos, but was not necessary for the germination of mature somatic embryos. The efficiency of somatic embryo germination was as high as 77% from semi-wild soybean and 60–64% from cultivated soybeans, showing that the plant regeneration system developed in this study was efficient and practical.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BR brassinolide - GA₃ gibberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS Sodium Lauryl Sulfate     

Rapid propagation of lemongrass (Cymbopogon flexuosus (Nees) Wats.) through somatic embryogenesis in vitro

Somatic embryos induced from callus cultures of lemongrass [Cymbopogon flexuosus (Nees) Wats.] on Murashige and Skoog medium supplemented with 5 mg/l of 2,4-D, 0.1 mg/l of NAA and 0.5 mg/l of Kn developed into plantlets when plated on a medium supplemented with 3 mg/l of BA, 1 mg/l of GA₃ and 0.1 mg/l of NAA. The regeneration potential of callus was retained for more than 2 years on the nutrient medium supplemented with comparatively lower levels of growth regulators (2,4-D at 2 mg/l, NAA at 0.1 mg/l and Kn at 0.25 mg/l). Approximately 30–35 plantlets were produced after two months of culture per 100 mg of callus inoculated. Regenerants were transplanted into soil and transferred to the field for assessment of various morphological and biochemical characteristics. The results of 1 year of field trials showed that plants derived from somatic embryoids were more uniform in all the characteristics examined when compared with the field performance of plants raised through slips by standard propagation procedures. Thus, a procedure has been developed for high frequency long term plant production of lemongrass through in vitro methods.Abbreviations 2,4-D 2,4 -dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberllic acid - MS Murashige and Skoog (1962) basal medium     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic 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     

Somatic embryogenesis and regeneration of plantlets in protoplast cultures from somatic embryos of coffee (Coffea canephora P. ex Fr.)

Somatic embryogenesis was achieved in protoplast cultures of coffee. Protoplasts were isolated from cell suspension-derived somatic embryos of Coffea canephora. After repeated subculture in a medium supplemented with 0.5 mg/l of each of kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA), microcalli developed. Transfer of these microcalli to a medium lacking growth regulators resulted in the formation of globular embryos. Upon subculture without growth regulators they grew to well-differentiated embryos, Eventually some of them developed to plantlets which were transferred to the greenhouse for further observation.     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk.

Summary Abies nordmanniana Lk. is used in short intensive rotations for Christmas tree production. Thus there is a high demand for development of advanced propagation and breeding methods. Somatic embryogenesis was easily induced from immature (precotyledonary) embryos collected in July 1989 with cytokinin as the sole plant growth regulator. The proliferating embryogenic cell masses were characteristic of conifer somatic embryogenesis and could be maintained on a simple basal medium containing 5 M benzylaminopurine. Auxin inhibited induction as well as proliferation. Proliferation was improved by up to 30 % by addition of L-glutamine and/or casein hydrolysate. Neither cytokinin concentration nor culture on 3 different basal media, differing markedly in their nitrogen composition, affected the proliferation rate. Embryos matured using a 4 week subculture on medium containing 10 M abscisic acid and subsequent transfer to medium devoid of plant growth regulators.Abbreviations TDZ thidiazuron (Schering) - BAP benzylaminopurine (Sigma) - KIN kinetin (Sigma) - 2,4-D 2,4-dichlorophenoxyacetic acid (Sigma) - ABA +/2-cis-4-trans-abscisic acid (Sigma) - CH casein hydrolysate (Sigma Type 1, acidic) - L-gln L-glutamine (Sigma) - EDTA ethylene diamine tetra acetic acid (Sigma)     

Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales

Basal media and plant growth regulators were tested for the promotion of somatic embryogenesis from immature wheat-rye hybrid embryos. Influence of growth regulators and chilling on plant regeneration were tested on two media. A medium containing four amino acids-glutamine, arginine, glycine and aspartic acid-as the nitrogen source, promoted the production of, on average, twice as much embryogenic callus as the other media, and somatic embryos developed well. The growth regulator dicamba was significantly better than 2,4-dichlorophenoxyacetic acid in promoting somatic embryogenesis and subsequent plant regeneration. Germination of somatic embryos on both regeneration media was enhanced by cold treatment. Supplementing 190-2 plant regeneration medium with a combination of -naphthaleneacetic acid + benzyladenine, indole-3-acetic acid + kinetin or indole-3-acetic acid + zeatin resulted in equally high germination rates.Abbreviations 190-2 Plant regeneration medium of Chuang & Jia - 2,4-d 2,4d Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-o-anisic acid - AA Amino acid medium of Müller & Grafe - IAA Indole-3-acetic acid - BA Benzyladenine - NAA -Naphthaleneacetic acid     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoog's semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 M indoleacetic acid and 0.046 M kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.Abbreviations MS Murashige and Skoog's medium - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - RH relative humidity