Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

Embryogenesis and plant regeneration of Medicago spp. in tissue culture

Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2–4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced. Respectively: Biotechnology Department, Alberta Research Council, Agriculture Canada, Beaverlodge, Alberta, and University of Alberta, Edmonton, Alberta, Canada     

Embryogenesis and plant regeneration from callus cultures derived from unpollinated ovaries of Hyoscyamus muticus L.

Unpollinated ovaries of Hyoscyamus muticus L. (commonly known as Egyptian henbane) were cultured on Murashige and Skoog and Bourgin and Nitsch media supplemented with various growth hormones to study the organogenesis, embryogenesis and regeneration of plantlets. Embryogenesis was reported for callus grown on both media containing 0.05 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Differentiation of roots and shoots from the calli also occurred in these media. Albinism or chlorophyll deficiency and variation in ploidy level were observed among the ovary-derived plantlets. Received: 7 April 1997 / Revised received: 2 August 1997 / Accepted: 2 September 1997     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

Isolation, culture and plant regeneration from mesophyll protoplasts of Digitalis obscura

High yields of protoplasts were obtained from mesophyll tissue of Digitalis obscura L. Osmotic potential of the isolation medium and Ca²⁺ were important in obtaining a high viability of the preparations. In different culture techniques employed, liquid-over-agar-solidified medium was superior to liquid medium alone. Agar plating technique was ineffective. On Murashige and Skoog modified medium with casein hydrolysate and several indoleacetic acid and benzyladenine combinations, isolated protoplasts underwent sustained mitotic division and produced calli. The calli formed shoots when transferred to regeneration media. Regenerated shoots could be easily rooted and developed into whole plants on half-strength Murashige and Skoog hormone-free medium.     

Embryogenesis and plant regeneration of pakchoi (Brassica rapa L. ssp. chinensis) via in vitro isolated microspore culture

Isolated microspores of various populations of three varieties of the Chinese cabbage pakchoi (Brassica rapa ssp. chinensis) were cultivated in vitro on NLN82 medium (Lichter 1982) and embryos and plantlets obtained with nine cultivars. The best embryo yield per bud was 57.4. A 33°C one day heat treatment was generally necessary to induce embryogenesis. Analysis of ploidy level through flow cytometry for two cultivars indicated that haploids were present.Abbreviations BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindol - NAA naphthalene acetic acid - DH doubled haploid     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Protoplast culture and plant regeneration in Lycium barbarum L.

Protoplasts were isolated from leaves of the woody plant Lycium barbarum L. and cultured in liquid nutrient medium TM-2 at a density of 10⁴–10⁵ cells ml^-1. After ten days of culture, regenerated colonies were transferred to the agar-solidified medium TM-3, and 5–7 days later to regeneration media PRM or TM-4. Formation of shoots was observed after 30–40 days. Completely formed and rooted plants were transferred to the soil. Cytological and morphological analysis of the regenerated plants revealed relative genetic stability of this species in the process protoplast — plant. The results obtained allow us to conclude that L. barbarum can be used in the experiments on somatic hybridization or direct gene transfer.     

Somatic embryogenesis and plant regeneration inPisum sativum L.

Somatic embryogenesis was induced in immature zygotic embryos of pea (Pisum sativum L.), synthetic auxins α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) being used. Only one (line HM-6) of 46 genotypes tested exhibited good potential for somatic embryogenesis. 2,4-D was found as the best somatic embryo inductor. Three different ways of somatic embryo conversion have been described. Plantlets from individual somatic embryos were micropropagated as somaclones and subsequently rooted. A sterile morphological mutant has been found within a group of fertile plants of T₀-generation. Sufficient amount of T₁-seeds is available for somaclonal variation studies.     

In vitro plant regeneration in Melia azedarach L.

Nodal explants of 3- 6-week-old seedlings cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) (17.75 μm) produced multiple shoots. Shoots were isolated and induced to root on 1/2-strength MS medium supplemented with indole-3-butyric acid (4.92 μm). In-vitro-rooted shoots resumed growth after a short period of acclimatization and resulted in plantlets which were successfully established in soil. In vitro flowering was observed in some of the nodal explants in the above medium, and also in cotyledonary leaves and internodal explants on MS medium supplemented with a combination of indole-3-acetic acid (IAA) (0.06 μm)+BA (4.44 μm) and IAA (0.06 μm)+kinetin (4.65 μm). Received: 25 October 1997 / Revision received: 1 May 1998 / Accepted: 15 May 1998     

Plant regeneration from hairy roots and calluses of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L.

Plants regenerated from hairy root culture and calluses of foxglove purple and periwinkle have been obtained. It was found that organogenesis in hairy root culture occurs spontaneously on hormone-free medium but with varying efficiency. The frequency of direct shoot formation from root cultures was up to 60% in Digitalis and 3.7% in Vinca. The addition of 1 mg/L BA, 0.1 mg/L NAA, and 5% sucrose to B5 medium increases regenerative capacity of periwinkle up to 19.1%. Regenerated plants showed morphological features typically seen in Ri-transgenic plants. They include growth and plagiotropism of the root system, increased shoot formation, changed leaf morphology, and short internodes.     

Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers

In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.     

Somatic embryogenesis and plant regeneration in Gloriosa L

Friable callus was initiated from shoot apices of Gloriosa superba L. on basal MS medium supplemented with 2, 4-D (4mg L(-1)) + Kn(5 mg L(-1)) + CH(10 mg L(-1)) + CW(20%). Subculture of callus on the same medium after 4-5 weeks showed induction of large number of somatic embryos, which was confirmed with histological studies. Development of embryoids in plantlet took place when the embryogenic callus was transferred to basal MS medium supplemented with BAP (5 mg L(-1)), CH(50 mg L(-1)) +CW(20%). Roots were developed by subculturing them on to the medium containing Kn or BAP (5 mg L(-1)) and IBA (4 mg L(-1)). Plantlets were successfully transferred to pots containing mixture of soil, sand and farmyard manure (2:1:1).     

Somatic embryogenesis and plant regeneration in Cichorium intybus L. (Witloof,Compositae)

Somatic embryogenesis of Cichorium intybus L. var. Carolus is induced using cubical pieces of mature tap roots with an intervening callus phase. A Murashige and Skoog's (MS) semi solid basal medium supplemented with 2,4-dichlorophenoxyacetic acid (0.02 or 0.2 mg/l) and benzylaminopurine (0.25 mg/l) and a liquid MS medium devoid of growth regulators are used respectively for induction of callus and somatic embryoids and for further development and germination. Regeneration from the nodular proembryonal stage to the full grown embryoids occurs following different morphological pathways depending on the physical and chemical environment of the culture. Further development of these embryos into plantlets and the possibilities of application of this technique in plantbreeding have been discussed.Abbreviations MS Murashige and Skoog medium - BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from cotyledons of walnut,Juglans regia L.

Immature cotyledons of open-pollinated seeds from five walnut (Juglans regia L.) cultivars were excised from fruits at 6–11 weeks after full pistillate bloom and grown on a sequence of media to induce somatic embryogenesis. Globular, heart, cotyledonary and complete somatic embryos were obtained. Embryogenic cultures were maintained for more than a year by repetitive embryogenesis in which the roots, cotyledons and hypocotyls of somatic embryos formed additional adventive somatic embryos. Mature somatic embryos required a cold treatment of 8–10 weeks at 2–4°C to overcome apical dormancy. Selected plantlets derived from these somatic embryos were grown to young plants in soil. In addition, somatic embryogenesis was induced in J. hindsii (Jeps.), Jeps., and in Pterocarya sp., another member of the Juglandaceae.