Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Summary Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.     

Somatic embryogenesis and plant regeneration from leaf tissue of jojoba

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength Murashige and Skoog basal medium (MS/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.35–4.52 μM) with 6-benzylaminopurine (1.33–4.43μM) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N′-phenylurea (1.21–4.03μM) or (E)-6-[3-(trifluoromethyl)-but-2-enylamino] purine (1.11–3.71μM) resulted in formation of embryogenic cultures and somatic embryos. After two 30-day subcultures, embryogenic cultures were transferred onto MS/2 medium supplemented with different auxins and cytokinins. Somatic embryo maturation, germination and plantlet formation were achieved using 1-naphthaleneacetic acid (3.75μM) or indole-3-butyric acid (3.44μM) in combination with BA (0.44 or 1.33μM) or F3iP (0.37 or 1.11μM). Histology confirmed each stage of development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. Ex Steud.

Embryogenic cultures of the common reed [Phragmites australis (Cav.) Trin. Ex. Steud.] were induced on Murashige and Skoog (1962)-based medium with 2% (w/v) sucrose, B5 vitamins and 4.5 μM 2,4-dichlorphenoxyacetic acid. Four independent culture lines, two initiated from stem nodes and two from roots, were established. These cultures underwent somatic embryogenesis. In one line of stem node origin, the somatic embryos germinated and developed into plants, following transfer of embryogenic cultures to Murashige and Skoog (1962)-based medium lacking growth regulators, with 108 ± 17 plants being recovered per 100 mg fresh weight of culture. In other lines, the somatic embryos developed roots, but not shoots. Shoot regeneration via somatic embryogenesis offers potential as anin vitro system for physiological studies, including assessments of the response of common reed to environmental pollutants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

Regeneration from anthers of adultCamellia japonica L

Summary Embryogenesis and callus formation inCamellia japonica L., cv. Elegans and cv. Ville de Nantes (>50 yr old), were higher when anthers at tetrad stage or early uninucleate microspore stage were used. Direct embryo formation was obtained, both in anthers and anther-petaloids. Embryogenesis only occurred under light on modified Murashige and Skoog medium supplemented with 6-benzylaminopurine (MS10). Embryo production was higher from petaloids (2.2 to 3.5 embryos/petaloid) than from anther locules (1.2 to 1.5 embryos/anther). However, petaloid-derived embryos aborted by Week 10 of culture. The embryogenic efficiency of anthers was 5.3% for cv. Elegans and 4.1% for cv. Ville de Nantes. Scanning electron microscopy images showed that anther-derived globular embryos were covered by a layer of material of smooth texture. Shoot regeneration efficiency by organogenesis was 6.8%. Regeneration by embryogenesis was 60% for cv. Ville de Nantes and 97.5% for cv. Elegans.     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.     

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd

Protolasts of Bupleurum scorzonerifolium were prepared from stem node-derived embryogenic calli with an enzyeme mixture, in which snailase was a necessary component. Follolwing cell wall regeneration protoplasts divided and directly formed somatic embryos which developed into plantlets. The conditions favorable to direct embryo formation were investigated, and the nature of the callus used for protoplast preparation was found to be a critical factor. The osmotic concentration and the composition of the culture medium including the phytohormone combinations were also important.     

Indirect somatic embryogenesis and plant recovery from cotton (Gossypium hirsutum L.)

Summary We describe a tissue culture procedure for somatic embryogenesis and plantlet regeneration in cotton (Gossypium hirsutum L. cv. Coker 312). Callused explants or individual globular embryos were transferred to basal media to induce somatic embryogenesis. To determine characteristic early indicators of successful germination and conversion, we identified six types of embryos that developed on basal media. Two of the six embryo types, designated as tulip-shaped and trumpet-shaped, could undergo conversion in preliminary tests, whereas the others had little or no developmental potential. Several media treatments designed to enhance the maturation of globular somatic embryos failed to increase the fraction of embryos which matured to form recoverable types. In efforts to improve plantlet recovery, tulip-shaped embryos were used in limited trials to contrast the effects of chemical and physical desiccation treatments on germination and conversion. The selective use of tulip-shaped somatic embryos, coupled with partial desiccation, seems to have augmented plant recovery. Growth habit, flowering, seed set, and lint production of most of the regenerated plants were comparable to seed-derived plants grown under the same conditions. Partial research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Plant regeneration from undeveloped ovules and embryogenic calli of Citrus: Embryo production,germination, and plant survival

Embryogenic cultures were initiated from undeveloped ovules of several polyembryonic Citrus species on a basal medium supplemented with either malt extract, 2,4-D alone, or 2,4-D in combination with BA or daminozide. Primary embryos of all responsive cultivars were harvested directly from ovule cultures; secondary embryo harvests were made from Handin orange ovule cultures and long-term embryogenic callus. Differences were observed among cultivars and treatments in percentage of responsive ovules and total number of embryos produced. The most effective treatment for embryo production varied among cultivars. Embryo germination and plant establishment frequencies were determined for this plant regeneration system. Differences among cultivars with respect to regenerate survival percentage were minimal. Plant regeneration via secondary or long-term callus-derived embryos was as efficient as from primary embryos. Critical factors influencing plant production and survival were the production of normal viable embryos, balanced germination, and successful acclimatization to the external environment.     

Somatic embryogenesis and plant regeneration inAzadirachta indica A. Juss

Summary Media for induction of somatic embryogenesis from immature cotyledonary tissues ofAzadirachta indica (Neem) were determined. Callus was initiated on Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ of indol-3 acetic acid, 1.0 mg·liter⁻¹ of 6-benzyl amino purine, and 1000 mg·liter⁻¹ of casein hydrolysate. Effect of kinetin was also studied for embryo induction. Carbohydrate source in the form of sucrose and glucose alone and in combination was tested for embryogenic efficiency. Seventy percent embryos showed germination. Healthy plants were potted in sand and soil. Histologic studies confirmed indirect somatic embryogenesis.     

High frequency plant regeneration from Aralia cordata somatic embryos

An efficient method of plant regeneration from Aralia cordatasomatic embryos was developed. Somatic embryos at early stages obtained through inflorescences–derived embryogenic cell suspension cultures were matured in liquid Murashige and Skoog (MS) medium containing various concentrations of abscisic acid (ABA). For plant regeneration, mature cotyledonary embryos were transferred to solid MS basal medium for 6 weeks. Plant regeneration frequency of the embryos matured from heart-shaped embryos was proportional to the concentration of ABA from 0.76 to 3.8 M. The highest frequency (60.7%) was obtained from 3.8 M ABA pretreatment. The survival rate of the plantlets after transfer to plastic pots containing vermiculite in the growth room was 90%. All plants transferred to soil in greenhouse survived. The results indicate that micropropagation procedure can be applied for an efficient mass propagation of Aralia cordata.     

Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 M) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.Published as Paper No. 785 in the journal series of the Experiment Station, HSPA     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of <Emphasis Type="Italic">Melia azedarach</Emphasis> (Meliaceae)

Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.