Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Efficient plant regeneration from suspension cells of <Emphasis Type="Italic">Allium cepa</Emphasis> L.

Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O·Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O·Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.Abbreviations BAP 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

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.     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

Effects of mutagens on somatic embryogenesis and plant regeneration in groundnut

The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm⁻³ 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10–50 Gy) or treated with 1–5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm⁻³ 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm⁻³) and 0.5 mg dm⁻³ 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm⁻³ BAP and 0.25 mg dm⁻³ naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

Cultural behavior of protoplasts from different organs of eggplant (solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 Em^-2s^-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl^-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl^-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×10³ protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×10³ and 1,220.4×10³ protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl^-1) + zeatin (0.5 mgl^-1) + NAA (1 mgl^-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl^-1) + IAA (0.2 mgl^-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl^-1) + NAA (0.5 mgl^-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.Abbreviations (IAA) Indol-3-acetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (NAA) naphthale-neacetic - (BAP) 6-benzylaminopurine - (MS) Murashige and Skoog basal medium - (CPW) Cell and Protoplast Washing solution     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.     

In vitro regeneration from bulb explants of Indian squill,Urginea indica Kunth

Callus cultures were established from bulb explants of diploid Urginea indica Kunth (Indian squill) on a modified basal medium of Murashige and Skoog (1962) supplemented with either 2 mg/l^-1 2,4-D+15% (v/v) CM or 4 mg/l^-1 2,4-D+2 mg/l^-1 NAA+2 mg/l^-1 KN+1 g/l^-1 YE. Shoot primordia developed after 2–3 subcultures in that medium. Increased growth of shoot primordia was obtained in media containing less auxins and vitamins. Rooted bulbous plantlets obtained were maintained in MS medium with 0.5% sucrose.Adventitious shoots were induced from adaxial epidermal cells of outer scales of regenerated bulbs used as secondary expiants in presence of 1 mg/l^-1 of 2,4-D with slightly higher concentration of the three vitamins of MS medium. From each scale leaf, approximately 400 bulblets were produced in 18 weeks in liquid culture. 90% of the plants transferred to potted soil have survived.     

A simple culture method for Brassica hypototyl protoplasts

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2–4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4–6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3–4 weeks.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4 — dichlorophenoxyacetic acid