Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

Somatic embryogenesis in sisal (Agave sisalana Perr. ex. Engelm)

A protocol has been developed for somatic embryogenesis and plant regeneration of sisal (Agave sisalana Perr. ex. Engelm). Embryogenic callus cultures were initiated from young shoots raised in vitro from the stem portion of the bulbil on medium supplemented with 1–2 mg l^-1 kinetin (KN) and 0.2–0.5 mg l^-1 -naphthaleneacetic acid plus KN or 1–1.5 mg l^-1 benzylaminopurine (BAP) or 0.25–0.5 mg l^-1 2,4-dichlorophenoxyacetic acid plus BAP or 0.5–1.0 mg l^-1 KN. Embryos at various developmental stages (globular-, heart- or torpedo-shaped) produced mature and germinating embryos on being transferred to a new medium containing 0–0.25 mg l^-1 KN. After 28 days, a maximum of 76% germinated embryos was obtained on a medium supplemented with 0.1 mg l^-1 KN. The capacity for embryogenesis remained constant in the callus upon subculturing on the same medium for more than 48 months. Histological observations showed a distinct multicellular origin for most of the somatic embryos as they developed from epidermal, sub-epidermal and inside callus cells, while a few of them originated from a superficial callus cell. Plantlets regenerated from embryos were transferred to the field where their survival rate was 100%.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indoleacetic acid - KN Kinetin - NAA -Naphthaleneacetic acidCommunicated by W. Barz     

Media optimization for maximum biomass production in cell cultures of pacific yew

Three cell lines of Taxus brevifolia Nutt. with differing growth rates were used to assess the effects of basal salt mixtures, carbohydrates, organic nitrogen additives, vitamin formulations, and plant growth regulators on callus growth. Gamborg's B5 major salts provided significantly better growth than all other salt formulations tested. The greatest biomass was obtained with 1% total carbohydrate. The best carbohydrate combination, 0.5% fructose + 0.5% sucrose, was significantly better than all other combinations of carbohydrates tested. A complex vitamin mixture was significantly better than any one previously published vitamin formulation. Greatest rates of callus growth were obtained with 4.14 M (1 mg l^-1 picloram, 0.46 M (0.1 mg l^-1 kinetin, and 0.38 M (0.1 mg l^-1) abscisic acid or 0.29 M (0.1 mg l^-1 gibberellic acid. Our final medium, TM5, is superior to published methods for the general callus culture of T. brevifolia. This medium has improved growth in three tested cell lines to provide doubling times of 3.5 to 5.6 days, an average 5.3-fold increase over our previously published medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid, 2ip-6-(,-dimethylamino)-purine - ABA abscisic acid - BA 6-benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Induction and growth of callus from immature inflorescences of “Zebra” bermudagrass as affected by casein hydrolysate and 2,4-D concentration

Summary Regeneration of grass plants through tissue culture is affected by many factors including genotype and type and concentration of medium components, particularly growth regulators. Objectives of this research were to characterize differentiation and growth of callus from immature inflorescence-explants of a variegated bermudagrass,Cynodon dactylon (L.) Pers. cv. “Zebra”, and to assess the effects of casein hydrolysate (CH) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations on development of total callus mass and the embryogenic (E) portion. Immature inflorescences about 6 mm in length were inoculated in petri dishes containing modified Murashige and Skoog (MS) agar medium. Two levels of CH (0, 200 mg L⁻¹) and four 2,4-D concentrations (1,3,5,7 mg L⁻¹) in subplots were tested. Regeneration of 57 plants via somatic embryogenesis was achieved. Both fresh callus weight and E callus, expressed as a percentage of total callus, were significantly affected by CH and 2,4-D treatments. Maximum callus fresh weight and E callus concentration were attained in medium with 3 mg L⁻¹ 2,4-D. Addition of CH to the medium increased fresh weight and E callus within each level of 2,4-D, but not to the same extent as indicated by a significant (P<.01) 2,4-D X CH interaction. Presence of CH and 3 mg L⁻¹ of 2,4-D maximized both fresh weight and percentage of E callus. Journal Article 5331 of the Agric. Exp. Stn., Oklahoma State University, Stillwater, OK. I. R. A. and C. M. T. are former Graduate Research Assistant and Professor, respectively, Department of Agronomy; and B. B. J. is Professor, Department of Botany and Microbiology, Oklahoma State University, Stillwater, OK 74078.     

Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants

Summary Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.Abbreviations NAA -napthaleneacetic acid - KT Kinetin - 2,4-D 2, 4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - BAP N⁶ benzylaminopurine - NFM NaCl-free medium     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Callus induction and plant regeneration of U.S. rice genotypes as affected by medium constituents

Summary This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorbitol or 4% sucrose alone, and 0.5 to 4 mg·L⁻¹ (2.26 to 18.10 μM) 2,4-D with or without 0.5mg·L⁻¹) (2.32 μM) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg·L⁻¹ (9.29 μM) kinetin combined with 0 or 0.1 mg·L⁻¹ (0.54 μM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg·L⁻¹ (2.26 μM) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration.     

Macrocyclic trichothecenes produced by Stachybotrys isolated from Egypt and Eastern Europe

Twenty seven isolates of Stachybotrys chartarum, S. albipes, S. kampalensis and S. microspora from Egypt and Eastern Europe were tested for production of macrocyclic trichothecenes. Twenty of the 27 isolates, grown on rice seeds, were toxic to brine shrimp larvae. Based on TLC and HPLC analyses, 5 macrocyclic trichothecenes (verrucarin J, roridin E, satratoxins F, G & H) as well as trichoverrols were identified. When grown in liquid culture on rice extract medium, only 3 isolates were toxic and produced verrucarin J, roridin E and satratoxins G & H. Extracts from mycelial mats were more toxic than culture filterates of two isolates grown on rice extract and both contained the same macrocyclic trichothecenes (285.5 mg/4 L), in addition to trichoverrols A & B (31 mg/4 L) found in mycelial mats only. When grown on 3% sucrose Czapek's medium supplemented with peptone and yeast extract (still cultures), all isolates were non-toxic to brine shrimp and no trichothecenes could be detected in the extracts.     

Callus initiation,growth and plant regeneration in Plantago ovata Forsk. cv. GI-2

The technique for callus initiation, growth and plant regeneration from cultured hypocotyl explants of Plantago ovata cv. GI-2 is described. Best initiation and growth of callus was achieved on Murashige & Skoog's medium containing 2,4-dichlorophenoxyacetic acid (1.0 mgl^-1) and kinetin (1.0 mgl^-1). The callus showed maximum shoot differentiation on medium containing kinetin (4.0 mgl^-1) and -naphthaleneacetic acid (0.01 mgl^-1). Root formation of shoots was best on half-strength medium supplemented with 3-indolebutyric acid. The regenerated plants were successfully transferred into pots.     

Production of phenolic compounds in callus cultures of tea plant under the effect of 2,4-D and NAA

The effect of hormone-like compounds at different concentrations: 2,4-D (2 × 10^?6; 2 × 10^?5; and 2 × 10^?4M) and 1-NAA (2 × 10^?7; 2 × 10^?6; 2 × 10^?5; 4 × 10^?5, and 6 × 10^?5 M) on the growth and production of phenolic compounds, including flavans and lignin, was investigated in callus culture of tea plant (Camellia sinensis L., a highly productive strain IFR ChS-2). The growth of the culture was vigorous, and production of phenolic compounds therein was efficient in the medium containing 2 × 10^?5 M 2,4-D. Substitution of 1-NAA for 2,4-D in all the cases decelerated the growth of the culture. These changes were more pronounced when 2 × 10^?7 and 2 × 10^?6 M 1-NAA was used; in this case, biomass accumulation decreased by 1.5–2.0 times as compared with control material growing on the medium with 2 × 10^?5 M 2,4-D. In the presence of 1-NAA, the content of total soluble phenolic compounds and flavans in the calli rose by 30% on the average as compared with control material. Accumulation of lignin remained essentially the same. Therefore, the replacement of 2,4-D with 1-NAA in the nutrient medium used for the growing of highly productive strain of tea plant callus did not induce considerable changes in its ability to produce phenolic compounds.     

Characterization and manipulation of embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L.)

Characterization and optimization of the embryogenic response from in-vitro-cultured immature inflorescences of rice (Oryza sativa L. sub-species indica and japonica) are described. Histological and morphological analyses revealed that the parenchymatous ground tissue present in the region between the second whorl of sterile bracts and the base of the fertile bracts, the embryogenically competent region (ECR), was involved in the embryogenic response. Initial cell divisions within the ECR occurred in the vicinity of the pro-vascular regions of the spikelet. Continued cell divisions resulted in groups of proliferating units and each single proliferating unit was the product of a coordinated behavior of neighboring cells functioning as a morphogenic group. Further proliferation of this embryogenic tissue was due to the development of cambium-like tissue(s) often forming an embryogenic stratum which under optimal culture conditions produced plants at a high frequency. The morphogenic pathways governing plant regeneration from spikelets of the immature rice inflorescence were dependent upon the growth-regulator composition of the culture medium. Three different modes of plant regeneration were observed: (i) direct plant regeneration, (ii) plant regeneration with an intervening callus phase (prolific non-embryogenic growth associated with unorganized, loose and mucilaginous tissue), and (iii) plant regeneration without an intervening callus phase (compact embryogenie tissue with highly organized growth). The efficiency of plant regeneration, via somatic embryogenesis without an intervening callus phase, was increased by optimizing the culture conditions. In a two-step procedure, immature inflorescences of rice were first cultured on a conditioning medium supplemented with 2.0 mg · 1^–1 2,4-dichlorophenoxyacetic acid + 1.5 mg · 1^–1 kinetin + 0.75 mg · 1^–1 -naphthaleneacetic acid for a period of two weeks. The conditioning medium, with the appropriate culture conditions, allowed redirection of partially differentiated cells of the ECR into embryogenically competent pro-embryogenic groups. Maturation of these pro-embryogenic groups was achieved by transferring them to an embryo proliferation medium, and plants could then be regenerated at a high frequency upon their transfer to the regeneration medium.Abbreviations CM conditioning medium - 2,4-D 2,4-dichlorophenoxyacetic acid - ECR embryogenically competent region - NAA -naphthaleneacetic acid - SEM scanning electron micrograph Thanks are extended to Stephanie Lara, Barbara Bricks, Kay Robbinson-Beers, James Haudenshield, Dave Bayer and Gene Nester, for their invaluable help during the course of this research. The research was partly supported by the Rockefeller Foundation through the Rice Biotechnology program as a grant to W.J.L. and a Postdoctoral Fellowship to J.R.R.     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (liliaceae)

Bulb scale and inflorescence explants of Urginea indica Kunth. (2n=20) were cultured in vitro on modified Murashige & Skoog's medium with different hormonal composition. Media containing 2,4-dichlorophenoxy-acetic acid (2,4-D) (2 and 4 mgl^–1) and -naphthalene-acetic acid (NAA) (2 mgl^–1) could induce callus in inflorescence explants. Combination of 2,4-D (4 mgl^–1) + NAA (2 mgl^–1) + Kinetin (2 mgl^–1) only could induce callus formation in scale explants. The bulb scale explants contained mostly diploid cells while the inflorescence explants contained cells with nuclear DNA content ranging from 2C to 64C. The lowest karyological heterogeneity was recorded in callus derived from bulb scale and in callus derived from inflorescence induced with NAA. The highest variability was recorded on media with 2,4-D alone. Induction of division, probably of the pre-existing polytenic nuclei in the inflorescence explant, has been suggested to be the cause of origin of polyploid cells in such cases.     

Callus induction and plant regeneration from gladiolus

A method for the initiation of callus capable of plant regeneration from in vivo grown cormels of gladiolus (Gladiolus x grandiflorus Hort.) is described. Sliced cormels of the large-flowering hybrid, Peter Pears were cultured in vitro on a modified Murashige and Skoog medium, supplemented with various auxins. Yellow callus, which was either friable or compact, could be induced on all media tested. Callus induced on media with naphthaleneacetic acid failed to proliferate. Callus induced on media with 9 mM 2,4-dichlorophenoxyacetic acid showed the best growth. Addition of micro-elements and vitamins increased the induction and growth of callus capable of plant regeneration. Explants taken from the middle part of the cormels had the highest competence for callus initiation. Callus was induced on several gladiolus hybrids and the South African species G. garnierii Klatt. Callus induction was genotype dependent and among the cultivars tested, Peter Pears and White Prosperity were superior with respect to callus production on the media with either 2,4-dichlorophenoxyacetic acid or picloram. Plants were regenerated from yellow compact callus of all genotypes on media containing zeatin and benzyladenine in various concentrations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog basal salt and vitamins (1962) - CI callus induction medium - NAA -naphthaleneacetic acid - BA 6-benzyladenine - picloram 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid - zeatin 6-[4-hydroxy-3-methylbut-2-enylamino]purine     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Somatic embryogenesis and plant regeneration of Nerium oleander

Leaf explants of Nerium oleander L. produced masses of callus when both an auxin and a cytokinin were included in the medium. Leaves cultured on the B5 medium of Gamborg et al. supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d; 9.05 M) plus benzyladenine (BA; 4.4 M) produced callus and profuse rhizogenesis was observed from callus developed from older leaves. On Murashige & Skoog medium (MS) with the same concentration of 2,4-d and BA, explants from young and mature leaves produced callus, but only that from young leaves was embryogenically competent. Globular somatic embryos were obtained when embryogenic cells were cultured on MS medium without growth regulators. Both normal and anomalous development of embryos occurred in either liquid or gelled medium. Plantlets were produced faster when mature embryos were cultured on either solid medium or placed on Sorbarod plugs soaked with this same medium but with 1% sucrose. Plantlets with three nodes were transferred to pots and acclimatized in a growth chamber and afterwards transferred to garden beds.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Somatic embryogenesis and plant regeneration inMedicago suffruticosa

A successful procedure has been designed for the regeneration of plantlets from leaf sections of the self-pollinating species,Medicago suffruticosa. Callus growth was promoted by a 4-week culture period on liquid Kao's medium containing 4.9 M benzyladenine and 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), followed by a 4-day treatment in which the benzyladenine was elevated to 44.4 M. Shoots/plantlets were observed after 3–4 weeks culture on growth regulator-free agar-solidified medium. Under these conditions, the regeneration frequency from callus was 18% and a histological study showed that this regeneration was through somatic embryogenesis. The growth regulator treatment, with a relatively high concentration of growth regulators (44.4 M benzyladenine) for a short time period (4 days), is important for inhibiting polyphenol compounds and for stimulating callus growth and plant regeneration.Abbreviations 2,4-d 2,4-dichiorophenoxyacetic acid - BA benzyladenine - NAA -napthaleneacetic acid