Shoot Induction Medium Research Articles

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20μl drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.

Production of Transgenic Soybean Plants Using Agrobacterium-Mediated DNA Transfer

Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.

High-frequency plant regeneration from cultured cotyledons of Arabidopsis thaliana

Wild-type plants of Arabidopsis thaliana strain "Columbia" regenerated at a high frequency from immature cotyledons cultured on a shoot-inducing medium containing 1.0 mg/l 6-benzylaminopurine and 0.1 mg/l 1-naphthaleneacetic acid. Cotyledon segments expanded rapidly and produced numerous shoots after 2-3 weeks in culture. Regeneration occurred in the absence of the original shoot apex. Hypocotyl segments from immature embryos produced root hairs and callus in culture but only rarely developed shoots. Hygromycin, kanamycin and G-418 inhibited cotyledon expansion and shoot formation in culture. Vancomycin was much less toxic to cotyledon segments than either carbenicillin or cefotaxime. Immature cotyledons therefore yield numerous regenerated plants that may be useful in future transformation studies.

Adventitious Shoot and Plantlet Formation from Cultured Pomegranate Leaf Explants

Abstract Callus from leaf explants of two pomegranate (Punica granatum L.) clones produced adventitious shoots after 2 months of in vitro culture. Leaf segment explants initially were cultured on a modified Murashige and Skoog (MS) basal medium supplemented with a range of 0.5-10 μm BA and 0.05-5 (μm NAA. Shoot elongation was stimulated most efficiently when the initial calli were transferred from the shoot induction medium to half-strength MS supplemented with 2 μm BA. Elongated shoots rooted easily on a medium of one-half MS and 0.1 μm NAA. Whole plants were recovered after transfer to vermiculite in a greenhouse. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA), 1-naphthaleneacetic acid (NAA).

Plant regeneration from cotyledon protoplasts of Lycopersicon pimpinellifolium.

Protoplasts of L.pimpinellifolium were isolated from cotyledons from about 10 day old seedlings which were grown under sterile conditions at 20°C and 4, 500 lux. Isolated protoplasts were cultured in a plating medium of TM-2 (SHAHIN, 1985) with liquid on agarose and, subsequently, on the second medium based on TM-3 (SHAHIN, 1985) under the conditions of 25°C and 400 lux. The calli brought up to about 1.0 mm calli were transferred onto the shoot induction medium of TM-4 (SHAHIN, 1985) where shoots never appeared. Calli were again transferred to modified MS medium (MURASHIGE and SKOOG, 1962) supplemented with ZR (Zeatin riboside) on which shoots were regenerated at the rate of 27% during the ten day culture from 45 days after plating of proto plasts.

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species ( S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg −1). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg −1). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.

In vitro control of caulogenesis by growth regulators and media components in embryonic explants of eastern white pine (Pinus strobus)

Horizontally oriented embryos of Pinus strobus (L.) produced shoots on Schenk and Hildebrandt medium containing cytokinin. Shoots developed primarily from cotyledons in contact with the medium. Seed pretreatments at 5 or 27 °C did not affect caulogenesis. N6-Benzyladenine (BA) and N6-(Δ2-isopentenyl)adenine (2iP) both induced caulogenesis, with BA being 10–20 times more potent than 2iP. High BA levels caused callus formation. BA exposures from 1 to 8 weeks were equally caulogenic with horizontal explants, but exposures longer than 2 weeks led to increased variability and callus formation. A 1-week, upside-down, vertical orientation during BA treatment increased the uniformity of cotyledon response and was as caulogenic as a 4-week horizontal BA exposure. Neither auxins nor triiodobenzoic acid induced or significantly enhanced shoot formation. Full-strength Schenk and Hildebrandt medium was superior to Murashige and Skoog medium for shoot induction. Dilution of Schenk and Hildebrandt medium had no significant effect on shoot production, but shoot elongation was suppressed on one quarter strength Schenk and Hildebrandt medium. Half-strength Murashige and Skoog medium was as caulogenic as Schenk and Hildebrandt medium. The NH4 level of the macronutrients was responsible for the difference between Schenk and Hildebrant medium and Murashige and Skoog medium. The higher NH4 concentration of Murashige and Skoog medium inhibited shoot formation.

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.

Rapid and efficient regeneration of plants from explants of Arabidopsis thaliana

A procedure is given for the regeneration in vitro of a previously difficult to regenerate plant, Arabidopsis thaliana (L.) Heynh. Leaf explants of Arabidopsis that are given a short exposure to a callus-inducing medium prior to incubation on a shoot-inducing medium exhibit high survivability and rapidly produce shoots. Some media combinations allow shoots to form on 100% of the explants and the majority of these explants form multiple shoots. The shoots are normal in appearance and most of them can be induced to form functional roots and retain fertility. This regeneration scheme should be of use in the development of an Arabidopsis transformation procedure and may also be applicable to other recalcitrant plant species.

Phenocritical times in the process of in vitro shoot organogenesis

Shoot organogenesis occurs when leaf explants of Convolvulus arvensis are cultured on Murashige and Skoog salts, sucrose, vitamins, and 0.05 mg/liter IAA with 7.0 mg/liter 2-isopentenyl adenine. Under the influence of this shoot inducing medium (SIM), the explants become competent for the organogenic effects of SIM and eventually become determined for shoot formation. The induction process includes five separate transient sensitivities to inhibitors. Such stage-specific inhibitions reflect phenocritical times in development rather than general metabolic toxicities. The phenocopying agents are tri-iodobenzoic acid (TIBA), sorbitol, ribose, ammonium ion, and acetylsalicylic acid. The process of in vitro shoot organogenesis from leaf explants is now seen to include a series of discrete steps which precede morphological differentiation. An initial dedifferentiation process results in the formation of competent callus tissue along the cut edges of the explant. Under the influence of the phytohormone balance in SIM, shoot organogenic induction proceeds. This process involves a time which is sensitive to inhibition by salicylates followed by a time sensitive to TIBA which is followed in turn by a time sensitive to sorbitol and culminates in cells or groups of cells determined for shoot formation. This process also includes a time sensitive to inhibition by ribose, although its place in the order of events is not yet firmly assigned. There is also a sensitivity to ammonium ion (or lack of nitrate) at or near the time the explant becomes determined for shoot production.

Isolation, Culture and Regeneration of Lettuce Leaf Mesophyll Protoplasts

Summary An efficient and rapid method for the isolation, culture and regeneration Of lettuce mesophyll protoplasts into whole plants is described. Two weeks after protoplast isolation, protoplast-derived calli were transferred to a medium for shoot induction. Shoot buds were visible 4 days after transfer at a frequency of about 20%. A third medium was employed for shoot growth and root formation. One hundred and fifty protoclones of six head lettuce cultivars have been produced. Fifteen protoclones have produced seeds by self pollination.

Surface structural analysis of cultured cotyledons of Douglas-fir

Cotyledons of Douglas-fir are triangular in cross section and possess two epistomatic surfaces with centrally located stomatal rows (commonly seven). After 1 week in culture on a medium inducing adventitious shoot formation (5 μM N6-benzylaminopurine and 5 μM α-naphthalene acetic acid (NAA)) or callus proliferation (5 μM NAA) cells of the hypodermal region immediately below the epidermis begin to elongate and divide rupturing the epidermis. Apical domes of adventitiously produced bud primordia emerge from the ruptured epidermis after 14–21 days in culture on shoot induction medium. Emergence of buds takes place preferentially on epistomatic cotyledonary surfaces. The large number of hypodermal cells that respond to shoot induction medium by forming adventitious shoots suggests further investigation of fundamental events associated with morphogenesis in cotyledon cultures of Douglas-fir.

Characterization of Kanamycin-Resistant Cell Lines of Nicotiana tabacum

Two kanamycin-resistant variants of Nicotiana tabacum were derived by culturing seedling leaf sections on a shoot-inducing medium containing kanamycin. The variants displayed a higher resistance to the structurally related antibiotic streptomycin than to kanamycin. The resistance phenotype was expressed when the tissue was cultured either as callus or as a differentiating tissue and was stably maintained in the absence of kanamycin. Plants regenerated from the variant lines had morphologically abnormal flowers and produced few viable seeds. These resistant lines are potentially useful for protoplast fusion or genetic modification experiments requiring selectable phenotypic markers.

The in vitro propagation of amaryllis (Hippeastrum spp. hybrids).

A new, rapid technique for the propagation of amaryllis (Hippeastrum spp. hybrids) by means of tissue culture is reported. Leaf bases, scapes, peduncles, inner bulb scales and ovaries were cultured successfully in vitro and plantlets were induced readily at various concentrations of growth regulators. Some plantlets also were produced in the absence of growth regulators. The most productive tissues for propagation were inverted scapes and peduncles, cultured in a modified Murashige and Skoog salt solution with added organic constituents and 1 mg per 1 (4.5 micron) 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg per 1 (4.4 micrometer) 6-benzylaminopurine (BAP). Plantlets induced axenically also grew roots on the generalized shoot-inducing medium so that no special rooting medium was required. Although friable callus was obtained from ovary tissue cultured on a medium containing 2 mg per 1 (11 micrometer) naphthaleneacetic acid and 4 mg per 1 (18 micrometer) BAP, it produced shoots after 8 weeks of further subculture on the same medium. An average of 10 rooted plantlets was obtained from each scape or peduncle explant on the shoot-propagating medium. Thus, if 45 explants are obtained from each bulb, 450 rudimentary plantlets could be obtained from each mother bulb in 8 weeks of culture. This is a substantial increase over present propagation methods.

<i>In vitro</i> shoot proliferation and development of micropropagation protocol from leaf disc of Gladiolus

The experiment was carried out during the period from July 2008 to March, 2009 at the Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. The present investigation was to be better source for shoot multiplication. The calli derived from leaf discs were cultured on shoot induction media containing different combinations and concentrations of BAP (1.0, 1.5, 3.0 and 6.0 mg/L) and Kn (0.5, 1.0, 1.5 and 2.0 mg/L). The highest percentage of shoot regeneration (91.66%) and the highest shoot length was 4.24 (cm) with a minimum number of days (13.33) was observed in the MS medium supplemented with 3.0 mg/L BAP and 0.5 mg/L Kn. Thus shoot multiplication in successive subculture was possible. The present investigation was carried out to study the in vitro shoot multiplication in gladiolus by using leaf disc as explants Keyword: Shoot proliferation; Micropropagation; BAP: 6-benzylamino purine; Kn: Kinetin; Leaf disc DOI: http://dx.doi.org/10.3329/jbau.v9i1.8739 JBAU 2011; 9(1): 21-26