Callus Initiation Medium Research Articles

Production and Characterization of Tissue Cultures of Four Crocus Species from the Carpathian Basin

AbstractWe aimed to produce tissue cultures and plant regeneration from endangered Crocus species: C. scepusiensis, C. tommasinianus, C. vittatus (“Verni” series of the genus) and C. banaticus. For initiation of cultures we used a plant growth regulator (PGR) combination used for in vitro culture of saffron and its relatives: 10 mg L-1 α-naphthaleneacetic acid (NAA) and 1 mg L-1 6-benzyladenine (BA). Shoot tips of young seedlings (C. scepusiensis) and corms (for the rest of species) were used as explants. C. scepusiensis explants developed into organogenic calli. On media with decreased NAA and with or without increased BA concentration, calli produced stigma-like structures and/or shoots and whole plants. In the other species, callus initiation medium induced callus formation with abundant somatic embryos. In C. tommasinianus, embryos developed shoots when auxin content of medium was decreased. In C. banaticus, a decrease of auxin with or without an increase in cytokinin content led to shoot or whole plant regeneration, as in C. scepusiensis. In the case of C. vittatus and C. banaticus, initiation and/or maintenance of cultures on indole-3-butyric acid (IBA) and increased sucrose concentration stimulated whole plant regeneration and in vitro cormlet development. C. scepusiensis and the rest of cultures (organogenic vs. embryogenic) differed at the biochemical level: C. scepusiensis cultures had higher (yet still low) enzymatic antioxidant (catalase, peroxidase) activities. With respect to catalase isoenzyme patterns, C. banaticus was different from the rest of cultures, demonstrating its distinct taxonomical position. Besides germplasm preservation use of the present cultures, they have a potential biotechnological value.

Characterization of Physical, Thermal and Spectral Properties of Biofield Treated Date Palm Callus Initiation Medium

Characterization of Physical, Thermal and Spectral Properties of Biofield Treated Date Palm Callus Initiation Medium

Characterization of Physical, Thermal and Spectral Properties of Biofield Treated Date Palm Callus Initiation Medium

The date palm is mainly cultivated for the production of sweet fruit. Date palm callus initiation medium (DPCIM) is used for plant tissue culture applications. The present work is intended to evaluate the impact of Mr. Trivedi’s biofield energy treatment on physical, thermal and spectral properties of the DPCIM. The control and treated DPCIM were evaluated by various analytical techniques such as X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) spectroscopy, particle size analyzer (PSA), surface area analyzer and ultra violet-visible spectroscopy (UV-vis) analysis. The XRD analysis revealed a decrease in intensity of XRD peaks of the treated sample as compared to the control. The crystallite size of the treated DPCIM (81.02 nm) was decreased with respect to the control sample (84.99 nm). The DSC analysis showed a slight decrease in melting temperature of the treated sample. Additionally, the latent heat of fusion of treated sample was changed by 45.66% as compared to the control sample. The TGA analysis showed an increase in onset degradation temperature of the treated sample (182oC) as compared to the control sample (142oC). This indicated the increase in thermal stability of the treated DPCIM. PSA results demonstrated an increase in average particle size (d50) and size showed by 99% of particles (d99) by 19.2 and 40.4%, respectively as compared to the control sample. The surface area analyzer showed a decrease in surface area of treated DPCIM by 13.4%, which was well supported by the particle size results. UV spectra of the treated sample showed the disappearance of absorption peak 261 nm in treated sample as compared to the control. Overall, the result showed that biofield energy treatment has a paramount influence on physical, thermal and spectral properties of DPCIM. Therefore, it is assumed that biofield treated DPCIM could be used as a better medium for plant tissue culture applications.

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).

English

In our approach to control reactive oxygen species produced as a result of oxidative stress experienced by cassava roots during harvesting, a phenomena which causes postharvest physiological deterioration in the roots, we transformed cassava variety TMS 60444 with the AtAOX1a gene, driven by the 35S promoter, using agrobacterium-mediated approach. Extracted genomic DNAs of putative transgenic lines were screened using polymerase chain reaction technique (PCR). Messenger RNA was extracted from selected PCR-positive lines for reverse transcription-PCR analysis for gene expression. To screen positive lines for gene function, leaf lobes from two transgenic lines with a line carrying an empty vector and the wild type were subjected to somatic embryogenesis (SE), a known oxidative stress process. The results show that the wild type, at 16 days after initiation (DAI) of the leaf lobes on callus initiation medium, had the highest (100%) number of leaf lobes that produced at least one observed organised embryogenic structure (OES). This was followed by PEV-3, the empty vector plant with 50% OES production, while PB-3 had the least percent (20%) of leaf lobes with OES. PB-3 line also had no OES at all in five out of the seven periods of data collection. During the period, the wild type recorded the highest attainable OES quality score of 2.0 (on a scale of 1-5 where 1=bad and 5=excellent) at the first initiation cycle. Both the transgenic lines and the empty-vector plantlet recorded quality score of 1.0. It seems AtAOX1a only hinders OES development, but exerts little effect on the quality, if OES does not degenerate after development. Key words: Genomic DNAs, reverse transcription-PCR, somatic embryogenesis (SE).

Effects of copper sulphate and cobalt chloride on <i>in vitro</i> performances of traditional indica rice (<i>Oryza sativa</i> L.) varieties in Sri Lanka

This study was carried out to investigate the effects of copper sulphate and cobalt chloride on the rate of callus proliferation and the in vitro regeneration in nine indica rice varieties and a Japonica variety. The disinfected seeds were inoculated in callus initiation medium composed of N6 basal elements, 1000 mg/l proline, 2 mg/l 2,4-D and 3% (w/v) sucrose to screen high responsive varieties. Calli of screened varieties were used to determine the effects of additives on in vitro responses in proliferation and regeneration media. Taipei 309, Pachcha Perumal, Heenati Vee and Suvandel were screened as high responsive varieties at P ≤ 0.05. The rate of callus proliferation in the screened varieties was significantly higher in the medium supplemented with 5 mg/l copper sulphate and 5-10 mg/l cobalt chloride together (P < 0.05). In regeneration, the highest number of normal plants with the least number of albino plants could be obtained in the media containing 5 mg/l copper sulphate in combination with 5 mg/l cobalt chloride. The results in this study indicate that the in vitro performance in Sri Lankan traditional indica rice varieties can be improved by using the media containing both copper sulphate and cobalt chloride. Key words: Rice; copper sulphate; cobalt chloride; Indica; Japonica DOI: 10.4038/jas.v4i3.1652 The Journal of Agricultural Sciences, 2009, vol. 4, no. 3 pp.132-141

Establishment of sorghum cell suspension culture system for proteomics studies

This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. Friable sorghum callus was initiated from young shoots under completely dark conditions on MS medium supplemented with 3 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.5 mg/L 1-naphthaleneacetic acid (NAA). Additionally, sorghum cell suspension cultures have been initiated from the friable callus masses in liquid medium with the same composition as the callus initiation medium. Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different protein expression profiles. The sorghum TSP proteome, which is highly complex, is best resolved when separated on large format, 18 cm, pH 4 - 7 isoelectric focusing (IEF) immobilised pH gradient (IPG) strips. On the other hand, the sorghum CF proteome (secretome) is less complex with most proteins being resolved on mini format, 7 cm, pH 3 - 10 IPG strips. Furthermore, narrowing down the pH range from 3 - 10 to 4 - 7 for the CF proteome resulted in improved protein spot resolution.

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.

Screening Wheat Genotypes for High Callus Induction and Regeneration Capability from Immature Embryo Cultures

Selecting the explant genotypes is crucial step in in vitro culture and Agrobacterium-mediated transformation system due to its host range specificity. Immature embryos of five winter and three spring wheat (Triticum aestivum L) cultivars were evaluated for tissue culture response in three callus initiation media. MS medium containing 2,4-0 (2 mg ml-1) plus B5 vitamins (MSB5), MS medium containing 2,4-0 (1 mg ml-1) with no vitamins (MS1GC) or MS medium containing picloram (2.2 mg ml-1) and 2,4-0 (0.5 mg ml-1) plus MS vitamins (CM4C) were used for callus initiation. Percentage of callus induction varied widely with the genotype and initiation medium used, with values ranging from 5.7% to 100%. Embryogenic capacity of genotypes was evaluated by number of somatic embryos formed from cultured immature embryos. Bob White (spring) and NE92458 (winter) were equal and most embryogenic; Pronghorn and 2137 (both winter) were the poorest. CM4C medium was found to be the best medium for initiating embryogenic callus among three culture media tested. A standard regeneration procedure was used. The genotypes with the highest regeneration efficiencies were Bob White, Fielder and NE92458, (1.8, 1.4 and 1.6 plantslexplant, respectively).

Callus growth and essential oil of nilam (Pogostemon cablin (Blanco) Bth.) on the treatment with -naphtalen acetic acid and kinetin

The objectives of the research were to study the effect of adding of α - naphtalen acetic acid (NAA) and kinetin on callus growth and essensial oil production from nilam (Pogostemon cablin (Blanco) Bth.) callus culture. The outline of the research was the callus growth and secondary metabolite production from plant’s body could be triggered by the occurrence of plant growth regulation. The addition NAA and kinetin in culture’s medium would influence cell proliferation and synthesis of protein, so that both can induce callus growth and secondary metabolism production from the cell that have been cultured. According to the research objectives, the research was done by using in vitro callus culture method to obtain callus from explant P. cablin leaf and to induce essensial oil production. In vitro culture process consist of two stages. First stage was the callus initiation medium induced the callus from explant. The experiment was done by medium Murashige-Skoog (MS) with 2,4-D 0,5 mg/l and kinetin 0.5 mg/l; and the second stage was the medium treatment induced callus growth and essensial oil production. The research used factorial completely randomized design with two factors (NAA concentration: 0mg/l, 0,5 mg/l, 1,0 mg/l, 2,0 mg/l; and kinetin concentration: 0 mg/l, 0,5 mg/l, 1,0 mg/l, 2,0 mg/l) with 3 replicates. The collected data were qualitative data (callus morphology include texture and colour callus) and quantitative data (callus growth rate, callus dry weight and essensial oil content). Data were analyzed by Anova and followed by DMRT 5% confidence level and correlation regretion. The result of the research indicated the treatment with addition plant growth regulation (NAA and kinetin) on Murashige-Skoog’s medium had significant effect on callus growth rate but it didn’t have significant effect on callus dry weight and the increase of produced essential oil.

Callus growth and anthraquinones production of Indian mulberry (Morinda citrifolia L.) in Murashige-Skoog’s medium (MS) supplemented with Ca2+ and Cu2+

The objectives of the research were to study the effect of Ca2+ and Cu2+ ions in Murashige-Skoog’s medium on callus growth and anthraquinones production from Morinda citrifolia callus. The outline of the research was that the callus growth and secondary metabolite production from plant’s body could be triggered by the occurrence of elicitor that is added to culture’s medium, as biotic or abiotic elicitors. The addition of Ca2+ and Cu2+ ions in culture’s medium as abiotic elicitor would cause the occurrence of metal’s ion competition and interaction toward cells that being cultured, furthermore, it would influence ion transport from or to cell cytoplasm. Finally, cytoplasm pH would be influenced, so that both of callus growth and secondary metabolite from the cell that being cultured will also affected. In this research, in vitro callus culture method to obtain callus from explant (Morinda citrifolia leaf) and to induced anthraquinone production was used. In vitro culture used in this research consisted of 3 stages. First stage was the basal medium for sterilant object, second stage was the callus initiation medium to induce callus, and third stage was the treatment medium to induce anthraquinone production from callus. The research used factorial completely randomized design with 2 factors (Ca2+ ions: 0 mgl-1, 440 mgl-1, 880 mgl-1 and Cu2+ ions 0 mgl-1, 2,5 mgl-1, 5 mgl-1), with 3 replicates. Data collected were qualitative data (explant sterilization and callus morphology) and quantitative data (callus growth rate, callus dry weight, and anthraquinone content). The data were analyzed using ANOVA, followed by DMRT with 5% confidence level. The result of the research indicated that the treatment with addition of Ca2+ and Cu2+ ions on MS medium did not have any significant effect on callus growth and anthraquinone production.

In vitro selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (Dendrocalamus strictus Nees)

Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l−1 (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l−1 (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na+, sugar, free amino acids, and proline but a slight decline was recorded in K+ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l−1 (9.0 μM) 2,4-D+0.5 mg l−1 (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l−1 (0.1 μM) α-naphthaleneacetic acid +0.1 mg l−1 (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl−1 casein hydrolyzate, 6.63 mg l−1 (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l−1 (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl−1 (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl−1myo-inositol, and 150 mgl−1 asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.

Wheat Immature Embryo Culture for Embryogenic Callus Induction

Four 2,4-D concentrations along with three embryo developmental stages were tested to determine the optimum morphogenesis of wheat immature embryo culture. Concentration of 2 mg L 2,4-D was found to be optimum level for 1 morphogenesis which was a good indicator of embryogenesis. Compact and nodular calli were observed in first and second developmental stages of 2 mg L 2,4-D concentration. In addition, five callus initiation media were tested to determine effects 1 of medium constituents on somatic embryogenesis of wheat (Triticum aestivum L.). The highest embryogenic callus formation was observed on MS+B5 medium (98.3%). Therefore, first and second developmental stages of 2 mg L 2,4-D concentration 1 along with MS+B5 medium is suitable for embryogenic callus production in wheat.

Induction of Highly Embryogenic Calli and Plant Regeneration in Upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) Cottons

To accomplish our objective of broadening the number of regenerable cotton lines, we developed a protocol capable of producing plants through somatic embryogenesis of diverse cotton species. Callus was initiated from hypocotyl and cotyledon explants on a callus initiation medium [CIM; modified MS with 1 mg L−1 kinetin and 2 mg L−1 naphthaleneacetic acid (NAA)]. Friable embryogenic callus was periodically selected and transferred onto callus selection/maintenance medium (CS/MM) [modified MS with 0.1 mg L−1 kinetin and 0.5 mg L−1 NAA]. The selected callus was then transferred into a liquid embryo initiation medium (EIM) (modified MS medium in which NH4NO3 was removed and KNO3 amount doubled) followed by transfer to solid embryo maturation media EMMS2 (0.5 mg L−1 NAA + 0.05 mg L−1 kinetin). The liquid step not only decreased the culturing time but also increased the number of embryos per gram of cultured tissue. Germinating somatic embryos were placed on MS medium with no hormones and plantlets were acclimatized before transfer to the greenhouse. Significant numbers of somatic embryos and their derived plantlets were obtained from a commercial cultivar of G. hirsutum, Deltapine 90 and G. barbadense accession GB‐35B126 (PI‐528306). The mean embryos per gram for Deltapine 90 on EMMS2 were higher than those previously reported for Coker 312. Highly significant differences were found between the two genotypes for both embryo and plant production. To our knowledge, this is the first report of regeneration of G. barbadense through somatic embryogenesis.

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.

Development of transgenic cabbage (Brassica oleracea var. Capitata) for insect resistance by Agrobacterium tumefaciens-mediated transformation

Transgenic head cabbage (Brassica oleracea var. capitata), resistant to diamondback moth (Plutella xylostella) larvae, was developed through Agrobacterium tumefaciens-mediated transformation with Bacillus thuringiensis (Bt) cry genes using a modified procedure. Factors important for transformation included cabbage cultivar; preculture and coculture of explants on a callus initiation medium; use of appropriate amount; and delay in initial application of selective agents. A total of 15 independent transformed lines with over 100 plants were obtained from several transformation experiments, representing an overall transformation efficiency of ∼1%. Cabbage plants transformed with a synthetic Bt gene, cry1Ab3, were all resistant to larvae of the diamondback moth, whereas all plants transgenic for cryIIa3, a wild-type Bt gene, were susceptible. As a first step towards testing the hypothesis that reduced exposure of Bt to target insects would delay the evolution of insect resistance to Bt, cry1Ab3 expression was put under the transcriptional control of the soybean wound-inducible vspB promoter and transgenic cabbage was obtained. Insect bioassay showed that such plants were all resistant to diamondback moth even without induction for the expression of Bt.

331 Effects of Explant Type, Sucrose Level and Callus Development Time on In Vitro Plant Regeneration of Sweetpotato

Several experiments were conducted to evaluate the influence of explant type, sucrose level, and callus development time on sweetpotato [Ipomoea batatas (L.) Lam] in vitro culture. Shoot tip, petiole, and leaf of Selection 75-96-1 was used as explants in Murashige and Skoog (MS) media with different plant growth regulators. Calli derived from shoot tip and petiole produced 42.1% and 10.3% somatic embryos, respectively, but the leaf failed to produce somatic embryos. The effect of sucrose level was determined using shoot tip as explants. Compared with 3% sucrose in the same plant growth regulators level medium during callus initiation and callus proliferation periods, 5% sucrose level suppressed root growth and improved shoot regeneration. The callus development time was measured by using shoot tips on callus initiation medium containing 1.5 mg/L alpha-Naphthaleneacetic acid (NAA) and 0.25 mg/L Kinetin (KIN) plus 5% sucrose. When explants were cultured for less than 6 weeks during callus initiation, then transferred onto plant regeneration medium, plant regeneration via organogenesis occurred; whereas, maintaining cultures for more than 12 weeks on the same callus initiation/proliferation medium, plant regeneration was favored via embryogenesis. Explant type and other factors affecting plant regeneration noted here could be applied to protoplast culture, somatic hybridization, and transformation in sweetpotato.

Efficient Plant Regeneration in Garlic through Somatic Embryogenesis from Root Tip Expiants

The present study was conducted to establish an efficient protocol of plantlet regeneration through somatic embryogenesis in garlic (Allium sativum L.). Root tips measuring 2 to 3 mm were excised and cultured on agar-solidified MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) for callus and embryo formation. The optimum concentration of 2,4-D was 0.5 μM. At a concentration higher than 1.0 μM, 2,4-D had an inhibitory effect on callus and embryo formation. Embryos germinated and formed rooted plantlets on MS solid medium containing 5.0 μM kinetin. The number of plantlets regenerated per root tip expiant depended on the concentration of 2,4-D in the callus initiation medium. The plantlets were established in the soil after acclimatization in a growth cabinet. Somatic embryos were morphologically characterized by scanning electron microscopy (SEM).

585 PB 100 IMPROVED REGENERATION FREQUENCY OF KENTUCKY BLUEGRASS FROM CALLUS CULTURES

Genetic transformation of Kentucky bluegrass (Poa pratensis L.) requires methods for high-frequency regeneration of plantlets from cultured cells. Regeneration of this important turf species has been reported, albeit at a low frequency from seed-derived callus. We tested the potential of 3 synthetic auxins, used in the callus initiation and growth medium, for their ability to promote regeneration in 3 bluegrass varieties. 10 μM 2,4-D promoted regeneration from 0 to 5% of calli, 30 μM and 60 μM picloram promoted regeneration from 0 to 8% of calli, and 10 to 60 μM dicamba, in combination with BA, promoted regeneration from 1% to 8% of calli. In a subsequent experiment, both 60μM and 90 μM dicamba, with 20 μM BA, promoted regeneration from 45% of calli averaged across varieties. These media were tested for the promotion of regeneration in 12 diverse bluegrass varieties. Although up to 45% of the calli from some varieties regenerated plantlets, the response of other varieties was markedly lower, indicating a genetic component in the response to these media.