One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for 'Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.

The effect of auxin on plant regeneration of wheat, barley and triticale

Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T0) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T0 and T1 transgenic plants. Mendelian segregation of herbicide resistance was observed in some T1 progenies. Interestingly, a majority of the T1 progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T1 transgenic wheat plants.

https://www.k-state.edu/wgrc/Publications/1998/6828.pdf

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

Work on improvement of durum wheat (Triticum turgidum L.) using tools of biotechnology is limited. Development of a reliable in vitro plant regeneration procedure for this important cereal is a prerequisite for its improvement by genetic transformation. Here, we report the effects of three growth regulators (GRs), 2,4-D (2,4-dichlorophenoxyacetic acid), picloram (4-amino-3,5,6-trichloropicolinic acid), and dicamba (3,6-dichloro-o-anisic acid), on callus induction and plant regeneration from scutellum cultures of four commercial durum cultivars: Ben, Maier, Munich, and Lebsock. Callus induction was obtained from isolated scutella cultured on modified Murashige and Skoog (MS) basal medium. After 4 wk of callus induction, all calli were plated on MS basal medium for regeneration. The regenerated plantlets were fertile, maintained the normal chromosome number (2n = 4x = 28) and structure as revealed by fluorescent genomic in situ hybridization (fl-GISH), and showed no apparent somaclonal variation. Genotype and callus induction medium played a dominant role in plantlet regeneration. Dicamba proved the best GR for inducing compact callus and also gave the highest proportion (0.16) of regenerated plants across the four cultivars. Overall, Maier gave the highest proportion (0.27) of plantlet regeneration when dicamba at 2.0 mg L -1 concentration was used for initial callus induction. These results will facilitate genetic transformation work with durum wheat.

Effects of Growth Regulators on In Vitro Plant Regeneration in Durum Wheat

O licopeno esta presente numa serie de frutas frescas e hortalicas principalmente na folhas de Barringtonia racemosa. A extracao tradicional do licopeno tem sido empregada no lugar da facil tecnica de propagacao como o processo de cultura de celulas de explantes de folhas. E nossa intencao demonstrar como o licopeno pode ser extraido atraves de cultura de tecido sob luz (iluminacao com lâmpadas fluorescentes brancas de 8.200 lux, 16 h por dia a 25o C) e escuro. Explantes de folhas de Barringtonia racemosa foram cultivados em meio modificado de Murashige e Skoog (MS) para plantas lenhosas e meio B5, suplementado com diferentes concentracoes de acido 2,4-Diclorofenoxiacetico (2,4-D). Condicoes otimas para inducao e manutencao de calos sob luz e escuro foram investigadas e avaliados o crescimento e acumulo de licopeno. Entre meios com diferentes concentracoes de 2,4 -D, calos friaveis de crescimento rapido tiveram inicio em tres semanas apos serem cultivados em meio basal WPM suplementado com 2.0 mg L-1 (peso por volume) de 2,4-D enquanto inducao de calos em explantes cultivados em todos os outros meios comecaram somente apos cinco semanas. Calos foram subrepicados a cada 15 dias. Calos amarelo-palido e verdes desenvolvidos respectivamente sob condicoes escura e de luz foram entao selecionados para avaliacao do teor de licopeno. Um metodo aperfeicoado de cromatografia liquida de alto desempenho foi usado para a determinacao quimica seletiva do teor de licopeno. A producao de licopeno induzida sob luz e tambem o nivel maximo de licopeno incubado em luz foi mais alto do que aqueles incubados no escuro. As melhores taxas de crescimento de calo e suspensoes de celulas foram obtidas respectivamente em meio WPM e B5. A producao de licopeno dependeu do crescimento como demonstrado pela analise do crescimento e teor de licopeno de ambos calos e cultura de celulas em suspensao.

/pdf/optimization-of-callus-and-cell-suspension-cultures-of-53kq493a0m.pdf

Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production

The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB5 medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB5 medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB5 medium supplemented with 20.70 μM picloram, 10.30 mM NH4NO3, 6.25 mM KH2PO4, 2.06 μM Na2MoO4, 0.55 μM CoCl2, and 26.64 μM glycine (for induction); MSB5 medium supplemented with 12.47 μM picloram, 10.30 mM NH4N...

https://www.jstor.org/stable/pdfplus/4293786.pdf

Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant indian barley (hordeum vulgare l.) in vitro

Basal media and plant growth regulators were tested for the promotion of somatic embryogenesis from immature wheat-rye hybrid embryos. Influence of growth regulators and chilling on plant regeneration were tested on two media. A medium containing four amino acids-glutamine, arginine, glycine and aspartic acid-as the nitrogen source, promoted the production of, on average, twice as much embryogenic callus as the other media, and somatic embryos developed well. The growth regulator dicamba was significantly better than 2,4-dichlorophenoxyacetic acid in promoting somatic embryogenesis and subsequent plant regeneration. Germination of somatic embryos on both regeneration media was enhanced by cold treatment. Supplementing 190-2 plant regeneration medium with a combination of α-naphthaleneacetic acid + benzyladenine, indole-3-acetic acid + kinetin or indole-3-acetic acid + zeatin resulted in equally high germination rates.

A. S. T. Immonen

Papers

Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales