Kenneth J. Scott

A comparison of scutellum callus and epiblast callus induction in wheat: The effect of genotype, embryo age and medium

All 35 randomly chosen cultivars of Triticum aestivum produced both epiblast callus and scutellum callus, indicating that the capacity to form callus from these two origins is common in this wheat. The induction frequencies of the two calluses varied considerably between different genotypes, ranging from 28% to 96% for scutellum callus and 18% to 94% for epiblast callus. T. aestivum varieties on average formed scutellum callus at a higher frequency than epiblast callus and no correlation was observed between the induction frequencies of the two calluses. T. durum formed only scutellum callus. Although maximum induction of embryogenic epiblast callus had previously been shown to occur at stages II and II of the embryo development, the highest induction of embryogenic scutellum callus occurred at stages I, II and III. The concentration of macroelements in the MS medium affected the induction frequency of embryogenic callus from both scutellum and epiblast. Both scutellum and epiblast callus formed typical embryoids but at a low frequency. The epiblast callus was clearly differentiated from the underlying tissue and therefore the meristematic tissue could readily be separated from non-embryogenic tissue. The embryogenic scutellum callus, on the other hand, was always mixed with some non-embryogenic tissue and could not readily be separated. The scutellum callus was generally larger than epiblast callus and produced more plantlets.

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 μg/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 μg/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.

The effect of macroelements in the induction of embryogenic callus from immature embryos of wheat (Triticum aestivum L.)

Abstract The effects of the concentrations of each of the five MS macroelements on the induction and morphology of embryogenic callus from immature embryos of wheat were investigated. Relatively high induction frequencies of epiblast callus were obtained over wide ranges of concentrations of the five salts: NH 4 NO 3 10–20 mM, KNO 3 0–40 mM, KH 2 PO 4 0.31 –10 mM, CaCl 2 0–24 mM and MgSO 4 0–48 mM. For induction of scutellum callus, the optimum concentration range of salts was narrower than that for induction of epiblast callus: KNO 3 0–20 mM, CaCl 2 0–24 mM and MgSO 4 0.19–12 mM. The presence of NH 4 NO 3 was essential for the proliferation of embryogenic callus and a relatively high concentration (10 mM) was found to be optimum for the induction of both scutellum and epiblast callus. The presence of CaCl 2 , MgSO 4 and KH 2 PO 4 was not essential for the initiation of epiblast callus but strongly affected the further development of this callus and its differentiation potential. Phosphate showed a strong stimulation of the formation of leaf structures on the callus. The frequency of white callus was enhanced by four of the five macroelements at high concentrations and as high as 60% of primary callus could form white callus on some media. As the formation of typical embryoids of wheat has been previously reported as occurring with white callus, this study may provide a useful base for further work on factors affecting the formation of typical embryoids in wheat callus.

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.

Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Hartog)

Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength of the macroelements, 5 μm 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25%. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome complement for wheat (2n = 42).

Structure and expression of a barley acidic β-glucanase gene

A barley acidic β-1,3-glucanase gene was recovered from a barley genomic library by homology with a partial cDNA of barley basic β-1,3-glucanase isoenzyme GII. The gene, Abg2, is homologous to the PR2 family of pathogenesis-related β-1,3-glucanase genes. The ABG2 protein has 81% amino acid similarity to barley basic β-1,3-glucanase GII. The ABG2 protein is encoded as a preprotein of 336 amino acids including a 28 amino acid signal peptide. A 299 bp intron occurs within codon 25. The mature ABG2 protein has a predicted mass of 32642 Da and a calculated isoelectric point of 4.9. The second exon of the Abg2 gene shows a strong preference for G+C in the third position of degenerate codons. The Abg2 gene was functionally expressed in Escherichia coli. Abg2 mRNA is constitutively expressed in barley root; leaf expression of Abg2 mRNA is induced by mercuric chloride and infection by Erysiphe graminis f. sp. hordei. Southern blot analysis indicates that Abg2 is a member of a small gene family.

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

SummaryNear-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)+RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [32P]-labelled cDNA prepared from poly(A)+RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.

The histological development of the regenerative tissue derived from cultured immature embryos of wheat (Triticum aestivum L.)

Histological studies were conducted on the embryogenic tissue of wheat (Triticum aestivum L.), using primary, subcultured and differentiating callus derived from the scutellum and epiblast of immature embryos. Structures with multiple strata were observed in the late stages of callus induction and on subcultured callus; these were characterized by a middle stratum of ground meristematic tissue with a shoot-forming stratum above and a stratum of large cells below. The shoot-forming protuberances appearing on the differentiating callus were morphologically similar to those observed on subcultured and primary callus and also to the expanding shoot meristem of the cultured embryos. This similarity suggests the continuity of the pre-existing shoot forming tissue during culture in vitro. Typical embryoids have been previously reported on both scutellum and epiblast callus hence this callus could be described as embryogenic. However, organogenesis also occured on the callus as some of the protuberances had a vascular connection to the basal callus. Some of the regenerative outgrowths on the callus were broadly based and were not of single cell origin.

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 μM 6-benzylamino purine (BAP), supplemented with 0.25 μM α-naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 μM indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.

Altered host gene expression in near-isogenic barley conditioned by different genes for resistance during infection by Erysiphe graminis f.sp. hordei

The effect of infection by Erysiphe graminis f.sp. hordei on changes in leaf mRNA populations of near-isogenic resistant and susceptible barley ( Hordeum vulgare ) was investigated using in vitro translation of poly A + RNA followed by two-dimensional electrophoresis and fluorography and by hybridization of infection-related cDNA clones to total leaf RNA. Results of in vitro translations indicated that 15 mRNA species from infected leaves exhibited new or enhanced translational activity when compared to that of non-inoculated controls. Of these, nine mRNAs (infection-related mRNAs) were detected 12h after inoculation in both resistant and near-isogenic susceptible plants. However, six mRNAs (resistance-related mRNAs) showed greater translational activities in polyA + RNA from infected leaves of resistant plants carrying either the Mla or Mlp genes when compared to those of near-isogenic susceptible plants. Three of these six mRNAs were common to cultivars carrying the Mla and Mlp genes. In addition, 11 mRNAs showed reduced translational activity in infected leaves, but none of these showed greater alterations in either the susceptible or resistant cultivars. Six cloned infected-related cDNAs were used to determine the amounts of specific mRNAs in near-isogenic barley differing in the Mla, Mlp or Mlk genes. In the susceptible hosts, four of the mRNAs were induced in a distinct biphasic pattern with accumulation occurring at 12–24 h and 48–72 h following inoculation. For the remaining two mRNAs, induction occurred 12–36 h after inoculation. A comparison of mRNA induction between resistant and susceptible near-isogenic cultivars showed a three-fold accumulation of two mRNAs in all of the resistant cultivars 24–48 h after inoculation. These data demonstrate that genes for resistance function as regulators for the synthesis of common host mRNAs during the early stages of infection.