Karen Caswell

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.

Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.

Transgene inheritance and silencing in hexaploid spring wheat

Abstract Inheritance and expression of the Act1D-uidA::nptII transgene cassette inserted into the genome of a spring wheat cultivar, ’Fielder’, was studied in T4 and T5 transgenic wheat lines. Southern blot and PCR analyses demonstrated that the transgene was inherited for five generations of selfed plants. The multiple integration pattern displayed in the T1 generation was maintained up to the T5 generation with no evidence of transgene rearrangement. There was no cytoplasmic effect on the inheritance of the transgene as observed by GUS histochemical assays in F1 seeds of reciprocal crosses (T4 crossed with untransformed ’Fielder’). Based on the histochemical GUS activity a clear Mendelian segregation ratio was not obtained in the F2 seeds of the crosses, although there was a tendency towards a two-locus insertion ratio. For one cross (A1//FD/A1), some of the transgenic plants produced low GUS and NPTII enzyme activities in seeds, even though Southern blot and PCR analyses indicated the presence of an intact transgene expression cassette. The transgene of these plants was methylated based on Southern blot analysis of genomic DNA restricted with methylation-sensitive enzymes. Northern blot analysis revealed that the plants with the methylated transgene did not accumulate the uidA::nptII fusion gene transcript.

Somatic embryogenesis from isolated scutella of wheat: effects of physical, physiological and genetic factors☆

Induction of high frequency somatic embryogenesis from in vitro culture of isolated scutella has been referred to as the Enhanced Regeneration System (ERS). In the present study, the importance of such factors as genotype, culture medium, explant size and cold pretreatment in the induction of somatic embryogenesis and regeneration response of isolated scutella was assessed in a complete block design experiment. Two species of wheat, Triticum aestivum and T. durum were compared and the findings between the two differed. The relative importance of genotype effect prevailed over all factors. In cultivars with similar embryogenic potential, the effect of explant size and cold pretreatment had major influence on the induction and regeneration frequencies. The use of an induction medium composed of amino acids and double Murashige and Skoog salt concentration, favorably increased the regeneration frequency in T. aestivum, whereas the regeneration frequency in T. durum decreased due to developmentally arrested embryos.

The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells.

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli β-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.

Wheat Starch Modification Through Biotechnology

Natural mutations that affect the amylose/amylopectin ratio in starch are unlikely to develop naturally in wheat due to its allohexaploid genome (2n =6x; AABBDD). One of the strategies to modify wheat starch structure involves identification of germplasms with null alleles for starch biosynthetic genes, followed by exchange of functional alleles with the identified null alleles through classical plant breeding. This technique has successfully been used to combine the three null alleles for granule-bound starch synthase I (GBSSI) to develop a wheat line that produces amylopectin-rich (>95%) starch (waxy starch). Another strategy to alter expression levels of starch biosynthetic genes employs recent advances in molecular biology and genetic engineering of wheat. For this approach, various monocot vectors have been developed that drive expression of wheat starch branching enzyme I (SBEI) cDNA sequences in the anti-sense orientation. Several of the wheat cell lines transformed with the anti-sense vectors express branching enzyme (BE) activity at a significantly lower level than non-transformed cells. One transgenic wheat plant expressing the anti-sense SBEI RNA produces a ten-fold lower level of BE activity in kernels than wild-type wheat. Analysis of starch produced from the transgenic plant shows that starch structure and properties have been altered.

Wheat (Triticum aestivum L.) somatic embryogenesis from isolated scutellum: Days post anthesis, days of spike storage, and sucrose concentration affect efficiency

SummaryTo improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l−1 casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.

RECOVERY AND CHARACTERIZATION OF TRANSGENIC PLANTS FROM TWO SPRING WHEAT CULTIVARS WITH LOW EMBRYOGENESIS EFFICIENCIES BY THE BOMBARDMENT OF ISOLATED SCUTELLA

SummaryTwo commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.