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Abstract

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1-gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1-gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.

Barley Genomics: An Overview

“…In contrast to the biolistic gene transfer technique [57], a more efficient Agrobacterium mediated barley genetic transformation method based on immature embryos was developed in spring barley [58]. In a recent attempt to further improve this technology, Kumlehn et al [59] developed a transformation method for winter barley based upon the infection with Agrobacterium of androgenic pollen cultures.…”

TRANSGENIC TOMATO PLANTS (Solanum lycopersicum L.): DIRECT METHODS OF GENE TRANSFER AND FACTORS AFFECTING TRANSFORMATION EFFICIENCY (review)

“…Авторами установлено, что на эффективность этих процессов влияют следующие факторы: генотип, возраст интактного экспланта, состав питательной среды для культивирования протопластов, а также рН буферного раствора (61). Однако трансгенные растения впоследствии были получены только для ограниченного числа культур, в том числе растений семейства Solanaceae -петунии (62), табака (63), а также для ячменя (64). Экспериментальная работа японских исследователей по генетической трансформации интактных каллусных клеток томата методом микроинъекции была опубликована одной из первых в 1988 году.…”

Science Behind Cotton Transformation

“…Major limitation of microinjection method involves the use of expensive micromanipulator and it is a time-consuming procedure. Furthermore, frequency of transformation is very low and dependent on the species, i.e., proved to be successful in tobacco [55], Petunia [53], Rape [56], and Barley [57].…”