Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic tr...

https://bioone.org/journals/in-vitro-cellular-and-developmental-biology---plant/volume-40/issue-1/IVP2003501/Invited-review--Factors-influencing-Agrobacterium-mediated-transformation-of-monocotyledonous/10.1079/IVP2003501.pdf

Factors influencing Agrobacterium -mediated transformation of monocotyledonous species

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (β-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.

Factors influencing successful Agrobacterium-mediated genetic transformation of wheat.

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-20-10-1222

Biofilm Formation, Epiphytic Fitness, and Canker Development in Xanthomonas axonopodis pv. citri

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium-mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium-mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.

Agrobacterium‐mediated transformation of cereals: a promising approach crossing barriers

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat (Triticum aestivum) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast (Saccharomyces cerevisiae) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley (Hordeum vulgare). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms.

/pdf/wheat-vacuolar-iron-transporter-tavit2-transports-fe-and-mn-luf0exzrud.pdf

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

/pdf/factors-influencing-agrobacterium-mediated-transient-5g8b53nt1f.pdf

Factors influencing Agrobacterium‐mediated transient expression of uidA in wheat inflorescence tissue

thereof was responsible for the crown gall phenotype (see review by Zupan and Zambryski, 1997). The implica tions for crop improvement and also as a tool for studies in basic plant biology have since been noted (Potrykus, 1990). Indeed, successful gene transfer tech niques that exploit this natural gene transfer system of Agrobacterium sp. soon became widely available for the routine transformation of crops such as cotton, lettuce, celery, flax, and potato (Grant et al., 1991). However, it was also noted that the graminaceous monocots, which include such major food crops as wheat, maize and rice, were recalcitrant in this system because this group of crop plants was not naturally susceptible to Agrobacterium sp. Other methods were, therefore, sought by which this group of crops could be transformed. Several have been investigated, with varying levels of success, including the electrical induction of cells to aid their reception of foreign DNA (Rhodes et al., 1989), electroporation of tissues with DNA (Shimamoto et al., 1989), micro injection of DNA into floral tillers (De la Pena et al., 1987), DNA uptake by germinating pollen (Luo and Wu, 1988), and by the bombardment of cells and tissues with DNA-coated particles (Vasil et al., 1992). However, research to make the Agrobacterium-based transformation method amenable to this group of crops has continued as the system is perceived to possess several advantages over other forms of transformation including (i) the ability to transfer large segments of DNA with minimal rearrange ment; (ii) the precise insertion of transgenes resulting in fewer copies of inserted genes; and (iii) simple technology with lower cost. One of the simplest available plant transformation systems involves the infiltration of Agro bacterium cells into Arabidopsis plants before flowering, and direct selection for rare transformants in the resulting seedling population (Chang et al., 1994). However, the features which make this possible, including the small plant size, the rapid generation time and the high seed A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the estab lishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of soni cation treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inocula tion/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The invest igation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

H. Wu

Papers

Factors influencing successful Agrobacterium-mediated genetic transformation of wheat.

Factors influencing Agrobacterium‐mediated transient expression of uidA in wheat inflorescence tissue

Factors influencing Agrobacterium-medi'iated transient expression of uidA in wheat inflorescence tissue

Transgenic approaches to study quality traits in cereals