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Highlights in BioScience                                                      
 

 

 
Highlights in BioScience                                    DOI:10.36462/H.BioSci.20183                            July 2018 | Volume 

 

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Citation: Youssef A. A. and Rslan W. 

M. (2018) Sugar Beet Improvement 

using Agrobacterium-mediated Transf- 

ormation technology. Highlights in 

BioScience, Volume 1.Article ID 

20183, dio:10.36462/H.BioSci.20183 

 

 

 

 

 

 

Received: June  1, 2018 

 

Accepted: July 17, 2018 

 

Published: July 30, 2018 

 

 

Copyright:© 2018 Youssef  and 

Rslan. This is an open access article 

distributed under the terms of the 

Creative Commons Attribution 

License, which permits unrestricted 

use, distribution, and reproduction in 

any medium, provided the original 

author and source are credited. 

 

 

 

Data Availability Statement: All 

relevant data are within the paper 

 

Funding: The authors have no support 

or funding to report. 

Sugar Beet Improvement using Agrobacterium-

mediated Transformation technology 

Abo-Bakr A. Youssef
1
 and Wessam M. Rslan

2
*

 

1, 2 
Agricultural Genetic Engineering Research Institute; Agricultural Research 

Center, Egypt. 
 

*To whom correspondence should be addressed: wessam.rslan@ageri.sci.eg 

 

Abstract 

 

Since discovering Agrobacterium tumefaciens distinctive capacity 

to incorporate a specified part of their transfer-DNA (T-DNA) into 

eukaryotic cells, the bacteria were commonly used for crop 

transformation originally of dicotyledonous crops and subsequently 

of nearly all organisms. To achieve this, the tumor-inducing (Ti) 

plasmid was changed to extract phytohormone and opine 

biosynthetic proteins (cytokinin and auxin) so as not to interfere 

with ordinary morphological growth. Overall, the conversion 

mediated by Agrobacterium was easier, more effective and less 

costly relative to other technologies. It also results in insertions with 

small copy count. Tumor development in crops has also proved the 

susceptibility of explants from field-grown sugar beet crops to 

Agrobacterium tumefaciens. Early efforts by Agrobacterium 

tumefaciens to transform sugar beet were unsuccessful, primarily 

owing to inability to regenerate crops from stably modified callus or 

suspended cells. A genotype-independent method was defined 

under which cotyledonary explants of various sugar beet genotypes 

are inoculated with Agrobacterium tumefaciens comprising whether 

kanamycin tolerance and GUS activity or kanamycin resistance, 

GUS activity and glyphosate tolerance. GUS expression, NPT dot 

blot as well as EPSPS assays verified the presence of transgenes; 

progeny showed Mendelian genetically modified inheritance and 

glyphosate tolerance at deadly concentrations to control plants. 

Unfortunately, there was no publication of technical information of 

the technique. Here we reviewed the concept Agrobacterium-

mediated transformation and how to be applicable 
 

Keywords: Agrobacterium tumefaciens, callus,  suspended cells, sugar beet, 

kanamycin resistance.  

 

 

 

 

Review Article                                                                             Open Access   



 
 

                                                                                                                                       Sugar Beet Improvement using Agrobacterium 

 

 

 
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Introduction 

Sugar beet is extremely prone to Agrobacterium 

tumefaciens as an instance (1–5). Though, genotype-

dependent susceptibility (1,3) can be enhanced by pre-culture 

explants before inoculation (6) or by extending the length of 

co-culture (1,3). Krens et al. (4) used Agrobacterium 

tumefaciens strains carrying an isolated cytokinine gene to 

enhance the development of sugar beet leaf discs, cotyledons 

and hypocotyls from two-week-old seedlings of nine distinct 

types. Cotyledon and hypocotyl explants generated low-

frequency shoots; however, inoculated leaf discs did not 

regenerate the shoot. It was impossible to confirm a stable 

transformation. 

Lindsey and Gallois (1) revealed Agrobacterium 

tumefaciens first effective regeneration of genetically 

modified sugar beet crops. Transgenic seeds were acquired 

from co-cultivated shoot-base cells with Agrobacterium 

strain LBA4404 containing a bi-nary plant with a kanamycin 

resistance gene (nptII) and either chloramphenicol acetyl 

transferase (cat) or β-lucuronidase (gusA) genes. The 

frequency of transformation depended on the kinds of 

explants, genotypes and circumstances of choice. 

Halluin et al. (2) who established crops resistant to 

broad-spectrum herbicides, glufosinate and sulfonylureas, as 

well revealed sugar beet transformation. Friendly callus, 

originating from cotyledons, hypocotyls, petioles as well as 

true leaves of 2-to 3-month-old seedlings cultivated in dark, 

was carefully cut and inoculated with Agrobacterium 

tumefaciens either a mutant acetolactate synthase (ALS) or a 

bialaphos resistance (bar) gene powered by multiple 

promoters, i.e. 35S CaMV, nos, TR1' or TR2.' This 

genotype-dependent protocol took nearly two years to get 

grown, which probably explains the morphologies of the 

aberrant plant. 

Latest studies utilizing Agrobacterium tumefaciens 

generated transgenic crops at frequencies that are adequate to 

produce crops used in experiments to evaluate herbicide 

tolerance and resistance to disease (7–12). For physiological 

and molecular research, Agrobacterium tumefaciens 

produced transgenic crops were also used. An endogenous 

sugar beet GUS (SB-GUS) enzyme has been contrasted to E. 

coli in leaf disc transformation (13). Variations in patterns of 

gene expression in roots have been investigated using distinct 

constructs (14) in another application. In another, 

development habit and accumulation of sugar were explored 

after transforming into a patatin gene promoter (15) with a 

bacterial cytokinin biosynthetic gene. Although various 

explants of sugar beet, i.e., shoot bases, petioles, leaves and 

callus, were used, cotyledon explants were often more 

effective in transformation with efficiencies varying from 0.1 

to 1.0 (4,7,9,10,16,17). 

With kanamycin selection (18), leaf lamina explants 

from shoots multiplied from apical meristems of four 

genotypes of sugar beet produced a 6.2 percent 

transformation level. Small (1–3 mm) bud tips from various 

shoot clumps from undeveloped floral buds of five distinct 

genotypes resulted in hygromycin-resistant shooting rates of 

13.3 to 30.6 percent. Over 50% of crops from shoot-base 

tissue kanamycin-resistant explants produced the hpt gene 

(19). Finally, a mixture of Agrobacterium and void 

infiltration has been used to promote the transfer of bacterial 

DNA to callus and crop tissue, resulting in more than 40% 

transgenic callus clones (20). 

 

Factors influencing transformation efficiency  

Genotype (19), explant origin (21), bacterial strain and 

incubation time (18) selection technique (22),type of 

promoter promoting selectable gene (22), light intensity (23), 

wound with pre-inoculation particle bombardment (10), use 

of acetosyringone  (3), pre-treatment with drugs (6) and , pre-

and co-cultivation interval (6). Despite important 

advancement, the transformation of sugar beet to 

Agrobacterium tumefaciens is still regarded to also be 

recalcitrant. Because of the failure to absorb and integrate 

DNA, it is impossible to be due to the small amount of 

morphologically competent regeneration cells. If they are 

integrated in big amounts of non-competent cells (24), 

connection to such competent cells may also be impeded. 

 

Development of transgenic herbicide tolerance 

Sugar beet competes badly with weeds, particularly in 

early growth, leading in dramatically elevated yield losses 

that can range from 50 percent to total losses (25) unless 

adequate weed control is attained. Conventional control 

measures involve herbicide spraying at distinct moments and 

distances to decrease these losses, making weed control 

programs complex and challenging. 

The development of transgenic sugar beet resistant to 

broad-spectrum herbicides is therefore a significant 

alternative. Herbicide tolerance is one of the first features 

that genetic engineering has effectively brought into several 

plant species; some types of herbicide tolerant (HT) have 

been on the market for further than a decade (26). HT crops 

have been produced using genes from microorganisms or 

higher plants that confer tolerance by: I changing the active 

site of the target protein in such a way that converted cells 

are less susceptible to herbicide; (ii) using an enzyme that 

transforms the core components of the herbicide into inactive 

compounds; or (iii) overproducing herbicide target proteins 

(27). 

Agrobacterium-mediated transformation and in vitro 

cell selection have been used to evolve HT sugar beets 



 
 

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tolerant either to non-selective, broad-spectrum herbicides, 

i.e., glyphosate, Roundup ® active ingredient and 

glufosinate, Basta ® active ingredient, Liberty ® and 

Herbiace ® main ingredient, or specific herbicides such as 

imidazolinone, chlorsulfurone and sulfonylurea (28). In 

plants, encoded in the nucleus, the enzyme 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS) 

performs a main part in chloroplast responses arising in 

synthesis of phenylalanine, aromatic amino acids, tyrosine, 

and tryptophan. 

After finding in the early 1970s that perhaps the analog 

amino acid glyphosate [N-(phosphonomethyl)-glycine] could 

preferentially inhibit EPSPS activity, shut down aromatic 

amino acid synthesis and ultimately kill the plant, strategies 

are established to produce glyphosate-tolerant plants (or 

Roundup Ready ®). EPSPS occurs only in crops and certain 

microorganisms, not in livestock or humans, probably 

explaining why glyphosate toxicity is exceptionally small in 

human cells (29). 

To create glyphosate-tolerant sugar beet, two methods 

listed above I and ii) have been utilized. In the first scenario, 

a gene Agrobacterium sp. was used for an altered protein, 

CP4-EPSPS, which is extremely glyphosate tolerant (30); in 

vitro assays and in vivo herbicide sprays (16) verified 

glyphosate tolerance. The second approach used a gene 

encoding Achromobacter sp. glyphosate oxidoreductase, 

GOX, and catalyzing glyphosate degradation in and out of 

non-toxic compounds, glyoxylate and aminomethylphospho-

nic acid. 

However, sugar beet plants were transformed in the 

second approach with both CP4-EPSPS nd GOX genes (9) 

and evaluated with distinct Roundup ® spraying systems in 

the greenhouse and field. Extremely tolerant transformants 

have been acquired without phytotoxic or any other harmful 

phenotypic effects (31). Inversely linked with the transgenic 

copy number (9), herbicide tolerance emerged. Glufosinate 

(ammonium salt L-phosphinothricin, PPT) and bialaphos 

(PPT plus two alanines; L-phosphinothricinyl-L-alanyl-L-

alanine) are extremely toxic to plant cells; they behave as 

competing glutamine synthetase inhibitors that are critical to 

the transformation of glutamine acid and ammonia onto 

glutamine. 

Inhibition contributes to toxic ammonia accumulation, 

leading to death of the cell. Enzymes encoded in bars and pat 

and extracted from various Streptomyces sp., detoxify PPT 

and have been broadly used as selectable markers for the 

production of transgenic HT plants. Bar, motivated by the 

proponents of CaMV 35S, nos, TR1' or TR2, was used to 

achieve glufosinate-tolerant sugar beet crops (2) assessed in 

the sector of gene flow and agricultural efficiency research 

(32). Like glyphosate, glufosinate has a very low toxicity to 

mammals (29). 

Joersbo (33) assessed both glyphosate-and glufosinate-

tolerant sugar beets  environmental efficiency, including 

economics and utilization flexibility. By deactivating the first 

enzyme in the pathway, acetolactate synthase (ALS), 

sulfonylurea compounds prevent the biosynthesis of clustered 

amino acids, valine, leucine and isoleucine. Sugar beet has 

been transformed with a mutant sulfonylurea-insensitive ALS 

gene (2). 

In vitro cell selection (34) was also acquired from 

mutant sulfonylurea-and imidazolin one-tolerant sugar beet 

crops. However, many HT transgenic beet varieties have 

been approved for discharge in the U.S. (1996, 1998, 2005), 

Canada (2001, 2005) and Japan (2007) (26). Roundup Ready 

sugar beets were grown for the first season in 2008 by sugar 

beet growers in Michigan. For ten years, Michigan State 

University researchers have been collaborating with these 

varieties to determine implementation rates and timing and 

policies to delay glyphosate-resistant weed growth. 

 

 

 

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