Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Molecular Epidemiology of Multidrug-Resistant Pneumococci among Ghanaian Children under Five Years Post PCV13 Using MLST

Antibiotic resistance in pneumococci contributes to the high pneumococcal deaths in children. We assessed the molecular characteristics of multidrug-resistant (MDR) pneumococci isolated from healthy vaccinated children under five years of age in Cape Coast, Ghana. A total of 43 MDR isolates were selected from 151 pneumococcal strains obtained from nasopharyngeal carriage. All isolates were previously serotyped by multiplex PCR and Quellung reaction. Susceptibility testing was performed using either the E-test or disk diffusion method. Virulence and antibiotic resistance genes were identified by PCR. Molecular epidemiology was analyzed using multilocus sequence typing (MLST). Vaccine-serotypes 23F and 19F were predominant. The lytA and pavB virulence genes were present in all isolates, whiles 14–86% of the isolates carried pilus-islets 1 and 2, pcpA, and psrP genes. Penicillin, tetracycline, and cotrimoxazole resistance were evident in >90% of the isolates. The ermB, mefA, and tetM genes were detected in (n = 7, 16.3%), (n = 4, 9.3%) and (n = 43, 100%) of the isolates, respectively. However, >60% showed alteration in the pbp2b gene. MLST revealed five novel and six known sequence types (STs). ST156 (Spain9V-3) and ST802 were identified as international antibiotic-resistant clones. The emergence of international-MDR clones in Ghana requires continuous monitoring of the pneumococcus through a robust surveillance system

Vacuolar Invertase Gene Silencing in Potato (<i>Solanum tuberosum</i> L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

<div><p>Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (<i>VInv</i>) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.</p></div

Molecular Characterization and Antibiotic Susceptibility of Non-PCV13 Pneumococcal Serotypes among Vaccinated Children in Cape Coast, Ghana

Preventive strategies involving the use of pneumococcal conjugate vaccines (PCVs) are known to drastically reduce pneumococcal disease. However, PCV vaccination has been plagued with serotype replacement by non-PCV serotypes. In this study, we describe the prevalence and molecular characteristics of non-PCV13 serotypes (non-vaccine serotypes, NVTs) from pneumococcal carriage isolates obtained from children < 5 years old in Cape Coast, Ghana, after PCV introduction. The isolates were subjected to antibiotic susceptibility testing and multilocus sequence typing (MLST), and molecular techniques were used to detect the presence of virulence genes. Serotypes 11A, 13, 15B, 23B, and 34 formed the top five of the 93 NVT isolates. As such, 20 (21.5%), 49 (48.4%), and 70 (74.3%) isolates were non-susceptible to penicillin, tetracycline, and cotrimoxazole, respectively. Sixteen (17.2%) multidrug-resistant isolates were identified. However, non-susceptibility to ceftriaxone and erythromycin was low and all isolates were fully susceptible to levofloxacin, linezolid, and vancomycin. Whereas pcpA, pavB, lytA, and psrP genes were detected in nearly all serotypes, pilus islet genes were limited to serotypes 11A, 13, and 23B. MLST for predominant serotype 23B isolates revealed three known and seven novel sequence types (STs). ST172 and novel ST15111 were the most dominant and both STs were related to PMEN clone Columbia23F-26 (ST338). In conclusion, non-PCV13 serotype 23B was the most prevalent, with characteristics of rapid clonal expansion of ST172 and ST15111, which are related to international clones of the pneumococcus. Continuous monitoring of NVTs in Ghana is, therefore, essential, as they have the potential to cause invasive disease, show high antibiotic resistance, and attenuate the effects of PCV vaccination

Sugar-end defect frequency was reduced in French fries prepared from <i>VInv</i>-silencing lines of Ranger Russet.

<p>Sugar-end defects are apparent on nearly half of the fries from Ranger Russet (A) and empty vector (B) control tubers. No sugar-end defect fries where observed in fries from lines 1632-1 (C) and 1632-4 (D) in which the <i>VInv</i> had been silenced using RNA interference. In (A) and (B), fries with sugar-end defects are on the right and fries without sugar-end defects are on the left.</p

Expression of <i>VInv</i> in the bud end and stem end of tubers from <i>VInv</i>-silencing lines of Russet Burbank (RBKx) relative to expression of <i>VInv</i> in untransformed Russet Burbank at harvest and after one, three and five months of storage.

<p>Note: ^§Asterisks indicate overall differences of least squares means between RBKx and Russet Burbank controls at the same tuber end across sampling times of harvest and one, three and five months of storage.</p><p>*, p<0.05;</p><p>**, p<0.01;</p><p>***, p<0.001.</p><p><i>VInv</i> expression was determined using <i>actin97</i> as a reference gene and results are presented as a percentage of the level in Russet Burbank controls.</p

Sucrose content in both ends of Russet Burbank potato tubers and in tubers from <i>VInv</i>-silencing lines (RBKx) of Russet Burbank at harvest and after one, three and five months of storage.

<p>Note: Data are means ± standard deviation of five independent tuber samples. Sucrose contents are shown as mg g⁻¹ fresh tuber weight.</p

Acrylamide content at stem end and bud end of fried strips prepared from Russet Burbank and three <i>VInv</i>-silencing lines three months after harvest.

<p>Note: Acrylamide contents are means ± standard deviation of five individual fried strips.</p><p>Note that lines RBK1 and RBK22 have strong suppression of <i>VInv</i> and RBK46 shows little suppression of <i>VInv</i>.</p

Fried potato slices processed from <i>VInv</i>-silencing lines of Russet Burbank potato had fewer sugar end defects than those processed from Russet Burbank controls.

<p>Fried strips were prepared from tubers at harvest (A) and after 1 (B), 3 (C) and 5 (D) months of storage. The numbers 1 to 6 represent tubers from Russet Burbank, RBK46, RBK1, RBK25, RBK27, and RBK22, respectively. Slices are positioned with the tuber stem end portion nearest to the bottom of each photograph.</p