High tryptophan rice with an improved eating quality

Franz Marielle Nogoy,Hye-Jung Lee,Marjohn Nino,Me-Sun Kim,Sothea Ouk,Yu-Jin Jung,Kwon-Kyoo Kang,Ill-sup Nou,Yong-Gu Cho 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Geneticists and rice breeders are continuing to address solutions to high cases of undernutrition and malnutrition in many parts of the world. Fortification with vitamins in rice is a feasible solution to directly reach consumers who suffer nutritional problems. In this study, we are working on tryptophan, a limiting amino acid in almost all protein sources which are of importance for human nutrition. The present high tryptophan rice lines are much higher tryptophan level in mature seeds than wild type, however, the grain quality is very low. We try to improve the eating quality of the current high tryptophan rice lines by crossing them to popular Korean varieties, Hopumbyeo and Samgwangbyeo. Insensitive lines for tryptophan feedback inhibition are screened by growing in medium containing amino acid analogues, 5-methyl tryptophan. In vitro screening of each progenies enables us to select in each generation the rice lines with tolerance to 5-methyl tryptophan. After a series of in vitro screening and phenotypic selection in the field, the F4 progeny containing the same mutation in ASA2 gene from its parent showed an improvement in its grain quality.

Current Applicable DNA Markers for Marker Assisted Breeding in Abiotic and Biotic Stress Tolerance in Rice (Oryza sativa L.)

( Franz Marielle Nogoy ),( Jae-young Song ),( Sothea Ouk ),( Shadi Rahimi ),( Soon Wook Kwon ),( Kwon-kyoo Kang ),( Yong-gu Cho ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.3

Abiotic and biotic stresses adversely affect rice (Oryza sativa L.) growth and yield. Conventional breeding is a very effective method to develop tolerant rice variety; however, it takes a decade long to establish a new rice variety. DNA-based markers have a huge potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection (MAS). The large number of quantitative trait loci (QTLs) mapping studies for rice has provided an abundance of DNA marker-trait associations. The limitations of conventional breeding such as linkage drag and lengthy time consumption can be overcome by utilizing DNA markers in plant breeding. The major applications of DNA markers such as MAS, QTL mapping and gene pyramiding have been surveyed. In this review, we presented the latest markers available for some of the most important abiotic and biotic stresses in rice breeding programs. Achieving a significant impact on crop improvement by marker assisted breeding (MAB) represents the great challenge for agricultural scientists in the next few decades.

Genomics Researches and Their Applications in Plant Breeding at PAG XXIV Conference

( Franz Marielle Nogoy ),( Shadi Rahimi ),( Kwon-kyoo Kang ),( Yong-gu Cho ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.1

The rise of whole genome sequences of different plants provided more understanding about the gene regulation and genome evolution in further studying plants. More and more pathways and networks are identified by novel gene discoveries. Therefore, the Plant and Animal Genome Conference (PAG XXIV) provides a good venue to share the recent progress in the area of plant research genome sequencing technologies in various plants. However, this information can make a powerful system for developing improved crop varieties. By the way, the genome annotation and assembly is an essential key for breeding of stress-tolerant plants. PAG XXIV demonstrated different works about the extensive use of genomic databases accompanied by bioinformatics tools to accelerate breeding methods, discovery of new approaches to genomics, further increasing biomass of bioenergy crops, and explaining the genetic mechanisms in plant growth and defense. This review article summarizes some of the researches in various plants of rice, corn, wheat, cottonwood, switchgrasses, Thinopyrum, wheatgrass and Arabidopsis presented in PAG XXIV with the focus on genome technologies and their applications in plant breeding.

Characterization of ‘GolSam’ Lines Developed from the Cross between Samgwang and 5MT Resistant Lines in Rice

( Franz Marielle Nogoy ),( Yu Jin Jung ),( Kwon Kyoo Kang ),( Yong-gu Cho ) 한국육종학회 2018 Plant Breeding and Biotechnology Vol.6 No.3

Rice grain quality is usually observed by its chalkiness and is affected by genetic effects of endosperm, cytoplasm and maternal plant. Controlling the chalkiness in rice can be a very challenging task because it is affected by genotype and environmental factors. The present study aimed to introduce 5-methyl tryptophan (5MT) resistance from the 5MT resistant mutants into Samgwang, a high grain and eating quality Korean variety by introgression of, resulting to elevated tryptophan content in grains. The progenies generated from single crosses of two different cross combinations were phenotyped based on agronomic traits and by 5MT growth inhibition test. Through direct PCR sequencing, the inheritance of single base mutation (F124V) in OsASA was selected among the progenies. The latter generations were used to analyze the grain and eating quality of the selected lines. Inbred lines (S4-10, S4-28, and S5-11) carrying the point mutation in OsASA and with reduced chalkiness plus good eating qualities were successfully generated. Tryptophan content in the milled grains of the selected lines showed 2 - 4 times higher (mg/100 mg) than the maternal parent. The three selected lines, S4-10, S4-28, and S5-11, were later renamed as GolSam-1, GolSam-2, and GolSam-3 respectively.

Plant microRNAs in molecular breeding

Franz Marielle Nogoy,Marjohn C. Niño,송재영,정유진,강권규,노일섭,조용구 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.1

MicroRNAs are small, endogenous, non-coding RNAs found in plants, animals, and in some viruses, which negatively regulate the expression of genes by promoting the degradation of target mRNAs or by translation inhibition. Ever since the discovery of miRNAs, its biology, mechanisms, and functions were extensively studied in the past two decades. Plant and animal miRNAs both regulate target mRNAs, but they differ in scope of complementarity to their target mRNA. Plant microRNAs are known to play essential roles in a wide array of plant development. To date, there are many studies giving evidence that the regulation of miRNA levels can reprogram plant responses to abiotic (physical environment) and biotic stresses (pathogen and herbivore). Most of these studies were first carried out in the model plant Arabidopsis thaliana. Recently, the trend of miRNA research is furthering its role in crop breeding and its evolutionary origin. In this review, we presented the dynamic biogenesis of microRNAs, the diverse functions of miRNAs in plants, and experimental designs used in studying microRNAs in plants, and most importantly, we presented the applications of microRNA-based technology to improve the resistance of crops in abiotic and biotic stresses.

고 함량 트립토판 생산 GM 벼 개발 및 전사체 분석

정유진,Franz Marielle Nogoy,조용구,강권규 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.3

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive α subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3- acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

Application of ZFN for Site Directed Mutagenesis of Rice SSIVa Gene

정유진,Franz Marielle Nogoy,이상규,조용구,강권규 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.1

Many successful studies on genome editing in plants have been reported and one of the popular genome editing technology used in plants is Zinc Finger Nucleases (ZFN), which are chimeric proteins composed of synthetic zinc finger-based DNA binding domain and a DNA cleavage domain. The objective of this research was to utilize ZFNs to induce a double-stranded break in SSIVa, a soluble starch synthase involved in starch biosynthesis pathway, leading to the regulation of the SSIVa expression. The isoform SSIVa is not yet well studied, thus, by modifying the endogenous loci in SSIVa, we can explore on the specific roles of this gene in starch biosynthesis and other possible functions it might play. In this study, we used ZFN-mediated targeted gene disruption in the coding sequence of the SSIVa rice gene in an effort to elucidate the functional role of the gene. Generation of transgenic plants carrying premature stop codons and substitution events, revealed no SSIVa mRNA expression, low starch contents and dwarf phenotypes. Remarkably, based on our analysis SSIVa gene disruption had no effect on other starch synthesis related genes as their expression remained at wild type levels. Therefore, the engineered ZFNs can efficiently cleave and stimulate mutations at SSIVa locus in rice to affect plant height, grain filling and starch content.

Brassica rapa Sec14-like protein gene BrPATL4 determines the genetic architecture of seed size and shape

김준기,이혜정,Franz Marielle Nogoy,유달아,김미선,강권규,노일섭,조용구 한국식물생명공학회 2016 JOURNAL OF PLANT BIOTECHNOLOGY Vol.43 No.3

Seed size traits are controlled by multiple genes in crops and determine grain yield, quality and appearance. However, the molecular mechanisms controlling the size of plant seeds remain unclear. We performed functional analysis of BrPATL4 encoding Sec14-like protein to determine the genetic architecture of seed size, shape and their association analyses. We used 60 T3 transgenic rice lines to evaluate seed length, seed width and seed height as seed size traits, and the ratios of these values as seed shape traits. Pleiotropic effects on general architecture included small seed size, erect panicles, decreased grain weight, reduced plant height and increased sterility, which are common to other mutants deficient in gibberellic acid (GA) biosynthesis. To test whether BrPATL4 overexpression is deleterious for GA signal transduction, we compared the relative expression of GA related gene and the growth rate of second leaf sheath supplied with exogenous GA3. Overexpression of BrPATL4 did not affect GA biosynthesis or signaling pathway, with the same response shown under GA treatment compared to the wild type. However, the causal genes for the small seed phenotype (D1, SRS1, and SRS5) and the erection of panicles showed significantly decreased levels in mRNA accumulation compared to the wild type. These results suggest that the overexpression of BrPATL4 can control seed size through the suppression of those genes related to seed size regulation. Although the molecular function of BrPATL4 is not clear for small seed and erect panicles of BrPALT4 overexpression line, this study provides some clues about the genetic engineering of rice seed architecture.

Low-Affinity Cation Transporter 1 Improves Salt Stress Tolerance in Japonica Rice

Marjohn C. Niño,Franz Marielle Nogoy,강권규,조용구 한국육종학회 2018 Plant Breeding and Biotechnology Vol.6 No.1

Plants adapt to hostile environmental condition through specialized cellular processes which require coordinatedregulation of multiple transport mechanisms. Low-affinity cation transporter (LCT) 1 is a membrane transporter gene exclusive only tomembers of the grass family, and the rice genome contained only one copy of the gene. Accumulating evidence highlighted theimportant role of this gene in the regulation of various cations transport into the plant cells including heavy metals. To furthercharacterize the role of this membrane transporter in planta, we overexpressed the OsLCT1 in japonica rice using CaMV 35S andscreened the transgenic plants for high salt toxicity at the seedling stage. A striking difference in the phenotype ofOsLCT1-overexpression plants and the wild-type was observed at seven days after treatment, where transgenic plants exhibitedmoderate tolerance reaction to salinity stress. Our findings suggest that OsLCT1 gene can be useful to develop new resistant varietiesagainst salinity stress.

Application and utilization of marker assisted selection for biotic stress resistance in hybrid rice (Oryza sativa L.)

송재영,Sothea Ouk,Franz Marielle Nogoy,Marjohn C. Niño,권순욱,하운구,강권규,조용구 한국식물생명공학회 2016 JOURNAL OF PLANT BIOTECHNOLOGY Vol.43 No.3

Development of disease resistant plant is one of the important objectives in rice breeding programs because biotic stresses can adversely affect rice growth and yield losses. This study was conducted to identify lines with multiple-resistance genes to biotic stress among 173 hybrid rice breeding lines and germplasms using DNA-based markers. Our results showed that one hybrid rice line [IR98161-2- 1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66)] possessed 5 bacterial blight resistance genes (Xa4, xa5, Xa7, Xa13 and Xa21) while two hybrid rice lines [IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66) and 7292s (IR75589-31-27-8-33S(S1)/ IR102758B)] possessed 3 bacterial blight resistance genes (Xa4, Xa7 and Xa21, and Xa3, Xa4 and xa5). Molecular survey on rice blast disease revealed that most of these lines had two different resistant genes. Only 11 lines possessed Pib, Pi-5, and Pi-ta. In addition, we further surveyed the distribution of insect resistant genes, such as Bph1, Bph18(t), and Wbph. Three hybrid breeding lines [IR98161-2-1-1-k1-3 (IR86409-3-1-1-1-1-1/IRBB66), IR98161-2-1-1-k1-2 (IR86409- 3-1-1-1-1-1/IRBB66), and 7292s (IR75589-31-27-8-33S(S1) /IR102758B)] contained all three resistance genes. Finally, we obtained four hybrid rice breeding lines and germplasms [IR98161-2-1-1-k1-2 (IR86409-3-1-1-1-1-1/IRBB66), Damm- Noeub Khmau, 7290s, and 7292s (IR75589-31-27-8-33S(S1)/ IR102758B)] possessing six-gene combination. They are expected to provide higher level of multiple resistance to biotic stress. This study is important for genotyping hybrid rice with resistance to diverse diseases and pests. Results obtained in this study suggest that identification of pyramided resistance genes is very important for screening hybrid rice breeding lines and germplasms accurately for disease and pest resistance. We will expand their cultivation safely through bioassays against diseases, pests, and disaster in its main export countries.