MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF AGRICUNTURAL SCIENCES LE HUNG PHONG RESEARCH ON BREEDING PARENT LINES TO RESIST TO BROWN PLANTHOPPER AND BACTERIAL LEAF BLIGHT FOR DEVELOPING TWO-LINE HYBRID RICE IN VIETNAM Major: Genetics and Plant Breeding; Code: 620111 SUMMARY OF DOCTORAL THESIS HA NOI - 2018 The thesis is completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES (VAAS) Science instructor: Associate Professor Dr Nguyen Tri Hoan Doctor Nguyen Nhu Hai Reviewer 1: Associate Professor Dr NGUYEN THI TRAM Reviewer 2: Associate Professor Dr NGUYEN THI NGOC HUE Reviewer 3: Associate Professor Dr NGUYEN VAN VIET The thesis is reported at the grassroots thesis dissertation council meeting at: Vietnam Academy of Agricultural Sciences (VAAS) The Thesis can be found at the library: Vietnam National Library; Library of Vietnam Academy of Agricultural Sciences; Library of Field Crop Research Institute25-30 Vũ V ĐÃ CÔNG BỐ LIÊN QUAN INTRODUCTION The urgency of the topic In recent years, hybrid rice have contributed significantly to the food security, making an important contribution to stabilizing production and socioeconomic development of our country, while the world economy has many changes However, the expansion of hybrid rice area faces many difficulties such as imported seeds with high cost and not being able to be active in seed sources and the set of selected varieties in the country are still few, there are not many varieties resistant to pests and diseases (resistance to brown plant hopper, bacterial leaf blight ) and unfavorable conditions for production The source of pest and disease-resistant parent materials for tolerance hybrid rice breeding is poor, and production still faces many difficulties and risks due to weather, pests Besides, the area of rice cultivation in our country has been significantly reduced due to the effects of climate change causing drought and saline intrusion The development of pests and diseases (such as bacterial blight, brown planthopper, white back planthoper, Rice ragged stunt virus, Rice grassy stunt virus, Southern rice black-streaked dwarf virus etc.) causing difficulties for forecasting work and causing great damage to rice production The report of the Plant Protection Department (MARD) in 2013, 2014, 2015, 2016 and 2017 shows that: Recently, rice bacterial blight, stripe and brown planthopper, white back planthopper Rice grassy stunt virus, Southern rice black-streaked dwarf virus appear and cause serious damage in both Winter-Spring and Summer / Summer-Autumn crops in the Northern provinces The results of the study "Research on breeding parent lines to resist brown planthoper and bacterial blight to develop two-line hybrid rice in Vietnam" will create parent lines with good resistance and high combining ability To diversify sources of materials to choose to create 2-line hybrid rice varieties to resist brown planthopper and bacterial leaf blight The objective of the project - Selected 1-2 lines of parents capable of resisting tolerance of bacterial leaf blight, 1-2 parental lines that are resistant to brown planthopper for the selection and creation of two lines of hybrid rice to resist brown planthopper and bacterial blight - Selected 1-2 new, high-yield hybrid combination F1 (7-8 tons / ha), good quality, resistant to bacterial blight, brown planthoppers for hybrid rice development in Vietnam The scientific and practical significance of the topic 3.1 Scientific significance - The research results of the thesis supplement the scientific basis and the method to select the creation of parents maternal lines of thermosensitive genic male sterile (TGMS) and breedingtwo lines hybrid rice with resistance to brown planthopper and bacterial blight in Vietnamconditions - Information on new lines of parental lines to brown planthoppers, bacterial blight risistance to contribute to the selection of breeds in using materials to breed and select hybrid rice two lines with resistance to blight and brown planthopper 3.2 Practical significance - Determining the source of materials for the hybridization of the father and mother line of two lines of hybrid rice resistant to bacterial leaf blight and brown planthopper - The lines of TGMS as AMS35S-KBL, AMS30S-KBL (resistance to bacterial leaf blight ), KR95S, KR142S (brown planthopper resistance), the male lines as RP8, RP088, RP3, R1028-KR, R116R (BPH resistance) have many agronomic characteristics learning, good sexual characteristics, high combined ability of yield, is a good material source to breeding hybrid rice varieties resistant to bacterial leaf blight and brown planthopper - F1 Hybrid rice two lines combination with resistance to blight, brown planthopper have a short growth duration, high yield, contributing to testing and expanding hybrid rice production in Vietnam New contributions of the topic - Successfully create 02 lines of TGMS (AMS35S-KBL, AMS30SKBL) with bacterial blight resistance gene, stable sterility, high rate of stigma exertion, short growth duration, high value of general combining ability about real productivity - Create lines of TGMS (KR95S, KR142S) and 05 lines (RP8, RP088, RP3, R1028-KR, R116R) These parental lines have a short duration, good agronomic characteristics, high combined yield ability with brown plant hopper resistance genes These parental lines are very good materials for selectin two line hybrid rice varieties tolerant to brown planthoppers in Vietnam - Selection of hybrid rice combinations of TGMS lines with bacterial leaf blight resistance genes, having higher yield than check varieties 10% is AMS35S-KBL / R100, AMS30S-KBL / R116 and AMS34S-KBL / SR18, resistance to blight score 3-5 Throught transferring the bacterial leaf blight resistance genes into the TGMS lines initially improved the resistance to blight disease of two-line hybrid rice HYT124 and HYT116 - Selecting 06 hybrid rice combinations of KR142S / SR3, KR142S / SR14, KR142S / SR18, KR95S / SR14, KR95S / AIQ6, KR95S / SR3 with short duration, high productivity (8.6-9.3 tons / ha) and resistance to brown planthopper Subjects and scope of research 5.1 Research subjects - Existing hybrid rice lines and parents of the Field Crop Research Institute which the author directly and participated in creating, good parent lines collected in the country and imported - The papolations of BC3 - BC6 from hybrid combinations for selecting parent lines in the direction of resistance to bacterial leaf blight and brown planthopper - Bactrial leaf blight resistant materials were identified from the International Rice Research Institute (IRRI) such as IRBB5, IRBB7, IRBB21, IRBB4, IRBB57, IRBB60, IRBB61, IRBB62, IRBB63, IRBB64 - Brown planthopper-resistant materials have been identified: The brown planthopper resistance lines of the Agricultural Genetics Institute are: IS1-2, IS1-3, E2-3, E3… and imported materials internal from IRRI like: Mudgo; ASD7; RathuHeenati; ARC 10550; Swarnalata; Chinsaba; T12; Pokkali; Ptb33; TN1 (infected) 5.2 Location and time of study In Hanoi and the Northern Delta provinces; 4/ 2012 to 4/ 2018 5.3 Limited scope of the topic - Select to create parental lines carrying resistance gene for bacterial leaf blight and brown plant hopper through hybridization and selection from intermediate materials - Select and purify elite lines with many characteristics of a good parent line to serve breeding - Evaluate the ability to combine the traits of selected lines and parents to serve hybridization, select 2-line hybrid rice varieties to resist brown leaf and planthopper - Test crossing to find new hybrids Structure of the thesis The main thesis has 132 pages in A4 size with 44 tables of data and figures The thesis consists of parts; Opening (6 pages), Chapter 1: Overview of research materials (40 pages) Chapter II Materials, content and research methods (17 pages) Chapter 3: Research and discussion results (67 pages), Conclusions and recommendations (2 pages) CHAPTER I OVERVIEW OF RESEARCH MATERIALS The thesis has consulted and reviewed 29 Vietnamese documents and 81 English documents Related contents include: 1.1 Scientific basis of the topic; 1.1.1 Hybrid advantages, genetic bases and hybrid superiority in rice; 1.1.2 Male sterility system in selecting two-line hybrid rice varieties; 1.2 Practical basis of the topic: 1.2.1 The situation of R&D of hybrid rice in the world; 1.2.2 Research on combining ability; 1.2.3 Situation of research to create hybrid rice seeds in the country With the collected data shows: * Heterosis: A phenomenon in which F1 hybrids originate from different parents, demonstrating superiority over their parents in vitality, productivity, quantity such as panicle size, number of seeds per panicle, number of effective panicle Hybrid advantage is shown only in the first generation (F1 generation), (Nguyen Cong Tan et al, 2002) In rice,hybrids between two indica and japonica subspecies exhibit maximum heterosis and are evaluated in descending order: Indica x japonica>indica x javanica> japonica x javanica>indica x indica> japonica x japonica>javanica x javanica (Yuan LP, 2006) * Heterosis in rice: Rice yield in rice expressed on vegetative growth and reproductive growth as root system (Lin and Yuan, 1980); in traits like, the ability to branch Lin and Yuan, 1980; Chang et al., 1971, duration (Nguyen Thi Tram and et al., 1994, Xu and Wang (1980), plant height (Singh SP and Singh HG, 1978), yield and functional components (Virmani et al., 1982) In morphological and biochemical traits (Chao, 1972; Deng, 1988), physiological traits such as photosynthetic intensity, respiratory intensity, leaf area (Murayama et al., 1984, NguyễnThịTrâm (1994), TUTL can be expressed at all stages of plant growth and development (Akita et al., 1986), in deep resistance diseases (Lin and Yuan, 1980) and unfavorable conditions (Senadhira and Virmani, 1987) * The situation of hybrid rice development in China: China was the first country to succeed in hybrid rice research in 1973, the process of developing hybrid rice in China divided into main stages: stage (19641975) and the second phase (1976-1990) was the research period to choose to create sterile lines and complete the 3-line hybrid rice system Phase (19902000), is the period of strategic development, initiation of super hybrid rice; Phase (2013-2020), the new development period, the development period of super hybrid rice reaches 16-19 tons / According to Yuan L.P (2017), the average yield of super hybrid rice reached 13.9 - 15.4 tons / in Longhui district in Hunan province in 2013 and Xupu district in Hunan province in 2014 From 1975 to present, there are more than 5000 Hybrid rice varieties are recognized and expand the cultivated area of over 500 million hectares, which is an important factor to ensure food security (Zeng et al., 2018) * Research on creating hybrid rice varieties resistant to Bacterial bacterial leaf blight disease: In order to improve the bacterial leaf blight resistance of hybrid rice varieties, many resistance genes (dominant resistance genes) such as Xa25, Xa21, Xa7, Xa4, Xa23 have been used to select the lines of parents resistant to blight By the backcross and selective method with the help of molecular markers, Chinese scientists have chosen to create many lines of hybrid rice parents with high resistance to blight In 2000, Chen et al (2000) successfully transferred the Xa21 gene into the Minghui63 restorer line Deng et al (2006) successfully transferred two Xa21 and Xa4 genes into the Mianhui 725 restorer line He et al (2007) succeeded in bringing together the two resistance genes Xa21, Xa7 into the Minghui63 restorer line The Philippine Rice Research Institute has successfully switched to MAS genes Xa21, Xa4, and Xa7 into two-line hybrid rice TGMS1 (PhilRiceGenbank Acc No PRT-1), (Chen et al., 2009) Dindo and colleagues succeeded in transferring the genes Xa7 and Xa21 into the hybrid parents of Mestizo1 and Mestiza to create pyramided genes including Xa4 / Xa7; Xa4 / Xa21; Xa4 / Xa7 / Xa21 at maintenance lines and restorer lines of Mestizo1 and Mestiza3, Dindo et al (two thousand and thirteen) Ji and the team transferred the Xa23 resistance gene from the CBB23 strain into the restorer line HN189 with the rice blast Pi1 gene, then selected two new restorer lines, HBH145 and HBH146 with higher resistance to blight HN189, Jiet al (2014) Luo and his colleagues successfully transferred genes of Xa4 and Xa21 blight resistance to Mianhui 725 (MH725) line which obtained Wanhui 421 (WH421) line with 96.9% genome compared to MH725 but high leaf resistance than the original line, Luo et al (2016) Ramalingam et al (2017), successfully transferred three genes (xa5, xa13 and Xa21) into the maintenance stream (CO 2B, CO 23B, and CO 24 B) Kumar and colleagues improved the resistance of the RPHR-1005 strain when transferring blight resistance genes Xa21, Xa33 and Pi2 from the DRRH-3 strain to the RPHR-1005 line and selected 10 lines in the ICF4 genome with all The above gene has a good resilience, Kumar et al (2016) Ji et al (2016), has gathered three rice blast resistance, bacterial leaf blight and BPH resistance genes into the three-line hybrid rice restorer line, and the HR13 strain carried all three resistance genes and four lines HR39, HR41, HR42 and HR43 The gene is resistant to blast and blight but there is no gene for brown planthopper resistance The Zhongzu 14 series is the result of all three rice blast resistance genes, bacterial leaf blight and brown plant hopper Xiao et al (2016), transferred a Xa23 gene and two genes for BPH resistance (Bph14 and Bph15) into the Huazhan restorer line to improve the resistance to bacterial leaf blight and BPH of Tianyouhuazhan variety (Ni et al., 2015), successfully improved the resistance to blast and bacterial leaf blight of P / TGMS Guangzhan63S series (GZ63S) but still retained the characteristics of the original maternal lines and F1 hybrids when collected Blast resistance gene Pi9 and Xa23 blight resistance gene into GZ63S * Researching to create hybrid rice varieties resistant to brown planthopper: Liet al's report (2011), showed that the team succeeded in transferring two Bph14 and Bph15 resistance genes into the restorer line of Minghui 63 (MH 63) and obtained two lines MH63-15 and MH63-14 & 15 H Ji et al (2013) published the results of cloning of BPH resistance gene Bph18 Ling and Weilin (2016) suggest that the genes Bph14, Bph26, Bph3 and Bph29 were evaluated as the best pest resistance gene According to Xiao et al (2016), BPH is the heaviest rice pest in Asia The authors transferred 13 genes and QTLs (Bph14, QBph3, QBph4, Bph17, Bph15, Bph20, Bph24, Bph6, Bph3, Bph9, Bph10, Bph18 and Bph21) into rice lines 9311 by back crossing with support of the instructions molecule All lines containing resistance genes reduced the development of brown backed rice plant hoppers at the plating stage Wang et al (2016) collected two Bph14 and Bph15 resistance genes into one Huahui 938 restorer line to improve resistance to brown plant hoppers Wang et al (2017) gathered Bph6 and Bph9 genes into line 9311, resulting in the creation of hybrid rice LuoYang69 with the progeny of 9311 carrying two highly resistant Bph6 and Bph9 genes with brown backed rice plant hoppers and unchanged from the original According to Fan et al (2017), chose to successfully create a hybrid line for hybrid rice with the large panical geneGn8.1, Bph6 and Bph9 BPH resistance genes and Rf3, Rf4, Rf5 and Rf6 recovery genes thanks to molecular markers * Research results and production of hybrid rice in Vietnam: According to the Report of the Department of Crop Production, Hybrid rice has so far been cultivated in most rice growing areas of our country, in which the northern provinces occupy a large area and mostly However, in recent years, hybrid rice production in the Northern provinces has continuously decreased in both area and yield, reducing both Spring and Summer crops, but the main cause is determined by the supply of hybrid seeds processing, high price of quality hybrid rice seeds, hybrid seed yield produced in the summer season is low because of pest infestation At present, we have mastered the technical process of F1 seed production and multiplication of the female lines of most of lines hybrids based on BoA and II32A –CMS lines The two line hybrids developed in Vietnam has been recognized by MARD In the period of 2005 2009, the production of hybrid rice varieties tended to decrease However, from 2015 up to now, the F1 seed production area has increased again, each year area of F1 seed production have reached more than 2200 with hybrid seed productivity 2.1 - 2.7 tons / (Department of Crop Production, 2016) However, the production of hybrid rice seeds in the Northern provinces faces many difficulties due to negative impacts of climate change, epidemics (black stripe dwarf in 2017, many areas are lost in Nam Dinh and some Northern province) so productivity is not high Production in the South Central Coast and Central Highlands provinces is more productive but the cost of seed is high and transportation costs to the North are also high, Large enterprises are not keen to invest in domestic production, these are The main cause of limited development of F1 seed production in our country today * Research results on pest resistance: After testing the standard lines carrying the blight resistance gene of IRRI with 11 bacterial lines in the Mekong Delta Rice Institute, the Vietnam Institute of Agricultural Science and the Hanoi Agricultural University No showed the lines IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7) and IRBB21 (Xa21) are well resistant to bacterial blight pathogens (6-9 lines) Breeding program to select breeders using molecular Marker to transfer the Xa4, xa5, Xa7, Xa21 genes into the hybrid rice lines of the above research units Vu Thi Thu Hien et al (2007), transferred the Xa21 11 - Evaluation of agronomic characteristics, yield of F1 hybrids by IRRI method, 1997 - Yield comparison experiments are applied according to the National Technical Regulation on cultivation value testing and use of QCVN 01-55: 2011 / BNNPTNT of Ministry of Agriculture and Rural Development 2.3.6 Methods of data analysis Kempthorne model analysis capabilities (1957) and Program analysis of variance "Line * Tester" Ver.2.0 and Ver.30 software by Nguyen Dinh Hien (1995, 1996) 2.3.7 Methods and criteria for evaluating pests and diseases Evaluation of agronomic, pest and yield characteristics according to Standard Evaluation System for Rice of the International Rice Research Institute (IRRI, 1996) CHAPTER III RESEARCH RESULTS AND DISCUSSION 3.1 Results of breeding and selection of parental lines resistant to brown planthopper 3.1.1 Results of evaluating materials resistant to brown planthopper: After evaluating the resource of BLB resistant genes and review the published documents in Vietnam and IRRI we chose CR203, IS1.2, E2, E3, E6, E7, RathuHeenati, Swarnalata, Pokkali as materials for for breeding parental lines resistant to BPH 3.1.2 Breeding, selecting parental lines resistant to brown planthopper The selection process of BPH resistant male lines is carried out according to Figure 2.1 (page 53) Through evaluation, selection and purification until 2014, there are most promising lines viz RP088-51, RP088-148, RP088-48, R253-KR, R1028- KR, RTQ5-KR, R838-KR, RP3, RP8 having many good characteristics of male lines, were selected for further and more specific studies: The results of evaluation of resistance to brown planthoper throught artificial infection, the resistance grades of brown planthopper infestation in 2017 at the Plant Protection Institute showed that: RP8 line is resistant to 12 brown planthopper (score 3), other lines with resistance genes are: RP088-148, RP088-48, RP3, R1028-KR med- resistance (score 5) Table 3.7 Results of evaluation of resistance grates to BPH of some parent lines in 2017 No Name Cross source Score Resistant grade D116ST/E3-4-1 D52S/Pok6-427 D59S/E3 TL6/E6-148-1 D116Str/E3 AP9 P20-5 Minhhui 63/IS1.2-8 R9311/IS1.2-3 R1028/E3-60-2 TL6/E7-48-3 AMS30S/RP3 AMS30S/R116 - 5 5 7 MR MR MR MR MR S S R 10 11 12 13 14 KR95S KR142S D59S-KR RP088-148 D116tr-KR AP9 P20-5 RP8 (APIRX14) RP3 R1028-KR RP088-48 HYT285* HYT116** TN1 5 MR MR R MR R SS 15 Ptb33 - R Note Female Female Female Male Female Male Male Male Male Male Male Hybrid Hybrid Check (S) Check (R) Source: Institute of Plant Protection, 2017; Note: * AMS30S / RP3 combination has male line (RP3) with BPH resistance gene; ** HYT116 is two-lines hybrid rice has male line of resistance BPH, R116 * Assessment of general combining ability (GCA) of new TGMS Table 3.9 The general combine ability (GCA) Testers (TGMS) Lines (Male Of some male lines Testers/ lines CT1-AMS35S CT2- AMS30S LSD0,01 LSD0,05 TH29 TH12 GCA -0,547 0,547 8,462 6,234 0,642 -1,425 13 lines) M415 M359 TH20 TR1-565 M385 RP088-48(R) RP3(MR) R1028-KR(MR) RP8(MR) K10-5 (with risistant BB gene) LSD0,01 LSD0,05 -4,742 -3,492 -4,325 -10,108 -2,425 4,958 7,858 6,858 3,008 3,192 8,879 6,541 Remark: By backcrossing and selecting the individuals ,the promising lines have been selected, they have good characteristics of male line such as high yield potential, good phenotype , high quantity of pollen After artificial screening for BPH only lines have moderate resistance to BPH , That is the RP8 line is resistant to brown plant hopper (score 3), the remaining lines such as RP088-148, RP088-48, RP3, R1028-KR have medium resistance grade (score 5) The new male lines with BPH resistance have a high general combining ability value, respectively RP3 (7,858), R1028-KR (6,858), RPO8848 (4,958) and RP8 (3,008) these GCA are statistical significance at LSD0,05 level The male lines not carry the BPH resistance in general have low general combining ability of the actual yield (negative value on GCA), 3.1.3 Results of breeding and selection of TGMS lines resistant to brown planthopper After the re-hybridization process and selection of sterile plants, in 2013 we selected 30 promising TGMS lines from some of the above hybrid combinations The lines of TGMS are compact plants, leaf colors from green to dark green, long grains, only lines are KR71-1, KR71-2, KR75-1 and KR31 had average seed size, the new TGMS lines are completely sterile in the 2013 crop conditions Test results of TGMS lines selected from KimS / E3 (KR67S) and D116ST / E3 (KR95S) by RM6997 molecular markers have identified plant 14 TGMS with BphZ gene from K67S and plant TGMS carry BphZ gene from KR95S (table 3.13) The TGMS lines with BphZ will be selected on the genetic background of KimS (AMS37S) and the genetic background of D116ST will be a good material for selecting two-line hybrids with resistance to brown planthopper Biotype and Biotype3 in Vietnam Table 3.14 Results of identification of resistance genes for BPH Bph3, TT 10 Bph6 and Bph9 in some new maternal lines were selected Materials Bph3 Bph6 Bph9 TT Materials Bph3 Bph6 Bph9 Rathuheenati + Rathuheenati + Swanalata + Swanalata + Pokali + Pokali + TN1 TN1 KR82-1 + 11 KR142-1 + KR82-2 + 12 KR142-2 + KR82-3 + 13 KR142-3 + D59S-1 + 14 KR95-1 + D59S-1-1 + 15 KR95-2 + D59S -3 + 16 KR95-3 + Source: Department of Biotechnology, FCRI; Note: +: Resistant homozygote +/-: Heterozygous resistance; -: infected; 0: not on the band From the results summarized in Table 3.13 and 3.14, lines of TGMS containing BPH resistance genes and the following symbols are selected: KR67S (KimS / E3), KR95S (D116ST / E3), KR142S (D52S / Pokkali), KR82S (D59S / E2 // D59S) and D59S-1-1 Among them, two lines of TGMS are KR95S (Bph3, BphZ), KR142S (Bph9) have many agronomic characteristics of good maternal line such as: higher panicle exertion rate, higher stigma exertion rate and longer panicle to use for further research Research results of some agro-biological characteristics of the TGMS line KR95S and KR142S in the spring and summer crop seasion of 2017 in Thanh Tri, Hanoi are presented in Table 3.17 (page 88) It show that: The two TGMS lines KR95S and KR142S have growth duration in the short-day group, the height of semi-dwarf plants These characteristics are recorded in both of the TGMS muntilication and F1 seed production Crops The rate of stigma 15 exersion of the two lines is quite high at 75.5% (KR95S) and 73.6% (KR142S), the KR95S line has a purple stigma and KR142S is white calour Both of them are typically sterile (WA), completely sterile temperatures are > 24.oC; The ability of branching is quite good, the number of effective panicle reaches 7.9 panicle / hill, the number of flowers / panicle reaches 159 - 142 flowers / panicle, grain shape of the two lines is average - long, the number of leaves / main stem of KR95S is 13.5 and KR142S is 15, these two TGMS lines will be good materials for breeding hybrid rice varieties with BPH resistance * Assessment of general combining ability (GCA) of new TGMS Assessing the GCA of the TGMS lines to select the female lines with high combining ability value, the selected female lines will have a higher probability for the hybrid with superiority Results of analysis for the GCA for yield and yield components of TGMS lines are presented in table 3.18, Table 3.18 The general combining ability (GCA) of parents lines For the yield components and productivity Rate of 1000 Panicles/ Grains/ empty grain hill panicle Par.lines grain weight Female (TGMS) lines KR142S 0.29 -10.25 2.29 -0.06 D60S -0.31 2.95 0.01 0.12 D67S 0.05 -3.05 -0.47 0.50 KR95S -0.03 10.35 -1.83 -0.56 LSD0,05 0.05 2.50 0.10 0.15 LSD0,01 0.06 3.34 0.13 0.20 Male lines SR3 -0.08 8.65 -0.74 0.06 SR14 0.12 7.4 -1.24 0.73 SR18 -0.18 6.65 -0.54 -0.12 AIQ6 -0.03 -3.35 1.17 -0.85 SR5 0.17 -19.35 1.34 0.18 LSD0,05 0.05 2.79 0.11 0.17 LSD0,01 0.07 3.74 0.14 0.22 Feature Theoryield Grain yield -1.56 -4.16 2.00 3.72 2.08 2.79 -0.625 -2.465 0.495 2.595 1.46 1.96 3.42 10.51 0.43 -5.51 -8.84 2.33 3.12 1.84 5.59 0.07 -2.79 -4.71 1.63 2.19 16 - Regarding the number of panicle/hill: The TGMS line -KR142S has the highest general combining ability (0.29) and is significant at the level of LSD0.01 (0.06), - The number of grains / panicle: The KR95S having BPH resistance gene has a very high GCA (10.35) and is significant at LSD0.01 (3.34), the D67S line has a value of 2.9, the other two lines have negative values - The 1000 grain weight: KR59S and KR142S carrying BPH resistance gene have negative value, while lines D60S and D67S have values of 0.12 and 0.5 are higher than expected meaning at the level of LSD0.01 (0.2) - In terms of actual yield: There are lines KR95S and D67S having positive values for GCA However, only KR95S have the highest positive value of 2.595 being significant at the level of LSD0.01 (1,96) 3.2 The result of breeding and selection of TGMS lines resistant to bacterial blight disease 3.2.1 Results of evaluation of materials for selection of parent lines resistant to BLB Evaluating 32 lines of genomic materials (NILs) carrying the blight resistance gene (on the genetic background of IR24 rice variety) at the Field Crops Research Institute showed that: The morphological characteristics of the lines not have much difference the lines, all have dark green leaves, Vcompact shape Of the 32 lines with bacterial blight resistance genes, most of them had head and beard in which IRBB13 line had the longest bearded head (2.5cm) The grain color and granularity of the line samples containing bacterial blight resistance genes are not significantly different, almost all lines of grain color from light yellow to yellow and elongated granular form 3.2.2 Results of transferring resistance to bacterial leaf blight into the mother line TGMS Electrophoresis the combinations with the M3 directive associated with the Xa7 gene Results of the V54 (AMS30S / IRBB7) electrophoresis using the M3 indicator showed that V54-1, V54-2, V54-3, V54-4, V54-5 all carried the Xa7 gene similar to the BB7 strain (Figure 3.7, page 100) 17 The study results showed that two Npb181 markers associated with Xa4 and M3 genes linked to Xa7 gene for a clear polymorphic range were used to identify the sterile line carrying the Bacterial leaf blight resistance genes In particular, using the Nbp181 directive identified 11 individuals of the V50 (AMS34S / BB60), V31 (AMS35S / BB60) and BL59 (AMS34S / BB60) lines carrying the BLB resistance gene Xa4 Using the M3 directive, the V54 line (AMS30S / IRBB7) was identified with individuals carrying the Xa7 gene Comment: Throught screening uniformed TGMS lines, we selected AMS34S similar TGMS lines containing Xa4 resistance gene, AMS35S similar TGMS containing Xa4 gene and the TGMS line similar to AMS30S contains the Xa7 gene All these identified TGMS lines having bacterial leaf blight resistance genes were improved from the original TGMS: AMS34S, AMS35S and AMS30S, these original TGMS are being exploited as the mother line of many hybrids in Vietnam Therefore, new improved BLB resistant TGTMS lines will be required firstly for crossing and selecting two line hybrid rice varieties resistant to BLB in our country The lines of TGMS AMS35S-KBL and AMS30S-KBL have a completely sterile temperature of ≥ 23.5 0C, stable sterility, high percentage of stigmata exertion (70-75.8%), rice husk opening angle wide, the duration from sowing to heading 10% in the summer season is 72 - 80 days, the degree of BLB infection is 3-5 score in artificial conditions The lines of TGMS AMS35S-KBL and AMS30S-KBL have many characteristics of good maternal flow (Table 3.22, page 103) selected by the method of individual selection of sterile lines from BC4F2 generation * GCA assessment of yield of new TGMS lines with BLB resistance genes Table 3.23 The combined ability value of some TGMS lines Tester and line GCA value M415 -1,679 Tester (male) TH29 1,679 LSD0,01 3,237 LSD0,05 2,337 Line (TGMS-female) 35S 0,474 18 36S 37S D64S D116ST 827S 35S-KBL 827S-KBL LSD0,01 LSD0,05 -0,459 -0,681 -1,113 -2,979 1,967 0,521 2,271 5,027 3,629 The TGMS lines were assessed for their GCA and their specific combining ability (SCA) for productivity by Line x Tester method and using LINE * TESTER Ver 3.0 variance analysis software program by Nguyen Dinh Hien (1996) Evaluation results of lines TGMS are: 35S, 36S, 37S, D64S, 116 T, 827S, 35S-KBL, 827S-KBL with two test lines: M415 and TH29 (two lines have been evaluated as two lines of females having a high degree of GCA and a good line for hybrid rice lines) showed that: The GCA of 35S-KBL and 827S-KBL has a higher positive value than other lines does not make sense at the level of LSD0,05 but higher than the original TGMS lines 3.3 Hybrid rice breeding towards resistance to bacterial blight, brown planthopper 3.3.1 Hybrid rice breeding towards resistance to brown planthopper The results of evaluating the components of productivity and productivity (Table 3.26, page 108) to estimate the potential of the variety, on that basis,the growers can apply appropriate technical measures to maximizing the advantages of each factor to gain better productivity for the crop Evaluating the number of effective panicle/hill of rice hybrids in the Spring season 2016 showed that most of the combinations have the average number of panicle / hill from 5.0 - 7.0 panicle / hill, only the number combination averaged 4.8 panicle /hill; The number of seed / panicle is quite high and varies from 139 to 209 seeds / panicle, of which the highest number of seeds / panicle is D60S / SR18 (209 seeds), 19 The combinations with female lines have the BPH resistant genes have seeds / panicle from 159 - 204 seeds / panicle, control TH3-3 has 161 seeds / panicle.; The weight of 1000 grains of the combinations ranged from 23.4 to 28.9 grams, weight of 1000 grains of hybrids using female having BPH resistance gene ranged from 24.8 to 28.7 grams, the control achieved 23.4 grams The theoretical yield of the combinations ranges from 9.52 to 13.6 tons / ha, the combinations with females carrying the BPH resistance genes range from 9.52 to 12 , 14 tons / ha, control reached 9.04 tons / ha; The actual yield of the combinations reached 7.67 - 9.76 tons / ha, the combinations with females having BPH resistance gene had the actual yield of 7.73 - 9.32 tons / ha, much higher than the TH3-3 (7.06 tons / ha) Results of pest monitoring (Table 3.27, page 109) show that: In the Spring 2016 season, most of the combinations have the level of bacterial and blight stripe infection from points 3-5 Among 10 hybrids of female with the gene that was resistant to BPH there were hybrids (number 8, 9) infected with score 3, hybrids (numbers 1, 12, 16, 17, 20) infected with score3-5 and hybrids (numbers 4, 5, 13) infected with score 5; About BLB disease, there are hybrids (numbers 1, 5, 8, 9) infected with score 3, hybrids (numbers 12, 16, 17, 20) infected with score 3-5 and (number 4, 13) infections with score 5; Other pests such as blast disease not appear * Assessment of specific combining ability (SCA) of new TGMS lines with BPH resistant gene: Table 3.33 The specific combining ability of new TGMS lines on actual yield Females KR142S D60S D67S KR95S Males SR3 3.70 -2.16 1.08 -2.62 SR14 -2.85 -4.71 7.03 0.53 20 SR18 2.27 3.01 -1.55 -3.75 AIQ6 -3.78 2.26 -2.99 4.51 SR5 0.65 1.59 -3.57 1.33 LSD0,05 3.27 LSD0,01 4.38 The analytical results in the table 3.33, show that SCA of actual yield of D67S / SR14 (7.03), followed by KR95S / AIQ6 (4,51) they are have statistical significance at the level of LSD0.01 (4,38) and KR142S / SR3 (3.7) has statistical significance at the level of LSD0.05 (3,27) combinations have positive values but no significant differences and 10 other combinations with negative values Hybrid with TGMS lines carrying BPH resistant genes and having the highest SCA value in terms of the actual yield were hybrid KR142S / SR3 (3.7) gained (89.4 quintals / ha), anf the hybrid KR95S / AIQ6 (4,51) obtained actual yield of 88.8 quintals /ha campared to 70.6 quintals / ha) of chech-TH33 It is expected that hybrds KR95S / AIQ6 and KR142S / SR3 will be most promising hybrids Conclusion : Through evaluation of hybrid combinations with the participation of female lines carrying BPH resistance genes in the Spring 2016, There are hybrids having actual yields from 85.1 - 93.2 quintals / being higher than control TH3-3 (70.6 quintals / ha) 20% or more However, only hybrids have TGMS lines carrying BPH resistant genes combining with high yields were selected for further yield trials in next season The selected hybrids are KR142S / SR3 (89.4 quintals / ha) , KR142S / SR14 (86.6 quintals / ha), KR142S / SR18 (86.2 quintals / ha), KR95S / SR14 (93.2 quintals / ha), KR95S / AIQ6 (88.8 quintals / ha) and KR95S / SR3 (86.3 quintals / ha) 3.3.2 Hybrid rice breeding towards resistance to bacterial blight Assessing the degree of BLB damage of hybrid combinations in the crop condition by artificial infection in the field (Table 3.35, page 119) shows that: Most of the hybrids usingTGMS lines are resistant to bacterial leaf blight 21 at score 5-7, while hybrids having female with BLB resistance genes have a lower degree of infection TGMS line AMS34S-KBLis used as female of hybrids (numbers 7, 9) have high resistance score 1-3,one hybrids(number 6, 8) have resistance score and one hybrid (numbers 10) have medium resistance -score 3-5 The female line AMS35S-KBL has hybrid with BLB high resistance- score 1-3 (number 29), hybrid having score (number 27) and hybrids having medium resistance- score 3-5 (numbers 26, 28, 30); The mother line AMS30S-KBL has hybrids resistance - score (number 16, 19, 20) and hybrids have medium resistance- score 3-5 (number 17, 18); the check have moderate resistance -score Results of evaluation of yield components and yield of hybrids in (Table 3.36) show that: number of panicle / m2 of combinations ranges from 224 - 315 panicle / m2 (Number 24), in which there are combinations (numbers 10, 12, 24) with the number of panicle / m2 being higher than 270 panicle/ m2, combinations (numbers 8, 14, 17, 18, 22, 28) reaching 250 - 266 panicle / m2 The number of grains / panicle of hybrids using AMS34S-KBL as female ranged from 121 to 165 seeds / panicle being higher than those of mother AMS34S (114-125 seeds / panicle); The hybrids using the AMS30SKBL have the number of filled grains / panicle varying from 114 to 130 grains lower than the hybrids of AMS30S (ranging from 119 to 161 seeds); The hybrid of the AMS35S-KBL have a number of filled grain / panicle ranging from 118 to 150 seeds, higher than the hybrid combinations of AMS35S (98 136 seeds) In the 2016 crop, the sterile seed rate of the hybrid are quite high, ranging from 10.2 to 23.8% The weight of 1000 grains of the hybrids fluctuates from 20.8 - 28.1 grams (number 30), the control TH3-3 is 23,7g - The theoritical productivity of the combinations ranges from 64.6 (number 17) - 10,08 tons / (No 28 - AMS35S-KBL / R100) - The highest actual yield is 72.1 quintals / of AMS35S-KBL / R100 (HYT124-KBL) compared the yield of 67.8 quintals /ha of AMS35S / R100 named HYT 124; The combination AMS30S / R116 is actually named 22 HYT 116 gained actual yield of 67.3 quintals / compared to combination AMS30S-KBL / R116 (HYT116-KBL) reaching 69.4 quintals / ha, Table 3.42 Specific combining ability (SCA) of new TGMS lines on Females Males AMS34S productivity (actuall Yield) AMS34SAMS30SAMS30S KBL KBL AMS35S AMS35SKBL SR14 2.43 1.37 -4.83 3.97 -0.81 -2.11 RP3 2.48 -0.78 1.12 -4.98 5.74 -3.56 R100 -3.36 -5.42 -5.62 0.48 4.50 9.40 SR18 0.93 5.97 4.37 -6.53 -2.51 -2.21 R116 -2.47 -1.13 4.97 7.07 -6.91 -1.51 LSD0,05 2.37 LSD0,01 3.16 Selecting combinations with high SCA value of high actuall yield are: AMS35S-KBL / R100 (HYT124-KBL) giving the highest SCA value of 9.4, combination of AMS30S-KBL / R116 (HYT116-KBL) is 7.07 and AMS34S-KBL / SR18 achieved SCA value of 5.97 Final results of our BLB breeding programme: Through breeding and evaluation of hybrid combinations in the direction of resistance to BLB in the season of 2016, we selected combinations with higher yield than check variety > 10% (yield> 6.5 ton / ha) are AMS35SKBL / R100, AMS30S-KBL / R116 and AMS34S-KBL / SR18 in which the most productive combination is AMS35S-KBL / R100 (called HYT 124-KBL) reaching 7.21 tons / ha, bacterial leaf blight resistance score 3-5; The second combination is AMS30S-KBL / R116 (called HYT 116 - KBL) with actual yield of 6.94 tons / ha, BLBresistance score compared to 5.95 tons / and resistance to BLB resistant score of check TH3-3 The evaluation also showed that the two improved hybrids HYT124-KBL and HYT116 - KBL have similar viz, characteristics of the original hybrids, but the ability to resist bacterial leaf blight are significantly improved (table 3.43, 3.44- page 128, 130) 23 CONCLUSIONS AND RECOMMENDATIONS Conclusion: 1- Created new TGMS lines having BPH resistant gene: KR95S have BPH resistant genes Bph3, BphZ and KR142S having gene Bph9 These TGMS have short growth duration in the summer season, pollen sterile stability, critical temperature to induce complete pollen sterility at >24 oC, ratio of stigma exertion >70 % KR95S have GCA for traits of grain number/panicle, theory yield and actual yield These TGMS lines are good female parents for breeding line hybrid rice to tolerate to BLB and BPH in Vietnam 2- Created new male parental lines tolerant to BPH (from medium resistance to high resistance), having good characteristics such as high yielding capacity, good phenotype, high pollen production These lines are RP8, RP08848 resistant to BPH (score 3); RP3 having gene Bph3 resistant to BPH (score5); R1028KR tolerant to BPH (score5) Through screening male papulation by the molecular markers, the individual plants of R116 having gene bph6 was sellected for use Four among five of the new BPH resistant lines have high general combining ability (GCA) for grain yield following in this order: RP3, R1028-KR, RPO88-48 and RP8 3- Created new TGMS lines having BLB resistant genes: AMS35SKBL (having Xa4) and AMS30S-KBL (having Xa7), these two lines have critical temperature to induce complete pollen sterility at >23.5oC, pollen sterile stability, ratio of stigma exertion (70 %-75.8%), level of BLB tolerance at artificial conditions (score3-5) AMS35S-KBL have positive value of GCA fot actual yield 4- Breeding hybrids used female-TGMS having BLB resistant genes and resistant to BLB (score 3-5), having actuall yield over check to be more than 10 percent They are AMS35S-KBL/R100, AMS30S-KBL/R116 and AMS34S-KBL/SR18, among them AMS35S-KBL/R100 (named HYT124KBL) gained highest yield (7.1 tons/ha) Second hybrid is AMS30SKBL/R116 (named HYT116 – KBL) have actuall yield of 6.94 tons/ha, resistant to BLB (score3) compared to yield of check (5.95 ton/ha) having resistant to BLB (score5) 24 5- Breeding hybrids involved female-TGMS having BPH resistant genes, having actuall yield ranged 8.62 – 9.32 tons/ha These yields are higher 20 % than check TH3-3 (7.06 ton/ha) They are KR142S/SR3 (8.94 tons/ha), KR142S/SR14 (8.66 tons/ha), KR142S/SR18 (8.62 tons/ha), KR95S/SR14 (9.32 tons/ha), KR95S/AIQ6 (8.88 tons/ha) KR95S/SR3 (8.63 tons/ha) 6- Though back crossing method combined with conventional method and supported by Molecular approach, the BLB and BPH resistant genes were transfered from the donors into the exsiting parental lines The hybrids based on these new improved parents showed similar phenotypic characteristics and yield compornents of the original hybrids However, they showed good resistant to BLB and /or BPH instead of susceptable to the BLB and/or BPH of the original hybrids Recommendation: - Continue to improve and apply the process of selecting parent lines to resist brown plant hopper, bacterial leaf blight by backross, combining traditional selection and selection through molecular markers to transfer resistance genes Brown plant hopper, bacterial leaf blight into the line of hybrid rice parents in Vietnam - Continue to purify and use parental lines selected to create hybrid rice varieties resistant to bacterial leaf blight, brown planthopper, - Continue to test and research on hybrid seed production techniques, multiply the mother lines of selected combinations for production LIST OF PUBLISHED REPORTS RELATED TO THE THESIS / Le Hung Phong, Nguyen Tri Hoan, Le Dieu My, Nguyen Thi Hoang Oanh Results of Breeding and creating two-line hybrid rice HYT 116, Journal of Vietnam Agricultural Science and Technology, No 2, 2017, pp 3-10 / Le Hung Phong, Nguyen Tri Hoan, Le Dieu My, Trinh Thi Lien Results of Breeding and creating two –line hybrid rice varieties HYT 124 with hight quality, Journal of Vietnam Agricultural Science and Technology, No 2, 2017, pp 10-18 / Le Hung Phong, Trinh Thi Lien, Nguyen Thi Hang, Nguyen Thu Trang, Le Dieu My, Nguyen Tri Hoan, Nguyen Nhu Hai Breeding of thermo-sensitive genic male sterility (TGMS) lines with bacterial blight resistant, Journal of Vietnam Agricultural Science and Technology, No 3, 2018, pp 3-7 / Le Hung Phong, Nguyen Thi Hoang Oanh, Nguyen Thi Hang, Nguyen Thu Trang, Le Dieu My, Nguyen Tri Hoan, Nguyen Nhu Hai Breeding of Brown planthopper resistant males for development of two-line hybrid rice in Vietnam, Journal of Vietnam Agricultural Science and Technology, No 4, 2018, page - ... dissertation council meeting at: Vietnam Academy of Agricultural Sciences (VAAS) The Thesis can be found at the library: Vietnam National Library; Library of Vietnam Academy of Agricultural Sciences;... Biotype3 in Vietnam Table 3.14 Results of identification of resistance genes for BPH Bph3, TT 10 Bph6 and Bph9 in some new maternal lines were selected Materials Bph3 Bph6 Bph9 TT Materials Bph3... Journal of Vietnam Agricultural Science and Technology, No 3, 2018, pp 3-7 / Le Hung Phong, Nguyen Thi Hoang Oanh, Nguyen Thi Hang, Nguyen Thu Trang, Le Dieu My, Nguyen Tri Hoan, Nguyen Nhu Hai Breeding