Summary of doctoral thesis: Research on selection and breeding of maize hybrid varieties with high yield, cold-endurability suitably to production conditions in Red River Delta

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Evaluating the growing, developing and agronomic characteristics of several inbred maize lines in order to select successfully the researched maize family which is early mature and cold endurable, high yield and high combination possibility for maize variety breeding and selecting affairs with cold-endurability and high yield; evaluating the agro-biological characteristics, genetic diversity between lines, creating a hybrid combination with cold-endurability and high yield.

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Nội dung Text: Summary of doctoral thesis: Research on selection and breeding of maize hybrid varieties with high yield, cold-endurability suitably to production conditions in Red River Delta

MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES ***************** PHAM THANH THUY RESEARCH ON SELECTION OF HIGH-YIELD, COLD-TOLERANT HYBRID CORN BREEDING SUITABLE FOR PRODUCTION CONDITIONS IN THE RED RIVER DELTA Ranch of Study: Genetic - Plant Breeding Code: 96 20 111 SUMMARY OF DOCTORAL THESIS Hanoi - 2021
This theisis is completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Science Supervisor: Prof. Dr. Sc. Tran Dinh Long Critic 1: .................................................................................................................... Critic 2: .................................................................................................................... The thesis will be defended before the Council for thesis assessment at Institutional-level held at Vietnam Academy of Agricultural Sciences. At……..o’clock……….day ……… month …….. year 2021 This Thesis can be referred to at: 1. National Library of Vietnam 2. Library of Vietnam Academy of Agricultural Sciences 3. Library of Maize Research Institute of Vietnam
1 INTRODUCTION 1. The urgency of thesis Maize (Zea mays L.) is one of three most important cereal grains which provide food for human and livestock. In addition, maize is also a foodstuff crop providing baby maize ear as high-ranking vegetable, white maize, sweet maize for eating freshly, making maize milk and drinks and canned foods. In addition, maize also provides material for factories producing food, foodstuff, pharmaceutical products, and light industry, especially ideal materials for biological energy. Maize is also the valuable agricultural commodity for export, and brings foreign currencies for many countries and territories. Maize was brought into our country about 300 years ago (Ngo Huu Tinh, 2009) and among food plants, maize is ranked in the second position after rice. However, the maize yield of our country is still lower than average yield of the world, for example: In 2013, its average yield was 4.43 tons/ha in comparison with 5.52 tons/ha average yield of world, equally to 80.25% (General Statistics Office of Vietnam, 2014); In 2018, it reached 4.72 tons/ha in comparison with 5.92 tons/ha, equally to 79.7% (General Statistics Office of Vietnam, 2019; FAO, 2018). About domestic maize yield, although the speed increases rather fast, it still does not satisfy because the consumption demand increases highly, for example, in the crop year 2016/ 2017, the maize demand of our country was 12.9 million tons, we imported 8.5 million tons of maize; in the crop year 2019/ 2020, the demand was 15.4 million tons, we imported 11.5 million tons (USDA, 2020). Therefore, we can see that the increase of domestic maize yield for satisfying domestic demand and limiting import is a challenge of agriculture. However, about area, the maize planting regions with big area and maize yield are currently narrowed, because several other plants with higher economic value replace a part of maize area (typically as Son La). Consequently, in addition to utilizing absolutely regions where maize can be planted, it should apply the modern, synchronous mechanizing; crop re-structuring, technological progress applying, variety breeding and selecting methods should be applied to have new seeds suitable to each crop and ecological region to have high yield and big maize output. The winter crop after two rice crops in Red River Delta is an especially specific crop. After ending the winter rice crop, a part of this rice area will be used to plant vegetable plants, the most of left area is usually not used. This is an opportunity to increase the maize planting area, to increase the maize yield in area with two rice crops in the Northern provinces; especially, now the Ministry of Agriculture and Rural Development has a guideline of developing biomass maize as green food for breeding animal in the winter crop in the Northern provinces. However, during the production process of Winter Crop, the temperature increases gradually in the final months of year, in some years, the temperature decreased to lower than 15oC and the cold spell could lengthen for several weeks, which has significant impacts on plant production in general and maize production in particular. Therefore, to develop strongly
2 the winter maize crop, it should mechanize from planting, caring, harvesting steps to drying, maintaining steps and the most important factor is new maize varieties with good cold- endurability and high yield. To exploiting totally the land potential of Winter Crop and improving the yield and to increase the maize output of Red River Delta, to satisfy the maize demand for animal food, especially biomass maize as green food for cattle in the cold winter of Northern Vietnam, to solve the matters on arranging plants, it is very important to strengthen the researching affairs to select and breed new early maturing maize varieties with good cold-endurability and high yield. Therefore, the scientific thesis “Research on selection and breeding of maize hybrid varieties with high yield, cold-endurability suitably to production conditions in Red River Delta“ is necessary. 2. Targets and requirements of thesis 2.1. Targets Selecting 1 - 2 potential maize hybrid combinations to develop the hybrid variety with high yield, cold-endurability, adding into variety for maize production in Red River Delta. 2.2. Requirements - Evaluating the growing, developing and agronomic characteristics of several inbred maize lines in order to select successfully the researched maize family which is early mature and cold endurable, high yield and high combination possibility for maize variety breeding and selecting affairs with cold-endurability and high yield; - Evaluating the agro-biological characteristics, genetic diversity between lines, creating a hybrid combination with cold-endurability and high yield; - Selecting 1 - 2 potential hybrid maize combinations to develop the hybrid variety with cold-endurability with high yield for producing maize in the winter crop in provinces of Red River Delta. 3. Scientific and actual meanings of thesis 3.1. Scientific meaning Providing the scientific information on researched maize lines which are early mature and cold endurable for selecting maize varieties suitably to winter crop maize production in Red River Delta. 3.2. Actual meanings - Selecting the potential researched maize lines for early maturing, cold- endurable maize variety selecting and breeding affairs. - Identifying VN158 hybrid combination which is early mature, cold- endurable, high yield, suitable to production conditions of winter crop in Red River Delta. 4. Research object and scope 4.1. Research object - The researched inbred maize lines are bred from different material sources. - Top cross combinations, diallel cross combinations, potential hybrid combinations.
3 4.2. Research scope - Experiments for evaluating and selecting lines. - Experiments for analyzing the genetic diversity of lines by SSR instruction. - Experiments for surveying hybrid combinations by top cross method and diallel cross method. - Experimental tests of author (comparing potential hybrid combinations), experimenting the cultivation values and maize variety using values. 5. New contributions of thesis - Thanks to results of growth and development properties, endurability, combination possibility and yield evaluating, we select 11 lines, including: C352, C16, C431, C838, C769, C608, C801, C855, C628, C783, C252, and then introduce to early maturing, cold-endurable maize variety breeding and selecting program. - Selecting and breeding VN158 hybrid combination (C431 x B67CT) with early average growing time, cold-endurability and high yield suitable to maize production in maize planting regions in the Northern Vietnam in general and in Red River Delta in particular. 6. Structure of thesis The thesis covers 127 pages, including: Introduction (4 pages); 3 content chapters; Chapter 1: Overview of document and scientific foundations (41 pages), Chapter: Researching materials, contents and methods (14 pages), Chapter 3: Findings and discussion (67 pages); Conclusion and petition (1 page); References 13 pages, with 41 tables (33 tables of figures), 10 illustrative figures and images. Referring 119 documents, in which 31 Vietnamese documents, 88 foreign language documents and website. CHAPTER 1 OVERVIEW OF DOCUMENT AND SCIENTIFIC FOUNDATIONS 1.1. Role and position of maize Maize is spread and planted in most of continents over the world and it plays a role as important cereal grain of human. We can say that maize is a cereal grain which feeds nearly 1/3 global population. This role is shown through main aspects: As food for human, as food for animal; As foodstuff; Providing materials for industry; As the export commodity source; In addition, in the condition of our country, maize also helps to use land effectively, destroying the rice monoculture. Beside material values, the maize is meaningful in the spiritual life, folk legends, decorative art, cuisine culture of Vietnam and the World. The data stated above show that maize plays an important role and position for economy, society and culture of the world and our country. It gives the life for human in both material and spiritual aspects. 1.2. Maize consumption and production situations in the world and our country 1.2.1. Maize consumption and production situation in the world The population of world is estimated is 9.73 billion people in 2050, and the population in urban area will occupy more than 75% (FAO, 2017), thus
4 the demands on maize, wheat and wt rice of the world in each year will be 3.3 million tons, increasing 800 million tons in comparison with current demand (about 2.5 million tons), the demands of maize will increase 50 - 60% (FAO, 2016). The maize yield of the world in the crop year 2017/2018 reaches 1,375.50 million tons, so much higher than wheat yield (763.18 million tons) and wet rice yield (495.07 million tons) (USDA, 2019). Until now, the maize production over the world is developing strongly and comprehensively. Maize really becomes one of important branch of world’s economy. The results stated above are originated initially from applying hybrid advantage broadly into selecting breeding varieties and simultaneously improving the cultivation technical methods. 1.2.2. Maize consumption and production situation inside our country About consumption, the demand of maize in our country increases dramatically. In each year, our country still must import a big number of maize, the number of imported maize in this year is always higher than previous year, for example: in the crop year 2016/2017, the maize demand inside our country was 12.9 million tons and we imported 8.5 million tons; In the crop year 2019/2020, the maize demand inside our country was 15.4 million tons and we imported 11.5 million tons (USDA, 2020). 80% imported maize are used as food and a few of maize are used in industry as producing beer, additives in pharmaceutics. About production, for recent years, the area of maize planting land has the tendency of decreasing, but the achievements on variety, technological progresses on cultivation and application of modern biological technologies make the maize yield of our country to increase and to catch gradually up with average yield of the world [31]. 1.3. Combination possibility, genetic foundations of characteristics selection and maize variety breeding and selection program 1.3.1. Genetic diversity and gene source of maize Maize is the cross pollinating plant, through impacts of natural and artificial selection processes which help maize to have so broad genetic diversity and to become one of most popular plant over the world (Zhang et al., 2015). Maize is currently planted in all continents and it adapts to most kinds of ecological climate, from temperate climate, subtropical climate to highly tropical climate and lowly tropical climate. 1.3.2. Selection of material for breeding line The efficiency of selection and hybridization process depends on the initial material source (Vasal and Srinivasan, 1999). According to Ngo Huu Tinh and Phan Xuan Hao (2005), one of progresses of hybrid maize development program in Vietnam for recent years is the selection of suitable initial material source, and then we can create the commercial hybrid variety for production activities. In the research on selection and breeding of maize variety which adapts to climate change, especially can be planted in conditions of winter crop in Red River Delta, the evaluation and screening of initial materials is necessary and
5 it is the premise and foundation of hybridizing successfully the hybrid maize varieties which can adapt to climate conditions of winter crop of this region. 1.3.3. Several inbred variety breeding methods From initial material source, there are many different methods to breed and develop the inbred line, such as: Self pollination; Full sib/Half sib; Back cross; Doubled haploid… 1.3.4. Combination possibility and combination possibility evaluation 1.3.4.1. Combination possibility Retaining or removing the inbred line based on combination possibility valuation results. The combination possibility evaluation can be conducted trially for initial material source of self-pollination and characteristics of line on combination possibility are formed early during the process of line breeding, and they are descended to next generations rather stably (generations S2 and S3, S4 are similar) (Ngo Huu Tinh, 2009). Therefore, the combination possibility is frequently tested in generation S2 or before S4. The combination possibility is identified through evaluation of general combination possibility and specific combination possibility. In selecting and breeding the hybrid maize variety, the inbred line breeding and line evaluation are conducted early and frequently to prevent from removing any potential lines. 1.3.4.2. Evaluation of combination possibility The inbred line is only effective when it has high combination possibility and several good agro-biological characteristics and it can satisfy the requirements of breeder. To evaluate the combination possibility of inbred lines, the most popular method is hybridizing trially by top cross and diallel cross in combination with hybrid plant (Zaidi et al., 2005; Ngo Huu Tinh et al., 1996): (a). Trial hybridizing period and trial plant: According to Bauman, the breeders evaluate the lines by trial hybridizing method in generations S3 and S4 about 60%; in the generation S5 or later about 22% (Bauman, 1981). The breeders usually select to select the trial plants which have no relationship with materials tested and preferably it belongs to the corresponding hybrid vigor group. To increase the reliability, we frequently use two or more trial plants with different genetic bases; (b). Top cross method, includes: (1) Total top cross; (2) Partial top cross; (c). Diallel cross method: to indentify the general combination possibility and specific combination possibility of parental line, simultaneously to collect the information on nature and to estimate the genetic index, we frequently use the diallel analyzing method of Griffing (1956). This method helps to identify the changed components caused by additive effects, dominance and overdominance of genes (Alvarado et al., 2014). 1.3.4.3. Genotype and environment interaction evaluation and combination possibility by GGE-Biplot GGE-Biplot is used to select the parental line with the highest general combination possibility and specific combination possibility or the best variety suitable to specific environment.
6 1.3.5. Selecting and breeding maize variety by traditional selection method in combination with biological technology application Application of molecular marker into selecting and breeding maize varieties suitable to disadvantageous conditions; evaluating the genetic diversity and estimating the hybrid vigor group of maize. In which, the morphological marker is used firstly, and it is the initial foundation in vegetation evaluation and classification, and ADN marker is currently used broadly in evaluating the genetic diversity and it is an effective tool in selecting and breeding plant varieties. The molecular markers as Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphism DNA (RAPD), Amplified Fragment Length polymorphism (AFLP), Simple Sequence Repeat (SSR)... have been applied much in selecting the molecular varieties. In which, SSR marker is effective and it has been used in many scientific studies in identifying varieties, genetic types, species generation, genetic diversity, genetic distance calculation to predict the hybrid vigor, identifying the genetic purification of lines and etc. 1.3.6. Selection index and application on maize variety breeding and selection The product between selection target and selection intensity shows the selection degree which should be paid attention to in selecting. More and more variety selectors apply the selection index in selecting lines and varieties of hybrid maize (Ribaut et al., 1997; Zaidi, 2000). Several characteristics are collected by CIMMYT from experiences of maize variety selectors and selection intensity values, they are: Grain yield with intensity: +5; number of maize ears on plant: +3; TP-PR difference: -2; number of green leaves: -2; dimension of tassel: -2; degree of withered leaves: -1 (Ribaut et al., 1997; Zaidi, 2000). 1.4. Research and usage of early-mature, cold-endurable hybrid maize variety 1.4.1. Scientific foundations of early maturity The growth and development process of maize relates to average temperature of day, lighting cycle and the temperature is the major factor (Olsen et al., 1993). The gross temperature that a maize plant need during its living cycle from seeding to maturing is much higher than other plants, from 1.700oC to 3.700oC depending on variety (Velican, 1956). According to Derieux (1979), the interventions of breeder into top splitting time of growth to select the early-maturity of maize are faster effective. Because in same seeding conditions with specific number of leaves, the top growth of early-mature maize frequently has fewer split leaves, which will promote fast the early maturity process of plant to transit to reproduction period. 1.4.2. Researching and using situation of early-mature hybrid maize variety in the world and in Vietnam 1.4.2.1. Researching and using situation of early-mature maize variety in the world The early-maturity of maize are paid much attention to by many scientists and breeders, in which they pay special attention to researching direction of
7 selecting and breeding early-mature hybrid maize varieties for production activities, because the early-mature hybrid maize varieties frequently have more stable and higher yield than lately-mature maize varieties in years when disadvantageous conditions happen suddenly (Kato A., 2002). Especially in regions where the extreme climate phenomena frequently happen and regions where the cultivation time is short, the early-mature varieties will be planted to avoid disadvantageous conditions better than lately-mature varieties. On the other hand, the early-mature varieties which are planted in short cultivation crop will have more stable and higher yield than lately-mature varieties (Troyer and Brown, 1976). When evaluating the “Turda” gene source for early-mature hybrid maize breeding and selecting program, Has I. (2012) concluded that the evaluation of early-mature maize’s gene source was very important in selecting and breeding the self-pollinating lines and new commercial hybrid maize varieties which could adapt to colder areas. The selection of early mature maize varieties was very necessary to maize planters in regions with low temperature but its yield did not decrease. 1.4.2.2. Researching and using situation of early-mature maize variety in Vietnam The freely-pollinating and early-mature maize variety TSB2 has been selected and bred from population of good individuals selected from early- mature population Suwan 2 hybridized with 6 other early-mature populations according to method: Good individuals selected from 6 populations are aed into mother row and father row are grains selected by improved method of maize/row to create the freely-pollinating and early-mature maize variety TSB2. By traditional self-pollination method with material of commercial hybrid maize variety CP999 and Cargil777, Bui Manh Cuong et al., selected and bred the early-mature hybrid maize variety LVN885 which are used broadly in production (Bui Van Hieu, 2019). 1.4.3. Roles of early-mature variety in agricultural production The early-mature variety plays an especially important role in agricultural production. It solves the multicropping matter and re-arranges the crop structure reasonably. On the other hand, the early-mature variety can avoid risks, natural disasters, disadvantages in climate conditions. The maize varieties with high yield and short growing time have very big meaning because they can be planted in many crops in year and increase the economic efficiency in area unit of cultivation land. According to Pswarayi and Vivek (2007), farmers plant early-mature hybrid maize varieties to increase income before planting main crop and this is especially important in regions with two crops per year. In Vietnam, the regions where wet rice is planted 2 crops/year can change and plant the early-mature maize varieties 3 crops/year, which will contribute into improving yield. On the other hand, the winter crop is the specific crop in the Northern Vietnam. After ending the winter rice crop, a part of this rice area will be used to plant vegetables, the most of left area is empty. This is an opportunity to increase the maize planting area and maize
8 yield and one of most effective and practical methods is creating an early- mature maize variety with high yield and adaptability suitable to region. 1.4.4. Researches on selection and breeding of cold-endurable maize varieties The major difficulty of maize variety breeder is the complication of maize’s adaptability to low temperature. If maize and several plants of Poaceae family met cold temperature at the pollination time, they will grain poorly or they cannot grain, which will decrease their yield seriously. In addition, low temperature lengthens the growing time and decreases the growing speed of plants. Maize can sprout at temperature below 10oC, thus it should ensure the optimal conditions for sprouting to reduce impacts of low temperature. 1.4.5. Demands on using early-mature and cold-endurable maize varieties The Northern Vietnam in general and Red Rive Delta in particular frequently happen the very cold climate in long time; the average temperature is lower than 150C at the beginning of Spring crop and at the end of Winter crop. It has very significant impacts on maize production in the region because maize is a kind of tropical plants which are sensitive to cold climate. The low temperature influences dramatically the sprouting period and growth and nutrition in sapling period of Spring maize crop. In addition, low temperature in long time can affect seriously the growth and accumulation of nutrition into grains in the Winter maize crop, consequently which decreases significantly the harvesting yield and product’s quality. Therefore, for this region, it is necessary to research, select and breed the early-mature or medium early-mature varieties with high yield, stability and pestilent insect endurability and adaptability to disadvantageous conditions, especially drought, coldness and hoar frost. 1.4.6. Research findings on Winter maize in Vietnam “Winter maize in two rice land in the Northern Delta” is one of two events creating a change during the process of maize development of our country. From the beginning of decade 80, century 20, there are many studies and tests on varieties and technologies to plant maize in the winter crop on wet land after two rice crops. With progresses on variety (early maturity, high yield, cold-endurability, flood-endurability…) and special technologies (making maize land, cultivating and caring on wet land, pestilent insect prevention…) and policies encouraging farmers to cultivate in Winter crop of Government and localities, the Winter maize has been confirmed and developed fast. Especially, in the Winter crop of 2020, Ministry of Agriculture and Rural Development has the guideline of preparing early and carefully the solutions and policies to create the breakthrough in biomass maize development in the Northern provinces because of demands on biomass maize to serve cattle breeding in the bigger and bigger scope, and then it can raise the production movement of Winter crop which frequently meets difficulties for recent years. Winter maize is one of economic solution with big importance which is suitable and satisfies absolutely the food and foodstuff, consumption goods and export goods programs of Vietnam because the Winter maize has added a
9 stable food plant crop with high yield and it provinces food for human and breeding activities and material for export commodity producing activities. 1.4.7. Maize production situation in Red River Delta Red River Delta is rather diversified in terms of crop, depending on each land foot to arrange crops suitably. However, for Winter maize, it is necessary to pay attention to fight against drought at the end of crop and using the early- mature variety. Currently, the maize yield of this region is 49.1 tons/ha, higher than average yield of the whole country (46.7 tons/ha) (Statistics Yearbook, 2017) (Table 1.4). Red River Delta is a land which is potential to develop agriculture, its winter crop is specific to land fund of nearly 200 thousand hectares in each year (Department of Crop Production, 2018). After ending the winter rice crop, a part of this rice area will be used to plant vegetables, and the most of left area is frequently empty, this is an opportunity to increase the maize planting area to increase the maize yield inside country, to decrease the imported maize quantity and to create employment for inhabitants and to contribute into improving the socio-economic efficiency for the whole region. 1.4.8. Conclusions drawn from the research review Maize is one of the main food crops, has a particularly important position in the structure of global agricultural production and has the highest growth rate in terms of productivity and output. In Vietnam, maize has the second most important position after rice, with achievements in research and breeding of hybrid maize varieties, farming techniques, fertilizers and consumption markets, etc. Corn production in the past time has fundamentally changed maize farming in our country and is an important driving force for Vietnam's maize area, productivity and output to increase continuously at a high rate during recent years. this. The combination of traditional methods and the application of biotechnology to select and create hybrid maize varieties suitable for each ecological region, resistant to a number of adverse conditions and associated with biological transformation. Plant structure in order to make the most effective use of natural resources (soil, climate, water sources) to gradually limit imports, towards self-sufficient domestic corn demand, contributing to raising income value for farmers, population and stable development, but winter maize after rice in the Red River Delta is a typical example (needs an appropriate set of varieties, high yield and cold tolerance). CHAPTER 2 RESEARCH MATERIALS, CONTENTS AND METHODS 2.1. Research materials - They include 53 self-pollinating ordinary maize lines of self-pollinating generation S8-S12: 49 research lines and 4 Test lines C88N, T8, T5, DF5. - Two trial plants T5 and B67CT. - The control varieties are the commercial hybrid maize varieties which are planted broadly in maize producing regions in the winter crop in Red River Delta, including LVN4, LVN99, DK9901, DK6919.
10 - Laboratory supplies and chemicals: Extraction buffer: (1M Tris-HCl, pH 8.0; 0.5M EDTA pH 8.0; 2.5N NaCl; 10% SDS), 5M Potassium acetate, Isopropanol, 70% ethanol, TE 0.1X (10mM Tris, pH8.0; 1mM EDTA, pH8.0). 2.2. Research contents (1) Evaluation and selection of cold-endurable and early-mature inbred maize lines: - Evaluating the agro-biological characteristics on growing time, morphological characteristics, resistance abilities, constituting components of yield and yield. - Screening the cold-endurable and early-mature lines by selection index. - Analyzing the genetic diversity of lines selected by SSR molecular marker. (2) Evaluating the combination possibility of maize lines and selecting the potential hybrid combinations: - Evaluating the combination possibility on yield of cold-endurable and early-mature lines. - Surveying and selecting the potential hybrid combination (top &diallel cross). (3) Experimenting and evaluating the potential hybrid combinations in provinces of Red River Delta: - Evaluating the agro-biological characteristics of potential hybrid combinations in Red River Delta. - Evaluating the stability on yield of potential hybrid combinations in Red River Delta. (4) Experimenting and developing new varieties: Basic experiment; Production experiment. 2.3. Research methods 2.3.1. Field experimenting designing method - It includes the line evaluation, inbred line development self-pollination tests, hybrid combination comparison and evaluation tests according to method of Gomez and Gomez (1984). - Hybrid combination evaluation tests in several environmental conditions according to method of Ketthaisong et al. (2015) and Khampas et al. (2015). - The control maize varieties in hybrid combination comparison and evaluation tests are LVN4, LVN99 originated from Maize Research Institute and varieties DK9901, DK6919 originated from Monsanto Company. 2.3.2. Inbred line breeding and selecting and inbred line evaluation methods - Selecting and breeding the inbred varieties by obligatory self-pollination method. - Inbred line evaluation method: the lines are maintained 2 crops/year in the research material group, arranging continuously 15 - 20 rows/line for hybridizing affairs. Evaluating lines arranged according to random complete blocks (RCBD), for 3 repeating times. For each formula, maize is seeded in 2 rows, each row is 5 m in length, the dimension is 70x25cm, 1 plant/hole and the targets are supervised according to guideline of QCVN 01-56: 2011/BNNPTNT.
11 2.3.3. Combination possibility evaluation method Combination possibility evaluation method on grain yield: Applying the top cross method. The top cross combinations (formula) between 26 lines and 2 trial plants are compared according to the diagram of random complete blocks, 3 repeating times, density of 57.000 plants/ha, the seeding dimension is 70 x 25 cm, 1 plant/hole. 2.3.4. Method of top cross and diallel cross combinations surveying The top cross and diallel cross combinations are evaluated according to diagram of random complete blocks, 3 repeating times, density of 57.000 plants/ha, the seeding dimension is 70 x 25 cm, 1 plant/ hole. 2.3.5. Line selecting method by selection index Using “Chondong” program in Quantitative Gene Program of Nguyen Dinh Hien and co-author (2007). It bases on data collected for characteristics paid attention to during the process of line evaluation. 2.3.6. Genetic diversity analyzing method by SSR molecular marker - Evaluating the genetic diversity by molecular marker with 22 SSR markers which have been classified and selected basing on the diversity in the genetic diversity evaluation test (Table 2.2). The information about SSR primers used is presented in Appendix 2 of the thesis. - Reading data and identifying the genetic similarity factor, subgrouping the genetic difference by UPGMA method, done according to maize gene analyzing procedure by SSR marker and analyzing data of Luz and Ellen (2004) by NTSYSpc 2.1 software (Rohlf, 2000). 2.3.7. DNA purification and extraction method PCR method; Electrophoresis method on gel Agarose. 2.3.8. Stability and adaptability evaluating method Evaluating the adaptability and stability according to Linear Regression Analysis Method of Eberhart S.A. and Russel W. (1966), by Quantitative Gene Program of Nguyen Dinh Hien and co-author (2007): Identifying the stability of yield (S2di ); Adaptability (Regression index (bi ). - Environmental index Ij: Ij = Li - Grand mean (L: Location), in which: If Ij > 0: advantageous environment; If Ij < 0: disadvantageous environment. I2j = Sum(i12 + …. + in2). - Regression index bi: bi = adaptibility index - regression index, in which: If bi > 1: advantageous; If bi = 1: broad; If bi < 1: Disadvantageous.  Yij Ij = [Y] x   Ij1   Yij.Ij  bi = I 2  Ijy.... j - Stability index S2di: S2di = stability index. If S d 2 i  0: stability; S2di 0: stable.  D   EMS  S2d =   -   L 2  r  In which: L-Location; D-Diff. If S2d0: correlative stable yield GE linear.
12 2.3.9. Experimenting method - Experimenting at grassroots level. - Experimenting the cultivation and using values of maize variety (VCU). 2.3.10. Experimental data processing and collection method - Date processing and collecting method: According to CIMMYT (1985) and Standard QCVN 01-56: 2011/BNNPTNT. Data are processed statistically by Excel and IRRISTAT 5.0 programs. - Evaluation on combination possibility of lines is calculated and processed by software of Quantitative Gene Program of Ngo Huu Tinh, Nguyen Dinh Hien (1996). 2.3.11. Supervision targets in line evaluation and comparison of hybrid combinations Including: Growing time; morphological targets of plants, tassel, maize ear; Constitutional factors of yield; yield evaluation; endurability against: Rhizoctonia solani Kuhn, ratio of rotten maize ear, cold-endurability.... 2.4. Research place and time 2.4.1. Research place - Evaluating materials, selecting the potential lines in Maize Research Institute (Dan Phuong - Hanoi). - Experimenting to evaluate the potential hybrid combinations in provinces of Red River Delta. - Experimenting varieties in National testing system in several province of Northern Vietnam. 2.4.2. Research time - From 2012 - 2014: Evaluating lines and selecting lines. - From 2015 - 2016: Choosing, experimenting at the grassroots level the hybrid combinations. - From 2017 - 2018: Evaluating the growing and developing abilities of potential hybrid combinations in the National test system. 2.5. Flowchart of the selection process An overview diagram of the process of line selection, cross- Figure 2.1. Diagram of maize breeding and testing of maize variety testing, hybridizing, line varieties is presented in Figure 2.1. selecting process
13 CHAPTER 3 FINDINGS AND DISCUSSION 3.1. Findings of early-mature, cold-endurable inbred maize line selection and evaluation 3.1.1. Happenings of climate factors in Red River Delta Analyzing deeply the impacts of climate factors of Red River Delta on growing and developing abilities of maize in the winter crop (Table 3.1), we see that: 3 factors (rainfall, temperature and humidity of air) change dramatically. The rainfall distributes unequally between months of year, the most lower level in in January (15.1 mm/month), the highest level in July reaches 311.4 mm/ month; the humidity of air is 26% at lowest level and 88% at highest level; The lowest temperature is 16,9oC (January) and the highest temperature is 40,7oC (June). In general, we can see that December and January is two months in Winter crop with climate conditions unsuitable for maize to grow and develop, because January and December are two months with lowest temperature and rainfall in year, thus the maize in the winter crop should be bred before and on 25 September, so that maize can have tassel before 20 November, and mature before 20 January, simultaneously it should pay attention to fight against drought at the end of crop. In selecting and breeding maize varieties, when we breed successfully any early-mature lines/ varieties with high yield in the winter crop regardless of disadvantageous climate conditions and low temperature, it is a success and these lines/ varieties will contribute into developing the winter crop in Northern Vietnam in general and Red River Delta in particular. Therefore, to select the cold- endurable maize lines, we conduct the agro-biological characteristics of maize lines researched in different crops in the winter crop. 3.1.2. Evaluation of agro-biological characteristics of 53 researched maize lines 3.1.2.1. Growing time of 53 researched maize lines Table 3.2 shows that research findings on growing time of 53 experimental maize lines, we can see: The experimented maize lines have rather different growing time. On average, it fluctuates from 99.7 days (C59) to 129.2 days (C649, C258) and the stable growing materials through sowing stages show that they can adapt well to disadvantageous conditions and they are divided into: Early-mature group (Growing time < 105 days) only includes 1 line C59 (99.7 days); Medium-mature group (Growing time 105 - 120 days) includes 40 lines; Lately-mature group (Growing time > 120 days). Therefore, 77% researched lines can be used as father or mother in selecting and breeding the early-mature or medium early-mature hybrid maize varieties (short-time) suitable to conditions of winter crop in Red River Delta. 3.1.2.2. Morphological characteristics of researched maize lines Table 3.3 evaluates several morphological targets of 53 researched maize lines. Including: - Height of plant: fluctuates rather significantly, from 96.2 - 170.8 cm, the lowest height is line C847 (96.2cm) and the highest height is line C491 (170.8cm).
14 - Height of insertion of upper ear of researched maize line fluctuates from 36 - 94%, in which only 2 materials have the ratio of upper ear < 40% is C28 and C524, 15 materials have the ratio of upper ear > 60% including C63, C124, C252, C269, C282, C318, C409, C491, C571, C608, C649, C691, C722, C777 and the control group is T5. Therefore, 36/53 researched lines have the height of insertion of upper ear suitable to next breeding and selecting affairs including 3 control lines. - Ratio of shattered plants: The diameter of plant foot of researched maize lines is evaluated in same period of plant height, it fluctuates from 1.3 (C28) - 2,2 cm (C354). - Number of leaves: The researched materials have rather high number of final leaves, on average from 16.8 (C352, C491) - 19.4 leaves/plant (C282) and all of they are materials keeping the long-lasting green leaves, the number of left green leaves on plant fluctuates from 9.8 - 12 leaves. Therefore, most of researched lines have their plant height and height of insertion of upper ear at average level and rather uniform, showing in change factor CV (%) smaller than 15%, its leaves are rather bushy (16.8 - 19.4 leaves/ plant) and stable in two experimenting crops. We can confirm that the researched lines are inbred lines and have enough conditions to conduct next experiments (Table 3.3). 3.1.2.3. Yield of researched maize lines The line yield is always the target paid much attention to by maize variety breeder because of its benefits in many aspects. The actual productivities of 53 experimental maize lines are collected in Table 3.4, in which most of lines have no significant change, the change factor CV (%) from 5.90 - 8.10% showing the genetic stability of researched maize lines. The line with lowest actual yield is C539 (1.430 tons/ha), lower than almost left materials at the meaningful level. The average yield of control lines reaches 2.184 - 2.311 tons/ha, belonging to average group among researched materials. The line with highest yield is C805, reaching 2.655 tons/ha, higher than 4 control varieties at the meaningful level. The lines C608, C777, C855 have the average yield higher than 2 control lines C88N and T8 at the meaningful level. We recognize that the experimental maize lines have rather high and stable yield through two experimental crops, they are good materials which can be exploited in selecting and breeding the hybrid maize varieties with high yield. The evaluation results of growing time, morphological characteristics and yield of researched lines are combined with desired gene screening results by Selection index program to select the most potential lines in group of 53 maize lines stated above. 3.1.3. Cold-endurable, early-mature line selecting results 3.1.3.1. Cold-endurable, early-mature line selecting results by selection index Based on data of seven targets supervised, including: growing time, yield plant height, height of insertion of upper ear, stalk diameter, number of leaves and number of green leaves in the period of mature maize ear to select the most potential lines serving the maize variety hybridizing affairs which are early-
15 mature and adapt to cold climate. For research contents of thesis, we have special attention to growing time, next grain yield and green leaf’s durability (number of green leaves in the period when plants have mature maize ears) of researched lines. Table 3.5. Selection targets and intensity of experimental maize lines Target Goal Intensity Desired goal value Growing time -1.5 9.0 108.4 Yield 2.0 8.0 26.3 Number of green leaves when 2.0 6.0 12.0 maize ears are mature The summary of researched maize lines through basic statistical results describes lines shown in Table 3.6. Table 3.6. Statistical data describing 53 researched lines Standard Change Smallest Biggest Target Average deviation factor value value Growing time 117.296 5.938 0.051 99.7 129.2 Yield 20.328 2.971 0.146 14.3 26.6 Plant height 132.751 19.271 0.145 96.2 170.8 Stalk diameter 1.790 0.220 0.123 1.3 2.2 Number of leaves 17.834 0.718 0.040 16.8 19.4 Number of green leaves 10.808 0.608 0.056 9.8 12.0 when maize ears are mature The potential line selecting results though selection index are shown in Table 3.7 with 30 lines selected. It identifies 26 lines suitable to researching direction and 4 control lines. 30 selected lines with selection index change from 3.84 to 10.56. The lines with small selection index show theses lines near to selection targets and vice versa. Among selected lines, 6 lines have the selection index lower than 4 control lines C777, C608, C783, C801, C571 and C855 with selection index which fluctuates from 3.84 - 6.67 (Table 3.). Table 3.7. Selection index and values of 30 selected lines green leaves when maize Plant height Number of Number of Selection Growing diameter ears are mature leaves Lines index Yield Stalk time No. 41 C777 3.84 107.8 25.9 142.0 2.04 18.2 11.2 34 C608 4.04 111.5 25.9 157.6 2.06 18.8 11.2 42 C783 4.53 115.3 23.8 120.2 1.84 17.2 11.8 44 C801 5.99 115.5 25.0 106.8 1.82 19.0 11.0 33 C571 6.56 115.7 23.6 116.0 2.12 17.8 11.0 49 C855 6.67 111.5 25.6 116.0 1.90 17.4 10.6 51 T8(TL) 7.14 117.2 21.8 130.2 1.82 18.2 11.4 46 C833 7.65 114.5 23.7 111.4 1.76 17.2 10.6 6 C50 8.04 111.3 20.0 109.4 1.38 16.8 11.2
16 53 DF5(TL) 8.39 116.7 23.1 122.4 1.60 17.6 10.6 4 C28 8.47 111.3 18.9 119.4 1.30 17.6 11.6 28 C475 8.53 120.7 24.6 168.0 1.66 18.6 10.8 22 C373 8.84 116.8 19.6 150.2 1.78 17.4 11.4 35 C628 9.04 115.2 21.4 148.2 2.16 18.0 10.6 50 C88N(TL) 9.17 114.7 22.2 97.6 1.72 17.4 10.4 40 C769 9.32 112.0 20.5 117.0 1.80 17.8 10.6 26 C431 9.49 112.0 19.0 138.6 1.74 17.6 11.0 21 C354 9.51 114.5 18.2 165.0 2.20 19.0 11.6 10 C124 9.66 122.0 20.1 135.2 1.84 18.2 11.8 16 C282 9.70 117.8 18.5 138.6 2.00 19.4 11.8 32 C541 9.85 118.2 24.1 106.4 1.98 17.2 10.2 13 C252 9.87 113.5 18.3 125.0 1.84 18.2 11.2 43 C795 9.89 119.7 20.8 147.4 2.10 18.0 10.8 3 C18 10.20 114.8 17.8 151.2 1.74 18.8 11.4 2 C16 10.30 110.0 17.4 116.6 1.32 17.4 11.4 20 C352 10.30 115.2 19.0 144.2 1.84 16.8 10.8 47 C838 10.42 119.2 23.1 113.2 1.80 17.0 10.2 52 T5 (TL) 10.49 119.0 22.8 107.2 1.91 18.4 10.2 17 C290 10.55 116.0 17.2 147.6 1.98 18.6 12.0 8 C63 10.56 108.5 19.0 129.4 1.66 17.2 10.6 Thanks to collected findings, we continue evaluating the reaction of 26 lines selected with 4 Test lines in next crops. 3.1.3.2. Growing time of 30 selected lines After selecting, 30 experimental maize lines (self-pollinating generation S8-S12) are continued evaluating the morphology. The growing time evaluation results of researched lines are shown in Table 3.8. In Spring crop, 2 lines have the growing time of 113 days similar to the control line which is mature firstly (C88N), 11 lines have the growing time > 120 days similar to 3 left Test lines, 13 lines have the growing time fluctuating from 115 - 119 days. In winter crop, 12 lines have the growing time from 108 - 113 days, shorter than 4 Test lines from 3 - 11 days; 11 lines have the growing time from 115 - 119 days similar to 4 Test lines; 3 lines have the growing time from 120 - 121 days longer than Test lines from 1 - 6 days (C475, C124, C795). We can see that 30 inbred maize lines are rather diversified in terms of genetics, with G-TP time and also growing time belonging to medium early-mature to lately-mature groups thus they may have good yield. 3.1.3.3. Morphological characteristics of 30 selected lines The morphological characteristics of 30 selected lines are presented in plant height; Ratio of of insertion of upper ear; Length of tassel; Number of final leaves. In general, 30 inbred lines in experiment have very good morphological characteristics, satisfying demands of selecting new hybrid maize varieties.
17 3.1.3.4. Constituent elements of yield and yield of 30 selected lines The maize lines in experiment have the average yield of 2.167 tons/ha (C777) - 3.433 tons/ha (C373, C628), in which there are 13 lines with high yield, reaching > 3.0 tons/ha equivalent to 3 control lines C88N, T8, T5; 8 lines have the yield lower than level of 3.0 tons/ha at the meaningful level. The lines with highest actual yield are C373, C628 (3.433 tons/ha), C28 (3.366 tons/ha), C354, C252 (3.333 tons/ha)... These lines have no outstanding constituent elements of yield but they still have high yield, which show their adaptability to very good cultivation and environmental conditions, thus they can promote all of their yield potentials of variety. 3.1.3.5. Endurability of 30 selected lines Evaluating preliminarily the endurability against main pestilent insects in maize plant of 30 inbred maize lines, the results in Table 3.11 show that the lines have rather good endurability against harmful factors thus they have few impacts on growth, yield and grain quality. 3.1.3.6. Kernel filling ability of 30 selected lines Evaluating the kernel filling ability based on 2 main targets: ratio of poorly- filled ear and ratio of dormant kernels, the data are presented in Table 3.12. The ratio of poorly-filled ear of researched materials fluctuates from 1.00 - 5.34%. The line C541 has the highest ratio of poorly-filled ears (5.34%), significantly higher than almost left materials with reliability 95%. In addition, there are 8 lines with ratio of poorly-filled ears > 3%, including: C855 (3.21%), C475, C124, C838 (3.33%), C783, C373 (3.38%), C63 (4.21%) and C282 (4.26%). The ratio of dormant kernels fluctuates from 0 - 5.33%. The remarkable line is the material line C431 without dormant kernels (0%), equivalent to two control lines T8, C88N. In addition, the line C801 has the ratio of dormant kernels 1.00%. Only line C16 has high ratio of dormant kernels >5% (5.33%). The research on selecting, breeding the early-mature maize variety adapting to cold climate is one of solution which reduces harmful effects of low temperature on maize production. We recognize that the materials in this research have the ratio of poorly-filled ear and ratio of dormant kernels which are generally evaluated as rather low and have few impacts on yield and quality of collected kernels, in combination of evaluation results of agro- biological characteristics, constituent elements of yield and kernel filling ability of maize ears in the harvesting period, thus they are used as a target in cold-endurable hybrid maize variety selecting and breeding affairs. Ending the line evaluation experiment, we select 30 potential maize lines which have characteristics as rather good growth, development, yield and disadvantageous climate conditions-endurability; these lines will be tested to evaluate the general combination possibility to find out lines with high combination possibility.
18 3.2. Genetic diversity evaluation and analysis results of 30 selected lines Using 22 locus SSR by program NTSYS is shown in Table 3.13 and Figure 3.4, shows that: the genetic similarity index of lines changes from 0.54 - 0.94, which proves that the lines are rather different in terms of genetic material, is the foundation to have hybrid combinations with high combination possibility or on the other hand they have high heterosis. We recognize that: in genetic similarity index 0.75, the researched maize lines are divided into 5 groups: Group I has 2 lines: C18 and C608; Group II has 12 lines: C475, C777, C16, C282, C354, C252, C28, C795, C855, C838, C373, C541; Group III has 6 lines: C124, C783, C571, C352, C88N, T8; Group IV has 9 lines: C628, C63, C833, C50, C769, C801, C290, DF5, C431; and a single line T5. Therefore, we can estimate that several hybrid pairs are able to have high heterosis, including: Group I can hybridize with Group II, III, IV; Group III Figue 3.4. Genealogical diagram can hybridize with Group I, II, IV and Group IV can hybridize of 30 researched maize lines based with group I, II, III. Especially, on analyzing 22 locus SSR line T5 can have high heterosis by UPGMA method with lines in 4 left groups. 3.3. Combination possibility evaluation results of researched lines 3.3.1. General combination possibility evaluation results 26 selected lines are evaluated their combination possibility in term of kernel yield by top cross method with 2 CT of T5 and B67CT. The results are: CT1 (T5) has the general combination possibility on yield, higher than CT2 (Table 3.14), in which the line C352 has the highest general combination possibility (gi = 13.807), next lines C16 (gi = 8.827), C431 (gi = 8.533), C838 (gi = 7.972), C801 (gi = 6,788), C769 (gi = 6.335) (Table 3.15). In addition, 5 lines C608, C783, C855, C628, C252 have high gi value that fluctuates from 3.595 - 4.780 (Table 3.15). In addition, the lines have high specific combination possibility variance C18 (2si = 70.193), C373 (2si = 64.432), C475 (2si = 54.794). The lines C18, C373, C475, C855 have the specific combination possibility Line * high trial plant with CT1 (T5), in which the lines C63, C431, C769, C571 and C50 reach high value with CT2 (B67CT)