RESEARCH ON SELECTING SOME TRIPLOD LINES CITRUS VARIETIES

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES LE QUOC HUNG RESEARCH ON SELECTING SOME TRIPLOD LINES CITRUS VARIETIES Major Field: GENETICS AND PLANT BREEDING Code: 62.62.01.11 DOCTORAL THESIS SUMMARY HA NOI - 2015 The Doctoral thesis was completed in VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Scientific Supervisors: 1. Prof.Dr. Do Nang Vinh 2. Assoc.Prof. Dr. Ha Thi Thuy Reviewer 1: Reviewer 2: Reviewer 3: Dissertation will be defended before committee in charge for doctoral dissertation in academy level, meeting place 󰜧󰜧󰜧 on 󰜧󰜧󰜧󰜧, 2015 The PhD thesis may be fount at: - Ha Noi National Library - Library in Vietnam Academy of Agricultural Sciences HA NOI - 2015 1 INTRODUCTION * Urgency of research Citrus trees are one of fruit crops which have high nutrition and economic value. Production of citrus fruit all over the world reached 85.6 ton in the years of 2012/2013, in which orange production accountes 50% (USDA, 2013). This number will be continued increasing because of the demand in some countries are highly expanded such as China, Korea, Russia, India, East Europe and Asia. In China, the consumption of fresh fruit has been singnificantly increasing, especially citrus fruits. In recent years, China has ranked top second biggest citrus fruit production after Brazil (Deng, 2008). Vietnam is one of the center in which Citrus are originated (Vo Van Chi, 1997; Pham Hoang Ho, 1992). The area of citrus in 2011 is 138,200 ha, about 18 percent of all fruit area and the production was estimated about 1.35 million tons (Department of Planting, Ministry of Agriculture and Rural Development, 2013). However, Vietnam still has to import a sheer amount of citrus fruit annually because of low productivity causing by diseases and the quality of varieties (low quality and containing many seeds). Selecting seedless citrus are being conducted in some commonplace following approaches. 󽟙 Mutating 󽟙 Mating between diploid lines (2x) and tetraploid lines (4x) to select triploid lines (3x) (Roose and Williams, 2000) 󽟙 Culturing endorsperm of immature seeds (Gmitter et al, 1990) 󽟙 Selecting triploid embryos in nature (3x) (Esen et al, 1971) 󽟙 Rescuing triploid embryos from dud seeds (Ollitrault et al, 1996) 󽟙 Implementing fusion between diploid protoplast and haploid protoplast cells (Ollitraul et al, 2000) 󽟙 Applying biotechnology techniques in embryo rescuing, protoplast fusion, selecting somaclonal variations (Froelicher et al, 2003; Grosser et al, 2000; Juárez et al, 1990; Ollitrault et al, 1998) In Vietnam, some well-known Citrus cultivars are widely grown widespread such as Xa Doai, Van Du, Citrus nobilis (King Mandarin), Bu orange, Phuc Trach, Dien, Doan Hung pummelos󰜧 These varieties are high quality and economic value but contains many seeds. To improve traits by selecting from these varieties based on the ability to generate heterosis, well-adapted and disease resistance is top priority. From the above judging, we have conducted a dissertation: 󰜝Research on selecting some triploid lines in citrus varieties󰜞 * Aims of research To produce a larger number of triploid lines of pummelos and King Orange (Citrus nobilis) as the initial resources for selecting varieties with non-seed and fewer seed varieties. 2 Testing, evaluating and selecting triploid pomelo and Citrus nobilis lines to identify promising lines and establish selecting approaches * Breakthrough of this research For the first time in Vietnam, we have selected a large number of triploid Citrus lines by mating (2x 󽞵 4x) and Citrus nobilis (2x 󽞵 2x) types including 92 triploid pummelo lines and 22 triploid Citrus nobilis lines. There are 2 King orange lines (2x) and 6 pummelo lines (3x), all are well growth, non-diseases. Two lines of 6 pummelo lines above have been produced seedless fruits after budded on 5 year old of sour pummelo. The fruit is round shape, yellow skin, succulent, sweet, yellow epicarp and 494,16 g on average. Confirming the ability of hybridizing method in order to make triploid pomelo lines and by budded on 5 year old of sour pummelo to shorten the growth of 3x lines compared with budded on 7 to 8 month Citrus hystrix D.C. * Contribution of the dissertation Scientific aspect: The project has provided a substantial number of selected triploid citrus lines from the native cultivars. From which, all triploid pummelo lines are well-growth, two of them have produced seedless fruits. Especially, the project knowledge was made a direction for further researching and selecting triploid Citrus nobilis and Citrus grandis in Vietnam. We have also successfully estabshed and applied technological procedure for selecting triploid citrus, for example, the success of mating 8 pairs between diploid and tetraploid to collect a large number of different triploid. We have also made integration between traditional method and biotechnology methods such as (rescuing embryo) and some techniques for quick selection (by budded on flowering citrus plants). All knowledge can be used as reference for researching and lecturing about citrus fruits. Reality aspect: The project has provided a substantial number of triploid lines as resources for mating between different types. There are 2 triploid pummelo lines and some Citrus nobilis lines from the specility citrus of Vietnam. They are being tested for confirmation as national cultivars. * Materials and scope of the research Material: Father plants provided pollen are Phuc Trach, Dien pummelos and King (Citrus nobilis), Van Du orange which are tetraploid types were selected by colchicine treatment belonging to the project: 󰜝Researching and selecting seedless citrus fruit plant from indigenous󰜞 (2001 to 2005). These tetraploid plants have been grown in Van Giang, Hung Yen province which also have conferred and authorized by Department of Planting, Ministry of Agriculture and Rural Development, 515/QD- TT-VPVH, 12 th , November, 2013. Mother plant (2x) was selected from healthy plants ,well-flowering Nam Roi, Phuc Trach, Dien pummelos and King oranges (Citrus nobilis). Triploid pummelo and Citrus nobilis lines also utilized and selected by rescuing embryos from dud seeds belonging to the project: 󰜝Researching and 3 selecting seedless pummelos, orange and grapefruit applied biotechnology󰜞 (2006 to 2010). Scope of the research: The research are conducted in Vietnam. In specific, the experiment were conducted at Key national of plant cell Lab (Agricultural Genetics Institute), Van Giang, Hung Yen practical farm, Vegetable and Fruit Research Institute and other fruit farms in some provinces such as Cao Phong (Hoa Binh province), Ham Yen (Tuyen Quang province). CHAPTER I OVERVIEW OF CITRUS 1.1. Methods for selecting seedless Citrus varieties Research for selecting Citrus fruits have been focused on comparing productivity, quality and disease resistance, especially with seedless trait are priority (Esen and Soost, 1971; Ollitrault et al, 1998; Ollitrault et al, 2000; Ollitrault et al, 2006; Koltunow et al, 1998; Ray, 2002; Deng X.X, 2006; Grosser et al, 2006; Jean Baptiste Bassenet et al, 2009). Some non-sticky skin, seedless, nutrient, storage and disease resistance traits are concerned by scientists (Ollitrault et al, 2000; Roose and Williams, 2000). Utilizing heterosis, multiploid, protoplast fusing and somaclonal variation were applied to select seedless lines with disease resistance trait serving beverage industry and fresh fruit consumption (Guo et al, 2004; Grosser and Gmitter, 2005; Gmitter et al, 2007; Febres et al, 2009). Some researches were applied transgenic technology to select varieties with disease resistance and quality traits (Gmitter et al, 2007). 1.2. Selecting seedless fruit by radioactive and natural mutation methods In 10 years, China has conferred 17 new Citrus cultivars, was selected by 2 methods: selecting natural variations and radioactive mutations, including, 3 seedless and 1 little seed grapefruits, 3 seedless and 1 little seed oranges, 2 seedless pomelo varieties. Furthermore, some mutated cultivars have ripen longer, some were earlier but all of them have higher quality and productivity than the control (Deng, 2000). 1.3. Hybridizing between diploid and tetraploid types The crucial strategy to produce seedless Citrus plants is implemented mating between tetraploid and diploid lines (Soost and Cameron, 1975; Starrantino and Recupero, 1981). For instance, the typicalness of sucessful results of generating 2 triploid pummelo cultivars Oroblanco and Melagold were made (Soost and Cameron, 1980, 1985). However, the process of selecting high quality seedless cultivars has coper with many difficulties because of the shortage of gene pool of tetraploid cultivars. In the mating, diploid cultivars was utilized as mother plants and tetraploid was used as fathers. Triploid embryos are not developed normally because the relative 4 between embryo and endosperm is disconnected. So, the scientists have rescued such embryos by culturing them in vitro (Gmitter, 1995). The germination of collected triploid seeds is hard, just few seeds from 2 to 10 seeds per 100 fertilized flowers were possobly. Some mating pairs (2x x 4x) produce small fruits with germinated seeds. However, these young fruits often fall down in June. Some scientists of California State (US) developed a technique to rescue fallen young fruits by culturing their embryos on high sugar and citric acid medium without regulator chemicals. The results showed that the number of plants was increased up to 3 times (Williams and Roose, 2000). 1.4. Selecting tetraploid cultivars for diploid and tetraploid plant mating Selecting tetraploid lines can be implemented by the different methods. The tetraploid lines (4x) randomly appear in nature with relatively high frequency. Cameron and Frost (1968) obtained 2.5 percent per 3,600 trees from various cultivars. Tachikawa et al, (1961) used colchicine in order to produce tetraploid Citrus and then hybridized with diploid plants. Tetraploid types are able to produce by protoplast fusing (Grosser et al, 2000). To summary, there are well-known methods to make tetraploid lines: randomly in nature, colchicine treatment, protoplast fusing. 1.5. Invitro propagation for selecting triploid Citrus a. Invitro embryo rescuing technique The target is rescuing embryos from mating pairs between 2x and 4x lines and 2x and 2x. Determining different developing stages of embryos is very important, Starrantino and Redupero (1981) has obtained triploid trees by rescuing embryos 3 to 4 months embryos after pollinating. Some can be done when fruits are ripen, by culture cause less developed seeds (Froelicher et al, 2003). The prevalent medium for embryo rescuing is MT (Murashige and Tucker, 1969). Some elements are added on medium for regeneration, for example; Adenine sulfate (25mg/l) and malt extract (500mg/l) made beneficial effects, some hormones support embryo germination and shoot induction such as GA3 (1mg/l) balanced with BAP (0.5 mg/l) + 0.5 mg/l kinetin + 0.1 mg/l NAA. The procedure of triploid embryo rescuing was developed by scientist of CIRAD-INRA (Froelicher et al, 2003). The optimum medium is MT (Murashige and Tulecker, 1969) + 30 g/l Sucrose + 25 mg/l adenine sulfate + 500 mg/l malt extract + 1 mg/l GA3 + 8 g/l agar, pH = 5.7 b. Rescuing triploid embryos obtained from 2n and 4n mating Hybridizing mother diploid plants with pollen from tetraploid plants have been conducted for a long time (Esen and Soost, 1972). However, the obtained embryos was not develop normally, just few seeds can be germinated (Cameron and Frost, 1968; Tachikawa et al, 1961; Esen and Soost, 1972). Some reports have suggested that uncompleted development of triploid seeds are related to endorsperm abnormality. So, to overcome this barrier, scientists have rescued embryos by culturing them on in vitro medium. 5 Starrantino and Recupero (1981) have cultured 585 embryos selected from 3 diploid mother lines with 6 tetraploid lines hybridizing pairs on MS medium by adding 500 mg/l malt extract and 25 mg/l adenine sulfate. They have collected 211 triploid plantlets and 89 unidentified chromosome number were collected. They showed that culturing embryos after 3 to 4 months pollinated could be generated other embryos which produced many plantlets. Oiyama and Kobayashi (1993) collected triploid plants by rescuing dud seeds from hybridizing pairs between single embryo grapefruit cultivar Clementin (Citrus reticulate), Miyauchi Iyokan (Citrus natsudaidai Hayata) with pollen of tetraploid tree Kawano Natsudaidai (Citrus natsudaidai Hayata. The medium for culturing is added MT 500 mg/l malt extract and 40 mg/l adenine sulfate after pollinating 7 months. Besides, Oiyama also have collected high ratio of triploid plants by culturing undifferentiated small embryos forming from dud seeds of ripen fruits on MT + 1 mg/l GA. Grapefruit Clementin used as the mother and father are tetraploid plant by hybridizing between sweet orange (Citrus sinensis) and three leaf orange (Poncirus trifoliata). 1.6. Methods for identifying different ploid types in Citrus By using FC machine has allowed us to identify quickly different ploid types. CHAPTER 2 MATERIALS AND METHODS 2.1. Materials and time schedule Father plants provided pollen are Phuc Trach, Dien pummelos and King (Citrus nobilis), Van Du oranges which are tetraploid types selected by colchicine treatment belonging to the project: 󰜝Researching and selecting seedless citrus fruit plant from indigenous󰜞 (2001 to 2005). These tetraploid plants have been grown in Van Giang, Hung Yen province which have also conferred and authorized by Department of Planting, Ministry of Agriculture and Rural Development, 515/QD-TT-VPVH, 12 th , November, 2013. Mother plant (2x) was selected from healthy plant, well-flowering Nam Roi, Phuc Trach, Dien pummelos and King orange (Citrus nobilis). Triploid pomelo and Citrus nobilis lines also utilized to select by rescuing embyros from dud seeds belonging to the project: 󰜝Researching and selecting seedless pummelo, orange and grapefruit applied biotechnology󰜞 (2006 to 2010). Time ranges from 2011 to 2014 2.2. Dissertation contents 2.2.1. Preparing initial materials for selecting seedless and fewer seed citrus varieties - Hybridizing between same, different varieties and different species - Rescuing embryos to make initial sources 6 - Assessment of agro-biological characteristics of some selected triploid lines 2.2.2. Evaluating agro-biological characteristics and selecting triploid pummelos and Citrus nobilis lines in nethouse - Assessment of agro-biological characteristics of triploid Citrus nobilis lines in nethouse - Assessment of agro-biological characteristics of triploid pummelo lines in nethouse 2.2.3. First steps for testing triploid pummelos and citrus nobilis lines - Testing for triploid Citrus nobilis lines budded on Citrus hystix - Testing for triploid pummelo lines budded on Citrus hystix - Evaluation results of triploid lines budded on 5 year old of sour pummelo plants and identifying some promising lines. 2.3. Methods 2.3.1. Preparing initial materials for selecting seedless and fewer seed citrus varieties Describing characteristics of diploid and tetraploid Citrus species for assessment and selection of promising individual plants with useful genetic traits as initial materials. No. Mating pairs Gender of plants 1 󰵁 PT2x × 󰵃 PT4x 󰵁Phuc Trach Pummelo(2x) × 󰵃Phuc Trach Pummelo(4x) 2 󰵁 BD2x × 󰵃 PT4x 󰵁 Dien Pummelo(2x) × 󰵃 Dien Pummelo(4x) 3 󰵁 NR2x × 󰵃 PT4x 󰵁Nam Roi Pummelo(2x) × 󰵃Phuc Trach Pummelo(4x) 4 󰵁 PT2x × 󰵃 BD4x 󰵁Phuc Trach Pummelo(2x) × 󰵃Dien Pummelo(4x) 5 󰵁 BD2x×󰵃 BD4x 󰵁Dien Pummelo (2x) × 󰵃Dien Pummelo (4x) 6 󰵁 NR2x × 󰵃 BD4x 󰵁Nam Roi Pummelo (2x)×󰵃Dien Pummelo (4x) 7 󰵁 CS2x × 󰵃 CS4x 󰵁 Cam Sanh (2x) × 󰵃 Cam Sanh (4x) C. nobilis 2x × C. nobilis 4x 8 󰵁 CS2x × 󰵃 VD4x 󰵁 Cam Sanh (2x) × 󰵃 Van Du Orange (4x) C. nobilis 2x × C. sinensis (L.) Osbek 4x 9 (control) 󰵁 CS2x × 󰵃 CS2x 󰵁 Cam Sanh (2x) × 󰵃 Cam Sanh (2x) C. nobilis 2x × C. nobilis 2x Hybridizing method: Flowers of mother plants in mating are on top of branches, healthy and earlier blossoming. Before blossoming 1 to 2 days, the flowers have to be removed anthers. Flowers with matured stigma need to be hybridized by selected father with rapid pollen and then covered with nylon packs to avoid other pollen. With small flowers, we have used specific small broom for hybridizing. After hybridizing, we removed all the non-hybridizing flowers to ensure the remained flowers not competing with nutrition. After hybridizing 2 to 3 weeks, disentangling nylon packs and marking positions of hybridized flowers. For natural hybridizing 7 pairs 󰵁 CS2x × 󰵃 CS2x, marking 30 flowers to observe fertilizing ability and remained fruit ratio. After fruit ripening, collecting fruits to assess of remained fruit ratio and number of seeds per fruit of different mating pairs were made. Hybridizing seeds have shown normal, less developed (containing embryos) and small seeds. Dud seeds with embryos of Citrus nobilis mating pairs have sterilized and cultured on suitable medium. Embryo rescuing medium is optimum for small and dud seeds (Ha Thi Thuy et al, 2009) that is basal medium (Murashige and Tucker, 1969) adding 1.0 mg/l GA3, 15% coconut liquid, 30 g/l sucrose and 5 g/l agar. Identifying ploid level by Flow Cytometry machine, following the method of Ollitrault et al, 1992 procedure and machine provider󰜚s guidance Partec, Germany. Solution for separating and dying samples was spplied by Partec, Germany. 2.3.2. Assessment of agro-biological characteristics of triploid pomelo and Citrus nobilis lines in nethouse and fields. Assessment of morphology was carried out following the guiden documents of International Plant Genetic Resources Institute document and procedure for testing pomelo, grapefruit and orange of Ministry of Agriculture and Rural Development (10TCN-2007). The traits are observed: shape of canopy, diameter of stem, branching, stipule density, leaf morphology, buds growth, morphology and time of flowers and fruits, factors contributing productivity and quality. The morphology and structure of flowers includes tension of petals, color of anthers and pollen have observed by naked eyes and microscope Leica DMLS2 at magnitude 40 x 0.65 and 100 x 1.25 magnification. Pollen surviving and pollen germination ratio have been assessed. Pollen germination was assessed with 10 repeats per one line and varieties. Flowers was collected and reserved in petri dishes before blossoming 1 to 2 days. When flowers blossom, they was dyed by aceto carmine (Domingues et al, 1999) and observed on microscope to evaluate pollen surviving. Identifying pollen germination ratio by culturing on artificial medium with 5 repeats followed the method of Shivanna, K.R, 2003. 2.4. Satiscal analysis Data are analyzed by excel 2007 CHAPTER 3 RESULTS AND DISCUSSION 3.1. Generating initial materials for selecting seedless and fewer seed citrus varieties 3.1.1. Hybridizing between two different ploids of same and different varieties and different species We have implemented 8 hybridizing pairs on 2011 flowering season. The results have shown on the table below. Mating pairs Fruit ratio (%) 󰵁 CS2x × 󰵃 CS4x 36.6 8 󰵁 BD2x × 󰵃 BD4x 33.3 󰵁 NR2x × 󰵃 BD4x 10.0 󰵁 PT2x × 󰵃 BD4x 󰵁 NR2x × 󰵃 PT4x 󰵁 BD2x × 󰵃 PT4x 󰵁 PT2x × 󰵃 PT4x 󰵁 CS2x × 󰵃 VD4x 󰵁 CS2x × 󰵃 CS2x 13.3 󰜔 23.3 Then, the seeds from hybridizing ripen fruits of 2x and 4x pummelo mating pairs have separated. The results are shown on the table below. Hybridizing pairs Number of seeds Number of seeds (control 2x) 󰵁 BD2x × 󰵃 BD4x 19.1 (2 seeds with normal embryos per 1 fruit) 76 (all seeds with normal embryos) 󰵁 NR2x × 󰵃 PT4x 64.3 (5.5 seeds with normal embryos per 1 fruit) 118 (all seeds with normal embryo) Seeds from 6 hybridizing pairs are almost empty and no embryos freom 89.5 to 91.4% which are useless. Seeds with embryos remained 8.6 to 10.5% which are sterilized to culture on suitable medium for plantlet regeneration. We are also conducted hybridizing between diploid and tetraploid varieties on orange especially King Mandarin (Citrus nobilis). This is commercial varieties and grown widespread in Northern provinces and Cuu Long delta area. However, they contain many seeds. The number of average seeds of hybridizing pairs have shown on the below table. Mating pairs Number of average seeds per one fruit 󰵁 CS2x × 󰵃 VD4x 18.7 󰵁 CS2x × 󰵃 CS4x 21.5 󰵁 CS2x × 󰵃 CS2x 22.1 Seeds per fruit of all hybridizing pairs are included normal embryo and no embryo seeds, very few of small seeds have been obtained. The number of small seeds and normal seeds of some pairs are shown in the following table. Pairs Number of small seeds Normal seeds 󰵁 CS2x × 󰵃 VD4x 3.88 󰵁 CS2x × 󰵃 CS4x 2.72 󰵁 CS2x × 󰵃 CS2x 2,75 72.03 󰜔 76.13% Small seeds with embryos are the least 7.63% in 󰵁 CS2x × 󰵃 CS4x pairs and the highest 15.47% in 󰵁 CS2x × 󰵃 VD4x pairs. Dud seeds with embryos in 󰵁 CS2x × 󰵃 CS2x are 3.40% and in 󰵁 CS2x × 󰵃 VD4x are 5.36%. Dud seeds without embryos in 󰵁 CS2x × 󰵃 CS2x and 󰵁 CS2x × 󰵃 CS4x are from 15.25% and 3.57% in 󰵁 CS2x × 󰵃 VD4x hybridizing pairs. [...]... sunshine in 3 first months Figure 3.21 Triploid Citrus nobilis lines budded on Citrus hystix were testing on fields flowering on Spring season 2014 3.3.2 Testing some triploid pummelo lines budded on Citrus hystix 217 triploid lines were selected from hybridizing, rescuing embryos from seeds and propagation by budded on Citrus hystix have grown for testing, evaluating and selection Characteristics of... These lines are consecutively for testing and evaluating 2 Some triploid pummelo and Citrus nobilis lines budded on Citrus hystix (7 moths) have shown difference about growth, flowering and fruits After budded plants are 6 to 7 years old, 2 triploid Citrus nobilis lines CS.06.02, CS.06.09 and 6 triploid pummelo lines PD.06.11, PD.06.14, PD.07.33, PD.07.34, PD.07.57, PD.07.76 has been blossomed but none... budded on Citrus hystix Triploid Citrus nobilis lines budded on Citrus hystix plants were grown in the field for testing which we have divided according canopy shape (umbrella, global, broom and eclipse shape) into two main types First, plants are vertical branching, small branching angle and canopy shape like broom as the same as control Citrus nobilis (2x) including some triploid Citrus nobilis lines. .. CS2x control including some triploid Citrus nobilis lines CS.05.01, CS.05.06, CS.06.02, CS.06.04, CS.06.09, CS.06.12 (iii) Category 3: middle types, horizontally branching, angle is small, shape of canopy is eclipse, cylinder not the same as control CS2x is CS.05.02, CS.06.06, CS.06.08, CS.07.01, CS.07.02, CS.07.03 Stipule density of triploid Citrus nobilis less than control CS2x Some Citrus nobilis lines. .. year triploid lines budded on Citrus hystix from 24th of January to 15th of February and have been completed on 23rd of Feb to 3rd of March earlier than spring buds of 7 year triploid lines budded on Citrus hystix on 14th of Feb to 3rd of March Summer buds formed intensively from 14th to 28th of May and finished from 1st to 15th of June as same as 7 year triploid pummelo lines budded on Citrus hystix... 67,53 9,87 8,16 15,43 14,57 1,89 1,62 1,13 1,14 1,67 1,12 0 0 23 Figure 3.24 Morphology and construction of fruits of triploid pummelo lines PT.05.01 budded on 5 year sour pummelo plants in 2013 CONCLUSION AND PROPOSALS CONCLUSION 1 We have selected triploid pummelo and Citrus nobilis lines by hybridizing between varieties pummelo, King and Van Du orange (diploid and tetraploid) From 8 hybridizing pairs... These lines are heterosis about phenotype, branching characteristics, variety about angle of branching, stipule density, healthy growth and development compared with diploid Dien and Phuc Trach and tetraploid pummelo control We are consecutively testing these lines on the field 3.3 First steps of testing some triploid pummelos and Citrus nobilis lines 3.3.1 Testing some triploid Citrus nobilis lines. .. appearance is the least and contributed 1 to 4 percent of total To sum up, from 22 triploid Citrus nobilis lines were tested and assessed, we have selected some potential lines compared with control Citrus nobilis (2n) including CS.05.03, CS.06.02, CS.06.12, CS.07.01, CS.07.03, CS.06.08 which are versatile about phenotype, healthy growth and development, non-disease Some lines started flowering in 2014... diploid Citrus nobilis and tetraploid control 1 2 3 4 Figure 3.10 Triploid Citrus nobilis lines in nethouse 1 2 3 4 Figure 3.11 Triploid pummelo lines in nethouse 3.2 Assessment of agro-biological characteristics and selection of triploid Citrus noblis and pummelos in nethouse 3.2.1 Assessment of agro-biological characteristics and selection of triploid Citrus noblis in nethouse 22 triploid Citrus nobilis... above control are the important genetic materials for selecting seedless citrus, are very diverse about genotype of each individual which has one genotype and will become a different triploid lines by propagation 11 12 3.1.3 Agro-biological characteristics assessment of triploid pummelos and Citrus nobilis lines selected from 2011 After characterizing some agro-biological traits of pomelo and Citrus . have conducted a dissertation: Research on selecting some triploid lines in citrus varieties * Aims of research To produce a larger number of triploid lines of pummelos and King Orange (Citrus. Testing for triploid Citrus nobilis lines budded on Citrus hystix - Testing for triploid pummelo lines budded on Citrus hystix - Evaluation results of triploid lines budded on 5 year old of sour. pummelo control. We are consecutively testing these lines on the field. 3.3. First steps of testing some triploid pummelos and Citrus nobilis lines 3.3.1. Testing some triploid Citrus nobilis lines