RESEARCH ON SELECTING SOME TRIPLOD LINES CITRUS VARIETIES

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CATION G 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/QDTT-VPVH, 12th, 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, 12th, 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 PT2x × PT4x Phuc Trach Pummelo(2x) × Phuc Trach 1 Pummelo(4x) BD2x × PT4x Dien Pummelo(2x) × Dien Pummelo(4x) 2 NR2x × PT4x Nam Roi Pummelo(2x) × Phuc Trach 3 Pummelo(4x) PT2x × BD4x Phuc Trach Pummelo(2x) × Dien 4 Pummelo(4x) BD2x× BD4x Dien Pummelo (2x) × Dien Pummelo (4x) 5 NR2x × BD4x Nam Roi Pummelo (2x)× Dien Pummelo (4x) 6 CS2x × CS4x Cam Sanh (2x) × Cam Sanh (4x) 7 C. nobilis 2x × C. nobilis 4x CS2x × VD4x Cam Sanh (2x) × Van Du Orange (4x) 8 C. nobilis 2x × C. sinensis (L.) Osbek 4x 9 CS2x × CS2x Cam Sanh (2x) × Cam Sanh (2x) (control) 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 13.3 23.3 PT2x × PT4x CS2x × VD4x CS2x × CS2x 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 76 (all seeds with normal embryos per 1 fruit) embryos) NR2x × PT4x 64.3 (5.5 seeds with normal 118 (all seeds with normal embryos per 1 fruit) 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 72.03 76.13% CS2x × CS4x 2.72 CS2x × CS2x 2,75 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. 9 From the above results, we have catalogued them into four groups including normal, small embryos and dud seeds with no embryos. Small and dud seeds with embryos were cultured on appropriate medium to regenerate plantlets and then these plantlets of ploid types were indentified. 3.1.2. Rescuing embryos to produce as source of initial material Medium for embryo rescuing have been modified to be suitable for culturing embryos of small and dud seeds (Ha Thi Thuy et al, 2009) are MT (Murashige and Tucker, 1969) adding 1.0 mg/l GA3 + 15% coconut liquid + 30 g/l sucrose + 5 g/l agar. The development of these seeds from hybridizing pairs (between diploid and tetraploid types of same and different cultivars) are shown very different. Ratio of germination of normal seeds of different variety pairs and different ploid level ( BD2x × PT4x) showed minimum by 81.8%. Otherwise, the germinated ratio of 5 hybridizing pairs of same and different varieties and two different ploid level were higher by 90%. The results for Citrus nobilis is lowest 33,3% to 44.4% for dud seeds and 75,0% to 88.9% for small seeds. The plantlets after rescuing embryos on invitro medium are on the below table. Same variety Number of plantlets PT2x × PT4x 24 BD2x × BD4x 18 CS2x × CS4x 21 CS2x × CS2x 7 Different variety BD2x × PT4x 9 NR2x × PT4x 20 PT2x × BD4x 17 NR2x × BD4x 15 Different species CS2x × VD4x 26 After culturing in vitro 1 month (plantlets height from 2.5 to 4.0 cm and had secondary roots), these plantlets have washed and grown in fresh sand trays containing 75 holes (2.5x2.5cm) each then after 15 days that are covered by plastic pieces. After 15 days, they were removed and we examined and counted the number of remained plantlets. The plantlets after transplanting are highly survived about 80% for pummelo lines and above 42,8% for Citrus nobilis. After one month transplanting, we have tested the ploid level of them and the results are shown on the belowtable. Same variety Number of triploid plantlets PT2x × PT4x 21 BD2x × BD4x 15 (2 tetraploid) CS2x × CS4x 9 CS2x × CS2x 1 10 Different variety BD2x × PT4x 9 NR2x × PT4x 18 PT2x × BD4x 15 NR2x × BD4x 14 Different species CS2x × VD4x 12 114 triploid plantlets and 2 tetraploid from 8 hybridizing pairs and the 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 nobilis lines, we have selected 8 triploid Citrus nobilis line CS.11.02, CS.11.06, CS.3x.11.01, CSV.11.02, CSV.11.05, CSV.11.08, CSV.11.11, CVS.11.12 and 9 triploid pummelo lines PT.3x.11.08, PT.3x.11.14, DT.3x.11.03, NT.3x.11.17, BD.3x.11.13, ND.3x.11.01, ND.3x.11.03, PD.3x.11.02, PD.3x.11.15. These are promissing lines and heterosis about growth, well-branching, varied phenotypes stipule density ranged from normal to dense compared with Dien, Phuc Trach pummelos, 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 lines have branched in many different types which are divided into 3 main categories. 13 (i) Category 1: in vertical, branching angle is small, the canopy is umbrella type as same as CS2x control including some triploid Citrus nobilis lines CS.05.03, CS.05.04, CS.05.05, CS.06.01, CS.06.03, CS.06.05, CS.06.07, CS.06.11, CS.07.04. (ii) Category 2: branching horizontally, angle of branching is larger, the canopy is flat different from 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 developed from germination of seeds have many stipules. 8 year of Citrus nobilis lines exhibited 2 to 6 primary branches and 6 to 14 secondary branches. The smallest primary branch diameter is 1.3cm belong to CS.05.06 and highest is 2.9cm CS.05.05. These CS.05.06, CS.05.01, CS.05.03 are highly branching. The highest height is 210.4 cm (CS.05.06 lines) and lowest is 142.8 cm (CS.05.04 lines). The stem diameter of Citrus nobilis lines are varied from highest 5 cm (CS.05.01) and lowest is 2.9 cm (CS.05.04). There are also different between canopy diameters from 70 cm (CS.05.04) to 165.6 cm (CS.05.06). 7 year of Citrus nobilis lines exhibited 2 to 6 primary branches and 6 to 14 secondary branches. The diameter of primary branches varied from 1.3 cm (CS.06.01) to 3.5 cm (CS.05.10). Some triploid Citrus nobilis lines CS.06.04, CS.06.11, CS.06.02, CS.06.10 and CS.06.12 are highly branching. The height of these triploid lines ranged from 90.7 cm (CS.06.12) to 222.8 cm (CS.06.04). The stem diameter of these lines are significantly different from 2.5 cm (CS.06.01) to 6.5 cm (CS.06.10). The canopy diameter are from 60.7 cm (CS.06.01) to 164.7 cm (CS.06.04). 6 year of Citrus nobilis plants exhibited 2 to 5 primary and 6 to 9 secondary branches. The smallest primary branch diameter is 1 cm (CS.07.03) and highest is 2.5 cm (CS.07.04). Some lines such as CS.07.02, CS.07.01, CS.07.04 is highly branching. The height ranged from 112.3 cm (CS.07.02) to 192.6 cm (CS.07.04). There is significant deviation about stem diameter from 2.5 cm (CS.07.01) to 4.4 cm (CS.07.04). The canopy diameter varied from 90.4 cm (CS.07.02) to 147.3 cm (CS.06.03). There are 4 shape types about morphology of leaf including oval shape (diploid and tetraploid Citrus nobilis leaves) and ovate, eclipsoid, are different from normal and tetraploid Citrus nobilis. However, the color of the leaves is dark green the same between 3 types of Citrus nobilis lines. The length of blade varied from 7.16 cm (CS.05.04) to 10.42 cm (CS.05.01), width ranged from 4.82 cm (CS.05.03) to 6.38 cm (CS.05.01). The leaflets are not significantly different between triploid lines. 14 The length of 8 year triploid Citrus nobilis line blades is from 7.16 cm (CS.05.04) to 10.42 cm (CS.05.01), the width is from 4.82 cm (CS.05.03) to 6.38 cm (CS.05.01). For 7 year triploid Citrus nobilis lines, the length of blades is from 7.56 cm (CS.06.05) to 9.96 cm (CS.06.08), the width from 4.04 cm (CS.06.04) to 5.58 cm (CS.06.10). For 6 year triploid Citrus nobilis lines, the length is from 5.84 cm (CS.07.02) to 8.58 cm (CS.07.04), the width is from 3.58 cm (CS.07.01) to 5.66 cm (CS.07.04). * Morphology of flower Citrus nobilis (2x) flowers: white, curled big petals with length and width 1820 x 7 mm, 5 small green sepals (3 to 4 mm) which are whorled and cover by layer of thin fiber. The petiole is small with diameter 1 mm. There are 20 stamens sticking together and separating at the top. The anther is ovate, yellow adhered back. The ovary is global, oil gland is small and comfortable scent. Stamens are longer than stigma. Triploid Citrus nobilis lines: white, curled big petals with size length and width 18-20 x 7 mm, 5 small green sepals (3 to 4 mm) which are whorled and cover by layer of thin fiber. The petiole is small. There are 24 to 26 stamens lengthened 8.2 mm, separating at the top with ovate, yellow, adhered back anthers. The ovary is global. Style length is 9.5 mm and little curled. Stigma is global. Flower with scent, stamens are shorter than stigma. Tetraploid Citrus nobilis (4x). Flowers are white, scent and 4 to 5 green sepals, 4 to 5 white petals lengthening 1.21 cm, white filaments, yellow and ovate anthers. The number of filaments is about 24 to 28 per flower, separating with the length is 8.0 mm. The length of style is 7.0 mm. Stigma is global. Stamens are longer than stigmas. The significant difference between other types of flower is implied the size of flower and the length of filaments. The size of triploid flowers is bigger than diploid and smaller than tetraploid type. The length of filaments of triploids is shorter than style. Two other ploid type are contradict. The flowering time of triploid Citrus nobilis start from 21st to 28th of January. All of flowers blossoming from 14th to 19 of February and completing from 19th to 24th of February. To recapitulate, from 22 triploid Citrus nobilis lines, we have selected 2 lines namely CS.06.02 and CS.06.09 which are flowering but no fruits producing from hybridizing pairs CS2x × CS2x to be continuing testing in the field. 15 1 2 3 Figure 3.14. Flower morphology of Citrus nobilis lines in 2013 1. Tetraploid 2. Triploid 3. Diploid 3.2.2. Agro-biological characteristics and selection of triploid pummelo lines in nethouse 44 triploid pummelo lines were selected from mating pairs BD2x × PT4x in 2006 were characterized some traits such stipule density, branching, branching angle, canopy shape to compare to control Dien and Phuc Trach pummelos. 7 year of triploid pummelo lines have 2 to 5 primary branches and 5 to 19 secondary. The diameter of primary branches ranged from 3.2 cm (PD.06.16) to 7.6 cm (PD.06.09). Some lines are highly branching for example PD.06.39, PD.06.28, PD.06.27. The height of triploid plants ranged from 165.5 cm (PD.06.16) to 313.20 cm (PD.06.11). The diameter of stem is not different between other lines. The diameter of canopy varied from 96.70 cm (PD.06.16) to 256.50 cm (PD.06.42) (Table 3.13). 6 year of triploid pummelo lines were varied about phenotypic types, canopy, stipules density and leaf types. In average, there are 2 to 8 primary branches and 6 to 24 secondary. The diameter of primary branches ranged from 1.5 cm (PD.07.04) to 6 cm (PD.07.71). Some triploid pummelo lines have highly branching ability such as PD.07.64, PD.07.65 and PD.07.58. The height is ranged from 150.0 cm (PD.07.48) to 297.7 cm (PD.07.47). The stem diameter of triploid are significantly different ranging from 4.1 cm (PD.07.28) to 9.3 cm (PD.07.38). The diameter of canopy varied from 115.4 cm (PD.07.28) to 312.4 cm (PD.07.53). There are 4 types about leaf tip morphology of 44 triploid pummelo lines (7 years plants) are , and 3 types of shape of leaf blade are ovate, eclipse and oval with dark green color. The length of blade is from 8.36 cm (PD.06.43) to 11.26 cm (PD.06.10). The width of blade ranged from 5.00 cm (PD.06.22) to 7.26 cm (PD.06.10). The leaflet of pummelo lines is seen more clearly than orange and grapefruit lines. The length of leaflets of triploid pomelo lines is relatively long and no significant difference between these lines. Results of observation about 93 triploid 6 year pummelo lines of leaf tip morphology have myriad shapes but most of all is the same above. The blade shapes are included mainly three types (ovate, eclipse and oval). The color of blade is dark 16 green. The length of blade varied from 7.92 cm (PD.07.24) to 12.50 cm (PD.07.04). The width of blade is ranged from 5.00 cm (PD.07.67) to 8.00 cm (PD.07.01) larger than Dien and Phuc Trach control pummelo plants. * Characteristics and morphology of flower Characteristics of diploid Dien pummelo (2x): flowers are very scent and 2 types of flowers (single and pack). Packs of flowers contain 5 to 7 flowers. Sepals are 4 to 5 green star-shape. Flowers have 4 to 5 white petals with length about 20.1 mm, on the petals containing small pockets of oil. Filaments are white. Anthers are orange yellow and ovate. There are 24 to 26 filaments per one flower, arranged in bundles with 3 to 5 filaments per one bundle. The style is 14.0 mm which is shorter than stigma 14.5 mm. The stigma is yellow green. Flowers characteristics of tetraploid Dien (4x) are very scent. There are 2 types of flowers; single and pack of flowers. There are 4 to 5 white petals with length about 25.3 mm. Filaments are white. Anthers are orange yellow, ovate with 25 to 26 per on flowers arranging in bundles, each bundles contains 3 to 5 filaments shorter than stigma (filaments 9.7 mm, stigma 14.2 mm). Flowers of diploid Phuc Trach pummelo (2x): flowers are very scent. There are 2 types of flowers; single and pack of flowers. Each pack of flowers contains 5 to 7 flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with length about 17.4 mm. On petals, there are spots containing pockets of oil. Filaments are white. Anthers are orange yellow and ovate arrange in bundles. Each bundles includes 3 to 5 filaments length about 14.6 mm shorter than stigma is 15.0 mm. Flowers of tetraploid Phuc Trach pummelo (4x): flowers are very scent. There are 2 types of flower single and pack of flowers. Each pack of flowers contains 5 to 7 flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with length about 26.8 mm. On petals, there are spots containing pockets of oil. There are 24 to 25 white ovate filaments. Anthers are orange yellow and ovate arrange in bundles. Each bundles includes 3 to 5 filaments length about 11.0 mm which is shorter than stigma is 15.3 mm. Flowers of triploid pummelo plants (3x): flowers are very scent. There are 2 types of flowers; single and pack of flowers. Each pack of flowers contains 6 to 8 flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with length about 20.3 to 24.8 mm and width from 7.2 to 9.9 mm. Filaments are white with 24 to 27 filaments each flower. Anthers are orange yellow and ovate arrange in bundles. Each bundles includes 3 to 5 filaments length from 10.6 to 13.2 mm shorter than stigma is from 11.4 to 14.5 mm. Pollen of diploid and tetraploid pummelo are able to germinate on in vitro medium. In contrast, pollen of triploid are very much but not able to germinate on in vitro medium. After observation on flowering time in 2 seasons 2012 and 2013. We have found that there no difference between triploid lines and control about flowering time. In 2012, flowers of triploid pummelo lines have started blossoming from 27th of 17 January to 3rd of Feb and finished from 25th of Feb to 2nd of March. In 2013, they started and finished earlier than 2012 about 3 to 5 days. 1 1 1 2 3 Figure 3.18. Flowering triploid pummelo lines in 2013 (1, 1, 1). Flowers of triploid lines, (2). Flowers of diploid Phuc Trach, (3). Flowers of tetraploid Phuc Trach In summary, we have assessed 137 triploid pummelo lines selected from hybridizing pairs BD2x × PT4x from which we have obtained 6 potential lines including PD.06.11, PD.06.14, PD.07.33, PD.07.34, PD.07.57, PD.07.76. After 6 7 years, these lines have been flowering but not fruiting. 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 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 CS.05.03, CS.05.04, CS.05.05. Second, plants are horizontally branching, large branching angle and global canopy shape different from control CS2x including CS.05.01, CS.05.02, CS.06.02. Stipule density of triploid Citrus nobilis lines is on average level different from the control Citrus nobilis (2x) without stipules. Stipule appearance reflects propagation progress and characteristics of varieties. Fruit plants have grown from seeds showing many stipule after propagation (grafting and layering) over years stipules are decreased, in some cases, no stipules left. Some triploid Citrus nobilis lines are selected by hybridizing and propagation by budded on Citrus hystix so they still have genetic characteristics from mother with average stipule density. Three year of Citrus nobilis lines height ranged from 112.47 cm (CS.06.11) to 231.53 cm (CS.07.03). The stem diameter is not different between lines. The canopy diameter is varied from 95.06 cm (CS.06.11) to 198.32 cm (CS.06.02). Growth assessment of experimented Citrus nobilis lines was shown by appearance of buds. In the first development stage (build-up stage), in one year, triploid Citrus nobilis plant has grown 3 to 4 bud times including spring, summer, 18 autumn buds and winter buds is rarely. Spring buds appear from 10th to 25th of February and finish in the end of February. Summer buds appear from 16th to 27th of May and complete from 10th to 15th of June. Autumn buds show up from 22nd to 28th of August and finish from 21st to 23rd of September. Growth of spring buds were observed frequently in the year and we concluded spring buds were appeared most constituted 70 to 75 percent of bud total in a year. Some parameters expressed spring bud growth such as length of buds from 11.07 cm to 26.60 cm, diameter from 0.4 cm to 0.66 cm, number of leaves from 5.33 to 11.67. Time for spring bud matured is about 30 to 35 days. Summer buds appeared less than spring because the nutrition was focused on spring bud growth. Summer buds constituted 5 to 9 percent of year bud total. Summer buds are relatively healthy and strong with length from 16.46 cm to 30.56 cm, diameter from 0.32 cm to 0.57 cm, number of leaves from 8.00 to 16.33, time for maturing from 35 to 40 days. Autumn buds contributed 12 to 14 percent of year bud total. Growth of autumn buds were shown by such parameters such as length from 12.27 to 21.67 cm, diameter from 0.26 to 0.43 cm, number of leaves from 7.00 to 12.67, time for maturing from 29 to 32 days. Rate of winter bud 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 (3 year plant) but not produced fruits because of raining, high humidity and no 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 branching, canopy shape of 217 triploid pummelo lines are very diverse. Stipule density of triploid lines is more than control such as Nam Roi, Phuc Trach, Dien (2x) pummelos and tetraploid control without stipules. Height of three year triploid pummelo plants (2013) ranged from 121.47 (PD.06.84) to 290.80 cm (PD.05.10). The stem diameter is from 3.10 (PD.05.07) to
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