Nghiên cứu chọn tạo và biện pháp nâng cao năng suất, phẩm chất dòng dưa hấu (citrullus vulgaris l ) tam bội in vitro tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Crop Science Code: 62 62 01 10 TRAN THANH TRUYEN INVESTIGATE ON BREEDING AND MEASURING TO IMPROVE YIELD AND QUALITY OF TRIPLOID WATERMELONS (Citrullus vulgaris L.) IN VITRO Can Tho, 2019 The thesis was completed at College of Agriculture and Applied biology, Can Tho University Scientific supervisors: Prof Dr Lam Ngoc Phuong The thesis is defended in front of the University Examination Council in Can Tho University Time:………… …………… Date:…………….…………… Reviewer 1: Reviewer 2: Further information of the thesis could be found at: Learning Resource Center of Can Tho University National library of Vietnam LIST OF PUBLIC WORKS Tran Thanh Truyen, Lam Ngoc Phuong and Ngo Phuong Ngoc 2014 Effect of Benzyl adenine (BA), Indole butyric acid (IBA) and activated charcoal on the shooting and emergence of triploid watermelon (Citrullus vulgaris Schrad.) in vitro Journal of Science, Can Tho University 2014: 168-172 Tran Thanh Truyen and Lam Ngoc Phuong 2017 The result of planting triploid seedless watermelon lines in Mekong Delta Journal of Agriculture and Rural Development (Crop), Volume 1: 108-114 CHAPTER 1: INTRODUCTION Watermelon (Citrullus vulgaris L.) is one of the most popular crops in the world Since the reign of King Hung Vuong, Vietnamese people have been growing watermelons as one of the traditional crops, till then, the production of watermelons in Vietnam keeps going on and it was ranked at the fifth in exports (FAOStat, 2012) Watermelon is an ideal tropical fruit for hot summer days, however, the significant disadvantage of the diploid watermelons is that they contain too many seeds (Donald, 2012) Therefore, seedless triploid watermelons (3X-triploid) have become a favorite choice due to its seedless convenience and sweet flavour Although the price for these seedless fruits is quite higher than the usual diploid watermelons, it is still acceptable for the customers (Marr and Gast, 1991; Maynard et al., 2002) Triploid watermelon is formed by the hybridization method crossing the tetraploid inbreds with diploid watermelon inbreds (Andrus et al., 1971; Kihara, 1975; Guo et al., 2011) The tetraploid watermelons are formulated by treating diploid seedlings with colchicine or oryzalin, in nurseries or in vitro (Kihara, 1951; Raza et al., 2003; Noh et al 2012, Lam Ngoc Phương and Nguyen Kim Hang, 2010) Nevertheless, tetraploid watermelon plants produce a small amount of seeds (lower 10 times than the diploid watermelon plant seeds) and have low seed rate germination In other words, growing tetraploid watermelon plants requires high investment costs (Tran Khac Thi et al., 2008) According to Compton and Gray (1992) and Compton et al., (1993), the method which adapts tissue culture techniques in tetraploid and triploid lines shows that it can reduce the time for propagation (1-2 years) and more convenient for the preservation process than the traditional one In the Mekong Delta, seedless watermelon production has been increased recently but its production expense is high-priced because of using imported varieties mostly, expensive seeds due to its high-demanding in preservation and losing germination easily (Marr and Gast, 1991) As a consequence, it is clearly that growing new seedless watermelon plants in Vietnam has become a necessary mission Therefore, I decided to carry out the research: "Investigate on breeding and measuring to improve yield and quality of triploid watermelons (Citrullus vulgaris L.) in vitro" 1.1 Aims of the thesis: Main objective: to hybrid triploid seeds of Vietnamese origin Specific objectives: (1) produce the tetraploid watermelon with colchicine and oryzalin in vitro (2) find the suitable environment for cloning and evaluate the ability to grow and develop the tetraploid watermelons in vitro, and to hybrid triploid seeds in the field conditions (3) find the suitable medium for clonal culture and select triploid watermelon lines for in vitro conditions, evaluate the growth, development, yield and fruit quality of two triploid (3x) cultures in field conditions (4) invest the effects of nitrogen fertilizer rates and plant density on yield and water quality of the tissue-cultured TriP1 triploid watermelon line 1.2 Meaning of the thesis: i) To produce triploid watermelons originated from Vietnam ii) To apply tissue and cell culture techniques to shorten selection time, multiply lines and reduce the cost of tetraploid and triploid seedlings iii) To identify the suitable environment for propagation of tetraploid and triploid watermelons iv) To investigate and evaluate the growth and fruit quality of two triploid watermelon lines forming transplanted tissue in the field v) To have initial evaluation of nitrogen fertilization and planting density on TriP1 triploid watermelons for field planting 1.3 New findings of the thesis i) Creating tetraploid seedlings by tissue culture and multi-nucleotide treatment; planting trees in the field ii) Hybriding triploid hybrids from tetraploid flowers with diploid pollen iii) Finding in vitro culture medium suitable for shoot multiplication, the formation of tetraploid, triploid, and rapid multiplication of seedlings; reduce the initial investment in seedless watermelon production iv) Investigating and evaluating the growth and fruit quality of two watermelon lines forming transplanted tissue in the field in Can Tho and Hau Giang v) Initially evaluating nitrogen fertilization rates and planting density is appropriate for the growth, yield and quality of the transplanted tissue culture in the field CHAPTER 2: LITERATURE REVIEW 2.1 The scientific basis to make polyploid with colchicine and oryzalin The chemical formula of colchicine is C22H25O6N which is extracted from a plant called Colchium autumnale L These plants are usually grown on the Mediterranean coast The pure colchicine powder is ivory white, soluble in water, alcohol, chloroform Colchicine is highly durable and can be sterilized in the autoclave, but it is easily decomposed in the light so it should be stored in the dark Colchicine is a poison that is paralyzing and should be very careful when working with it (Tran Thuong Tuan, 1992) Oryzalin has the chemical formula as C12H18N4O6S, which is a Dinitroaniline herbicide It is an orange-yellow solid with a melting point of 141-142°, its molecular weight is 346.35 and it is dissolved in water at 2.5 ppm under 25°C Oryzalin can easily soluble in organic solvents such as acetone, ethanol, methanol and acetonitrile, but less soluble in benzene and xylene It is insoluble in hexane Oryzalin is used to stimulate multiplicity in many crops (Morejohn et al., 1987) Oryzalin has been shown to inhibit cell division in plant species and was first registered in the United States in 1974 The effect of oryzalin on cell division is similar to that of colchicine, which affects the disulphite bond of the protein and the ribose molecule of the ribonucleic acid In the process of cell division, the decongestant stops working because it forms a microtubule-chemical complex, which prevents later division of the cell and the later partisation of the cell while chromosomes and chromate formations are still normal In the middle of the meridians, the chromosomes are not distributed on the equatorial plane of the spindle, the chromosomes are shortened and thickened Disagreement of the opposite-polar chromosomes in the lateral phase did not occur, the cell did not divide, although the number of chromosomes was doubled The size of these cells is larger than that of normal cells When stopping the action of chemicals, the cell is able to divide normally and form tetraploid cells (Tran Thuong Tuan, 1992; Dolezel et al., 2004) However, the activity associated with microtubules of oryzalin is tighter than colchicine Plants treated with oryzalin at micromolar concentrations (μM) gave the same results as those treated with colchicine at millimolar concentrations (Dolezel et al., 2004) On the other hand, oryzalin mainly penetrates through the cut surface and enters the epidermis and through the cuticle on the surface of the leaf cells (Allum et al., 2007) 2.2 Determination of polyploidy Shape characteristics, growth and development: leaf morphology is a useful criterion for identifying tetraploid sources before recombinant selection (Jaskani et al., 2005) The tetraploid watermelon leaves are larger, thicker and darker than the diploid plants’ (Kihara, 1951) In addition, the length width ratio of tetraploid leaves (1.00±0.02 and 1.02±0.02) were lower than those of diploid leaves (1.28±0.02 and 1.30±0.02) on both Giza and Giza 21 watermelons under in vivo conditions (Nasr et al., 2004), the tetraploid leaf area was larger than the diploid leaves of all observed varieties The TPS watermelon has larger flowers, the fruit weight is also large, average kg and has about 70 seeds/fruit, dark red fruit pulp, hard and sugar ratio about 12.5% (Karchi et al., 1981) In addition, shell thickness and number of seedlings of tetraploid plants were higher than those of diploid (Ahmad et al., 2013) Flowering time is also later than diploid (Pradeepkumar, 2011) Stomatal coefficients/leaf area and stomatal length: the difference in stomatal number is the criterion to distinguish diploid (2n) and tetraploid (4n) plant cells as well as to identify polymorphisms (Hamill et al., 1992) The size of stomatal cells on tetraploid sugarbaby watermelon (as determined by nucleotide DNA analysis) is greater than that of diploid plants (Thayyil et al., 2016) This method is fast, inexpensive, does not require sophisticated equipment and has a high accuracy rate (in some cases up to 90%, Cohen and Yao, 1996) However, this is just an indirect method for evaluating polyploidy If the plant is in the state of polyploidy, this method is not reliable It should be combined with other methods (Chen et al., 2006) Counting Chromosomes: Chromosomal counting is one of the most direct and accurate methods to determine polyploidy levels, but early and preliminary selection will be time-consuming In addition, chromosome counting will also be timeconsuming, laborious, and difficult to perform on plants with large chromosomes and small cell sizes In particular, this method is difficult to implement for watermelons because of the small chromosome size (Jaskani and Khan, 2000) Analysis of flow cytometry with Partec Ploidy Analyzer The method of analysing flow cytometry is more advantageous than chromosome counting (Leus, 2005; Loureiro et al., 2005) It is convenient to sample preparation and is quick to carry out because it requires only a small amount of sample One of the reasons flow cytometry is more advantageous is because it only requires a small sample size and is quick to prepare The method is a simple analytical process that performs analysis on multiple samples at the same time Different types of tissue can be analyzed such as leaves, roots, stems, petals, seeds and fruits, etc without cell division stage identification The cost of sample analysis by analysing the flow cytometry is acceptable The initial cost for the analyzer is noticeable, however, the materials and chemicals involved are inexpensive Currently, in the world, flow cytometry analysis is one of the most important tools in evaluating the multiplicity of produced seed specimens, and many researchers study into this method (Faten et al., 2012; Jaskani et al., 2005) However, this method meets many obstacles such as expensive equipment and unobservable characteristics of the chromosomes 2.3 Triploid watermelons (seedless watermelons): triploid watermelon was first produced by Kihara and Nishiyama in 1939 by using colchicine to produce 4x form and hybridize with diploid pollen For watermelon to grow to normal size without hollow hearts inside it is necessary to perform artificial pollination (Mark Arena, 2012; Johnson, 2014) Therefore, it is essential to plant alternately between 25% and 33% of diploid watermelon plants to ensure fruit pollinating success (Olson et al., 2012; Fiachino and Walters, 2003) Triploid watermelon hybridization requires four steps: (1) selection of diploid varieties, (2) production of tetraploid plants, (3) development of pure tetraploid and diploid varieties and (4) cross-breeding of triploid hybrids (tetraploid mother plant x diploid pollen) and planting triploid hybrids (pollination will complement diploid pollen) (Wehner, 2008) 2.4 The Role of Micropropagation in Crops: micro-propagation is a powerful tool to multiply herbaceous plants (medicinal plants, fruit trees, food crops, ornamental plants, etc.), reducing the cost of F1 hybrids, especially for triploid hybrids such as seedless watermelon It is highly beneficial to compare with the use of other traditional methods, contributing to the protection of food security and the fight against global climate change This method has overcome the limitations of annual seed production such as: poor grain yield, high price, low seed germination and weak seedlings (Nguyen Bao Toan, 2005; Lam Ngoc Phuong, 2012) 2.5 Impacts of planting density and nitrogen fertilizer on yield and quality of watermelons: watermelon planting density varies in each region and with each variety The shorter the distance, the higher plant density, which will increase the yield of seedless watermelons compared to low density (Motsenbocker and Arancibia, 2002; Walters, 2009; Strang et al., 2005) Fertilizers are significant in increasing yield and quality of watermelons Providing adequate protein will result in effective photosynthesis, strong growth and dark green leaves Increasing the nitrogen concentration will raise the yield of fruit, Brix degree and lycopene content in watermelons However, when the nitrogen content is too high, watermelon plants will grow strong leaves, but may affect tree stiffness As a result, the plants become succulent, so they are vulnerable to pests and diseases The plants will also fail to fruit, and the fruits will take long time to ripen, become tasteless and are difficult to store after harvested Protein deficiency results in poor growth, shortness, small leaves and small fruits (Pham Hong Cuc, 2007) CHAPTER 3: MATERIALS AND METHODS Experimental study consists of main contents (Figure 3.1) and each period has its own experiments: 3.1 Induction of tetraploid watermelon in vitro by using colchicine and oryzalin on diploid watermelon varieties 3.1.1 Experiment 1: Induction of tetraploid watermelon in vitro by using colchicine Materials: watermelon seeds of two clean-bred diploid varieties including TPS, TPT In which: TPS variety is derived from Sugar Baby variety with round or oval dark green fruit and red flesh TPT variety is originated from the breed of Thanh Long variety with oval green fruit with dark green stripes and red flesh These varieties were provided by the Biophysics-Biochemistry Laboratory The watermelon seeds of the two clean-bred diploids were sterilized and cultured in MS environment in vitro Experimental design: the experiment is carried out under the methodology of one completely randomized factor formula, which includes levels of colchicine processing time (4, 6, days) and one controlled factor (without treatment) with reduplications, each repeats with pots, each pot with 10 top bud samples in vitro Monitoring indicators: indicators on the effects of colchicine on growth (shoot height, shoot/sample quantity, leaf quantity) and on tetraploid plantlets identification after treatment (amount of stomata/mm2, the proportion of plantlets with polyploidy phenotype and determination of tetraploid watermelon by flow cytometry) 3.1.2 Experiment 2: Induction of tetraploid watermelon in vitro by using oryzalin Materials: watermelon seeds of four clean-bred diploid varieties including TPS, TPB, TPT, TPX In which: TPS and TPT varieties are used as in experiment TPB variety is originated from Bao Long variety with oval dark green fruit with stripes and red flesh TPX variety is originated from Xuan Lan variety with oval light green fruit with green stripes, yellow flesh These varieties were provided by the BiophysicsBiochemistry Laboratory The watermelon seeds of the four clean-bred diploid varieties were sterilized and cultured in MS environment in vitro Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula, which includes diploid watermelon varieties TPS, TPB, TPT, TPX and levels of processing oryzalin (48 and 54 hours) and one controlled factor (without treatment) with reduplications, each repeats with pots, each with 10 top bud samples in vitro Monitoring indicators: similar to experiment 3.2 Experiment 3: Evaluation of shooting and growth of two tetraploid varieties TPT and TPS in medium supplemented with BA Materials: the tissue-cultured single shoots of two tetraploid watermelon varieties, TPT and TPS, were selected by analysing flow cytometry, cultured in MS environment weeks before the materials experiment Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula with treatments including tetraploid watermelon varieties TPT and TPS, and three BA concentration levels (0; 0.5; mg/L) and reduplications, each with pots, each pot with samples Monitoring indicators: number of shoots increased/sample, shoot height, leaf/sample increased 3.3 Experiment 4: Evaluation of shooting and growth of two tetraploid varieties TPB and TPX in medium supplemented with BA Materials: the tissue-cultured single shoots of two tetraploid watermelon varieties TPB and TPX (simlar to experiment 3) Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula with treatments including tetraploid watermelon varieties TPB and TPX, two BA concentration levels (0.5; mg/L) and reduplications, each with pots, each pot with samples Monitoring indicators: similar to experiment 3.4 Experiment 5: Evaluation of rooting and growth of tetraploid variety TPT in medium supplemented with IBA and NAA Materials: single shoots of tetraploid watermelon variety TPT tissue-cultured in MS environment after weeks with relatively identical height and leaf quantity Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula with treatments, including two IBA concentration levels (0 and mg/L) and three NAA concentration levels (0; 0.2; 0.5 mg/L) and reduplications for each treatment, each with pots, each pot with samples Monitoring indicators: root quantity (root), root length (cm) 3.5 Experiment 6: Evaluation of rooting and growth of tetraploid variety TPS in medium supplemented with IBA and NAA Materials: single shoots of tetraploid watermelon variety TPS tissue-cultured in MS environment after weeks with relatively identical height and leaf quantity Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula with treatments, including two IBA concentration levels (0 and mg/L) and three NAA concentration levels (0; 0.2; 0.5 mg/L) and reduplications for each treatment, each with pots, each pot with samples Monitoring indicators: similar to experiment 3.6 Experiment 7: Evaluation of rooting and growth of four tetraploid varieties in medium supplemented with IBA Materials: single shoots of four tetraploid varieties tissue-cultured in MS environment after weeks with relatively identical height and leaf quantity Experimental design: the experiment is carried out under the methodology of one completely randomized factor formula, including tetraploid watermelon varieties with reduplications, each reduplication with pots, each pot with samples Monitoring indicators: similar to experiment 3.7 Experiment 8: Evaluation and selection of tetraploid varieties tissuecultured and hybridization of triploid seeds on fields Materials: seedlings of four tetraploid watermelon varieties domesticated on experimental fields in Can Tho University Experimental design: the experiment is carried out under the methodology of one factor of latin square formula including tetraploid watermelon varieties tissuecultured with reduplications in such a way that each reduplication has all tissuecultured tetraploid watermelon varieties correlative to 10 plantlets/varierty/reduplication Experimental location: Hau Giang province Monitoring indicators: growth indicator (vine length, number of leaves/vine quantity, and fruit indicators (successful hybridized fruit quantity, fruit weight, yield) and fruit qualities (fruit pale thickness, Brix degree, triploid seeds/fruit/variety) 3.8 Experiment 9: Evaluation of shooting and growth of triploid varieties tissue-cultured in medium supplemented with BA Materials: Collected from experiment 8, single shoots of triploid watermelon seeds are sterilized and transferred into MS environment in vitro In which, TriP1, TriP2, TriP3, TriP4 are hybridized seeds from tetraploid mother varieties TPX, TPT, TPB and TPS Experimental design: The experiment is carried out under the methodology of two completely randomized factors formula with 16 treatments including four BA concentration levels (0; 0.5; mg/L) and triploid watermelon varieties Each treatment has reduplications, each reduplication with pots, each pot has samples Monitoring indicators: number of shoots increased, shoot height increased 3.9 Experiment 10: Evaluation of shooting and growth of TriP1 triploid watermelon variety in medium supplemented with BA and activated charcoal Materials: tissue-cultured TriP1triploid watermelon variety Experimental design: The experiment is carried out under the methodology of two completely randomized factors formula with treatments including four BA concentration levels (0; 0.5; mg/L) and two activated charcoal concentration levels (0 g/L) Each treatment has reduplications, each reduplication with pots, each pot has samples Monitoring indicators: number of shoots increased, shoot height increased 3.10 Experiment 11: Evaluation of rooting and growth of the three triploid watermelon varieties in vitro in medium supplemented with IBA Materials: tissue-cultured triploid watermelon varieties: TriP2; TriP3; and TriP4 Experimental design: the experiment is carried out under the methodology of one completely randomized factor formula including triploid watermelon varieties (TriP2; TriP3; and TriP4) in environment supplemented with IBA mg/L with reduplications, each reduplication has pots, each pot has samples Monitoring indicators: root number/sample quantity, root length, shoot height 3.11 Experiment 12: Evaluation of root development and growth of TriP1 triploid watermelon varieties in medium supplemented with IBA and activated carbon Materials: tissue-cultured TriP1 triploid watermelon variety Experimental design: the experiment is carried out under the methodology of two completely randomized factors formula with 10 treatments, including five BA concentration levels (0; 0.2; 0.5; and mg/L) and two activated charcoal concentration levels (0 and g/L) with reduplications Each reduplication has pots, each pot has samples Monitoring indicators: root/sample quantity, root lenght, shoot height 3.12 Experiment 13: Evaluation of the growth, yield, productivity and quality of two triploid watermelon (3x) varieties tissue culture in the field Materials: seedlings of tissue cultured triploid watermelon varieties: TriP1, TriP2, TriĐC (control) domesticated on experimental fields in Can Tho Univeristy In which, TriĐC is Mat Troi Do seed, which belongs to Syngenta Company (growing time: 60-62 days (dry season) or 65-67 days (rainy season), average weigth: 3-4kg/per fruit, Brix degree: 12-13%, red flesh, high adaptability, can be planted year round, easy to fruit) Experimental design: the experiment is carried out under the methodology of one completely randomized factor formula including treaments and hybridized triploid watermelon varieties (TriP1, TriP2) respectively and tissue cultured control seed variety (TriĐC) after weeks of domestication, with reduplications, each reduplication with 20 plantlets/variety Experimental location: Can Tho (autumn-winter crops, 2013) and Hau Giang (winter-spring crops, 2013) Monitoring indicators: similar to experiment 3.13 Experiment 14: Study on the impact of nitrogen fertilizer (N) content and planting density on yield and quality of TriP1 triploid watermelon tissue cultured Materials: seedlings of tissue-cultured TriP1 triploid watermelon variety domesticated on experimental fields in Can Tho Univeristy variety had an average stomatal index on diploid plantlets of 233.7 ± 24.4 stomata/mm2, while the TPB was 333.3 ± 24.3 stomata/mm2; TPT was 273.3 ± 17.8 stomata/mm2; TPX variety was 324.2 ± 25.7 stomata/mm2; while the average stomata index in lower polyploidy plantlets: TPS variety was 139.5 ± 19.1 stomata/mm2, TPB variety was 187.6 ± 27 stomata/mm2, TPT variety was 184.4 ± 27.6 stomata/mm2, TPX variety was 208 ± 27.2 stomata/mm2 The results showed that the polyploidy plantlets had less stomatal index due to the longer stomatal length than that of the diploid plantlets This result is consistent with the study of Lam Ngoc Phuong and Nguyen Kim Hang (2010) Nguyen Van Hien (2000) also reported that the stomatal index of TPS diploid watermelon is higher than that of polyploidy plantlets, but the stomatal size is shorter 4.1.2 Polyploidy TPS and TPT watermelons rate (%) after processed with colchicine: polyploidy plant rate was determined by polyploidy phenotype (big stems, big and dark green leaves) combined with counting stomatal index to regconize polyploidy plantlets The results showed that high rates of polyploid formation occured in colchicine treatments, and vice versa Specifically, for TPS variety, the rate of polyploid was highest in the 4-day colchicine treatment regimen (32%) and this rate decreased as the colchicine treatment time increased In which, days treatment was 18% and days was 12% For the TPT variety, the highest polyploid rate was observed in the 6-day colchicine treatment (25%), in the 4-day colchicine treatment, which had a lower rate (19%) and 8-day treatment for the lowest rate (12%) 4.1.3 Polyploidy TPS, TPB, TPT TPX watermelons rate (%) after processed with oryzalin: polyploidy plant rate after processed with oryzalin is determined by polyploidy phenotype (big stems, big and dark green leaves) combined with lower stomatal index compared with control shoots The results showed that high rates of polyploid formation occured in oryzalin treatments The rate increased when the processing time increased compared with unprocessed treatments (0% polyploidy plantlets), statistically significant difference 1% Specifically: TPS variety, in 48 hours, reached 20.7% polyploidy plantlets and in 54 hours reached 29.1% Similarly, TPB variety reached 12.9% in 48 hours and 16.7% in 54 hours TPT variety, in 48 hours reached 12.5% and 54 hours reached 19.3% TPX variety, in 48 hours of processing reached 15.1% and 54 hours reached 21.4% 4.1.4 Determination of tetraploid watermelon by flow cytometry Treatment with oryzalin: after flow cytometry testing, leaf samples in oryzalin treated treatments were analyzed with a 1C peak of 62.78-average cell DNA content in a total of plantlets analysis In the 54-hour treatment, the tetraploid count was 4% with a 1C peak of 121.67 (double DNA content compared with diploid), which was 7% of the 74 polyploid In the treatment of oryzalin 48 hours did not give the tetraploid but only the multiply plants accounted for 12% of the 59 polyploid analysis Treatment with colchicine: The results of the analysis showed that in the noncolchicine treatment the samples were diploid In treatments with colchicine and days, there were no tetraploid and polyploid (corresponding to 114 specimens and 83 specimens analyzed) In the day colchicine treatment, the tetraploid plant was 9% complete in 44 plantlets for analysis In conclusion, the multiplicity of diploid watermelon shoots with colchicine and oryzalin resulted in tetraploid and polyploidy The results also showed that tetraploid 10 ratios increased with increased treatment time in both chemicals In that, with short treatment time on both chemicals not create tetraploid, but with days colchicine makes up 9% tetraploid and with oryzalin in 54 hours make up 4% tetraploid, tested by flow cytometry This is similar in the study by Nasr et al (2004) with a duration of days for high tetraploid yield with colchicine levels of 2000 and 2500 μM/L and oryzalin concentrations of 100 μM/L, while at the same concentrations but with and days without tetraploid plant However, this rate was low and similar in Koh's study (2002) The tetraploid effect was 3.3% -5.5% when treating watermelon shoots with oryzalin (5-60 μM) 4.2 Evaluation of shooting and growth of two tetraploid varieties TPT and TPS in the medium supplemented with BA 4.2.1 Number of shoots increased: at weeks after culture (WAC), Table 4.1 showed that the tetraploid watermelons had a statistically significant increase of 5%, of which TPS was 3.1 shoots and TPT gaining 2.1 shoots Concentration of BA also influenced the number of shoots increased, statistically significant difference of 1% In which, medium supplemented with BA 0.5 mg/L and mg/L gave higher shoots (3.33.6 shoots) than non BA supplemented shoots for the lowest shoots 1.0 shoots Interaction between tetraploid watermelon and BA concentration for shoots increased from 0.7 to 4.3 shoots, but not statistically significant Table 4.1: Number of shoots increased in tetraploid watermelon medium BA (mg/L) at different concentrations at WAC BA concentration (mg/L) Tetraploid watermelon (A) 0,5 TPT 0,7 2,4 TPS 1,4 4,3 Average (BA) 1,0b 3,3a FA * FBA ** FA xBA ns CV (%) 36,9 on supplemented culture 3,3 3,9 3,6a Average (A) 2,1b 3,1a Note: Numbers with the following letters are the same in the same column or row or in the column and row are not statistically different from the Duncan test; ns: not statistically different, *: statistically different at 5% significance level, **: statistically different at 1% significance level 4.2.2 Shoot height: at WAC, shoot height increased between two distinct tetraploid watermelons at a significance level of 5%, of which the TPT line was 1.25 cm and the TPS line was 0.27 cm The medium with or without supplement BA increased shoot height not significantly different, ranging from 0.34 cm to 1.17 cm Similarly, there was no interaction between BA levels and tetraploid watermelons on shoot height, ranging from 0.23 cm to 2.12 cm In conclusion, medium supplemented with BA levels of 0.5 mg/L and mg/L gave higher shoot multiplication, but height and leaf numbers were not significantly different from those without supplementation Similar results were found in Nguyen Thi Phuong Thao et al (2010) and Veysi Okumus et al (2011) 4.3 Evaluation of shooting and growth of two tetraploid varieties TPB and TPX in medium supplemented with BA 4.3.1 Number of shoots increased: at WAC, BA concentrations affected the number of shoots increased, in which the medium supplemented with BA concentration 11 1.0 mg/L achieved 2.2 shoots higher than that of the medium supplemented with 0.5 mg/L BA gain 1.4 shoots, statistically significant difference at 1% level However, between two different tetraploid watermelons, the number of shoots increased without statistically significant difference (1.8 shoots) Similarly, there was no interaction between tetraploid watermelon and BA concentration on the number of shoots increased 4.3.2 Shoot height increased: at WAC, medium supplemented with BA 0.5 and mg/L gave rise height of 0.16 cm and 0.27 cm respectively, but not statistically different Similarly, between two different tetraploid watermelon varieties, the shoot height was not statistically different Interactions between tetraploid watermelon and BA concentrations did not affect the height of shoots Thus, the medium supplemented with BA 0.5 and 1.0 mg/L gave effective shooting on both tetraploid TPB and TPX lines In addition, the interaction between BA concentration and tetraploid watermelon for shoot multiplication is not statistically significant The above results showed that MS medium supplemented BA mg/L suitable for shoot multiplication on tetraploid lines under in vitro conditions 4.4 Evaluation of rooting and growth of tetraploid variety TPT in medium supplemented with IBA and NAA 4.4.1 Root number: Table 4.2 showed that at WAC, supplemented with mg/L IBA supplementation for high root numbers (6.3 roots) was statistically significantly different at 1% additional IBA (1.9 roots) However, medium without NAA gave the highest number of roots (6.5 roots) statistically significant difference at 1% level compared with medium supplemented with NAA (0.2-0.5 mg/L) for the lowest root numbers (2.4-3.6 roots) The interaction between IBA and NAA auxin influences root formation, IBA mg/L does not incorporate NAA for the highest number of roots (12.8 roots), statistically significant difference 1% compared with the remaining treatments (control treatment for the lowest roots-0.2 roots) Table 4.2: Root number of TPT tetraploid watermelon shoot on IBA and NAA supplemented media at different concentrations in WAC NAA (mg/L) concentration IBA concentration (mg/L) 0,2 0,2c 2,0bc 12,8a 2,8b a Average (NAA) 6,5 2,4b FIBA ** FNAA ** FIBA x NAA ** CV (%) 33,2 0,5 3,7b 3,5b 3,6b Average (IBA) 1,9b 6,3a Note: Numbers with the following letters are the same in the same column or row or in the column and row are not statistically different from the Duncan test; **: statistical difference at 1% significance level 4.4.2 Root length: at WAC, medium supplemented with IBA mg/L for high root length (3.55 cm), statistically significant difference at 1% level compared to nonIBA medium (1.35 cm) However, with no NAA medium supplemented for high root lengths (3.05 cm), statistically significant difference was 1% compared with medium supplemented with NAA (0.2-0.5 mg/L) for root length reaching 2.02-2.28 cm 12 The interaction between the two types of auxin IBA and NAA influenced the root length, statistically significant difference of 1% In which, IBA mg/L did not incorporate NAA for the highest root length (5.78 cm), statistically different from the other treatments, in which control treatments for low root length most (0.33 cm) In conclusion, both IBA and NAA factors affected the growth and development of roots on the tetraploid watermelon shoots In particular, medium supplemented with IBA 1mg/L gave high rooting and root length 4.5 Evaluation of rooting and growth of tetraploid variety TPS in medium supplemented with IBA and NAA 4.5.1 Root number: at WAC, medium supplemented with IBA mg/L, the result showed that there were large number of roots (8.1 roots) which was statistically significant difference of 1% compared with the control medium (4.5 roots) NAA concentrations affected the number of roots on the shoots of TPS tetraploid watermelons, in which medium supplemented with 0.5 mg/L NAA gave the highest number of roots (7.2 roots) statistically significant difference 1% compared with the control medium (4.8 roots), but not different from the medium supplemented with NAA 0.2 mg/L (6.8 roots) Interactions between IBA and NAA concentrations had a significant effect on the number of roots, statistically significant at 1% In details, IBA mg/L did not supplement NAA with the highest number of root (9.0 roots), statistically different from the control treatments for the lowest number of root (0.5 roots) and treatments supplemented with NAA 0.2 mg/L without IBA (6.3 root), but not different from the other treatments 4.5.2 Root length: at WAC, medium supplemented with IBA 1mg/L for high root length (3.03 cm), statistically significant difference at 1% compared with no supplemented IBA medium (1.73 cm) Medium was not supplemented and medium was supplemented with NAA (0.2-0.5 mg/L) gave non-statistically significant in root length, ranging from 2.27 cm to 2.51 cm There was an interaction between two types of IBA and NAA auxin on root length, 1mg/L IBA did not incorporate NAA for the highest root length (4.31 cm), statistically significant difference of 1% with all remaining treatments, in which the control treatment gave the lowest root length (0.43 cm) In general, the TPS tetraploid watermelons in medium supplemented with IBA 1mg/L gave a high rooting efficiency (number of roots, root length), and the shoot growth is also good (shoot height, number of leaves) In conclusion, MS medium supplemented with mg/L IBA gave a high rooting efficiency compared with no IBA supplemented medium and also higher than NAA supplemented medium (0.2-0.5 mg/L) This result is similar in the study of Khalekuzzaman et al (2012) which found that the IBA medium of mg/L gave the highest rooting efficiency (100%), the highest number of roots (12 roots) and the highest root length (7.0 cm) on Elite F1 watermelon Similarly, according to Okumus et al (2011), the medium supplemented with IBA mg/L also gave high rooting efficiency on the various watermelon lines 4.6 Evaluation of rooting and growth of four tetraploid varieties in medium supplemented with IBA 4.6.1 Root number: at WAC, different watermelon lines gave different number of roots, in which the TPT line gave the highest number (5.0 roots), statistically significant difference at 1% compared with the other lines (2.0-2.3 roots) 13 4.6.2 Root length: four different watermelon lines also gave different root length at WAC, of which the TPT gave the highest root length (1.38 cm), statistically significant difference at 5% compared with the other lines In conclusion, MS medium supplemented with IBA mg/L gave the effect of rooting on all tetraploid watermelon lines However, the difference in rooting efficiency between the tetraploid lines can be effectively attributed to the formation of tetraploid lines as follows: TPT>TPX> TPS>TPB 4.7 Evaluation and selection of tetraploid varieties tissue-cultured and hybridization of triploid seeds on fields 4.7.1 Vine length, number of leaves: at 21 days after the top had been cut, the TPB variety gave the lowest plant length (129.32 cm), statistically significant different at 5% compared with the TPT (208.02 cm) and TPS (205.5 cm), but not different from the TPX variety (179.91 cm) There was a strong increase in number of leaves in varieties of tetraploid watermelon varieties tissue culture Specifically, the TPX variety was 33.1 leaves; the TPS variety was 34.2 leaves; the TPT variety was 33.1 leaves, and the TPB variety was 30.0 leaves, but between them there is no statistical difference Based on statistical results at the time of growth, we can rank order of the growth of tetraploid watermelon varieties as follows: TPS> TPT> TPX> TPB 4.7.2 Number of hybrids succeeded: using the hybridization method by hand, after picking to harvest and harvesting all tetraploid watermelon lines produced triploid seeds However, the number of succeeded hybrids was low and varied in each line: the TPX line was 35 trees; the TPS line was 23 plantlets; the TPT line was 21 plantlets; and the TPB line was 15 plantlets out of 40 hybrids per line 4.7.3 Fruit weight and yield: the fruit weight of tetraploid watermelon lines ranged from 1.27 kg to 1.44 kg but no significant difference was found between them The yields of tetraploid watermelon lines ranged from 22.29 tons/ha to 26.53 tons/ha, but they were not statistically different 4.7.4 Fruit qualities: Table 4.3 showed that there was a statistically significant difference at 1% between the tetraploid lines tissue culture of Brix degree The TPX line gave the highest Brix degree (9.3%), statistically different from the other tetraploid lines, with TPS and TPB giving the lowest Brix degree (8.2%) There was a statistically significant difference at 5% between the tissue culture tetraploid lines of pale thickness In particular, the TPB gave the highest pale thickness (0,86 cm) which was different from the TPT with the lowest fruit pale thickness (0.73 cm), but not different from the TPS (0.85 cm) and the TPX line (0.81 cm) 4.7.5 Triploid seeds/fruit: triploid seeds are seeds with a slightly inflated top on the shell compared with diploid and tetraploid seeds, often possessing a convex part of the shell (Figure 4.1) This was the distinguishing feature between triploid and diploid seeds Table 4.3 showed that the solid triploid seeds/fruits of the other tetraploid lines at a 1% level of significance, in which the TPT line gave the highest number of solid seeds (57.5 seeds), which was statistically different from the remaining lines (34.3-40.0 seeds) In brief, the four lines of tetraploid tissue culture grown in Hau Giang showed little difference in their ability to grow (vine length, leaf width, fresh vine weight) Fruit quality (Brix degree, fruit pale thickness) is statistically different The combination of growth, yield and fruit quality showed that the order of the tetraploid lines from high to 14 low is as follows: TPX>TPT>TPB>TPS and all four lines were successfully crossed to produce triploid seed Table 4.3: Brix degree (%), fruit pale thickness (cm), and number of seeds/fruit of the four tetraploid watermelon lines tissue cultures grown in Hau Giang Tetraploid lines Brix degree (%) Fruit pale thickness (cm) Number of seeds/fruit TPX 9,3a 0,81a 37,3b TPS 8,2c 0,85a 34,3b b b TPT 8,9 0,73 57,5a TPB 8,2c 0,86a 40,0b F ** * ** CV (%) 1,32 3,89 14,72 Note: Numbers followed by similar symbols in the same column are not statistically different by LSD; *: statistical difference at 5% significance level, **: statistical difference at 1% significance level TPX TPS TPB TPT Figure 4.1: Triploid seeds produced by the four tetraploid varieties watermelon 4.8 Evaluation of shooting and growth of triploid varieties tissue-cultured in medium supplemented with BA 4.8.1 Number of shoots increased: at WAT, incremental shoots of triploid lines differed statistically at 1% significance level The TriP1 line showed the highest number of shoots (3.3 shoots), which is statistically different from TriP2 and TriP4, but not significantly different from TriP3 Concentration of BA increased the number of shoots increased, statistically significant difference was 1%, in which the medium supplemented with BA 0.5 mg/L gave the highest increase (3.9 shoots) difference was found between BA mg/L (3.7 shoots) and non-BA supplemented with the lowest shoot growth (0.8 shoots), but not different from that of the medium supplemented with BA mg/L (3.8 shoots) There was a significant interaction between triploid watermelon with BA concentration on shoot increment, statistically significant difference at 1% level The TriP3 lines, whose medium was supplemented with BA mg/L and TriP4 tissue cultured which was supplemented with BA 0.5 mg/L gave the highest increase (4.7 shoots) Statistically significant difference was found between medium with no supplementation of BA in all four triploid lines tissue culture, demonstrating the lowest increase (0.7-1.0 shoots) observed 15 4.8.2 Shoot height increase: at WAC, increase in shoot height displayed by the lines of triploid watermelons had statistically significant difference at 1% level The TriP2 line gave the highest increase in height (1.26 cm), which was statistically different from the other three lines Concentration of BA influenced height gain whereas medium without BA supplementation demonstrated the highest increase in height (1.36 cm), statistically significant difference at 1% level compared to medium supplemented with BA (0.5; 1, mg/L) The interaction between BA concentration and triploid watermelon yields maintain statistically significance at 1% increase in height Treatment of TriP2 line tissue culture on medium not supplemented with BA gave highest shoot height of 2.71 cm and reduced shoot height in treatments with supplemented BA (0.5-2 mg/L) on all triploid watermelon lines In hindsight, medium supplemented with BA growth regulator (0.5-2 mg/L) gave 34 times higher shoot multiplication than non-supplemented medium in all lines of triploid watermelon, especially MS medium supplemented with BA mg/L for high shoot multiplication In addition, growth was also different in different triploid lines, in which the TriP1 and TriP2 lines gave relatively higher shoots but leaf numbers and shoot height compared with the other two lines 4.9 Evaluation of shooting and growth of TriP1 triploid watermelon variety in medium supplemented with BA and activated charcoal 4.9.1 Number of shoots quantity: in WAC, BA concentrations in cultured medium affected the number of shoots increased by triploid watermelons Specifically, at mg/L BA gain 2.9 shoots, statistically significant difference of 1% compared with control and 0.5 mg/L BA (1.4-2.6 shoots) but not significantly different from medium supplemented with BA 1.0 mg/L (2.8 shoots) Activated charcoal influences the multiplication factor The average of non-supplemented medium was 2.6 shoots, statistically significant difference of 1% compared to medium supplemented with 2.3 shoots There was a significant interaction between BA and activated charcoal concentrations at the 1% significance level Specifically, the BA treatment of mg/Lno activated charcoal the highest number of shoots (3.3 shoots) compared to the other treatments but not significantly different from the BA treatment of 1.0 mg/L-no activated charcoal (3.0 shoots) In addition, the control treatment was not supplemented with carbon and did not supplement BA with the lowest shoot (1.2 shoots) Thus, in general, the medium was supplemented with carbon to reduce the multiplication factor This may be due to the fact that activated charcoal adsorbs the number of shoots, but also absorbs organic compounds, growth regulators in the culture medium (George, 1993) In micro-propagation tetraploid and diploid watermelon, MS medium supplemented with mg/L BA was highly effective for shooting (Kapiel et al., 2004) However, the number of shoots obtained was low compared to many reports of watermelon shoots (Compton et al., 1992) (5-11 shoots); Lam Ngoc Phuong and Nguyen Bao Ve (2006) (6-8 shoots) Although, treatment with mg BA/L gave the most shoots, but the shoots was buds The use of high concentrations of BA for shoot propagation has been recommended by scientists for morphological disturbances, such as the formation of multiple shoots, shoot shoots, the formation of different forms of leaves and stems Due to the high dose of chemicals added to the environment The first experiments on triploid watermelon BA concentration of mg/L produce abnormal shoots (Lam Ngoc Phuong and Nguyen Bao Ve, 2006) 16 4.9.2 Shoot height quantity: at WAC, it was found that activated charcoal did not affect the shoot height of TriP1 triploid watermelon However, the BA concentration influenced the shoot height difference at 1% level Specifically, at the control level for the highest increase (1.4 cm), the statistical difference was 1% level compared with medium supplemented with BA concentration (0.5-2 mg/L) The field has a BA concentration of mg/L for the lowest increase (0.94 cm) This means that when the BA increases, the height decreases This result was also reported earlier when the growth regulator of height of watermelon shoot growth was limited (George, 1993) In general, mg/L BA was suitable for multiplication of TriP1 triploid watermelon At this point, exogenous BA has effect on the shooting, stimulating side shoots so the culture pattern gradually grow small shoots then develop leaves However, BA shoot stimulation was not much as the plantlets were in the rejuvenation stage, so the non-BA medium gave higher leaf height and number In conclusion, for both experiments, the medium supplemented with BA gave high shoot multiplication efficiencies, 2-4 times higher than that without BA supplementation According to Vu Van Trong et al (2007) and Le Duy Thanh (2000) noted that in addition to the nutrient supply to the culture medium, the addition of one or more growth regulators is necessary to stimulate growth and development Organizing, providing good vitality for the tissues, effecting on protein synthesis and enhancing the activity of some enzymes However, this will lead to the phenomenon of abnormal growth, small leaves, crooked, loss of chlorophyll (Debergh and Maene, 1981) and to reduce this phenomenon must reduce the cytokinin content in the cultured medium or in combination with the auxin group (Compton and Gray, 1993) Therefore, the MS medium supplemented with 1.0 mg/L BA was suitable for the shoot growth of triploid watermelon, the number of shoots healthy, without the hyperhydricity, although the shoot height was relatively low due to cytokinine The focus was on lateral tissue fragmentation, rather than on cell stretching and different shoot multiplication effects on different triploid melon varieties At the same time, concentration of BA 1.0 mg/L and activated charcoal 2.0 g/L gave outstanding number of leaves, good tree height after weeks of culture 4.10 Evaluation of rooting and growth of the three triploid watermelon varieties in vitro in medium supplemented with IBA 4.10.1 Root number: at WAC, there was root formation in all three triploid watermelon lines cultured on MS medium supplemented with IBA 2mg/L, statistically significant difference at 5% level TriP2 gave the highest number of roots of 2.9 distinct roots compared to the other two 4.10.2 Root length: at WAC, the root lengths of different watermelon triploid tissue cultures were statistically significant at 1% level In particular, TriP2 gave the highest root length of 2.68 cm different from the other two varieties 4.10.3 Shoot length quantity: there was a statistically significant difference of 1% after weeks of culturing on the height increment of triploid watermelon varieties In particular, the TriP2 variety gave the highest shoot height (1.59 cm) different from the other two triploid varieties In conclusion, culture of triploid watermelon varieties in vitro in MS medium supplemented with mg/L IBA resulted in rooting effect However, the root effect (number, length) was low and the effect was different between 17 different triploid varieties, in which TriP2 gave the highest number of roots, root lengths with shoot height and leaf number 4.11 Evaluation of root development and growth of TriP1 triploid watermelon varietie in medium supplemented with IBA and activated carbon 4.11.1 Root number: Table 4.4 showed that in WAC, there were statistically significant differences in the number of roots between different IBA concentrations and the presence or absence of activated charcoal supplementation In particular, the concentration of IBA from 0.5 to mg/L gave the highest number of distinct roots of 5% compared to the other two concentrations The roots had higher (2 g/L) roots (6.4 roots) than the non-activated charcoal medium (3.2 roots), statistically different at 5% There was a statistically significant 5% interaction between IBA and activated carbon In which the IBA 0.5 and mg/L supplemented with g/L gave the highest number of roots (7.2 and 7.5 roots respectively) statistically different for all treatments no carbon and IBA 0.2 mg/L with carbon, but not with IBA and supplemented with IBA mg/L supplemented with activated charcoal g/L Table 4.4: Root number of TriP1 triploid watermelon varietie in different medium supplemented IBA and activated charcoal after WAC Activated charcoal (g/L) (A) IBA concentration Average (B) (mg/L) (B) e abc 1,6 6,0 3,8b 0,2 1,9e 5,2bcd 3,5b cd a 0,5 4,5 7,2 5,8a cd a 1,0 4,3 7,5 5,9a 2,0 3,8d 6,3ab 5,1a b a Average (A) 3,2 6,4 F(IBA) * F(than) * F(IBA × coal) * CV (%) 27,57 Note: Figures followed by the following symbols are the same in the same column or row or in the column and row, which are not statistically different from the Duncan test; *: statistically significant difference at 5% significance level 4.11.2 Root length: there was a statistically significant differences in root length after weeks of culture between the mediums with 6.61 cm carbon and 2.08 cm with no carbon; as well as high levels of IBA (0.5, 1, mg/L) and low IBA The results are consistent with many results in vitro There was a significant interaction between IBA and activated charcoal concentrations on root length, statistically significant difference at 5% significance level The g/L supplementation carbon gave the highest number of roots compared to the non-carbon supplemented medium with IBA concentrations, the control medium gave the lowest root length Thus, there was a significant difference in rooting rates between treatments with carbon and no carbon, as well as between treatments with high levels of IBA and low IBA, and this difference was statistically significant Therefore, in carbonaceous medium at any IBA concentration, root numbers and root lengths remain distinct from no carbon supplement Similarly, in high IBA medium (0.5, 1, mg/L) also gave the high root number and root length, significantly different from low IBA concentrations, whether or not there was carbon 18 4.11.3 Shoot height: at WAC, the shoot height was higher (4.53 cm) than that without activated charcoal (3.35 cm), no statistically significant difference of 5% IBA concentrations from to mg/L gave no statistically significant increase in shoot height There was a significant interaction between IBA and activated charcoal concentrations on shoot height, statistically significant difference at 5% significance level In these treatments, supplementation of carbon at all concentrations of IBA and non-charcoal treatment with IBA 0.2-1.0 mg/L gave the highest shoot height and control treatments were the lowest (2.6 cm) In conclusion, in the above two experiments, mediums supplemented with IBA and 2.0 g/L activated charcoal increased the root effect on triploid watermelon compared with non-IBA medium Roots as well as root length increased with increasing IBA levels from 0.2 to 1.0 mg/L but tended to decrease with increasing to 2.0 mg/L, thus using IBA mg/L to compare the root effect on three triploid watermelon lines for low root numbers Therefore, medium containing 0.5 mg/L IBA supplemented with activated charcoal 2.0 g/L will be the suitable root-stimulating medium for watermelon triploid in vitro conditions A combination of shooting and rooting experiments found that different triploid varieties gave different shoot and rooting abilities In particular, the triploid TriP1 and TriP2 triploid watermelon varieties gave higher propagation and root formation than the TriP3 and TriP4 varieties with higher shoot height and leaf numbers Therefore, two triploid TriP1 and TriP2 varieties were selected for further investigation of the possibility of growing in the field compared to the “Mat Troi Đo” triploid watermelon variety of tissue culture 4.12 Evaluation of the growth, yield, productivity and quality of two triploid watermelon (3x) varieties tissue culture in the field 4.12.1 Vine length: Table of results 4.5 showed that in Can Tho, the length of three varieties/ triploid watermelon tissue culture was statistically significant at 1% at 21 days after the top had been cut In particular, the TriP1 variety gave the highest vine length of 242.51 cm, the TriDC variety was the lowest of 166.93 cm However, in Hau Giang, the length of the three varieties/triploid watermelon tissue cultures was not statistically different, ranging from 186.6 cm to 240.4 cm Table 4.5: Vine length (cm) and number of leaves/vine of the three varieties of triploid watermelon tissue culture grown in Binh Thuy, Can Tho and Chau Thanh, Hau Giang Cần Thơ Hậu Giang Varieties Number of Number of Vine length (cm)) Vine length (cm)) leaves/vine leaves/vine TriĐC 166,93c 46,6b 204,2 33,9 TriP1 242,51a 73,6a 186,6 27,7 TriP2 230,19b 68,4a 240,4 34,3 F ** ** ns ns CV (%) 1,14 8,17 11,83 10,31 Note: Numbers with similar letters in the same column are not statistically different by LSD; ns: not statistically different, **: statistically different at 1% significance level 4.12.2 Number of leaves/vine: in Can Tho, the number of leaves/vine of the triploid watermelon varieties differed significantly at the 1% level in 21 days after the top had been cut In this, the TriP1 variety gave the highest number of leaves/vine 73.6 leaves, different from the TriDC variety with the lowest number of leaves/vine 46.58 19 leaves, but not different from TriP2 variety However, in Hau Giang, the number of leaves/vine of the three varieties/triploid watermelon tissue culture was not statistically different, ranging from 27.7 leaves to 34.3 leaves (Table 4.5) Regarding the growth of the two varieties of triploid watermelon compared to the control variety (TriDC), can be arranged as follows: TriP1> TriP2> TriĐC in Can Tho The results of Enujeke (2013) also showed a difference in the length and number of leaves/vine of the six watermelon varieties studied in 2011-2012 However, the growth of these three varieties is not different when grown in Hau Giang 4.12.3 Fruit weight: Table 4.6 showed that in Can Tho, between watermelon varieties, fruit weight difference is not significant, ranging from 2.14 kg to 2.2 kg However, in Hau Giang, TriP1 yielded the highest fruit weight of 2.24 kg, significantly different at 5% compared to the reference variety (1.73 kg) and TriP2 (1.55 kg) 4.12.4 Yield: Table 4.6 showed that in Can Tho, yields between the three triploid watermelon varieties were not statistically significant, ranging from 31.76 to 36.54 tons/ha Similar results were reported by Joe et al (2012) when comparing varieties of seedless watermelons This finding is consistent with Strang et al (2004) comparing eight mini seedless watermelon in Kentucky found no statistically significant difference in fruit weight between them The study by Lam Ngoc Phuong and Nguyen Thanh Thinh (2009) also found that both seedless V1 and V2 seedlings in vitro weighed 3.42 kg and 2.85 kg, respectively However, in Hau Giang, yields of three different varieties of triploid watermelon tissue culture were statistically significant at the 5% level In particular, TriP1 yielded the highest yield of 36.91 (tons/ha), which was significantly different from the reference variety (28.49 tons/ha) and TriP2 (25.56 tons/ha) Table 4.6: Fruit weight (kg) and yield (ton/ha) of three triploid watermelon tissue culture varieties grown in Binh Thuy, Can Tho and Chau Thanh, Hau Giang Can Tho Hau Giang Varieties Fruit weight (kg) Yield (ton/ha) Fruit weight (kg) Yield (ton/ha) TriDC 2,20 36,54 1,73b 28,49b a TriP1 2,17 35,48 2,24 36,91a b TriP2 2,14 31,76 1,55 25,56b F ns ns * * CV (%) 2,52 9,95 6,65 6,47 Note: Numbers accompanied by similar symbols in the same column are not statistically different by LSD; ns: not statistically different, *: statistically significant difference at 5% significance level 4.12.5 Fruit quality Brix degree: Table 4.7 showed that in Can Tho, all three varieties of watermelon triploid tissue cultured for Brix degree ranged from 8.73% to 9.69%, the difference was not statistically significant Similarly, at Hau Giang Brix degree of varieties of triploid watermelon tissue culture ranged from 8.52% to 9.67% and also displayed no difference that is statistically significant According to Maynard (2001) sweetness is one of the major quality elements in watermelon and they are related to the TSS The combined results from several studies indicate that the Brix degree in watermelon triploids ranged from 8.31 to 13.4% (Kee and Ernest, 2005) Fruit pale thickness: Table 4.7 showed that the fruit pale thickness of all three varieties of the watermelon triploid tissue culture varied from 0.81 mm to 0.95 mm, but this difference was not statistically significant when grown in Can Tho Similar results 20 were obtained in Hau Giang The varieties of three watermelon triploid tissue culture variations ranged from 0.82 mm to 0.94 mm, but not statistically significant Some studies have shown that the watermelon's hull thickness makes the shells less dense and useful during rough transportation, but the watermelon pale thickness varies with the variety (Thomas et al., 2012) Table 4.7: Fruit quality of three triploid watermelon tissue culture varieties grown in Binh Thuy, Can Tho and Chau Thanh, Hau Giang Can Tho Hau Giang Varieties Brix degree Fruit pale thickness Brix degree Fruit pale thickness (%) (mm) (%) (mm) TriDC 8,98 0,81 8,52 0,94 TriP1 8,73 0,96 9,67 0,82 TriP2 9,69 0,85 9,24 0,83 F Ns Ns Ns Ns CV (%) 14,77 16,22 4,91 27,72 Note: Numbers followed by similar symbols in the same column are not statistically different by LSD; ns: not statistically different, *: statistically significant difference at 5% significance level From the above results, the growth (vine length, number of leaves/vine) in Can Tho of TriP1 variety is better than TriP2 and control varieties but in Hau Giang all varieties are equivalent In fruit weight and yield, in Can Tho all three varieties are equivalent, but in Hau Giang TriP1 is better than TriP2 and control The fruit quality (Brix degree and pale thickness) of all three varieties showed similar results in both survey sites (Figure 4.2) Figure 4.2: Cross section of two triploid strains TriP1, TriP2 and TriDC tissue cultured 4.13 Study on the impact of nitrogen fertilizer (N) content and planting density on yield and quality of TriP1 triploid watermelon tissue cultured 4.13.1 Fruit weight: Table 4.8 showed that the difference in nitrogen yield was statistically significant at 1%, in which nitrogen fertilization of 200 kg/ha resulted in higher fruit weight (2.20 kg) than 150 kg N/ha (1.33 kg) Planting density also affected fruit weight, statistically significant difference at 1% level, with 10,000 plantlets/ha for fruit weight (2.11 kg) higher than low density (8,750 plantlets/ha) (1.42 kg) Similarly, the interaction between nitrogen content and plant density affects fruit weight, statistically significant difference of 1% The M2N2 treatment (10,000 plantlets/ha+200 kg N/ha) gave the highest fruit weight (2.65 kg) statistically different from the other treatments, including the M1N1 treatment (8,750 plantlets/ha +150 kg N/ha) for the lowest fruit weight (1.09 kg) 4.13.2 Yield: Table 4.8 showed that the amount of nitrogen that influences the statistically significant difference in productivity is 1% In particular, application of 200 21 kg N/ha gave higher yield than 150 kg N/ha In inorganic fertilizers, protein is the most important fertilizer, providing sufficient protein to increase photosynthesis activity, strong growth leads to increased yields, when increasing protein yield to 1%, the yield will increase 0.25% (Pham Hong Cuc, 2007, Nguyen Le Hiep, 2010) Planting density affects the statistically significant difference at 1% significance level In particular, high density yielded 19.42 tons/ha higher than low plant density of 13.06 tons/ha This result was similar in the study of Hoang Thi Thai Hoa et al (2012) also found that with planting density of 9,000 plants/ha, the yield and economic efficiency were higher with planting density of 6,000 to 8,000 plants/ha Similarly, there was a significant interaction between nitrogen content and plant density on the yield, statistically significant difference at 1% significance level Specifically, M2N2 gave the highest yield (23.19 tons/ha), statistically different from the other three treatments, with the lowest yield (10.85 tons/ha) Table 4.8: Fruit weight, yield and fruit qualities of TriP1triploid watermelon tissue cultured with different content figures of nitrogen fertilizer and planting density Fruit Fruit pale Yield Brix degree Factor weight (kg) (ton/ha) (%) thickness (cm) Nitrogen fertilizer (N) N1 1,33b 13,25b 9,18b 0,80 a a (kg/ha) N2 2,20 19,23 10,45a 0,97 M1 1,42b 13,06b 9,51b 0,92 Planting density M2 2,11a 19,42a 10,12a 0,85 M1N1 1,09d 10,85c 8,96 0,94 M1N2 1,75b 15,28b 10,07 0,90 N x density M2N1 1,57c 15,66b 9,41 0,65 M2N2 2,65a 23,19a 10,82 1,04 FN ** ** ** ns Fdensity ** ** * ns F(N x density) ** ** ns ns CV (%) 3,11 3,03 4,13 22,9 Note: Figures followed by similar symbols are the same in the same column or row or in the column and row are not statistically different from the Duncan test; ns: not statistically different, *: statistically different at 5% significance level, **: statistically different at 1% significance level In particular, N1: 150 kg N/ha, N2: 200 kg N/ha, M1: 8,750 plantlets/ha and M2: 10,000 plantlets/ha 4.13.3 Fruit quality Brix degree: Table 4.8 showed that the amount of nitrogen has an effect on the Brix degree, an accumulated statistically significant difference of 1% In particular, fertilization of 200 kg N/ha for Brix degree (10.45%) was higher than that of 150 kg N/ha (9.18%) A study on atermelon planted in Cantho showed that the amount of nitrogen fertilizer increased from 100 to 200 kg N/ha, the Brix degree also increased from 10.84% to 11.16%, but not different (Tran Thi Ba et al., 2004) Similarly, planting density also has an effect on Brix degree, which is statistically different at 5% Specifically, the density of N1 (10,000 plantlets/ha) for Brix degree was 10.12% higher than density N2 (8,750 plantlets/ha) reaching 9.51% However, the interaction between nitrogen content and plant density for Brix degree varied from 8.96% to 10.82%, but not statistically significant Fruit pale thickness: Table 4.8 showed that application of 150 kg N/ha and 200 kg N/ha for pale thickness ranged from 0.80 cm to 0.97 cm, but not statistically 22 significant Planting density for pale thickness ranged from 0.85 cm to 0.92 cm, but not statistically significant Similarly, the interaction between nitrogen content and planting density for the pale thickness varied from 0.65 cm to 1.04 cm; but not statistically different In general, the amount of fertilizer of 150-200 kg N/ha with planting density of 8,750 plantlets/ha and 10,000 plantlets/ha affect fruit yield and yield Therefore, the combination of 200 kg N/ha and density of 10,000 plantlets/ha for high productivity and fruit quality This finding was consistent with the study by Maluki et al (2015) also found that the increase in nitrogen content (40, 80, 120 kg N/ha) for fruit yield, yield and total dissolved solids was significantly higher in both trials year 2012 -2013 on TPS variety As protein improves the photosynthetic activity of the plant, it helps the plant to grow well, lacking nitrogen for short, small leaves, small fruit (Nguyen Manh Chinh and Nguyen Dang Nghia, 2006; Jalali and Jafari, 2012) In addition, increasing plant density means narrowing the distance between plants in the same row will increase the yield of seedlings in all four varieties tested in 2006 and 2007, although the increase in density increases the cost of production, but they will yield higher returns after harvest (Walters, 2009) In retrospect, two varieties of polyploidization colchicine and oryzalin chemicals in combination with tissue culture technology yielded a tetraploid ratio of 4%-9% as the primary source of crosslinking triploid watermelon Micro-propagation in vitro had overcome the drawbacks of tetraploid and triploid seeds (including difficulty of storage, poor germination, etc.) and can produce genetically identical seedlings as well as shorten the time coupled with disease immunity This approach consistently yields greater economic benefits than traditional cloning methods (Figure 4.3) Diploid seeds germination in vitro Treatment buds with colchicine 0.01% in days or oryzalin 0.004% in 54 Determination of tetraploid watermelon by flow cytometry Multiplication of tetraploid watermelon varieties in vitro - MS+BA (1.0 mg/L) - MS+IBA (1.0 mg/L) Planting tetraploid varieties tissue cultured and hybridization of triploid seed Triploid watermelon seeds Multiplication of triploid variety in vitro - MS+BA (1.0 mg/L) - MS+ IBA (0.5 mg/L) + 2g/L of activated charcoal Triploid seedlings tissue cultured 23 CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions (1) Polyploidization treatment with 0.01% colchicine and 0.004% oryzalin in vitro condition on top of diploid watermelon bud growth produced tetraploid In details, colchicine treatment in days reached 9% tetraploid and treatment of oryzalin in 54 hours reached 4% tetraploid (2) The MS medium supplemented with BA 1.0 mg/L gave good result in shoot growth and IBA mg/L supplementation gave good rooting efficiency on all four tetraploid lines, however, each line for different sensitivities The tetraploidy tissue culture planted in Hau Giang have successfully crossed the watermelon hybrid seeds (3) The MS medium supplemented with 1.0 mg/L BA was suitable for multiplication of triploid watermelon with healthy shoots, without hyperhydricity The medium supplemented with IBA 0.5 mg/L and activated charcoal 2.0 g/L was suitable for rooting of triploid watermelons varieties Two triploid varieties TriP1 and TriP2 offer high sensitivity in vitro condition Two triploid varieties TriP1, TriP2 and control varieties tissue culture were cultivated in the field in Binh Thuy, Can Tho and Chau Thanh, Hau Giang The results showed that in Can Tho, the growth (leaf length, leaf number/vine, leaf size) of the TriP1 was higher than that of the control variety and TriP2, but fruit weight, yield and fruit quality (Brix degree and fruit pale thickness) were not statistically different In Hau Giang, the TriP1 variety had the same growth rate as the control variety and TriP2 variety, while fruit weight and yield (2.2 kg-36.91 tons/ha, respectively) were higher than the reference variety (1.73 kg-28.94 tons/ha) and the TriP2 (1.55 kg-25.56 tons/ha), and the same fruit qualities Figure 4.3: Production process of triploid watermelon tissue (4) With the amount of 200 kg nitrate fertilizers per hectare combined with the density of 10,000 plantlets/hectare, the cultures yield was higher than 150 kg N/hectare with the density of 8,750 plantlets/hectare 5.2 Recommendations Colchicine and oryzalin are effective in the formation of tetraploid on diploid watermelon shoots in vitro, in which oryzalin exhibits tetraploidal activity at low concentrations and shorter duration than colchicine, but results are not high Proposed further study to raise the tetraploid ratio on diploid watermelon buds by oryzalin Application of the "production process of triploid watermelon tissue" to breed and multiply triploid watermelon lines, contributing to the diversification of watermelon sources originating in Vietnam Further expansion of testing for development of triploid watermelons TriP1 and TriP2 on other ecoregions in the Mekong Delta 24 ... triploid watermelons for field planting 1.3 New findings of the thesis i) Creating tetraploid seedlings by tissue culture and multi-nucleotide treatment; planting trees in the field ii) Hybriding... the tetraploid watermelons in vitro, and to hybrid triploid seeds in the field conditions ( 3) find the suitable medium for clonal culture and select triploid watermelon lines for in vitro conditions,... seedlings; reduce the initial investment in seedless watermelon production iv) Investigating and evaluating the growth and fruit quality of two watermelon lines forming transplanted tissue in