General objective: To get mutant flowering flowers that have more number of petals than the study samples, larger flowers and more aromatic. To evaluate growth culture technique to generate calli and shoots for irradiation of gamma ray;
MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Biotechnology Code: 62 42 02 01 DAO THI TUYET THANH INDUCING OF MUTATION TUBEROSE (Polianthes tuberosa L.) LINES BY IRRADIATING WITH 60Co GAMMA RAYS IN VITRO Can Tho, 2018 THIS STUDY WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisor: Assoc Prof Doctor NGUYEN BAO TOAN The dissertation was defended at the university examination committee At.………………………………………., Can Tho University At……… hour ….…, on date…… month… … year…… Referee 1: Referee 2: Referee 3: The dissertation is available at Libraries: Central library of Can Tho University National library of Vietnam PUBLISHED PAPERS Nguyen Bao Toan, Nguyen Quang Thuc and Dao Thi Tuyet Thanh, 2014 60Co Gamma treatment at different irradiated doses on shoot clusters of two (Polianthes tuberosa) varieties in vitro Can Tho University Journal of Science (4): 41 - 46 (in Vietnamese) Dao Thi Tuyet Thanh and Nguyen Bao Toan, 2016 Effects of 60 Co gamma doses on the growth and development of in vitro tuberose shoot clusters (Polianthes tuberosa L.), appearance of abnormal structures and LD50 determination Can Tho University Journal of Science Part B: Agriculture, Aquaculture and Biotechnology 45: 25 - 32 (in Vietnamese) Dao Thi Tuyet Thanh, Le Thi Ngoc Quy and Nguyen Bao Toan, 2017 Study on growth and flower diversity of single petal tuberose clones (Polianthes tuberosa L.) irradiated with 60Co gamma rays by tissue culture Journal of Vietnam Agricultural Science and Technology 2(75): 47 - 52 (in Vietnamese) Dao Thi Tuyet Thanh and Nguyen Bao Toan, 2017 Study on genetic diversity of tissue cultured tuberose lines (Polianthes tuberosa L.) irradiating with Co60 by using ISSR marker Journal of Vietnam Agricultural Science and Technology 6(79): 20 - 24 (in Vietnamese) Dao Thi Tuyet Thanh and Nguyen Bao Toan Effects of gamma radiation doses on the growth, flowering and phenotypes of tuberose (Polianthes tuberosa L.) lines propagated by tissue culture Journal of Biotechnology (Accepted, in Vietnamese) Chapter I: INTRODUCTION Necessity of the dissertation Polianthes tuberosa L is one of the most popular cut flowers in the tropics and subtropics In Vietnam, it helps farmers get more income than rice and other crops Thus, it has been included in the crop restructuring program and considered a poverty reduction crop in the provinces: Tien Giang, Dong Thap, Can Tho and An Giang Nowadays, there are only two varieties of tuberoses with petals and 12 petals which are mainly cultivated in the Mekong Delta However, the propagation of tuberose is mainly rooted through generations leading to serious degeneration, pest infestation and significant reduction in productivity Therefore, the demand for new varieties is exigent In addition, breeding of tuberose in the traditional way has encountered some limitations due to high incompatibility because the flowers have stigmas and stamens which are not ripe at the same time and this is the reason why their seeds are not created under natural conditions (Estrada-Basaldua et al., 2011) Moreover, only the single petal flowers can produce seeds, but it is difficul for the seeds to sprout Perhaps, the mutation is the best way to breed a new tuberose variety Among physical mutagenic agents, gamma rays are most widely used because of their effectiveness (Matsumara et al., 2010) This technique increases genetic variation in some type of flowers such as changes in color, shape, growth characteristics, etc (Xu et al., 2012) Furthermore, in vitro culture should be applied to increase the number of irradiated samples Propagation of tuberose in vitro has been experimented (Huynh Thi Hue Trang et al., 2007; Hutchinson et al., 2004) Whereas, growth culture and irradiation are the effective methods to make plants uncontaminated, multiply rapidly and mutate This combination is successfully applied on palms, apples, potatoes, sweet potatoes and pineapples (Ulukapi and Nasircilar, 2015) and can be selected properly to produce tuberose varieties On the other hand, petals play an important role in flowering, pollination and cross-fertilization… For ornamental plants, the number of petals is related to the flower pattern When in vitro culture is combined with gamma ray treatment, changes to the number of rose and daisy petals have been reported (Usenbaevard and Imankulova, 1974; Kahrizi et al., 2012; Nagatomi, 2001) Thus, it is possible for tuberose to induce new source of variations in the characteristics of large numbers of petals by in vitro and gamma ray processing Until now, in Vietnam, there have not been any new tuberose variety with many petals created by traditional methods as well as by other modern biotechnological techniques For these reasons, it is necessary to carry out the study on "Inducing of mutation tuberose (Polianthes tuberosa L.) lines by irradiating with 60Co gamma rays in vitro" Research objectives 2.1 General objective: To get mutant flowering flowers that have more number of petals than the study samples, larger flowers and more aromatic 2.2 Specific objectives: (1) To evaluate growth culture technique to generate calli and shoots for irradiation of gamma ray; (2) To identify lethal dose of 50% (LD50) of gamma rays (60Co) on callus and rhizomes of tuberosa after 150 days; (3) To observe the phenotypic abnormalities of the domesticated stage; (4) To choose to mutant tuberose lines that increase the number of petals (more than 12 petals) with large size and fragrant odour by traditional breeding methods Research subjects and scope of the study - Two tuberose varieties including of the single and double petal flower are being cultivated in An Giang province - Investigating mutant line characteristics in the field over generations Location and duration This study was carried out from October 2013 to August 2017 at Can Tho University Experiments of irradiation treatment were done at the Radiation Technology Department of Dalat Nuclear Research Institute, Lam Dong province The single petal tuberose variety/lines were planted in Can Tho City and the double petal ones were cultivated in Tien Giang province New contributions of the dissertation - Using the meristem culture technique to initiate the materials for irradiating with the 60Co gamma ray; computing the lethal dose 50% (LD50) of the gamma rays on tuberose callus or shoot clusters of two tuberose varieties in vitro - Observing the abnormal structures of leaf, stem and shoot of the two tuberose varieties after 150 day culturing at the in vitro stage and after 60 day cultivating at field Besides, recording the growth, flowering parameters, the variations of petal numbers, the aroma of these flower types after 180 day on the field - Selecting at least from to mutant tuberose lines which were increasing of the petal number (22 and 36 petals), of the size and fragrance over the 2nd flowering - Assessing the genetic diversity by PCR - ISSR method to prove the differences of DNA among the two mutant tuberose lines and the two control varieties such as determining the appearance or the absence of DNA bands, sequencing the ITS1/4 regions to compare the DNA sequences and identify the mutant types as replacement, deleting and inserting one or more new nucleotides - This study has constructed the procedure of mutant tuberose line selecting by 60Co gamma treatment in vitro Outline of the dissertation The dissertation includes 142 pages of introduction, literature review, materials and methods, results and discussion, conclusion and recommendation, references and annexes, and also contains 35 tables, 35 figures and 277 references Chapter III: MATERIALS AND METHODS 3.1 Plant material - The tuberose varieties are growing well in An Giang province (Figure 3.1) Figure 3.1: The two tuberose varieties a: The single petal tuberose variety (HĐ ) with petal flowers and fragrant odour, short length of flower spike, small leaves and b: The double petal one (HK ) with 12 petal flowers and fragrant odour, higher length of flower spike, bigger leaves a b 3.2 Experimental methods 3.2.1 Research content 1: Inducing materials for irradiating with 60 Co gamma rays in vitro 3.2.1.1 Meristem culture of tuberose - Explant preparation, sterilization and using the MS basal medium for the initial medium (Murashige and Skoog, 1962) (Huynh Thi Hue Trang et al., 2007) * Experimental parameters: The survival rate of meristems after 30 day culturing (%) 3.2.1.2 Inducing tuberose clusters of calli and shoots - Transplanting the survival meristem of the tuberoses after every 30 day for times (M5) The culture medium was the basal medium (BM) including MS medium supplemented with thiamine; pyridoxine; nicotinic acid; 1.0 mg/l riboflavin for each; agar (8 g/l); sucrose (30 g/l) (Huynh Thi Hue Trang et al., 2007) Then, this mixture was adjusted to pH 5.8 before being autoclaved at 121oC for 30 and this medium was contained in nylon bags (Figure 3.2) a b Figure 3.2: Culture Figure 3.3: Petri dishes had cultured samples medium in a nylon before irradiating by gamma rays Note: a: calli samples and b: clusters of shoots bag - Using BM medium and additives of plant growth regulators for callus and shoot initiation media containing 1.0 mg/l NAA and BA 4.0 mg/l (Huynh Thi Hue Trang et al., 2007) 3.2.2 Research content 2: Determining the effects of 60Co gamma doses of two tuberose varieties/lines using the 50% lethal dose (LD50) in vitro * Preparing clusters of calli/shoots dedrived from the single petal tuberose variety/double petal one - Clusters of calli with 1.0 cm2 in size were cultured in petri dishes which contained 20 ml BM medium, culturing 10 samples/dish and in total of 60 dishes (Figure 3.3a) - Clusters of shoots were cut off leaves and roots in the average height of 1.0 - 1.2 cm, each of them which had 1.0 cm diameter were cultured in petri dishes (Figure 3.3b) 3.2.2.1 Experiment 1: Effects of 60Co gamma ray doses on the growth and the development of single petal tuberose callus clusters The experiment was laid out in a completely randomized design (CRBD) with a factor, 10 treatment, replications and included fifty explants per replication The non-irradiated treatment was the control one The 60Co gamma rays with the dose rate was 1.58 kGy/hour The experiment was shown in Table 3.1 After irradiating, clusters of calli were cultured in BM medium supplemented with 1.0 mg/l NAA and 6.0 mg/l BA (Le Ly Vu Vi et al., 2014) for shoot regeneration and multiplication, then they were transplanted at every 30 day (culturing cluster of callus/shoot/nylon bag) Table 3.1: Arranging gamma ray doses and treatments in the single petal tuberose variety Variety HĐ Control T1 T2 10 T3 Gamma ray dose (Gy) 15 20 25 30 T4 T5 T6 T7 40 T8 50 T9 60 T10 * Experimental parameters: - The percentage of clusters of calli regenerated into shoots after 30 day culturing (%) Visual observation clusters of shoots after 150 day irradiating by gamma rays - Death rate (%) = (Total of death shoots/Total of shoots) x 100 The clusters of calli were regarded as death when they could not induce any shoot or new shoot and get lost chlorohyll - Calculating LD50 followed Randhawa’s description (2009) - Shoot length (cm), number of shoots/cluter, number of leaves/clusters of shoots - Abnormal clusters of shoots which recorded at leaves, roots, shoots, stunt shoots types 3.2.2.2 Experiment 2: Effects of 60Co gamma ray doses on the growth and development of the double petal tuberose shoot clusters This was laid out similarly to the experiment (Table 3.2) Table 3.2: Arranging gamma ray doses and treatments in the double petal tuberose variety Variety Control HK T1 T2 10 T3 Gamma ray dose (Gy) 15 20 25 30 T4 T5 T6 T7 40 T8 50 T9 60 T10 After irradiating, clusters of shoots were transfered to shoot multiplied medium as in Experiment (culturing cluster of shoot/nylon bag) * Experimental parameters: - Death rate (%), calculating LD50, shoot length (cm), number of shoots, number of leaves and types of abnormal shoots after 150 day culturing were indicated as Experiment The death clusters of shoots were recorded as having no chlorohyll or inducing no new shoot 3.2.2.3 Mass multiplication and root formation - Using BM for the shoot multiplication medium, supplemented with 1.0 mg/l NAA and 6.0 mg/l BA (Le Ly Vu Vi et al., 2014); subculturing three times in every 30 days, but culturing 10 clusters of shoots/nylon bags - When the number of shoots were about 500, then transfering all of them into the root formation medium which was BM medium plus 4.0 ml/l atonik (Le Ly Vu Vi et al., 2014) in 60 days, subculturing in every 30 days and 10 shoots/nylon bag until the appearance of root 3.2.3 Research content 3: Determination of plantlet phenotype diversities at the acclimatization stage - Acclimatization of the tuberose plantlets was performed as the same as Nguyen Minh Kien (2011) * Experimental parameters: The frequency of abnormal plantlets (%): striped or curl leaves… after 30 day growing 3.2.4 Research content 4: Screening several tuberose lines having an increase of the number of petals, the flower size and fragrance by the traditional propagation method 3.2.4.1 Experiment 3: Evaluating of the growth and development of single petal tuberose variety/lines after irradiating by 60Co gamma ray on the field a The first times of growing (VĐM1) - This experiment was conducted in a factorial randomized completely block design in three replications in 500 m2 plots size with treatments The treatments included the bulb control (plants dedrived from bulbs), the in vitro control (plants dedrived from non-irradiated in vitro culture) and gamma ray doses from 5; 10; 15; 20; 25 and 30 Gy Every replications grew 50 plants The bulb control had bulb with diameter of 1.0 - 1.2 cm whereas the in vitro control or irradiated plantlets had 3.0 - 6.0 leaves, 6.0 - 10.0 cm Table 4.2: Shoot number, shoot height and leaf number of the double petal tuberose after irradiating by gamma rays at 150 day culturing Day 150 Shoot Shoot Shoot Leaf Shoot Leaf height height number number number number (cm) (cm) 3.4 1.1 2.2 7.3a 5.4b 18.0b ab a 3.4 1.1 2.2 6.9 7.0 18.6ab bc b 10 3.4 1.0 2.2 6.3 6.0 20.1ab abc a 15 3.5 1.1 2.3 6.7 6.6 21.7a c c 20 3.3 1.1 2.3 6.1 6.2 10.5c c b 25 3.5 1.1 2.2 6.1 5.6 21.1a d d 30 3.4 1.1 2.2 4.8 2.7 4.6d de d 40 3.5 1.1 2.2 4.2 2.4 9.9c ef e 50 3.4 1.1 2.1 3.6 1.1 4.7d f e 60 3.4 1.1 2.1 3.4 1.2 3.2d F ns ns ns * * * CV (%) 6.4 3.6 8.6 10.6 13.3 21.8 Note: Values within the columns followed by different letters are significantly different at 5% probability level Gamma ray dose (Gy) 4.2.2.4 Effects of 60Co gamma ray doses on double petal tuberose abnormal structures The in vitro control and Gy treatments had no mutation in leaves or shoots There were the total of abnormal phenotypes including shoots with twitch leaves (10 Gy); getting lost the chlorophyll and the earlier root information at 15 Gy shoots; the stunt shoots with saw-edged leaves at 20 Gy dose There were the phenotypes with similar shoots, but those with scrolling leaves at the doses ranging of 25; 30; 40 and 50 Gy Only at 60 Gy dose, all the shoot clusters turned brown 4.2.3 Mass multiplication and root formation In the media for the single petal tuberose shoot multiplication, the total number of shoots produced per an explant was shown large, and they were green and vigor The average number of induced shoots was 4.5 from 14 the first shoot after 30 days of this stage, except for the 25 and 30 Gy treatments For the double petal tuberose variety/lines, the number of shoots produced per explant was larger (the average of 6.5 shoots) in the same medium at the in vitro control, and 10 Gy treatment The others gave the smaller shoot numbers (about 2.5 - 3.5 shoots) Both of 40 and 50 Gy doses of tuberose varieties had weaken shoots and died before root information The rate of rooting about 95% for the single petal tuberose varieties/lines whereas about 97% for the double petal ones recorded in all of the treatments after 60 day culturing 4.3 Research content 4.3.1 The death rate of single petal tuberose variety/lines The bulb control treatment had the lowest death rate of the plantlet numbers (3 plantlets) The in vitro control treatment had the highest rate about 10% (30 plantlets) the opposite 4.3.2 The death rate of the double petal tuberose variety/lines In the double petal tuberose varieties/lines, the bulb control treatment still gave less of dead plantlets than the others did (just 3.0%) The obtained results indicated that the in vitro control and Gy treatments gave the death percentage increasing up to 8.5 and 7.2% (over 20 plantlets), respectively 4.3.3 The abnormal phenotypes of single/double petal tuberose varieties/lines All the mutant shoots with the above characteristics could not be regenerated into the normal plants at the in vitro stage and most of them gradually died The alive plantlets indicated that the differences of leaf and shoot phenotypes were not clearly recognized 4.4 Research content 4.4.1 The growth and flowering of single petal tuberose varieties/lines on the field (M1) - The death rate of plantlets 15 The higher the dose of radiation got, the higher the death rate of plantlets was after 60 day cultivating Plantlets irradiated at the in vitro control treatment, and 10 Gy showed the equal death rates of 4.0% The 25 and 30 Gy doses were lethal to most of the plantlets (about 8%) - Shoot number, leaf number, bulb number and bulb diameter The growth of irradiated explants decreased when a higher dose of irradiation of gamma ray was treated During the cultivation, this emphasis of growth was shown by both in number of shoots and number of leaves produced The number of shoots produced on Gy dose plantlets was a little smaller, whereas that of shoots sharply increased on the bulb control plantlets The leaf numbers varied on different doses, from the average of 48.9 leaves to that of 130.3 leaves The average bulb number of the 25 Gy was 14.5, the maximum value, and that of untreated plants was 13.5 The 25 Gy dose plants has the largest number of bulbs but the shoot and leaf numbers showed a significant difference compared to that among the treated plants and the two control ones The biggest diameter of bulbs was about 4.1 cm, corresponding to the 15 and 25 Gy tuberose lines (Table 4.3) Gamma ray dose (Gy) Bulb control In vitro control 10 15 20 25 30 F CV (%) Shoot Leaf Bulb Bulb number number number diameter (cm) 9.4a 107.8b 13.5ab 3.0d b a b 7.3 130.3 13.0 3.4c de c c 5.1 89.7 11.5 3.3cd bc ab c 7.0 121.0 10.9 2.7e cd c d 5.6 89.6 8.3 4.1a de e e 4.2 48.9 6.4 3.1cd de cd a 4.7 80.9 14.5 4.1a e d d 3.8 67.0 9.5 3.7b * * * * 14.1 9.1 7.1 4.4 Table 4.3: The growth parameters of single petal tuberose lines after 180 day cultivating Note: Values within the columns followed by different letters are significantly different at 5% probability level - Flowering time, inflorescence height, floret number/spike and floret diameter The bulb control, 10 and 25 Gy treatments took minimum days (147.0) to initiate flowering as compared to the rest of the treatments, 16 whereas the in vitro control; 5; 20 and 30 Gy ones took maximum days (175.0) to produce flowers in its 25% plants As far as the height of inflorescence was concerned, the 20 and 25 Gy lines produced inflorescence with the maximum height (118.0 and 122.7 cm) excelling those of the other treatments Regarding the number of florets/spike, the 25 Gy plants produced 41.3 florets, more than those produced by the rest of the treatments In spite of having the highest spike, the 20 Gy plants had the least number of florets (28.0) Data related to diameter of flower illustrated that bigger sized flowers (3.6 cm) were produced by 25 Gy line as compared to the rest of the treatments, whereas smaller sized ones (1.8 cm 2.9 cm) were produced at and 30 Gy doses Because of the unopened flowers, the Gy plants had florets with the smallest diameters (Table 4.4) Table 4.4: Flowering characteristics of single petal tuberose varieties/lines at the M1 generation Gamma ray Flowering Inflorescence Floret Floret diameter dose (Gy) time (days) height (cm) number/spike (cm) Bulb control 147.7a 91.0d 36.0abc 3.0b bc cd cd In vitro control 175.3 103.0 31.3 3.0b bc bc bcd 175.0 107.3 34.0 1.8c a bc abc 10 152.7 108.7 36.0 3.5a 15 162.0ab 108.0bc 40.7ab 3.5a bc ab d 20 177.7 118.0 28.0 3.1b a a a 25 155.7 122.7 41.3 3.6a c bc cd 30 181.3 110.0 30.7 2.9c F * * * * CV (%) 5.8 6.3 10.7 6.8 Note: Values within the columns followed by different letters are significantly different at 5% probability level - The abnormal characteristics of the single petal tuberose varieties/lines after irradiating with 60Co gamma + At leaf part: There were leaf phenotypes of single petal tuberose lines after 60 day cultivating Both of the control treatments with normal 17 leaves; the and 20 Gy lines with leaves of anthocyanin pigment color The others had the differences at the below positions of leaves The 10 Gy line had a small leaf to the right and a bigger one to the left; the 15 Gy plants had white streak along the two sides of leaves; the 25 Gy plants had the sticky leaves at the one third outside and the leaves form to lines and the 30 Gy plants had lowest small leaves, thick and wide + At stem and bulb part: Treated plants with varying in stem and bulb The 15 Gy plants had stem with skewing leaves; the 20 Gy plants perfectly aligned and at the 25 Gy dose, there was the earlier induction of bulblets + At flower part: Because of the unopened florets, it would be impossible to count the petal numbers of the Gy line There were 75% of the spikes with 6; and petals appeared at the 15; 25 and 30 Gy plants (Figure 4.3) + The fragrancy: Concerning with an increase of the petal numbers, the aroma of these lines was evaluated by marks In contrast, the unopened flowers had no fragrancy Although there were changes of petal numbers, the double petal type was not induced as expectation So, these mutant lines were selected and observed properly a b c d Figure 4.3: The distinct flower types of treated plants varying on petal numbers at single petal tuberose variety/lines Note: a: The bulb control (the petal floret ); b: 15 Gy (the and petal florets); c: 25 Gy (the and petal florets) d: 30 Gy (the and petal florets) 18 4.4.2 The growth and flowering of double petal tuberose varieties/lines on the field (M1) - The death rate of plantlet When all rooted plants were transplanted to the field, it was observed that only the bulb control plants were able to survive 100% whereas the in vitro control ones were dead at a high rate (4.0%) The survival percentage of the 15 and 25 Gy plants were the same (about 3.3%) and the 10 and 30 Gy doses gave the equality of the death plants, but showed the different death rates - Shoot number, leaf number, bulb number and buld diameter An average number of shoots of the controls, 25 and 30 Gy doses was 4.0 and that of treated plants from to 20 Gy had the maximum value (6.0 shoots) Both of the control plants had 19.0 leaves in average whereas this parameter of the 5; 10 and 20 Gy plants were 36.8, 40.5 and 35.9 cm, respectively The least leaf numbers gained at the 25 Gy dose (as 50% as the leaf numbers of 10 Gy dose) However, the highest dose (30 Gy) induced the larger leaf number than the 15 Gy one did Data pertaining to an average number and diameter of bulbs showed that maximum bulb number (14.0) was produced in and 15 Gy plants with the minimum size of bulbs (3.8 cm) On the contrary, the in vitro control treatment, the doses of 10; 20 and 30 Gy had the bigger size of bulbs (4.0 cm) and was less bulbs (Table 4.5) Gamma ray Shoot dose (Gy) number The bulb control 3.9b The in vitro control 5.8a 10 15 20 25 30 F CV (%) 6.0a 5.7a 6.1a 6.2a 4.3b 3.6b * 10.4 Leaf number abcd 28.9 29.3abcd 36.8ab 40.5a 21.3cd 35.9abc 19.3d 23.8bcd * 26.6 Bulb Bulb diameter number (cm) ab 13.9 9.1d 13.0ab 7.7d 14.4a 10.7c 12.4b 7.9d * 8.1 19 ab 3.8 4.2a 3.8ab 3.8ab 3.8ab 4.1ab 3.6b 3.9ab * 6.6 Table 4.5: The growth parameters of single petal tuberose varieties/lines after 180 cultivating Note: Values within the columns followed by different letters are significantly different at 5% probability level - Flowering time, inflorescence height, floret number/spike and floret diameter The maximum number of days (188.7) was taken by the 15 Gy dose, but this number gradually decreased for the others; whereas the minimum one was taken by the bulb control and 10 Gy treatments (156.0) On the other hand, the minimum height of inflorescence was about 64.3 cm at the 30 Gy plants, whereas the maximum one was obtained in plants irradiated with the 15; 20 and 25 Gy doses (100.0 cm) It was also observed that the height of inflorescences increased from 82.3 to 97.7 cm with the doses of 0; and 10 Gy The results showed that the number of florets per spike were changed, depending on the doses of gamma rays The minimum number of florets (38.7) was obtained in the 10 Gy plants, which was statistically at par with the others such as the in vitro control and Gy, about 52.0 florets per spike The diameter of florets got its least value at the both of controls; 5; 10 and 30 Gy treatments (about 4.0 cm), but there was an increase of this parameter (up to 5.0 cm) at the other doses (Table 4.6) Table 4.6: Flowering characteristics of the single petal tuberose varieties/lines at the M1 generation Gamma ray dose (Gy) Flowering time (days) Bulb control In vitro control 157.7d 161.0cd 163.0cd 156.0d 188.7a 177.7ab 170.0bcd 175.0abc * 4.5 10 15 20 25 30 F CV (%) Inflorescence height (cm) 97.7b 89.2b 90.2b 82.3b 102.3a 106.3a 104.7a 64.3c * 17.8 Floret number/spike Floret diameter (cm) 37.0b 51.7a 52.3a 38.7b 48.0a 45.7a 46.0a 45.3a * 54.6 4.1bc 4.1bc 4.2bc 3.8cd 5.1a 5.3a 5.2a 4,4b * 9.1 Note: Values within the columns followed by different letters are significantly different at 5% probability level 20 - The abnormal characteristics of the double petal tuberose varieties/lines after irradiating with 60Co gamma + At leaf part: The variation of shape and color of leaves in treated plants was fluctuant The bulb controls gave slender leaves At the in vitro control; and 10 Gy treatments, the xanthocyanin pigment appeared at leaves and the small leaves were induced to the right side; the properties of size, shape, number and position of leaves at the 10; 20; 25 and 30 Gy plants were recorded Besides, the 15 Gy dose gave the leaves having sticky interior part Only the 20 Gy plants had yellow streaks alongside the leaves + At stem and bulb part: There were of stem and bulb phenotypes for mutant double petal tuberose varieties/lines The bulb control treatment had symmetric leaves; the and 10 Gy plants had the different numbers of lopsided leaves Only the 15 Gy plants had earlier developed bulbs + At flower part: There were 18 different phenotypes in the irradiated double petal type varieties There was no abnormal plant in the number of petals, but two plants with yellow stamens appeared (2.3%) at the in vitro control treatment The dose of Gy did not change the petal number but those petals were long, aromatic, having a green streak with a frequency of 4.6% The mutant types appeared mostly at doses of 10 and 15 Gy At 10 Gy, there was a reduction in the number of petals from 12 to 4; 5; 6; and 10 petals and fragrancy with low frequencies At irradiated higher doses as 15; 20 and 25 Gy, there were the duplication and triplication of the amount of petals The dose of 15 Gy induced the flowers with 13; 16; 17; 18 and 19 petals (1.1%) The dose of 20 Gy had flowers with the number of petals increased, up to to 22 petals (5.8%), 36 and more than 40 petals (3.5%) When irradiated with the high dose of 30 Gy, the plants had distorted shape, bluish white flowers and not fully opened and loss of aroma (about 80%) There could be less than 12 petals (4.6%) or up to more than 40 petals (62.1%) (Figure 4.4; Figure 4.5; Figure 4.6) 21 Yellow stamen a b c d e f Green streak g h Figure 3.4: The distinct flower types of treated plants varying on the number of petals at double petal tuberose variety/lines Note: a and c: the 12 petal floret at and Gy; b: the petal floret at 10 Gy; d: the less than 12petal floret at 30 Gy; e, f and g: the 24 petal floret and more than 24 petal floret at 15, 20 and 25 Gy and h: the 4, 5, and 10 petal floret at 10 Gy a b c Figure 3.5: The abnormal types of double petal tuberose with the open and fragrant flower at the M1 generation Note: a: 15 Gy (the 24 petal floret); b: 20 Gy (the 36 petal floret; c: 20 Gy (the 40 petal floret) + The fragrancy: It could be possible to have categories depending on the number of petals The 1st group consists of 4; 5; 6; and 10 petals, the 2nd group with 13; 14; 16; 17; 18; 19; 22 or 24 petals, the 3rd group with more than 24 petals and the 4th one with unopened flower The tuberose lines at 20; 25 and 30 Gy had no fragrancy However, the aromatic level of tuberose lines of the 1st group were supposed with marks, so they have more fragrancy than the others The mutant lines belonging to the 3rd and 4th group were evaluated with mark 4.4.3 M2 generation (M2) Among the tuberose lines with an increase of petals (more than 12 petals), the two double tuberose lines were selected, having 22 and 36 petals and being satisfied with the purpose of the second planting on the field 22 (Figure 4.6) From the obtained results, the growth and the development data of them was gathered and given in Table 4.7 b a c d Figure 4.6: The two local tuberose varieties and the mutant tuberose lines at M2 generation Note: a and b: the bulb control plant with and 12 petal floret; c and d: the mutant tuberose lines with 22 and 36 petal floret Table 4.7: The agronomic characteristics of the selected tuberose varieties/lines at M2 generation Parameters The single petal tuberose variety The double petal tuberose variety The 22 petal tuberose line The 36 petal tuberose line Shoot number 6.0 5.0 7.0 8.0 Leaf number Leaf number / inflorescence Leaf length (cm) 8.0 9.5 18.0 14.0 12.0 24.5 15.0 20.0 49.9 45.0 46.5 43.0 Leaf width (cm) 1.2 1.4 1.3 0.9 Internode height (cm) 6.9 8.0 15.3 10.0 Bulb number 4.0 3.0 4.0 6.0 Bulb diameter (cm) 3.0 3.8 4.1 4.2 Flowering time (days) 147.7 120.0 123.0 145.0 Inflorescence height (cm) 91.0 97.7 141 133.0 Spike height (cm) 35.0 42.1 46.0 39.0 Floret number/spike 36.0 37.0 38.0 40.0 Flower longevity (days) 7.0 14,0 16.0 20.0 Floret diameter (cm) 3.0 4.1 5.1 5.4 23 4.4.4 DNA divergence analysis among tuberose varieties and mutants tuberose lines 4.4.4.1 Genetic diversity evaluation by using ISSR - PCR technique The results showed that the total of 84 bands generated with the average of 21.0 ± 5.89 bands/primer and all bands were observed as polymorphic by using four ISSR primers The size of bands scored in all the genotypes were in the range of 150 to 3.000 bp The number of amplified bands generated by individual ISSR varied from 13 (primer 808) to 27 (3A39 primer) bands with an average 3.16 bands per primer and the percentage of polymorphic bands was 100% 4.4.4.2 Genetic diversity evaluation by the differences of DNA band position From the ISSR profiles, new bands or the absence of bands were observed among mutants in comparison with double petal type variety Primer UBC 873 generated 23 bands of DNA from 200 - 3.000 bp For the mutant tuberose line with 22 petals, two new bands appeared at 250 bp and 1.000 bp, and DNA bands were absent at 350; 850; 1.250 and 3.000 bp Also, for the 36 petal tuberose line, whereas the DNA bands were absent at 300; 350; 850 1.250 bp, and were present at 1.000 and 2.750 bp Primer P23SR1 induced the new bands at 500; 600; 800; 1.750 and 2.000 bp in the two mutant lines Besides, these lines had the difference of the DNA positions to be distinguished from each other Further primers of 3A39 and 808 identified the two mutant lines based on the presence and the absence of a band with the control (Figure 4.7) 24 M 3.000 bp M 3.000 bp 2.000 bp 900 bp 600 bp 1.250 bp 850 bp 300 bp b a M M 1.000 bp 1.250 bp 600 bp 700 bp c d M: Marker 100 bp , : new DNA bands , Figure 4.7: ISSR profiling pattern of tuberose genotypes with ISSR primers Note: a) Primer UBC 873; b) Primer P23SR1; c) Primer 3A39; d) Primer 808 1: the single petal tuberose variety, 2: the 22 petal tuberose line, 3: the 36 petal tuberose line, 4: the double petal tuberose variety : absent DNA bands c The result of the genetic relationship between the tuberose varieties/lines The banding pattern of ISSR markers scored in the form of binary data was used for computing Jaccard’s similarity index values obtained by pairwise comparison and presented in Table 4.8 The similarity coefficient based on four ISSR markers ranged from 0.375 to 0.786 Of the considered genotypes, the lowest similarity index was recorded (about 0.375) between the double petal type tuberose variety and the mutant 22 petal tuberose line, hence they can be seen as most divergent The genotypes single petal one and the mutant 22 petal one gave the highest similarity index (0.786), so they can be considered closely related ones 25 Table 4.8: The values of Jaccard’s dissimilarity coefficient for four genotypes of tuberoses The single petal tuberose variety The single petal tuberose variety The 22 petal tuberose line The 36 petal tuberose line The double petal tuberose variety 1.000 0.643 0.786 0.429 The 22 petal tuberose line 1.000 0.571 0.375 The 36 petal tuberose line 1.000 0.500 The double petal tuberose variety 1.000 The dendrogram based on ISSR arranged the four genotypes into two main clusters at similarity index of 0.43 (Figure 4.8) The first cluster consisted of genotypes, the single petal type tuberose variety and the two mutant tuberose lines The second cluster had only one genotypes, the double petal type one These two mutant genotypes had a close association with the single petal genotypes in spite of they were derived from the double one This could be the limitation of PCR - ISSR technique The single petal tuberose variety The 36 petal tuberose line The 22 petal tuberose line The single petal tuberose variety variety Figure 4.8: The dendrogram shows the genetic relationship among tuberose genotypes, using UPGMA analysis of ISSR data 4.4.4.2 Sequencing the ITS region of tuberose varieties/lines The BLAST results on the NCBI showed that the ITS sequences of the and 12 petal cultivars as well as the two mutant tuberose lines with 22 and 36 petals were highly homologous to those of the two species of the genus Polianthes spp about 95 - 99% When aligning the DNA sequences between the two single and double varieties, there were found 12 different nucleotide positions When comparing between the two mutant ones with 22, 36 petals and the original 26 varieties, there were found 32 and 33 different nucleotide positions in sequences with the mutant types for the replacement of one nucleotide or a nucleotide fragment by removing or inserting Chapter V: CONCLUSION AND SUGGESTION 5.1 Conclusion (1) The results of the above experiments showed that the base medium supplemented 1.0 mg/l NAA and 4.0 mg/l BA was very suitable for inducing calli and shoot clumps as experiment materials; (2) The obtained LD50 value of single petal tuberose variety was about 10 - 15 Gy (the counting value was 10.96 ± 2.96 Gy), whereas the one of double petal tuberose variety was about 20 - 25 Gy (the counting value was 22.91 ± 4.01 Gy); (3) At the in vitro stage, the higher radiation doses were, the lower number of shoots got, shoot height and the number of leaves were the same as in all varieties/lines and (4) The single petal tuberoses at the 25 and 30 Gy had no double flower although there was an increase of petal and fragrancy In double petal tuberoses, among the variations of phenotypes were the two fragrant mutant lines with 22 and 36 petals at the 20 Gy dose, having the most potential ability for production The process of inducing mutant tuberose lines for irradiating by 60Co gamma in vitro is described in the following schema: 27 5.2 Suggestion Continuing to frequently observe the agronomic characteristics of the new mutant tuberose lines Then, rapidly propagating in quantities of these lines for production and trade 28 ... tuberose (Polianthes tuberosa L.) lines by irradiating with 60Co gamma rays in vitro" Research objectives 2.1 General objective: To get mutant flowering flowers that have more number of petals... Determining the effects of 60Co gamma doses of two tuberose varieties /lines using the 50% lethal dose (LD50) in vitro * Preparing clusters of calli/shoots dedrived from the single petal tuberose. .. inducing mutant tuberose lines for irradiating by 60Co gamma in vitro is described in the following schema: 27 5.2 Suggestion Continuing to frequently observe the agronomic characteristics of the