Summary of doctoral thesis: Inducing of mutation tuberose (Polianthes tuberosa L.) lines by irradiating with 60Co gamma rays in vitro

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

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:

1 Central library of Can Tho University

2 National library of Vietnam

PUBLISHED PAPERS

1 Nguyen Bao Toan, Nguyen Quang Thuc and Dao Thi Tuyet

clusters of two (Polianthes tuberosa) varieties in vitro Can Tho University

Journal of Science (4): 41 - 46 (in Vietnamese)

2 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)

3 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)

4 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)

5 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

1 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 do 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 6 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"

2 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 1 to 2 mutant tuberose lines that increase

the number of petals (more than 12 petals) with large size and fragrant odour by traditional breeding methods

3 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 2 generations

4 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

5 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 1 to 2 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

6 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

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 5 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 min and this medium was contained in

nylon bags (Figure 3.2)

Figure 3.1: The two tuberose varieties

a: The single petal tuberose variety (H Đ ) with 6 petal flowers and fragrant odour, short length of flower spike, small leaves and b: The double petal one (H K ) with 12 petal flowers and fragrant odour, higher length of flower spike, bigger leaves

Note: a: calli samples and b: clusters of shoots

- 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)

doses of two tuberose varieties/lines using the 50% lethal dose (LD 50) in

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 60 Co 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, 5 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 1 cluster of callus/shoot/nylon bag)

Table 3.1: Arranging gamma ray doses and treatments in the single petal tuberose variety

- 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 60 Co gamma ray doses on the growth and development of the double petal tuberose shoot clusters

This was laid out similarly to the experiment 1 (Table 3.2)

Table 3.2: Arranging gamma ray doses and treatments in the double petal tuberose variety

culturing were indicated as Experiment 1 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 60 Co gamma

ray on the field

a The first times of growing (V Đ M 1 )

- This experiment was conducted in a factorial randomized completely block design in three replications in 500 m2 plots size with 8 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

height and 1.0 - 2.0 shoots Every treatment was marked 5 points with 3 plants for the average data in statistics

- The experiment diagram was shown in Figure 3.4a The cultivation

process was done as Le Ly Vu Vi et al (2014)

* Experimental parameters:

- The death rate of plantlets after 60 day growing

- The growth parameters at 180 day planting: number of shoots, number of leaves on the flowering time; number of bulbs and bulb diameter (cm)

- The flowering parameters: Days to flowering when 25% plants induced inflorescence (days); inflorescence length (cm); floret number; flowering diameter (cm) Recording the abnormal types in leaves, bulbs, flowers and the fragrance (when 75% plants had inflorescences)

- Carrying to choose mutant lines by individual selection method, then naming them: tuberose line + petal numbers + irradiated dosage

- Evaluation the fragrance by sensory (smelling method) Aromatic level was assessed by the average of 10 persons which was established as a convention: 0 mark: no fragrance; 1 mark: normal fragrance và 2 marks: more fragrance The bulb control treatment was the control Each group of plants was estimated for 3 spikes when the lowest floret pair had completely opened

b The second times of growing (V Đ M 2 )

If there were some mutant tuberose lines having an increase of the petal number (more than 12 petals), flower size and fragrance, they would have grown the second times In contrast, they would have continuously stored for other purposes

* Experimental parameters when 100% plants had inflorescence:

The growth and flowering parameters, abnormal petal number types and the level of aroma were assessed likely to Part 3.2.4.1.a

Figure 3.4: The field experiment diagrams

3.2.4.2 Experiment 4: Evaluating the growth and the development

of the double petal tuberose variety/lines after irradiating with 60 Co

gamma ray on the field

a The first times of growing (V K M 1 )

This was laid out similarly to Experiment 3 but every replication grew

30 plants The plantlets had 3.0 - 5.0 leaves, 5.0 - 10.0 cm height and 1.0 - 2.0 shoots Every treatment was marked 3 points with 3 plants for collecting the data

- The experiment diagram was shown in Figure 3.4b and the cultivation process was conducted similarly to Experiment 3

* Experimental parameters: These were performed as the same as in Experiment 3, Part 3.2.4.1a

b The second times of growing (V K M 2 ): This was carried out

similarly to Part 3.2.4.1b

* Experimental parameters when 100% plants had spikes: These

were done as in Part 3.2.4.1b

3.2.4.3 DNA divergence analysis among tuberose varieties and mutants tuberose lines

a Genetic diversity evaluation by using ISSR - PCR technique

Using 14 ISSR primers (Mengli et al., 2012; Khandagale et al., 2014)

(Table 3.3)

Table 3.3: List of ISSR primers used for research

Primer Base sequence (5’- 3’) Primer Base sequence (5’- 3’)

UBC873 GACAGACAGACAGACA P23SR1 GGCTGCTTCTAAGCCAAC

- The polymorphic DNA amplification products were visualized and determined the positions of new or absent DNA bands in the two mutant lines comparing with the control bands (the double petal type variety) (bp) Cluster analysis was performed on molecular similarity matrices using the Unweighted Pair Group Method using Arithmetic Means (UPGMA) algorithm, from which dendrograms depicting similarity among genotypes were drawn using NTSYS-pc 2.1 Software (Rohlf, 2000)

b Sequencing ITS region: Using the pairs of bacteria primers (White

Chapter IV: RESULTS AND DISCUSSIONS

4.1 Research content 1

4.1.1 The meristem survival rates of two tuberose varieties

The survival rates of single petal tuberose variety were about 40% whereas the survival rates of double petal one had reached to 60% after 30

day culturing

4.1.2 Callus and shoot clusters multiplication

The alive meristems had been transfered into the shoot multiplication medium during 180 day period so that there were enough the samples for gamma radiation experiments There were appearances of calli and shoots for both of the varieties at the same time

4.2 Research content 2

4.2.1 Effects of 60 Co gamma ray doses on the growth and the development of single petal tuberose callus clusters

4.2.1.1 Shoot regenerated rate

At the 60 Gy dose, there was no any shoot initiated from calli The 50

Gy tuberose plants gave the lowest rate of the shoot regeneration (about 6.0%) On the contrary, the highest average numbers of shoots were induced

at the in vitro control treatment (90%)

4.2.1.2 The death rate of callus/shoot cluster

There were 22% death rates of the calli/shoot clusters at the lowest irradiation dose (5 Gy) The death rates were about 50% when there was an increase of the radiation doses from 15 Gy to 50 Gy and at the 60 Gy, all of them died

4.2.1.3 50% lethal dose (LD 50 ): The LD50 value of single petal

tuberose variety had the counting value of 10.96 ± 2.96 Gy (Figure 4.1)

4.2.1.4 Shoot number, shoot height and leaf number

The application of 5 and 10 Gy of doses gave the best shoot number, shoot heightand leaf length of tuberose However, the treatments of 30 and

40 Gy showed less numbers of growth parameters When plants were irradiated with 50 and 60 Gy, there was no increase of shoot number (Table 4.1)

Gamma ray

dose (Gy)

Shoot number

Shoot height (cm)

Leaf number

There were 4 abnormal structures of single petal tuberose variety due

to the impact of irradiating by different 60Co gamma doses The in vitro

control treatment had a normal shoot type; shoots with leaves sticking together and scrolling at some doses such as 5; 10; 15 and 20 Gy, and at the

Table 4.1: Shoot number, shoot height and leaf number of single petal tuberose after irradiating with gamma rays at 150 day culturing

Note: Values within column followed by different letters are significantly different at 5% probability level

percentage death (in probits) against log dose was plotted and

the dose corresponding to probit

of single petal tuberose variety at