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

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:

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
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).
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

1

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.
2


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
3


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 6
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 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).
4


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, 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).
5


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

Control
5
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 1 (Table 3.2).
Table 3.2: Arranging gamma ray doses and treatments in the double
petal tuberose variety
Variety
Control
HK
T1

5
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 1 (culturing 1 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
6


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 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 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
7


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 (VĐM2)
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.

8


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 60Co
gamma ray on the field
a. The first times of growing (VKM1)
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 (VKM2): This was carried out
similarly to Part 3.2.4.1b.

9


* 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’)

3A01
3A07
3A21

3A39
3A42
3A62
UBC873

(GA)8TC
(AG)7CTT
(TG)7ACC
(CA)7GTA
(GACA)4C
(TG)7ACT
GACAGACAGACAGACA

808
836
840
842
855
857
P23SR1

(AG)8C
(AG)8YA
(AG)8YT
(AG)8YG
(AC)8YT
(AC)8YG
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
et al., 1990):
ITS1: 5’ - TCCGTAGGTGAACCTGCGG - 3’
ITS4: 5’ - TCCTCCGCTTATTGATATGC - 3’
- Testing the ITS by BLAST (Basic Local Alignmet Search Tool) on
NCBI (National Center for Biotechnology Information) database to be sure
that these sequences were Polianthes spp. Aligning these to determine the
positions and types of DNA mutants.
10


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 60Co 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 (LD50): The LD50 value of single petal
tuberose variety had the counting value of 10.96 ± 2.96 Gy (Figure 4.1).

11


Probit

Figure 4.1: The graph of
percentage death (in probits)
against log dose was plotted and
the dose corresponding to probit
of single petal tuberose variety at
150 day culturing

Linear regression

Note:

Log-dose

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 height and 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).
Table 4.1: Shoot number,
Gamma ray
Shoot
Shoot height
Leaf
dose (Gy)
number
(cm)
number shoot height and leaf
0
3.2b
6.4a
14.7b
a
ab
number of single petal
5
4.2
6.2
18.3a
b
ab

10
3.5
5.9
17.5a
tuberose after irradiating
15
2.5c
5.4b
11.0c
with gamma rays at 150
20
1.9d
3.9c
7.9d
cd
c
e
25
2.0
3.8
3.7
day culturing
30
40
50
60
F
CV (%)

0.9e

0.5e
0.0f
0.0f
*
22.9

3.7c
0.7d
0.1d
0.0d
*
21.4

2.3ef
1.2fi
0.07i
0.0i
*
26.0

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

4.2.1.5. Effects of 60Co gamma ray doses on the single petal
tuberose abnormal structures
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

12


doses 25; 30 and 40 Gy, shoots had twitch and scrolling leaves. The albino
shoot type appeared only at the doses of 40 and 50 Gy.
4.2.2. Effects of 60Co gamma ray doses on the growth and
development of the double petal tuberose shoot clusters
4.2.2.1. The death rate of shoot cluster
At the lower dose of 40 Gy, the death rate of shoot clusters was less
than 50%. This increased dramatically at 50 Gy dose and reached to 100%
at 60 Gy.
4.2.2.2 50% lethal dose: The LD50 value of double petal tuberose
variety had the counting value of 22.91 Gy ± 4.01 Gy (Figure 4.2).
Probits

Linear regression

Figure 4.2: The graph of
percentage death (in probits)
against log dose was plotted
and the dose corresponding to
probit of double petal tuberose
variety at 150 day culturing
Note:

Log-dose

4.2.2.3 Shoot number, shoot height and leaf number

The shoot numbers had multiplied at doses of 5; 15 and 25 Gy during
the culture time. Besides, the shoot height gained maximum values at 5 and
15 Gy treatments. Data regarding number of leaves showed the maximum
values (about 21 leaves) in case of 52 Gy dose. The obtained results also
indicated that the number of leaves still increased althought there was no
increase of shoots and of their height at 60 Gy dose in 150 day culturing
(Table 4.2).

13


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
0
150
Shoot
Shoot
Shoot
Leaf
Shoot
Leaf
height
height
number
number number
number
(cm)
(cm)
0

3.4
1.1
2.2
7.3a
5.4b
18.0b
ab
a
5
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 5 Gy treatments had no mutation in leaves or
shoots. There were the total of 6 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, 5 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 3
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 5 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
4.4.1. 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, 5 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 5 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
5
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 và 2.9 cm) were produced at
5 and 30 Gy doses. Because of the unopened flowers, the 5 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
5
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 7 leaf phenotypes of single petal tuberose
lines after 60 day cultivating. Both of the control treatments with normal
17


leaves; the 5 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 3 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 5 Gy line. There were 75% of
the spikes with 6; 7 and 8 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 2 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 6 petal floret ); b: 15 Gy (the 7 and 8 petal
florets); c: 25 Gy (the 7 and 8 petal florets) và d: 30 Gy (the 7 and 8 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 0 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 5 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

5
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;
5 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 5 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

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; 5 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 5 of stem and bulb phenotypes

for mutant double petal tuberose varieties/lines. The bulb control treatment
had symmetric leaves; the 5 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 5 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; 9 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 0 and 5 Gy; b: the 6 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, 6 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 4 categories depending
on the number of petals. The 1st group consists of 4; 5; 6; 9 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 2 marks, so they have
more fragrancy than the others do. The mutant lines belonging to the 3rd and
4th group were evaluated with 0 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