INTRODUCTION

* Thesis imperative task
Seedless citrus fruit was loving at home and abroad market.
Seedless or few seed sitrus fruit caused by threefold chromosome
mutation, by pollen or ovule sterity or by self-incompatibility
included the self-incompatibility caused by self-pollination
(Jackson and Futch, 1997; Jackson and Gmitter, 1997) [72] [71].
In the world, almost planted citrus variety was selected from
natural mutation and only small ratio was formed from cross
breeding program. In world market, many citrus varieties appeared
from natural mutation selection, such as Navel orange, Satsuma
mandarin…(Vu Cong Hau, 1996) [40] and few citrus pieces was
discovered in our nation such as Nam Roi pomelo, seedless Mat
orange (Tran Thi Oanh Yen et al., 2005) [35].
Sanh orange and Duong mandarin adjusted to Mekong Delta
condition. Plants gave high yield, delicious quality, and got good
price so that they were planted widely. However many seed in fruit
was the reason that reduced the using and selling of fresh fruit. Fruits
researches on Sanh orange and Duong mandarin noted that there had
been many seedless or few seed cases (less than 5 seed per fruit).
Hence, Can Tho university co-operated with Vinh Long Science and
Technology Deparment for finding and studying seedless Sanh
orange and Duong mandarin in Mekong delta. That subject had the
result that two seedless Duong mandarin were discovered from
Duong mandarin population in Tan Phuoc commune, Lai Vung town,
Dong Thap province (Nguyen Bao Ve et al., 2007) [13].
Thus, subject “Study of seedless characteristic and morphology
characteristics of seedless duong mandarin in the Mekong Delta” was
carried out for having basic to develope the new seedless Duong
mandarin in Mekong delta

and fruits of the two seedless Duong mandarin plants were
homogeneous and not different to the seedy Duong mandarin plant. It
was able to identify the two seedless Duong mandarin plants by
RAPD technique with the aid of DNA markers. There was the
proximity close relationship between the two seedless Duong
mandarin plants and they were closed to the normal seedy Duong
3
mandarin plant; The cause of producing completely seedless fruits on
these two seedless Duong mandarin plants was the “late
development” of ovule; The seedlessness characteristic of the two
seedless Duong mandarin plants was stable through croping time, in
intercrop condition and due to cross pollination with other species of
citrus, at three different cultural regions in Mekong Delta, in three
grafting generations and on three kinds of rootstocks (Mat orange,
Tau lime and Hanh); These seedless Duong mandarin varieties gave
good fruit yield and quality in Mekong Delta.
+ Reality significant
In 5 years, research results helped to determine the stability of
seedless characteristic of the two seedless Duong mandarin fast, other
characteristics are basically not different to normal seedy Duong
mandarin. They are valuable scientific basises for the early
development of these precious varieties in widely production.
* Main object and range of thesis
+ Main object of thesis are two seedless Duong mandarin were
discovered in the Mekong Delta.
+ Range of thesis:
- Observate on the mother plant and progeny scion grafting plants.
- Three different cultural regions (Dong Thap, Can Tho, Vinh
Long) in Mekong Delta.
- In the period from 2007 to 2011.

Tran Thuong Tuan et al. (1999) [38] indicated that Duong
mandarin had simple leaf devision, evergreen, its color was green
with mark shape. According to Pham Hoang Ho (2003) [22], Duong
mandarin leaf have stalk but petiole is absent, leaf blade had many
conspicuous oil sacks. Its leaves often have chopped tip (Duong
Hong Dat, 2000 [3]
Citrus blossom was single or clusters, grew from axillary, be
usually bisexual flowers (Tran Thuong Tuan et al., 1994; Nguyen
Bao Ve and Le Thanh Phong, 2004; Tran Van Hau, 2009) [37]12]
[39]. Full bloom have 1.3 - 1.5 cm petal length, with 5 sepals. Five
white thick petals is alternate with the others and with the sepals.
According to Pham Hoang Ho (2003) [22], Duong mandarin
fruit had from round to slightly flattened shape, with knob at bottom
and thin peel. Duong mandarin fruits has a bright yellow and smooth
5
in ripeness stage. Each fruit had average of 10.7 sections, easily
separated and peeled by hand.
1.3 CITRUS POLLEN AND OVULE CHARACTERISTIC
Pollen have outer wall which consisted of two membrane
layers: the outer membrane and the inter membrane. According to Ha
Thi Le Anh (2005), [5], inside the wall, there are cytoplasm and two
nucleus: the big round somatic nucleus, which will germinate to
produce pollen tubes and smaller oval reproductive nucleus, which
will became two sperm.
Spiegel-Roy and Goldschmidt (1996) [125] considered that the
ovule of citrus have contrary attached format that the hole opposite to
the axis of the ovary. Matured ovule including embryonic stem,
embryo sac with eight cores and two ovule sheath. According to
Jackson and Gmitter (1997) [71], the sooner or later mature
(maturity, ripeness) depend on variety characteristic. This feature can

stimulation (Spiegel-Roy and Goldschmidt, 1996)[125]. On citrus,
strong sterity combined with Autonomic parthenocarpus to become
the necessary condition for production seedless fruit (Ollitrault et al.
2007) [101]. Eventhough sterity is capable, seedless fruit can not be
produced because of incapable autonomic parthenocarpus. Ollitrault
et al. (2007) [101] considered that the Autonomic parthenocarpus,
caused seedlessness without pollination, was a key process in citrus.
According Ollitrault et al. (2007) [101], several varieties of male
sterility and self-incompatibility can not form seedless fruit because
autonomic parthenocarpus was absent. Therefore, the autonomic
parthenocarpus ability are indispensable characteristics for the
production of seedless fruits and this seem to be popular on citrus.
Mooney (1997) [89] found that triploid Citrus have great
commercial potential because of the high degree of seedless, but the
frequency of triploid plants were very low in the wild. Reed (2003)
[113] thought that self-incompatibility is the disfunction of the male
and female gametes to form seeds when self-pollinated occurs, the
mismatch of the male gametes on the same species stigmas, or
prevent the growth of the pollen in the same species styles. The self-
incompatibility will create seedless fruit when they were not planted
with the other spieces.
Self male sterility can result from abnormal development of the
stamens leaded to deficiencies in the pollen development process.
Tran Thuong Tuan (1992), [36] indicated that unviable male gametes
caused of the male sterility. Female sterility is a very important trait
7
which closely related to the seedlesss characteristics and heritability
(Yamamoto et al., 1995; Yamamoto et al., 2001) [149] [148].

CHAPTER 2

CHAPTER 3
RESULTS AND DISCUSSIONS
3.1 MOPHOLOGY CHARACTERISTIC AND GENETIC
RELATIONSHIP OF SEEDLESS DUONG MANDARIN
The results showed that canopy got elliptical shaped, vertical
posture, branch density were sparse, scion direction and branching
devision much, sparse spine density, spine is straight and short,
smooth tree-trunk and angle of branching were moderate. In general,
the mophology characteristics of the two seedless Duong mandarin
were same to normal seedy Duong mandarin.
Leaf morphological characteristics between the two seedless
Duong mandarin is same to each other and not different with seedy
Duong mandarin. The flowers characteristics of two seedless Duong
mandarin are the same and similar to seedy mandarin. Time from the
onset of bud to the blooming of the two seedless Duong mandarin is
about 11 - 12 days, from blooming to stigma fall is about 2 - 3 days,
the same to normal seedy Duong, mandarin (control). These shows
that development period and capable blooming period for receiving
pollen in two seedless Duong mandarin is normal, and not different
to seedy Duong mandarin (control). The flower development of
seedless Duong mandarin No. 1 is same with seedless Duong
mandarin No. 2 and be same to seedy Duong mandarin(control)
Fruits of two seedless Duong mandarin and fruits of seedy
Duong mandarin have the same shape. They are big circular and
rather flat. Bottom of fruit have knob, top of fruit got slightly
concave. Peel transfer to smooth yellow Green when ripe. Basically,
the morphological characteristics between the two seedless Duong
mandarin are same, and not different to seedy Duong mandarin
(completely seedless characteristic).
In short, except the completely seedless characteristic,


Downside

Cross side

10

Figure 3.5 Seedless Duong mandarin PCR record with A13 primer
M: standard meter 1 kb; 1: seedless Duong mandarin No.1; 2: seedless
Duong mandarin No.2; 3: seedy Duong mandarin (control).
Figure 3.8 shows two seedless Duong mandarin and seedy
Duong mandarin as control fusion to be a main branch. It included
seedy Duong mandarin, seedless Duong mandarin No. 1 and seedless
Duong mandarin No. 2 with the high coefficient (0.87). Two seedless
Duong mandarin constitute a secondary branch and they have the
same coefficient (0.92). Thus, the two seedless Duong mandarin had
the close genetic relationship to each other and they have the close
genetic relationship to seedy Duong mandarin.

Figure 3.8 Branch diagram of seedless Duong mandarin
1: seedless Duong mandarin No. 1; 2: seedless Duong mandarin No. 2;

SEEDLESS DUONG MANDARIN
3.2.1 Pollen characteristic
Observations results showed that the pollen germination and
pollen tube length of the seedless Duong mandarin No. 1 and
seedless Duong mandarin No. 2 seed were not significant different to
seedy Duong mandarin (control). Force pollination results also
showed that using natural pollen of seedless Duong mandarin No. 1
and No. 2 in seedy Duong mandarin, the number of full seeds/ fruit,
flat seeds/ fruit were no significant differences to each other and to
seedy Duong mandarin (control). It showed that pollen of seedless
Duong mandarin were unsterile. Thus, seedless characteristics of the
two seedless Duong mandarin is not caused by pollen.
3.2.2 Ovule characteristic
3.2.2.1 Ovule Developement
* Before full bloom stage
In seedy Duong mandarin, ovule appeared early and their
quantity was stable on 5 flower stage (from 10.4 to 10.8 ovule). In
contrary, there are completely no ovule in seedless Duong mandarin
No. 1 and No.2 till full bloom stage. Late development characteristic
in full bloom stage may help to form seedless fruit in seedless Duong
mandarin No.1 and No.2
Table 3.18 and Table 3.19 show that in small flowers (flower
diameter is 3.5 mm), the ovule size of seedy Duong mandarin was
quite large (203 µm length and 130 µm width) and rapidly developed
in 5 mm. from this moment till the full bloom stage, the ovule size of
seedy Duong mandarin virtually unchanged (396 - 404 µm length and
12
246 - 258 µm width). At this time, the ovule had the pear shape
(Figure 3.14).
The stability of the size of the ovule in the period before and on


X

Sd

Seedless No.1 0 - 0 - 0 - 0 - 0 -
Seedless No.2 0 - 0 - 0 - 0 - 0 -
Seedy (control) 203

20

279

28

331

48

396

35

404

21

X
: average; Sd: standard deviation;


23
X
: average value; Sd: standard deviation;
13
Figure 3.14 Ovary Cross slices before blooming of seedless Duong
mandarin; zoom 100 time.
Flower
with
4 mm
diameter

Flower
with
4.5 mm
diameter

Flower
with
5 mm
diameter
Blooming
Seedless No. 1 Seedless No. 2 Seedy (control)
Flower
with

diameter
100 µm
Seedless No.
1

Seedless No.
2
Seedy (control)

15
flower side (petal fall: 411 µm, stigma fall: 443 µm, fruit sixe at 2.5
mm: 470 µm). The change of this size may be due to the process of
developing seeds after the fertilized. Meanwhile, in two seedless
Duong mandarin there are few ovules appear, but the size is smaller,
petal fall had the range of 77-81μm length and in the range of 63 - 66
µm width, in stigma fall, the length is in the range of 127 - 131 µm
and at fruit size 2.5 mm had length in 191 - 199 µm. The results
showed that, in seedless Duong mandarin No. 1 and No. 2, till 2.5
mm diameter fruit, ovule is still developing, has not yet reached
mature size. So maybe, ovule of two seedless Duong mandarin still
immature at this time.
In summary, the ovule of two seedless Duong mandarin had the
same "late development" characteristics to the ovule of seedy Duong
mandarin. May be the ovule characteristics "late development" is
cause for seedless fruit is seedless Duong mandarin.
3.2.2.2 Pollent tube appearance in ovary
* Self pollination condition

styles
Below
styles
Ovary Seedless No. 1 Seedless No. 2 Seedy (control)
17
pollinate with Mat orange pollen), most parts of the female structure
(stigma, above styles, below styles and ovary) were recorded that
there were the presence of pollen tubes (93.3 to 100%), difference
was not significant by χ2 testing.
3.2.2.3 Ovule fertilization bability
Seedless Duong mandarin No. 1 and No. 2 had fruits with
absolutely no-pollinated seeds in 3 types of pollination. Meanwhile,
the seedy Duong mandarin with 3 types of pollination have the
formation of seeds, number of full seeds/fruit was in range of 9.07 to
11.53 and the number of immature seeds/ fruit was 1.93 to 2.03
3.2.3 Discussion about seedless Cause
Flowering stage is the best time for pollination, fertilization and
forming seed. At this time, in the seedy Duong mandarin, through
morphological anatomy cross-sectional ovary, most ovule is mature,
perhaps due to this characteristic made famous commercial Duong
mandarin in the Mekong Delta has many seeds. Meanwhile, ovule of
two seedless Duong mandarin were still immature. By the petal fall
stage (about 2 - 3 days after blooming), the ovule of two seedless
Duong mandarim appeared, but they got small size and far from
mature size. So, whether pollination at this time (very difficult to
occurs due to petal fall, dried stigma, color changed and no longer to
receive pollen anymore), then fertilization is difficult to implement

No. 2 in all 3 experiments regions (Dong Thap, Can Tho, Vinh
Long). At the time of 3 days after blooming (petal fall), in all 3
experiments regions, the ovule in seedless Duong mandarin No. 1
and No. 2 ovules developed more than blooming stage, but there are
few ovule with small size.
All harvest fruits were completely seedless . Meanwhile, the
seedy Duong mandarin in three point Lai Vung - Dong Thap, Can
Tho city and Tra On Vinh Long sequential of full seed/ fruit is 8.56,
10.26 and 12.22; and immature seeds / fruit was 2.33, 2.08 and 2.36.
In short, absolutely seedlessness characteristics of two seedless
Duong mandarin may have genetic ability and maintain stable in
clonal generations (on Mat orange rootstock) in 3 cultivation regions
(Dong Thap, Can Tho and Vinh Long) of Mekong Delta.
3.3.3 Stability of seedlessness in grafting generations
Ovary observation results showed that do not have the presence
of the ovule at the blooming stage. At three days after blooming stage
there are no ovule with the mature ovule morphology and size
(ripening). Two seedless Duong mandarin through three generations
1 a 1 b
19
on Mat rootstock gave absolutely no seeds in the fruit of all
treatments.
In short, absolutely seedlessness characteristics of seedless
Duong mandarin No. 1 and No. 2 have the genetic ability and
maintain stability in the three grafting generations on Mat orange
rootstock.
3.3.4 Stability of seedlessness on different rootstock
At the blooming stage and 3 days after blooming stage, threr are
no ovule with mature ovule morphology and size in all treatments.
Full seeds in all treatments was 0.00. Immature seeds also have

and No.2 by grafting on 3 types of rootstock (Mat orange, Tau lime
and Hanh), planted in regions that had comfortable condition for
growing citrus in Mekong Delta and especially can be intercropped
with other citrus that absolutely seedlessness characteristic remained
stable.
However, accordiing to Jackson and Gmitter (1997) [71]
sooner or later maturity characteristics of the ovule can be affected by
a number of agents inside and outside. So, there may be some factors
that make the development of seedless Duong mandarin No. 1 and 2
ovule mature earlier and the time that stigmas receive pollen will be
longer (the period from blooming to petal fall for about 2 - 3 days in
seedless Duong mandarin), then this time can occur fertilization and
formation of seeds in seedless Duong mandarin. This issue should be
studied further.
3.4 YIELD POTENTIAL AND FRUIT QUALITY OF SEEDLESS
DUONG MANDARIN
3.4.1 Growing of tree
Diameter of rootstock, scion diameter, the ratio of rootstock
diameter and scion diameter, plant height, canopy width through
croping time showed that there are no difference between three
treatments. Therefore, the growth of seedless Duong mandarin No. 1
No. 2 seed is not equivalent to each other and do not different with
seedy Duong mandarin which were grown popular in the Mekong
Delta.
3.4.2 Yield
Percentage of fruit set can be used to evaluate the yield
potential of seedless Duong mandarin No. 1 and No. 2. Table 3.32
shows the percentage of fruit set of three treatments with the general
decreasing trend in the period from 30 - 90 days after flowering, but
21

mandarin tend to be lower than the seedy Duong mandarin (control).
22
Table 3.33 Total fruit per tree, fruit weight and Yield of seedless
Duong mandarin
Treatment
(Duong mandarin)
Total fruit

per tree
Fruit
weight (g)
Yield
(kg/tree)
Seedless No. 1

157

98,9

15,0 ab

Seedless No. 2 131 88,0 11,3 b
Seedy (control) 177 117,2 20,5 a
F

ns


citrus in the Mekong Delta.
23
3.4.3 Fruit quality
Flesh of seedless Duong mandarin No. 1 and No.2 and flesh of
seedy Duong mandarin have color of orange, fruit flesh color is same
to each other (Figure 3.32). Fruit core is quite vesicle, fruit juice is
very sweet and fragrant.
Figure 3.32 Cross slice of seedless Duong mandarin
Brix degree of them are in range of 9.24-9.47%, pH is in range
of 4.70-4.88 and quantity of vitamin C is in range of 35.2-36.5
mg/100g. They all are not significantly different among 3 treatments.
Due to those results showed that fruit quality of seedless Duong
mandarin No.1 and No.2 are not different with seedy Duong
mandarin fruit quality.
In short, the growth of seedless Duong mandarin No. 1 and
No. 2 are normal, equivalent tantamount to seedy Duong mandarin.
Both seedless Duong mandarin No. 1 and seedless Duong mandarin
No. 2 have delicious fruit quality, equivalent to each other and not
different to seedy Duong mandarin, which is famously fragrant,
delicious and sweet in the Mekong Delta, more specially that they are
seedless. Although the yield of seedless Duong mandarin lightly
lower than seedy Duong mandarin, seedless Duong mandarin have
the development potential in suitable citrus growing conditions
regions of Mekong Delta with the good yield potential and quality.
CHAPTER 4
CONCLUSION AND SUGGESTION
4.1 CONCLUSION
- The morphology characteristics such as branches, leaves,
flowers and fruits of the two seedless Duong mandarin plants were
homogeneous and not different to the seedy Duong mandarin plant. It

aimed to produce varieties and develope widely cultivation in
Mekong Delta.
- Researching and evaluating the level of parthenocarpy character
of seedless Duong mandarin. Concurrently, it is essential to research
approriate cultivation techniques for seedless Duong mandarin
production.
- Understanding more factors (plant hormones, nutrients,…)
have affected on stability of completely seedless characteristic of
seedless Duong mandarin fruit.
- In following researchs, finding out hereditary characteristics
of seedless Duong mandarin, it’s able to use SSR (Simple Sequence
Repeat) and SCAR technique (Sequense Characterized Amplifield
Region) because of their specialization.