MINISTRY OF EDUCATION
AND TRAINING
MINISTRY OF EDUCATION
AND TRAINING
MINISTRY OF AGRICULTURE
AND RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
LE QUOC HUNG
RESEARCH ON SELECTING SOME TRIPLOD LINES CITRUS
VARIETIES
Major Field: GENETICS AND PLANT BREEDING
Code: 62.62.01.11
DOCTORAL THESIS SUMMARY
HA NOI - 2015
The Doctoral thesis was completed in
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
Scientific Supervisors: 1. Prof.Dr. Do Nang Vinh
2. Assoc.Prof. Dr. Ha Thi Thuy
Reviewer 1:
Reviewer 2:
Reviewer 3:
Dissertation will be defended before committee in charge for doctoral
dissertation in academy level, meeting place 󰜧󰜧󰜧 on 󰜧󰜧󰜧󰜧, 2015
The PhD thesis may be fount at:
- Ha Noi National Library
- Library in Vietnam Academy of Agricultural Sciences
HA NOI - 2015
1
INTRODUCTION
* Urgency of research
Citrus trees are one of fruit crops which have high nutrition and economic

value. Production of citrus fruit all over the world reached 85.6 ton in the years of
2012/2013, in which orange production accountes 50% (USDA, 2013). This number
will be continued increasing because of the demand in some countries are highly
expanded such as China, Korea, Russia, India, East Europe and Asia. In China, the
consumption of fresh fruit has been singnificantly increasing, especially citrus fruits.
In recent years, China has ranked top second biggest citrus fruit production after
Brazil (Deng, 2008).
Vietnam is one of the center in which Citrus are originated (Vo Van Chi, 1997;
Pham Hoang Ho, 1992). The area of citrus in 2011 is 138,200 ha, about 18 percent of
all fruit area and the production was estimated about 1.35 million tons (Department
of Planting, Ministry of Agriculture and Rural Development, 2013). However,
Vietnam still has to import a sheer amount of citrus fruit annually because of low
productivity causing by diseases and the quality of varieties (low quality and
containing many seeds).
Selecting seedless citrus are being conducted in some commonplace following
approaches.
󽟙 Mutating
󽟙 Mating between diploid lines (2x) and tetraploid lines (4x) to select triploid
lines (3x) (Roose and Williams, 2000)
󽟙 Culturing endorsperm of immature seeds (Gmitter et al, 1990)
󽟙 Selecting triploid embryos in nature (3x) (Esen et al, 1971)
󽟙 Rescuing triploid embryos from dud seeds (Ollitrault et al, 1996)
󽟙 Implementing fusion between diploid protoplast and haploid protoplast
cells (Ollitraul et al, 2000)
󽟙 Applying biotechnology techniques in embryo rescuing, protoplast fusion,
selecting somaclonal variations (Froelicher et al, 2003; Grosser et al, 2000;
Juárez et al, 1990; Ollitrault et al, 1998)
In Vietnam, some well-known Citrus cultivars are widely grown widespread
such as Xa Doai, Van Du, Citrus nobilis (King Mandarin), Bu orange, Phuc Trach,
Dien, Doan Hung pummelos󰜧 These varieties are high quality and economic value

but contains many seeds. To improve traits by selecting from these varieties based on
the ability to generate heterosis, well-adapted and disease resistance is top priority.
From the above judging, we have conducted a dissertation: 󰜝Research on selecting
some triploid lines in citrus varieties󰜞
* Aims of research
To produce a larger number of triploid lines of pummelos and King Orange
(Citrus nobilis) as the initial resources for selecting varieties with non-seed and fewer
seed varieties.
2
Testing, evaluating and selecting triploid pomelo and Citrus nobilis lines to
identify promising lines and establish selecting approaches
* Breakthrough of this research
For the first time in Vietnam, we have selected a large number of triploid
Citrus lines by mating (2x 󽞵 4x) and Citrus nobilis (2x 󽞵 2x) types including 92
triploid pummelo lines and 22 triploid Citrus nobilis lines.
There are 2 King orange lines (2x) and 6 pummelo lines (3x), all are well
growth, non-diseases. Two lines of 6 pummelo lines above have been produced
seedless fruits after budded on 5 year old of sour pummelo. The fruit is round shape,
yellow skin, succulent, sweet, yellow epicarp and 494,16 g on average.
Confirming the ability of hybridizing method in order to make triploid pomelo
lines and by budded on 5 year old of sour pummelo to shorten the growth of 3x lines
compared with budded on 7 to 8 month Citrus hystrix D.C.
* Contribution of the dissertation
Scientific aspect: The project has provided a substantial number of selected
triploid citrus lines from the native cultivars. From which, all triploid pummelo lines
are well-growth, two of them have produced seedless fruits. Especially, the project
knowledge was made a direction for further researching and selecting triploid Citrus
nobilis and Citrus grandis in Vietnam. We have also successfully estabshed and
applied technological procedure for selecting triploid citrus, for example, the success
of mating 8 pairs between diploid and tetraploid to collect a large number of different

triploid. We have also made integration between traditional method and
biotechnology methods such as (rescuing embryo) and some techniques for quick
selection (by budded on flowering citrus plants). All knowledge can be used as
reference for researching and lecturing about citrus fruits.
Reality aspect: The project has provided a substantial number of triploid lines
as resources for mating between different types. There are 2 triploid pummelo lines
and some Citrus nobilis lines from the specility citrus of Vietnam. They are being
tested for confirmation as national cultivars.
* Materials and scope of the research
Material:
Father plants provided pollen are Phuc Trach, Dien pummelos and King
(Citrus nobilis), Van Du orange which are tetraploid types were selected by
colchicine treatment belonging to the project: 󰜝Researching and selecting seedless
citrus fruit plant from indigenous󰜞 (2001 to 2005). These tetraploid plants have been
grown in Van Giang, Hung Yen province which also have conferred and authorized
by Department of Planting, Ministry of Agriculture and Rural Development, 515/QD-
TT-VPVH, 12
th
, November, 2013. Mother plant (2x) was selected from healthy plants
,well-flowering Nam Roi, Phuc Trach, Dien pummelos and King oranges (Citrus
nobilis). Triploid pummelo and Citrus nobilis lines also utilized and selected by
rescuing embryos from dud seeds belonging to the project: 󰜝Researching and
3
selecting seedless pummelos, orange and grapefruit applied biotechnology󰜞 (2006 to
2010).
Scope of the research:
The research are conducted in Vietnam. In specific, the experiment were
conducted at Key national of plant cell Lab (Agricultural Genetics Institute), Van
Giang, Hung Yen practical farm, Vegetable and Fruit Research Institute and other
fruit farms in some provinces such as Cao Phong (Hoa Binh province), Ham Yen

(Tuyen Quang province).
CHAPTER I
OVERVIEW OF CITRUS
1.1. Methods for selecting seedless Citrus varieties
Research for selecting Citrus fruits have been focused on comparing
productivity, quality and disease resistance, especially with seedless trait are priority
(Esen and Soost, 1971; Ollitrault et al, 1998; Ollitrault et al, 2000; Ollitrault et al,
2006; Koltunow et al, 1998; Ray, 2002; Deng X.X, 2006; Grosser et al, 2006; Jean
Baptiste Bassenet et al, 2009). Some non-sticky skin, seedless, nutrient, storage and
disease resistance traits are concerned by scientists (Ollitrault et al, 2000; Roose and
Williams, 2000). Utilizing heterosis, multiploid, protoplast fusing and somaclonal
variation were applied to select seedless lines with disease resistance trait serving
beverage industry and fresh fruit consumption (Guo et al, 2004; Grosser and Gmitter,
2005; Gmitter et al, 2007; Febres et al, 2009). Some researches were applied
transgenic technology to select varieties with disease resistance and quality traits
(Gmitter et al, 2007).
1.2. Selecting seedless fruit by radioactive and natural mutation methods
In 10 years, China has conferred 17 new Citrus cultivars, was selected by 2
methods: selecting natural variations and radioactive mutations, including, 3 seedless
and 1 little seed grapefruits, 3 seedless and 1 little seed oranges, 2 seedless pomelo
varieties. Furthermore, some mutated cultivars have ripen longer, some were earlier
but all of them have higher quality and productivity than the control (Deng, 2000).
1.3. Hybridizing between diploid and tetraploid types
The crucial strategy to produce seedless Citrus plants is implemented mating
between tetraploid and diploid lines (Soost and Cameron, 1975; Starrantino and
Recupero, 1981). For instance, the typicalness of sucessful results of generating 2
triploid pummelo cultivars Oroblanco and Melagold were made (Soost and Cameron,
1980, 1985). However, the process of selecting high quality seedless cultivars has
coper with many difficulties because of the shortage of gene pool of tetraploid
cultivars.

In the mating, diploid cultivars was utilized as mother plants and tetraploid was
used as fathers. Triploid embryos are not developed normally because the relative
4
between embryo and endosperm is disconnected. So, the scientists have rescued such
embryos by culturing them in vitro (Gmitter, 1995).
The germination of collected triploid seeds is hard, just few seeds from 2 to 10
seeds per 100 fertilized flowers were possobly. Some mating pairs (2x x 4x) produce
small fruits with germinated seeds. However, these young fruits often fall down in
June. Some scientists of California State (US) developed a technique to rescue fallen
young fruits by culturing their embryos on high sugar and citric acid medium without
regulator chemicals. The results showed that the number of plants was increased up
to 3 times (Williams and Roose, 2000).
1.4. Selecting tetraploid cultivars for diploid and tetraploid plant mating
Selecting tetraploid lines can be implemented by the different methods. The
tetraploid lines (4x) randomly appear in nature with relatively high frequency.
Cameron and Frost (1968) obtained 2.5 percent per 3,600 trees from various
cultivars.
Tachikawa et al, (1961) used colchicine in order to produce tetraploid Citrus
and then hybridized with diploid plants. Tetraploid types are able to produce by
protoplast fusing (Grosser et al, 2000). To summary, there are well-known methods
to make tetraploid lines: randomly in nature, colchicine treatment, protoplast fusing.
1.5. Invitro propagation for selecting triploid Citrus
a. Invitro embryo rescuing technique
The target is rescuing embryos from mating pairs between 2x and 4x lines and
2x and 2x. Determining different developing stages of embryos is very important,
Starrantino and Redupero (1981) has obtained triploid trees by rescuing embryos 3 to
4 months embryos after pollinating. Some can be done when fruits are ripen, by
culture cause less developed seeds (Froelicher et al, 2003).
The prevalent medium for embryo rescuing is MT (Murashige and Tucker,
1969). Some elements are added on medium for regeneration, for example; Adenine

sulfate (25mg/l) and malt extract (500mg/l) made beneficial effects, some hormones
support embryo germination and shoot induction such as GA3 (1mg/l) balanced with
BAP (0.5 mg/l) + 0.5 mg/l kinetin + 0.1 mg/l NAA.
The procedure of triploid embryo rescuing was developed by scientist of
CIRAD-INRA (Froelicher et al, 2003). The optimum medium is MT (Murashige and
Tulecker, 1969) + 30 g/l Sucrose + 25 mg/l adenine sulfate + 500 mg/l malt extract +
1 mg/l GA3 + 8 g/l agar, pH = 5.7
b. Rescuing triploid embryos obtained from 2n and 4n mating
Hybridizing mother diploid plants with pollen from tetraploid plants have been
conducted for a long time (Esen and Soost, 1972). However, the obtained embryos
was not develop normally, just few seeds can be germinated (Cameron and Frost,
1968; Tachikawa et al, 1961; Esen and Soost, 1972). Some reports have suggested
that uncompleted development of triploid seeds are related to endorsperm
abnormality. So, to overcome this barrier, scientists have rescued embryos by
culturing them on in vitro medium.
5
Starrantino and Recupero (1981) have cultured 585 embryos selected from 3
diploid mother lines with 6 tetraploid lines hybridizing pairs on MS medium by
adding 500 mg/l malt extract and 25 mg/l adenine sulfate. They have collected 211
triploid plantlets and 89 unidentified chromosome number were collected. They
showed that culturing embryos after 3 to 4 months pollinated could be generated
other embryos which produced many plantlets.
Oiyama and Kobayashi (1993) collected triploid plants by rescuing dud seeds
from hybridizing pairs between single embryo grapefruit cultivar Clementin (Citrus
reticulate), Miyauchi Iyokan (Citrus natsudaidai Hayata) with pollen of tetraploid
tree Kawano Natsudaidai (Citrus natsudaidai Hayata. The medium for culturing is
added MT 500 mg/l malt extract and 40 mg/l adenine sulfate after pollinating 7
months. Besides, Oiyama also have collected high ratio of triploid plants by culturing
undifferentiated small embryos forming from dud seeds of ripen fruits on MT + 1
mg/l GA. Grapefruit Clementin used as the mother and father are tetraploid plant by

hybridizing between sweet orange (Citrus sinensis) and three leaf orange (Poncirus
trifoliata).
1.6. Methods for identifying different ploid types in Citrus
By using FC machine has allowed us to identify quickly different ploid types.
CHAPTER 2
MATERIALS AND METHODS
2.1. Materials and time schedule
Father plants provided pollen are Phuc Trach, Dien pummelos and King
(Citrus nobilis), Van Du oranges which are tetraploid types selected by colchicine
treatment belonging to the project: 󰜝Researching and selecting seedless citrus fruit
plant from indigenous󰜞 (2001 to 2005).
These tetraploid plants have been grown in Van Giang, Hung Yen province
which have also conferred and authorized by Department of Planting, Ministry of
Agriculture and Rural Development, 515/QD-TT-VPVH, 12
th
, November, 2013.
Mother plant (2x) was selected from healthy plant, well-flowering Nam Roi,
Phuc Trach, Dien pummelos and King orange (Citrus nobilis). Triploid pomelo and
Citrus nobilis lines also utilized to select by rescuing embyros from dud seeds
belonging to the project: 󰜝Researching and selecting seedless pummelo, orange and
grapefruit applied biotechnology󰜞 (2006 to 2010).
Time ranges from 2011 to 2014
2.2. Dissertation contents
2.2.1. Preparing initial materials for selecting seedless and fewer seed citrus
varieties
- Hybridizing between same, different varieties and different species
- Rescuing embryos to make initial sources
6
- Assessment of agro-biological characteristics of some selected triploid lines
2.2.2. Evaluating agro-biological characteristics and selecting triploid pummelos

and Citrus nobilis lines in nethouse
- Assessment of agro-biological characteristics of triploid Citrus nobilis lines
in nethouse
- Assessment of agro-biological characteristics of triploid pummelo lines in
nethouse
2.2.3. First steps for testing triploid pummelos and citrus nobilis lines
- Testing for triploid Citrus nobilis lines budded on Citrus hystix
- Testing for triploid pummelo lines budded on Citrus hystix
- Evaluation results of triploid lines budded on 5 year old of sour pummelo
plants and identifying some promising lines.
2.3. Methods
2.3.1. Preparing initial materials for selecting seedless and fewer seed citrus
varieties
Describing characteristics of diploid and tetraploid Citrus species for
assessment and selection of promising individual plants with useful genetic traits as
initial materials.
No.
Mating pairs
Gender of plants
1
󰵁 PT2x × 󰵃 PT4x
󰵁Phuc Trach Pummelo(2x) × 󰵃Phuc Trach
Pummelo(4x)
2
󰵁 BD2x × 󰵃 PT4x
󰵁 Dien Pummelo(2x) × 󰵃 Dien Pummelo(4x)
3
󰵁 NR2x × 󰵃 PT4x
󰵁Nam Roi Pummelo(2x) × 󰵃Phuc Trach
Pummelo(4x)

4
󰵁 PT2x × 󰵃 BD4x
󰵁Phuc Trach Pummelo(2x) × 󰵃Dien
Pummelo(4x)
5
󰵁 BD2x×󰵃 BD4x
󰵁Dien Pummelo (2x) × 󰵃Dien Pummelo (4x)
6
󰵁 NR2x × 󰵃 BD4x
󰵁Nam Roi Pummelo (2x)×󰵃Dien Pummelo (4x)
7
󰵁 CS2x × 󰵃 CS4x
󰵁 Cam Sanh (2x) × 󰵃 Cam Sanh (4x)
C. nobilis 2x × C. nobilis 4x
8
󰵁 CS2x × 󰵃 VD4x
󰵁 Cam Sanh (2x) × 󰵃 Van Du Orange (4x)
C. nobilis 2x × C. sinensis (L.) Osbek 4x
9
(control)
󰵁 CS2x × 󰵃 CS2x
󰵁 Cam Sanh (2x) × 󰵃 Cam Sanh (2x)
C. nobilis 2x × C. nobilis 2x
Hybridizing method: Flowers of mother plants in mating are on top of
branches, healthy and earlier blossoming. Before blossoming 1 to 2 days, the flowers
have to be removed anthers. Flowers with matured stigma need to be hybridized by
selected father with rapid pollen and then covered with nylon packs to avoid other
pollen. With small flowers, we have used specific small broom for hybridizing. After
hybridizing, we removed all the non-hybridizing flowers to ensure the remained
flowers not competing with nutrition. After hybridizing 2 to 3 weeks, disentangling

nylon packs and marking positions of hybridized flowers. For natural hybridizing
7
pairs 󰵁 CS2x × 󰵃 CS2x, marking 30 flowers to observe fertilizing ability and
remained fruit ratio.
After fruit ripening, collecting fruits to assess of remained fruit ratio and
number of seeds per fruit of different mating pairs were made. Hybridizing seeds
have shown normal, less developed (containing embryos) and small seeds. Dud seeds
with embryos of Citrus nobilis mating pairs have sterilized and cultured on suitable
medium. Embryo rescuing medium is optimum for small and dud seeds (Ha Thi Thuy
et al, 2009) that is basal medium (Murashige and Tucker, 1969) adding 1.0 mg/l
GA3, 15% coconut liquid, 30 g/l sucrose and 5 g/l agar.
Identifying ploid level by Flow Cytometry machine, following the method of
Ollitrault et al, 1992 procedure and machine provider󰜚s guidance Partec, Germany.
Solution for separating and dying samples was spplied by Partec, Germany.
2.3.2. Assessment of agro-biological characteristics of triploid pomelo and Citrus
nobilis lines in nethouse and fields.
Assessment of morphology was carried out following the guiden documents of
International Plant Genetic Resources Institute document and procedure for testing
pomelo, grapefruit and orange of Ministry of Agriculture and Rural Development
(10TCN-2007). The traits are observed: shape of canopy, diameter of stem,
branching, stipule density, leaf morphology, buds growth, morphology and time of
flowers and fruits, factors contributing productivity and quality.
The morphology and structure of flowers includes tension of petals, color of
anthers and pollen have observed by naked eyes and microscope Leica DMLS2 at
magnitude 40 x 0.65 and 100 x 1.25 magnification. Pollen surviving and pollen
germination ratio have been assessed. Pollen germination was assessed with 10
repeats per one line and varieties. Flowers was collected and reserved in petri dishes
before blossoming 1 to 2 days. When flowers blossom, they was dyed by aceto
carmine (Domingues et al, 1999) and observed on microscope to evaluate pollen
surviving. Identifying pollen germination ratio by culturing on artificial medium with

5 repeats followed the method of Shivanna, K.R, 2003.
2.4. Satiscal analysis
Data are analyzed by excel 2007
CHAPTER 3
RESULTS AND DISCUSSION
3.1. Generating initial materials for selecting seedless and fewer seed citrus
varieties
3.1.1. Hybridizing between two different ploids of same and different varieties and
different species
We have implemented 8 hybridizing pairs on 2011 flowering season. The
results have shown on the table below.
Mating pairs
Fruit ratio (%)
󰵁 CS2x × 󰵃 CS4x
36.6
8
󰵁 BD2x × 󰵃 BD4x
33.3
󰵁 NR2x × 󰵃 BD4x
10.0
󰵁 PT2x × 󰵃 BD4x
󰵁 NR2x × 󰵃 PT4x
󰵁 BD2x × 󰵃 PT4x
󰵁 PT2x × 󰵃 PT4x
󰵁 CS2x × 󰵃 VD4x
󰵁 CS2x × 󰵃 CS2x
13.3 󰜔 23.3
Then, the seeds from hybridizing ripen fruits of 2x and 4x pummelo mating
pairs have separated. The results are shown on the table below.
Hybridizing pairs

Number of seeds
Number of seeds
(control 2x)
󰵁 BD2x × 󰵃 BD4x
19.1 (2 seeds with normal
embryos per 1 fruit)
76 (all seeds with normal
embryos)
󰵁 NR2x × 󰵃 PT4x
64.3 (5.5 seeds with normal
embryos per 1 fruit)
118 (all seeds with normal
embryo)
Seeds from 6 hybridizing pairs are almost empty and no embryos freom 89.5 to
91.4% which are useless. Seeds with embryos remained 8.6 to 10.5% which are
sterilized to culture on suitable medium for plantlet regeneration.
We are also conducted hybridizing between diploid and tetraploid varieties on
orange especially King Mandarin (Citrus nobilis). This is commercial varieties and
grown widespread in Northern provinces and Cuu Long delta area. However, they
contain many seeds. The number of average seeds of hybridizing pairs have shown
on the below table.
Mating pairs
Number of average seeds per one fruit
󰵁 CS2x × 󰵃 VD4x
18.7
󰵁 CS2x × 󰵃 CS4x
21.5
󰵁 CS2x × 󰵃 CS2x
22.1
Seeds per fruit of all hybridizing pairs are included normal embryo and no

embryo seeds, very few of small seeds have been obtained. The number of small
seeds and normal seeds of some pairs are shown in the following table.
Pairs
Number of small seeds
Normal seeds
󰵁 CS2x × 󰵃 VD4x
3.88
󰵁 CS2x × 󰵃 CS4x
2.72
󰵁 CS2x × 󰵃 CS2x
2,75
72.03 󰜔 76.13%
Small seeds with embryos are the least 7.63% in 󰵁 CS2x × 󰵃 CS4x pairs and
the highest 15.47% in 󰵁 CS2x × 󰵃 VD4x pairs. Dud seeds with embryos in 󰵁 CS2x
× 󰵃 CS2x are 3.40% and in 󰵁 CS2x × 󰵃 VD4x are 5.36%. Dud seeds without
embryos in 󰵁 CS2x × 󰵃 CS2x and 󰵁 CS2x × 󰵃 CS4x are from 15.25% and 3.57% in
󰵁 CS2x × 󰵃 VD4x hybridizing pairs.
9
From the above results, we have catalogued them into four groups including
normal, small embryos and dud seeds with no embryos. Small and dud seeds with
embryos were cultured on appropriate medium to regenerate plantlets and then these
plantlets of ploid types were indentified.
3.1.2. Rescuing embryos to produce as source of initial material
Medium for embryo rescuing have been modified to be suitable for culturing
embryos of small and dud seeds (Ha Thi Thuy et al, 2009) are MT (Murashige and
Tucker, 1969) adding 1.0 mg/l GA3 + 15% coconut liquid + 30 g/l sucrose + 5 g/l
agar. The development of these seeds from hybridizing pairs (between diploid and
tetraploid types of same and different cultivars) are shown very different.
Ratio of germination of normal seeds of different variety pairs and different
ploid level (󰵁BD2x × 󰵃PT4x) showed minimum by 81.8%. Otherwise, the

germinated ratio of 5 hybridizing pairs of same and different varieties and two
different ploid level were higher by 90%. The results for Citrus nobilis is lowest
33,3% to 44.4% for dud seeds and 75,0% to 88.9% for small seeds.
The plantlets after rescuing embryos on invitro medium are on the below table.
Same variety
Number of plantlets
󰵁 PT2x × 󰵃 PT4x
24
󰵁 BD2x × 󰵃 BD4x
18
󰵁 CS2x × 󰵃 CS4x
21
󰵁 CS2x × 󰵃 CS2x
7
Different variety
󰵁 BD2x × 󰵃 PT4x
9
󰵁 NR2x × 󰵃 PT4x
20
󰵁 PT2x × 󰵃 BD4x
17
󰵁 NR2x × 󰵃 BD4x
15
Different species
󰵁 CS2x × 󰵃 VD4x
26
After culturing in vitro 1 month (plantlets󰜚 height from 2.5 to 4.0 cm and had
secondary roots), these plantlets have washed and grown in fresh sand trays
containing 75 holes (2.5x2.5cm) each then after 15 days that are covered by plastic
pieces. After 15 days, they were removed and we examined and counted the number

of remained plantlets.
The plantlets after transplanting are highly survived about 80% for pummelo
lines and above 42,8% for Citrus nobilis. After one month transplanting, we have
tested the ploid level of them and the results are shown on the belowtable.
Same variety
Number of triploid plantlets
󰵁 PT2x × 󰵃 PT4x
21
󰵁 BD2x × 󰵃 BD4x
15 (2 tetraploid)
󰵁 CS2x × 󰵃 CS4x
9
󰵁 CS2x × 󰵃 CS2x
1
10
Different variety
󰵁 BD2x × 󰵃 PT4x
9
󰵁 NR2x × 󰵃 PT4x
18
󰵁 PT2x × 󰵃 BD4x
15
󰵁 NR2x × 󰵃 BD4x
14
Different species
󰵁 CS2x × 󰵃 VD4x
12
114 triploid plantlets and 2 tetraploid from 8 hybridizing pairs and the above
control are the important genetic materials for selecting seedless citrus, are very
diverse about genotype of each individual which has one genotype and will become a

different triploid lines by propagation.
11
12
3.1.3. Agro-biological characteristics assessment of triploid pummelos and Citrus
nobilis lines selected from 2011
After characterizing some agro-biological traits of pomelo and Citrus nobilis
lines, we have selected 8 triploid Citrus nobilis line CS.11.02, CS.11.06,
CS.3x.11.01, CSV.11.02, CSV.11.05, CSV.11.08, CSV.11.11, CVS.11.12 and 9
triploid pummelo lines PT.3x.11.08, PT.3x.11.14, DT.3x.11.03, NT.3x.11.17,
BD.3x.11.13, ND.3x.11.01, ND.3x.11.03, PD.3x.11.02, PD.3x.11.15.
These are promissing lines and heterosis about growth, well-branching, varied
phenotypes stipule density ranged from normal to dense compared with Dien, Phuc
Trach pummelos, diploid Citrus nobilis and tetraploid control.
3.2. Assessment of agro-biological characteristics and selection of triploid Citrus
noblis and pummelos in nethouse
3.2.1. Assessment of agro-biological characteristics and selection of triploid Citrus
noblis in nethouse
22 triploid Citrus nobilis lines have branched in many different types which are
divided into 3 main categories.
4
Figure 3.10. Triploid Citrus nobilis lines in nethouse
1
2
3
1
3
2
4
Figure 3.11. Triploid pummelo lines in nethouse
13

(i) Category 1: in vertical, branching angle is small, the canopy is umbrella
type as same as CS2x control including some triploid Citrus nobilis lines CS.05.03,
CS.05.04, CS.05.05, CS.06.01, CS.06.03, CS.06.05, CS.06.07, CS.06.11, CS.07.04.
(ii) Category 2: branching horizontally, angle of branching is larger, the canopy
is flat different from CS2x control including some triploid Citrus nobilis lines
CS.05.01, CS.05.06, CS.06.02, CS.06.04, CS.06.09, CS.06.12
(iii) Category 3: middle types, horizontally branching, angle is small, shape of
canopy is eclipse, cylinder󰜧not the same as control CS2x is CS.05.02, CS.06.06,
CS.06.08, CS.07.01, CS.07.02, CS.07.03.
Stipule density of triploid Citrus nobilis less than control CS2x. Some Citrus
nobilis lines developed from germination of seeds have many stipules.
8 year of Citrus nobilis lines exhibited 2 to 6 primary branches and 6 to 14
secondary branches. The smallest primary branch diameter is 1.3cm belong to
CS.05.06 and highest is 2.9cm CS.05.05. These CS.05.06, CS.05.01, CS.05.03 are
highly branching. The highest height is 210.4 cm (CS.05.06 lines) and lowest is 142.8
cm (CS.05.04 lines). The stem diameter of Citrus nobilis lines are varied from
highest 5 cm (CS.05.01) and lowest is 2.9 cm (CS.05.04). There are also different
between canopy diameters from 70 cm (CS.05.04) to 165.6 cm (CS.05.06).
7 year of Citrus nobilis lines exhibited 2 to 6 primary branches and 6 to 14
secondary branches. The diameter of primary branches varied from 1.3 cm
(CS.06.01) to 3.5 cm (CS.05.10). Some triploid Citrus nobilis lines CS.06.04,
CS.06.11, CS.06.02, CS.06.10 and CS.06.12 are highly branching. The height of
these triploid lines ranged from 90.7 cm (CS.06.12) to 222.8 cm (CS.06.04). The
stem diameter of these lines are significantly different from 2.5 cm (CS.06.01) to 6.5
cm (CS.06.10). The canopy diameter are from 60.7 cm (CS.06.01) to 164.7 cm
(CS.06.04).
6 year of Citrus nobilis plants exhibited 2 to 5 primary and 6 to 9 secondary
branches. The smallest primary branch diameter is 1 cm (CS.07.03) and highest is 2.5
cm (CS.07.04). Some lines such as CS.07.02, CS.07.01, CS.07.04 is highly
branching. The height ranged from 112.3 cm (CS.07.02) to 192.6 cm (CS.07.04).

There is significant deviation about stem diameter from 2.5 cm (CS.07.01) to 4.4 cm
(CS.07.04). The canopy diameter varied from 90.4 cm (CS.07.02) to 147.3 cm
(CS.06.03).
There are 4 shape types about morphology of leaf including oval shape (diploid
and tetraploid Citrus nobilis leaves) and ovate, eclipsoid, are different from normal
and tetraploid Citrus nobilis. However, the color of the leaves is dark green the same
between 3 types of Citrus nobilis lines.
The length of blade varied from 7.16 cm (CS.05.04) to 10.42 cm (CS.05.01),
width ranged from 4.82 cm (CS.05.03) to 6.38 cm (CS.05.01). The leaflets are not
significantly different between triploid lines.
14
The length of 8 year triploid Citrus nobilis line blades is from 7.16 cm
(CS.05.04) to 10.42 cm (CS.05.01), the width is from 4.82 cm (CS.05.03) to 6.38 cm
(CS.05.01).
For 7 year triploid Citrus nobilis lines, the length of blades is from 7.56 cm
(CS.06.05) to 9.96 cm (CS.06.08), the width from 4.04 cm (CS.06.04) to 5.58 cm
(CS.06.10).
For 6 year triploid Citrus nobilis lines, the length is from 5.84 cm (CS.07.02)
to 8.58 cm (CS.07.04), the width is from 3.58 cm (CS.07.01) to 5.66 cm (CS.07.04).
* Morphology of flower
Citrus nobilis (2x) flowers: white, curled big petals with length and width 18-
20 x 7 mm, 5 small green sepals (3 to 4 mm) which are whorled and cover by layer of
thin fiber. The petiole is small with diameter 1 mm. There are 20 stamens sticking
together and separating at the top. The anther is ovate, yellow adhered back. The
ovary is global, oil gland is small and comfortable scent. Stamens are longer than
stigma.
Triploid Citrus nobilis lines: white, curled big petals with size length and width
18-20 x 7 mm, 5 small green sepals (3 to 4 mm) which are whorled and cover by
layer of thin fiber. The petiole is small. There are 24 to 26 stamens lengthened 8.2
mm, separating at the top with ovate, yellow, adhered back anthers. The ovary is

global. Style length is 9.5 mm and little curled. Stigma is global. Flower with scent,
stamens are shorter than stigma.
Tetraploid Citrus nobilis (4x). Flowers are white, scent and 4 to 5 green sepals,
4 to 5 white petals lengthening 1.21 cm, white filaments, yellow and ovate anthers.
The number of filaments is about 24 to 28 per flower, separating with the length is
8.0 mm. The length of style is 7.0 mm. Stigma is global. Stamens are longer than
stigmas.
The significant difference between other types of flower is implied the size of
flower and the length of filaments. The size of triploid flowers is bigger than diploid
and smaller than tetraploid type. The length of filaments of triploids is shorter than
style. Two other ploid type are contradict.
The flowering time of triploid Citrus nobilis start from 21
st
to 28
th
of January.
All of flowers blossoming from 14
th
to 19 of February and completing from 19
th
to
24
th
of February.
To recapitulate, from 22 triploid Citrus nobilis lines, we have selected 2 lines
namely CS.06.02 and CS.06.09 which are flowering but no fruits producing from
hybridizing pairs 󰵁 CS2x × 󰵃 CS2x to be continuing testing in the field.
15
3.2.2. Agro-biological characteristics and selection of triploid pummelo lines in
nethouse

44 triploid pummelo lines were selected from mating pairs 󰵁 BD2x × 󰵃 PT4x
in 2006 were characterized some traits such stipule density, branching, branching
angle, canopy shape to compare to control Dien and Phuc Trach pummelos.
7 year of triploid pummelo lines have 2 to 5 primary branches and 5 to 19
secondary. The diameter of primary branches ranged from 3.2 cm (PD.06.16) to 7.6
cm (PD.06.09). Some lines are highly branching for example PD.06.39, PD.06.28,
PD.06.27. The height of triploid plants ranged from 165.5 cm (PD.06.16) to 313.20
cm (PD.06.11). The diameter of stem is not different between other lines. The
diameter of canopy varied from 96.70 cm (PD.06.16) to 256.50 cm (PD.06.42) (Table
3.13).
6 year of triploid pummelo lines were varied about phenotypic types, canopy,
stipules density and leaf types. In average, there are 2 to 8 primary branches and 6 to
24 secondary. The diameter of primary branches ranged from 1.5 cm (PD.07.04) to 6
cm (PD.07.71).
Some triploid pummelo lines have highly branching ability such as PD.07.64,
PD.07.65 and PD.07.58. The height is ranged from 150.0 cm (PD.07.48) to 297.7 cm
(PD.07.47). The stem diameter of triploid are significantly different ranging from 4.1
cm (PD.07.28) to 9.3 cm (PD.07.38). The diameter of canopy varied from 115.4 cm
(PD.07.28) to 312.4 cm (PD.07.53).
There are 4 types about leaf tip morphology of 44 triploid pummelo lines (7
years plants) are , and 3 types of shape of leaf blade are ovate, eclipse and oval with
dark green color. The length of blade is from 8.36 cm (PD.06.43) to 11.26 cm
(PD.06.10). The width of blade ranged from 5.00 cm (PD.06.22) to 7.26 cm
(PD.06.10). The leaflet of pummelo lines is seen more clearly than orange and
grapefruit lines. The length of leaflets of triploid pomelo lines is relatively long and
no significant difference between these lines.
Results of observation about 93 triploid 6 year pummelo lines of leaf tip
morphology have myriad shapes but most of all is the same above. The blade shapes
are included mainly three types (ovate, eclipse and oval). The color of blade is dark
1

2
3
Figure 3.14. Flower morphology of Citrus nobilis lines in 2013
1. Tetraploid 2. Triploid 3. Diploid
16
green. The length of blade varied from 7.92 cm (PD.07.24) to 12.50 cm (PD.07.04).
The width of blade is ranged from 5.00 cm (PD.07.67) to 8.00 cm (PD.07.01) larger
than Dien and Phuc Trach control pummelo plants.
* Characteristics and morphology of flower
Characteristics of diploid Dien pummelo (2x): flowers are very scent and 2
types of flowers (single and pack). Packs of flowers contain 5 to 7 flowers. Sepals are
4 to 5 green star-shape. Flowers have 4 to 5 white petals with length about 20.1 mm,
on the petals containing small pockets of oil. Filaments are white. Anthers are orange
yellow and ovate. There are 24 to 26 filaments per one flower, arranged in bundles
with 3 to 5 filaments per one bundle. The style is 14.0 mm which is shorter than
stigma 14.5 mm. The stigma is yellow green.
Flowers󰜚 characteristics of tetraploid Dien (4x) are very scent. There are 2
types of flowers; single and pack of flowers. There are 4 to 5 white petals with length
about 25.3 mm. Filaments are white. Anthers are orange yellow, ovate with 25 to 26
per on flowers arranging in bundles, each bundles contains 3 to 5 filaments shorter
than stigma (filaments 9.7 mm, stigma 14.2 mm).
Flowers of diploid Phuc Trach pummelo (2x): flowers are very scent. There are
2 types of flowers; single and pack of flowers. Each pack of flowers contains 5 to 7
flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with
length about 17.4 mm. On petals, there are spots containing pockets of oil. Filaments
are white. Anthers are orange yellow and ovate arrange in bundles. Each bundles
includes 3 to 5 filaments length about 14.6 mm shorter than stigma is 15.0 mm.
Flowers of tetraploid Phuc Trach pummelo (4x): flowers are very scent. There
are 2 types of flower single and pack of flowers. Each pack of flowers contains 5 to 7
flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with

length about 26.8 mm. On petals, there are spots containing pockets of oil. There are
24 to 25 white ovate filaments. Anthers are orange yellow and ovate arrange in
bundles. Each bundles includes 3 to 5 filaments length about 11.0 mm which is
shorter than stigma is 15.3 mm.
Flowers of triploid pummelo plants (3x): flowers are very scent. There are 2
types of flowers; single and pack of flowers. Each pack of flowers contains 6 to 8
flowers. Each flowers has 4 to 5 green star-shape petals and 4 to 5 white petals with
length about 20.3 to 24.8 mm and width from 7.2 to 9.9 mm. Filaments are white
with 24 to 27 filaments each flower. Anthers are orange yellow and ovate arrange in
bundles. Each bundles includes 3 to 5 filaments length from 10.6 to 13.2 mm shorter
than stigma is from 11.4 to 14.5 mm.
Pollen of diploid and tetraploid pummelo are able to germinate on in vitro
medium. In contrast, pollen of triploid are very much but not able to germinate on in
vitro medium.
After observation on flowering time in 2 seasons 2012 and 2013. We have
found that there no difference between triploid lines and control about flowering
time. In 2012, flowers of triploid pummelo lines have started blossoming from 27
th
of
17
January to 3
rd
of Feb and finished from 25
th
of Feb to 2
nd
of March. In 2013, they
started and finished earlier than 2012 about 3 to 5 days.
In summary, we have assessed 137 triploid pummelo lines selected from
hybridizing pairs 󰵁 BD2x × 󰵃 PT4x from which we have obtained 6 potential lines

including PD.06.11, PD.06.14, PD.07.33, PD.07.34, PD.07.57, PD.07.76. After 6 󰜔 7
years, these lines have been flowering but not fruiting. These lines are heterosis about
phenotype, branching characteristics, variety about angle of branching, stipule
density, healthy growth and development compared with diploid Dien and Phuc
Trach and tetraploid pummelo control. We are consecutively testing these lines on the
field.
3.3. First steps of testing some triploid pummelos and Citrus nobilis lines
3.3.1. Testing some triploid Citrus nobilis lines budded on Citrus hystix
Triploid Citrus nobilis lines budded on Citrus hystix plants were grown in the
field for testing which we have divided according canopy shape (umbrella, global,
broom and eclipse shape) into two main types.
First, plants are vertical branching, small branching angle and canopy shape
like broom as the same as control Citrus nobilis (2x) including some triploid Citrus
nobilis lines CS.05.03, CS.05.04, CS.05.05.
Second, plants are horizontally branching, large branching angle and global
canopy shape different from control CS2x including CS.05.01, CS.05.02, CS.06.02.
Stipule density of triploid Citrus nobilis lines is on average level different from
the control Citrus nobilis (2x) without stipules. Stipule appearance reflects
propagation progress and characteristics of varieties. Fruit plants have grown from
seeds showing many stipule after propagation (grafting and layering) over years
stipules are decreased, in some cases, no stipules left. Some triploid Citrus nobilis
lines are selected by hybridizing and propagation by budded on Citrus hystix so they
still have genetic characteristics from mother with average stipule density.
Three year of Citrus nobilis lines󰜚 height ranged from 112.47 cm (CS.06.11) to
231.53 cm (CS.07.03). The stem diameter is not different between lines. The canopy
diameter is varied from 95.06 cm (CS.06.11) to 198.32 cm (CS.06.02).
Growth assessment of experimented Citrus nobilis lines was shown by
appearance of buds. In the first development stage (build-up stage), in one year,
triploid Citrus nobilis plant has grown 3 to 4 bud times including spring, summer,
Figure 3.18. Flowering triploid pummelo lines in 2013

(1, 1, 1). Flowers of triploid lines, (2). Flowers of diploid Phuc Trach,
(3). Flowers of tetraploid Phuc Trach
1
1
1
3
2
18
autumn buds and winter buds is rarely. Spring buds appear from 10
th
to 25
th
of
February and finish in the end of February. Summer buds appear from 16
th
to 27
th
of
May and complete from 10
th
to 15
th
of June. Autumn buds show up from 22
nd
to 28
th
of August and finish from 21
st
to 23
rd

of September.
Growth of spring buds were observed frequently in the year and we concluded
spring buds were appeared most constituted 70 to 75 percent of bud total in a year.
Some parameters expressed spring bud growth such as length of buds from 11.07 cm
to 26.60 cm, diameter from 0.4 cm to 0.66 cm, number of leaves from 5.33 to 11.67.
Time for spring bud matured is about 30 to 35 days.
Summer buds appeared less than spring because the nutrition was focused on
spring bud growth. Summer buds constituted 5 to 9 percent of year bud total.
Summer buds are relatively healthy and strong with length from 16.46 cm to 30.56
cm, diameter from 0.32 cm to 0.57 cm, number of leaves from 8.00 to 16.33, time for
maturing from 35 to 40 days.
Autumn buds contributed 12 to 14 percent of year bud total. Growth of autumn
buds were shown by such parameters such as length from 12.27 to 21.67 cm,
diameter from 0.26 to 0.43 cm, number of leaves from 7.00 to 12.67, time for
maturing from 29 to 32 days. Rate of winter bud appearance is the least and
contributed 1 to 4 percent of total.
To sum up, from 22 triploid Citrus nobilis lines were tested and assessed, we
have selected some potential lines compared with control Citrus nobilis (2n)
including CS.05.03, CS.06.02, CS.06.12, CS.07.01, CS.07.03, CS.06.08 which are
versatile about phenotype, healthy growth and development, non-disease. Some lines
started flowering in 2014 (3 year plant) but not produced fruits because of raining,
high humidity and no sunshine in 3 first months.
3.3.2. Testing some triploid pummelo lines budded on Citrus hystix
217 triploid lines were selected from hybridizing, rescuing embryos from seeds
and propagation by budded on Citrus hystix have grown for testing, evaluating and
selection. Characteristics of branching, canopy shape󰜧 of 217 triploid pummelo
lines are very diverse. Stipule density of triploid lines is more than control such as
Nam Roi, Phuc Trach, Dien (2x) pummelos and tetraploid control without stipules.
Height of three year triploid pummelo plants (2013) ranged from 121.47
(PD.06.84) to 290.80 cm (PD.05.10). The stem diameter is from 3.10 (PD.05.07) to

Figure 3.21. Triploid Citrus nobilis lines budded on Citrus hystix were
testing on fields flowering on Spring season 2014
19
7.03 cm (PD.06.63). The canopy diameter is from 119.73 (PD.06.10) to 257.23 cm
(PD.07.73).
In build-up stage, plants have 4 types of buds including spring, summer,
autumn and winter buds. Spring buds formed on February developing most from last
year autumn buds. Summer buds appeared from 13rd to 20
th
of May and finished 1
st
to 5
th
of June. Autumn buds are from 15
th
to 20
th
of August and finished 12
th
to 15
th
of
September. Winter one is from 25
th
to 27
th
of October and finished 16
th
to 19
th

of
November.
Spring buds contributed 70 to 75 percent of year bud total with some
parameters such as length 13.56 to 30.96 cm, diameter from 0.5 to 1.16 cm, number
of leaves from 6.33 to 14.00, time for maturing of buds from 30 to 35 days (Table
3.30).
Summer buds constituted 5 to 9 percent of year bud total less than spring buds
with length from 21.55 to 50.80 cm, diameter from 0.50 to 0.93 cm, number of leaves
from 12.33 to 27.00, time for maturing of buds from 35 to 40 days.
Autumn buds are 12 to 14 percent of total with length of buds from 11.45 to
36.77cm, diameter from 0.43 to 0.83 cm, number of leaves per bud from 7.5 to 17.00,
time for maturing of buds from 29 to 32 days. Winter buds are the least from 1 to 4
percent of year bud total.
To summary, from 217 triploid pummelo lines which we have selected some
potential lines with high heterosis to compared with parent including PP.07.06,
D.07.82, PD.06.72, PD.06.48 and PD.05.12 which are continuing to test in the
following years.
3.3.3. Assessment of triploid pomelo plant budded on 5 year sour pummelo plant
and identifying some triploid seedless pummelo
From 2006 to 2010, we have collected many triploid pomelo lines and it takes
7 to 10 years to be able to assess these lines about some characteristics of flower and
fruit traits. So we have budded on 5 year sour pummelo plant be fruited 2 year ago in
order to select seedless and fewer seed plant as quickly as possible.
Triploid pummelo lines were budded 15
th
of June, 2009. It takes 22 to 27 days
for forming a new bud. Survived rate of these triploid lines is above 95 percent. In
2011, after budding 24 months, the height of some triploid lines ranged from 113 to
148 cm, canopy diameter from 79.5 to 145 cm, stem diameter from 3.8 to 4.1 cm. In
general, these lines are able to grow relatively fast.

In 2013, most of triploid pummelo lines have produced 3 budding times:
spring, summer and autumn buds. Spring buds of triploid pomelo lines budded on 5
year sour pummelo plants was produced from 5
th
to 15
th
of February 6 to 7 days later
than 7 year triploid lines budded on Citrus hystix from 24
th
of January to 15
th
of
February and have been completed on 23
rd
of Feb to 3
rd
of March earlier than spring
buds of 7 year triploid lines budded on Citrus hystix on 14
th
of Feb to 3
rd
of March.
Summer buds formed intensively from 14
th
to 28
th
of May and finished from 1
st
to 15
th

of June as same as 7 year triploid pummelo lines budded on Citrus hystix
20
plants. Autumn buds formed from 17
th
to 19
th
of August and finished from 12
th
to 15
th
of September as same as 7 year triploid pummelo lines.
The quality of buds is very important with growth and development of plants
and influence directly on productivity of fruits. Fruits often develop from spring
buds. The more spring buds are, the more fruits are produced. Summer and autumn
buds bring nutrition to nurture fruits and to become fruit buds in next year.
Spring buds of triploid pomelo lines budded on 5 year sour pummelo plant
have grown with length of branches from 16.8 to 22.7 cm, diameter from 0.35 to 0.45
cm and number of leaves from 7.5 to 9.4 less than budded on Citrus hystix with
length from 16.2 to 29.43 cm, diameter 0.85 to 1.16 cm and number of leaves from
8.0 to 14.
Summer buds of triploid pomelo lines budded on 5 year sour pomelo plant
have grown with length of branches from 19.5 to 26.8 cm, diameter from 0.38 to 0.48
cm and number of leaves from 7.4 to 13.2 less than budded on Citrus hystix with
length from 30.26 to 46.86 cm, diameter 0.73 to 1.03 cm and number of leaves from
15.33 to 24.
Autumn buds of triploid pummelo lines budded on 5 year sour pummelo plant
have grown with length of branches from 11.5 to 19.8 cm, diameter from 0.30 to 0.45
cm and number of leaves from 5.3 to 10.6 less than budded on Citrus hystix with
length from 18.73 to 26.40 cm, diameter 0.61 to 0.80 cm and number of leaves from
9.33 to 13.33.

Regarding to observation on flowers and fruits, we have see that 4 of 15
triploid pummelo lines budded on 5 sour year pummelos have blossomed in 2012
season after 3 years. While 15 lines from seed germination have been grown for 6
years still not flowering.
Characteristics and morphology of flowers and flower pack: flowers of triploid
pummelo PT05.01 and PD.05.05 are very scent and have 2 types; single and pack of
flowers. A pack of flowers has 5 to 7 flowers. Sepals are like star-shape, 4 to 5 geen
wings. Petals are 4 to 5 white wings, length 19.6 to 20.7 mm, with green spots
containing liquid. Style is white with 24 to 26 piece per flower, anthers is ovate and
orange yellow, arranging in bundles of 3 to 5 styles shorter than stigma (style are
length 11.3 to 11.6 mm, stigmas are 12.1 to 12.3 mm). Stigma is backward conical
shape, yellow green.
Table 3.36. Characteristics of flowers of triploid pummelo lines budded on 5
year sour pummelo plants in 2012 (after 32 months)
Characteristics
PT.05.01
PD.05.05
Dien pummelo (control)
Diameter of sepal
Large
Large
Large
Length of petal (cm)
1,96±0,06
2,07±0,05
2,01±0,08
Width of petal (cm)
0,76±0,04
0,95±0,03
0,92±0,05

Length/width ratio (cm)
2,58±0,08
2,18±0,07
2,18±0,09
21
Length of style (cm)
1,13±0,02
1,16±0,04
1,40±0,05
Petiole of sepal
Yes
Yes
Yes
Color of anther
Yellow
Yellow
Yellow
Pollen surviving
No
No
Yes
Length of stigma (cm)
1,21±0,01
1,23±0,03
1,45±0,04
Stigma shape
Straigth
Straigth
Straigth
Table 3.37. Flowering time of triploid pummelo lines budded on 5 year sour

pummelo plants
Citrus lines
Time for
flower bud
Time of
flowering
blossom
Time for
whole
flowering
blossom
The end of
flowering
In 2013 (after 44 months)
Dien pummelo
(control)
20/01-25/01
08/02
14/02
18/02-22/02
PT.05.01
24/01-28/01
10/02
18/02
21/02-25/02
PD.05.01
25/01-29/01
11/02
15/02
20/02-23/02

PD.05.05
28/01-31/01
13/02
19/02
23/02-27/02
PN.05.03
23/01-26/01
9/02
16/02
20/02-25/02
After observation time of flowering in 2012 and 2013 seasons, we have found
that no significant difference between time for flowering of triploid pummelo lines
and control are recorded.
In 2012, triploid pummelo lines started flowering on 27
th
of January to 5
th
of
February and finished from 25
th
of Feb to 2
nd
of March. In 2013, these lines started
flowering from 21
st
to 28
th
of January and finished 19
th
to 24

th
of March 3 to 5 days
earlier than 2012 because of warmer climate.
Table 3.38. Fruit ratio of triploid pummelo lines budded on 5 year sour pomelo
plants in 2013 (after 46 months of budding)
Citrus lines
Total flowers
per plant
Total of
falling fruit
Remained
fruits
Fruit ratio
(%)
Dien pummelo
467
458
9
1,93
Figure 3.24. Flowers and fruits of triploid pummelo lines budded on 5
year sour pummelo plants in Hung Yen 2013
22
control
PT.05.01
437
429
8
1,83
PD.05.01
312

312
0
0
PD.05.05
406
399
7
1,72
PN.05.03
208
208
0
0
In 2013, there have been 4 triploid pomelo lines flowering and fruit ratio
reached from 1.72 to 1.83% lower than diploid Dien pummelo control (1.92%).
Productivity reflects adaptable ability with environment and regional climate of
triploid pummelo lines. Some characteristics of fruits and productivity of triploid
pummelo lines was shown on below table.
Table 3.39. Productivity of triploid pummelo lines budded on 5 year sour
pummelo plants in 2013
Triploid
pummelo lines
Fruit Weight
(g)
Average of
fruit per
plant
Theoretical
productivity
per plant

(kg)
Theoretical
productivity
(ton per ha)
Dien pummelo
control
271,61
7,00
1,90
0,76
PT.05.01
494,16
8,00
3,95
1,58
PD.05.05
232,18
7,00
1,63
0,65
PT.05.01 lines budded on 5 year sour pummelo plants has produced every 8
fruits per plant. Fruits of this lines are big with weight 494.16 g per fruit, theoretical
productivity recordes is about 1.58 ton per ha. PD.05.05 lines has produced 7 fruits
per plant with weight 232.18 g per fruit, theoretical productivity is about 0.65 ton per
ha. Fruits of this lines are small.
Table 3.40. Characteristics of morphology and construction of fruits of triploid
pummelo lines budded on 5 year sour pummelo in 2013
Citrus
lines
Weight

of fruit
(gr)
Fruit
diameter
(cm)
Height
of
fruit
(cm)
Number
of
segments
Thick
of
skin
(cm)
Diameter
of
segment
(cm)
Core
diameter
(cm)
Number
of seeds
Dien
pummelo
(control)
271,61
9,15

8,26
12,23
0,96
1,37
1,86
67,53
PT.05.01
494,16
10,26
9,87
15,43
1,89
1,13
1,67
0
PD.05.05
232,18
8,66
8,16
14,57
1,62
1,14
1,12
0
23
CONCLUSION AND PROPOSALS
CONCLUSION
1. We have selected triploid pummelo and Citrus nobilis lines by hybridizing
between varieties pummelo, King and Van Du orange (diploid and tetraploid). From
8 hybridizing pairs at 2 different ploid level PT2x × PT4x, BD2x × PT4x, NR2x ×

PT4x, BD2x × BD4x, PT2x × BD4x, NR2x × BD4x, CS2x × CS4x, CS2x × VD4x
and natural hybridizing pair CS2x x CS2x. We have collected 157 plantlets from
embryos rescuing including 121 plantlets that were checked ploid levels. We have
collected 114 triploid plantlets and 2 tetraploid plantlets. After 2 years, assessing
about agro-biological characteristics, we have obtained 7 triploid Citrus nobilis lines
CS.11.02, CS.11.06, CSV.11.02, CSV.11.05, CSV.11.08, CSV.11.11, CVS.11.12 and
9 triploid pomelo lines PT.3x.11.08, PT.3x.11.14, DT.3x.11.03, NT.3x.11.17,
BD.3x.11.13, ND.3x.11.01, ND.3x.11.03, PD.3x.11.02, PD.3x.11.15. These are
promising lines and heterosis compared with parents about growth, strong
development, branching, varied phenotypes and stipule density from middle to dense.
These lines are consecutively for testing and evaluating.
2. Some triploid pummelo and Citrus nobilis lines budded on Citrus hystix (7 moths)
have shown difference about growth, flowering and fruits. After budded plants are 6
to 7 years old, 2 triploid Citrus nobilis lines CS.06.02, CS.06.09 and 6 triploid
pummelo lines PD.06.11, PD.06.14, PD.07.33, PD.07.34, PD.07.57, PD.07.76 has
been blossomed but none of them produced fruits.
3. Two triploid pummelo lines were budded on mature 5 year sour plants which were
blossomed and fruited (seedless fruits) in the 4th and 5th year after budding.
Triploid pummelo lines PT.05.01 (from mating pair PT2x x PT4x) have been
produced fruits (8 fruits per plant), with weight about 494.16 g per fruit, theoretical
productivity is about 1.58 ton/ha, big global fruits, yellow skin after ripening,
seedless, yellow succulent, sweet, non-bitter fruit flesh, edible part constituting 45 to
55 percent.
Triploid pummelo lines PD.05.05 (from mating pair BD2x x PT4x) have been
produced fruits (7 fruits per plant), with weight about 232.18 g per fruit, theoretical
productivity recorded is about 0.65 ton/ha, small global fruits, yellow skin after
ripening, seedless, yellow succulent, sweet, non-bitter fruit flesh, edible part
constituting 45 to 55 percent.
Figure 3.24. Morphology and construction of fruits of triploid pummelo lines
PT.05.01 budded on 5 year sour pummelo plants in 2013