RESEARCH ON SELECTING SOME TRIPLOD LINES CITRUS VARIETIES

Mutating Mating between diploid lines 2x and tetraploid lines 4x to select triploidlines 3x Roose and Williams, 2000 Culturing endorsperm of immature seeds Gmitter et al, 1990 Selecting

AND TRAINING AND TRAINING AND RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

Scientific Supervisors: 1 Prof.Dr Do Nang Vinh

2 Assoc.Prof Dr Ha Thi Thuy

The PhD thesis may be fount at:

- Ha Noi National Library

- Library in Vietnam Academy of Agricultural Sciences

HA NOI - 2015

* Urgency of research

Citrus trees are one of fruit crops which have high nutrition and economicvalue Production of citrus fruit all over the world reached 85.6 ton in the years of2012/2013, in which orange production accountes 50% (USDA, 2013) This numberwill be continued increasing because of the demand in some countries are highlyexpanded such as China, Korea, Russia, India, East Europe and Asia In China, theconsumption of fresh fruit has been singnificantly increasing, especially citrus fruits

In recent years, China has ranked top second biggest citrus fruit production afterBrazil (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 ofall 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 lowproductivity causing by diseases and the quality of varieties (low quality andcontaining many seeds)

Selecting seedless citrus are being conducted in some commonplace followingapproaches

Mutating

Mating between diploid lines (2x) and tetraploid lines (4x) to select triploidlines (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 protoplastcells (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 valuebut contains many seeds To improve traits by selecting from these varieties based onthe 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

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 wellgrowth, non-diseases Two lines of 6 pummelo lines above have been producedseedless 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 pomelolines 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 linesare 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 differenttriploid We have also made integration between traditional method andbiotechnology methods such as (rescuing embryo) and some techniques for quickselection (by budded on flowering citrus plants) All knowledge can be used asreference 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, TT-VPVH, 12th, November, 2013 Mother plant (2x) was selected from healthy plants

515/QD-,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

selecting seedless pummelos, orange and grapefruit applied biotechnology (2006 to2010).

Scope of the research:

The research are conducted in Vietnam In specific, the experiment wereconducted at Key national of plant cell Lab (Agricultural Genetics Institute), VanGiang, Hung Yen practical farm, Vegetable and Fruit Research Institute and otherfruit 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 comparingproductivity, 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; JeanBaptiste Bassenet et al, 2009) Some non-sticky skin, seedless, nutrient, storage anddisease resistance traits are concerned by scientists (Ollitrault et al, 2000; Roose andWilliams, 2000) Utilizing heterosis, multiploid, protoplast fusing and somaclonalvariation were applied to select seedless lines with disease resistance trait servingbeverage industry and fresh fruit consumption (Guo et al, 2004; Grosser and Gmitter,2005; Gmitter et al, 2007; Febres et al, 2009) Some researches were appliedtransgenic 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 2methods: selecting natural variations and radioactive mutations, including, 3 seedlessand 1 little seed grapefruits, 3 seedless and 1 little seed oranges, 2 seedless pomelovarieties Furthermore, some mutated cultivars have ripen longer, some were earlierbut 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 matingbetween tetraploid and diploid lines (Soost and Cameron, 1975; Starrantino andRecupero, 1981) For instance, the typicalness of sucessful results of generating 2triploid pummelo cultivars Oroblanco and Melagold were made (Soost and Cameron,

1980, 1985) However, the process of selecting high quality seedless cultivars hascoper with many difficulties because of the shortage of gene pool of tetraploidcultivars

In the mating, diploid cultivars was utilized as mother plants and tetraploid wasused as fathers Triploid embryos are not developed normally because the relative

between embryo and endosperm is disconnected So, the scientists have rescued suchembryos by culturing them in vitro (Gmitter, 1995).

The germination of collected triploid seeds is hard, just few seeds from 2 to 10seeds per 100 fertilized flowers were possobly Some mating pairs (2x x 4x) producesmall fruits with germinated seeds However, these young fruits often fall down inJune Some scientists of California State (US) developed a technique to rescue fallenyoung fruits by culturing their embryos on high sugar and citric acid medium withoutregulator 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 Thetetraploid lines (4x) randomly appear in nature with relatively high frequency.Cameron and Frost (1968) obtained 2.5 percent per 3,600 trees from variouscultivars

Tachikawa et al, (1961) used colchicine in order to produce tetraploid Citrusand then hybridized with diploid plants Tetraploid types are able to produce byprotoplast 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 and2x 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, byculture 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; Adeninesulfate (25mg/l) and malt extract (500mg/l) made beneficial effects, some hormonessupport embryo germination and shoot induction such as GA3 (1mg/l) balanced withBAP (0.5 mg/l) + 0.5 mg/l kinetin + 0.1 mg/l NAA

The procedure of triploid embryo rescuing was developed by scientist ofCIRAD-INRA (Froelicher et al, 2003) The optimum medium is MT (Murashige andTulecker, 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 beenconducted for a long time (Esen and Soost, 1972) However, the obtained embryoswas not develop normally, just few seeds can be germinated (Cameron and Frost,1968; Tachikawa et al, 1961; Esen and Soost, 1972) Some reports have suggestedthat uncompleted development of triploid seeds are related to endorspermabnormality So, to overcome this barrier, scientists have rescued embryos byculturing them on in vitro medium

Starrantino and Recupero (1981) have cultured 585 embryos selected from 3diploid mother lines with 6 tetraploid lines hybridizing pairs on MS medium byadding 500 mg/l malt extract and 25 mg/l adenine sulfate They have collected 211triploid plantlets and 89 unidentified chromosome number were collected Theyshowed that culturing embryos after 3 to 4 months pollinated could be generatedother embryos which produced many plantlets.

Oiyama and Kobayashi (1993) collected triploid plants by rescuing dud seedsfrom 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 7months Besides, Oiyama also have collected high ratio of triploid plants by culturingundifferentiated small embryos forming from dud seeds of ripen fruits on MT + 1mg/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 fruitplant from indigenous (2001 to 2005)

These tetraploid plants have been grown in Van Giang, Hung Yen provincewhich have also conferred and authorized by Department of Planting, Ministry ofAgriculture and Rural Development, 515/QD-TT-VPVH, 12th, 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 andgrapefruit 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

- 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 innethouse

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 pummeloplants and identifying some promising lines

1 PT2x × PT4x Pummelo(4x)Phuc Trach Pummelo(2x) × Phuc Trach

2 BD2x × PT4x Dien Pummelo(2x) × Dien Pummelo(4x)

3 NR2x × PT4x Pummelo(4x)Nam Roi Pummelo(2x) × Phuc Trach

4 PT2x × BD4x Pummelo(4x)Phuc Trach Pummelo(2x) × Dien

5 BD2x× BD4x Dien Pummelo (2x) × Dien Pummelo (4x)

6 NR2x × BD4x Nam Roi Pummelo (2x)× Dien Pummelo (4x)

7 CS2x × CS4x C nobilis 2x × C nobilis 4x Cam Sanh (2x) × Cam Sanh (4x)

8 CS2x × VD4x C nobilis 2x × C sinensis (L.) Osbek 4x Cam Sanh (2x) × Van Du Orange (4x)

9

(control) CS2x × CS2x C nobilis 2x × C nobilis 2x Cam Sanh (2x) × Cam Sanh (2x)

Hybridizing method: Flowers of mother plants in mating are on top ofbranches, healthy and earlier blossoming Before blossoming 1 to 2 days, the flowershave to be removed anthers Flowers with matured stigma need to be hybridized byselected father with rapid pollen and then covered with nylon packs to avoid otherpollen With small flowers, we have used specific small broom for hybridizing Afterhybridizing, we removed all the non-hybridizing flowers to ensure the remainedflowers not competing with nutrition After hybridizing 2 to 3 weeks, disentanglingnylon packs and marking positions of hybridized flowers For natural hybridizing

pairs CS2x × CS2x, marking 30 flowers to observe fertilizing ability andremained fruit ratio.

After fruit ripening, collecting fruits to assess of remained fruit ratio andnumber of seeds per fruit of different mating pairs were made Hybridizing seedshave 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/lGA3, 15% coconut liquid, 30 g/l sucrose and 5 g/l agar

Identifying ploid level by Flow Cytometry machine, following the method ofOllitrault 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 ofInternational Plant Genetic Resources Institute document and procedure for testingpomelo, 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 offlowers and fruits, factors contributing productivity and quality

The morphology and structure of flowers includes tension of petals, color ofanthers and pollen have observed by naked eyes and microscope Leica DMLS2 atmagnitude 40 x 0.65 and 100 x 1.25 magnification Pollen surviving and pollengermination ratio have been assessed Pollen germination was assessed with 10repeats per one line and varieties Flowers was collected and reserved in petri dishesbefore blossoming 1 to 2 days When flowers blossom, they was dyed by acetocarmine (Domingues et al, 1999) and observed on microscope to evaluate pollensurviving 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 Theresults have shown on the table below

BD2x × BD4x 19.1 (2 seeds with normal

embryos per 1 fruit) 76 (all seeds with normalembryos) NR2x × PT4x 64.3 (5.5 seeds with normal

embryos per 1 fruit) 118 (all seeds with normalembryo)Seeds from 6 hybridizing pairs are almost empty and no embryos freom 89.5 to91.4% which are useless Seeds with embryos remained 8.6 to 10.5% which aresterilized 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, theycontain 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

From the above results, we have catalogued them into four groups includingnormal, small embryos and dud seeds with no embryos Small and dud seeds withembryos were cultured on appropriate medium to regenerate plantlets and then theseplantlets 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 culturingembryos of small and dud seeds (Ha Thi Thuy et al, 2009) are MT (Murashige andTucker, 1969) adding 1.0 mg/l GA3 + 15% coconut liquid + 30 g/l sucrose + 5 g/lagar The development of these seeds from hybridizing pairs (between diploid andtetraploid types of same and different cultivars) are shown very different

Ratio of germination of normal seeds of different variety pairs and differentploid level ( BD2x × PT4x) showed minimum by 81.8% Otherwise, thegerminated 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

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

BD2x × BD4x 15 (2 tetraploid)