citrus breeding, breeding of new varieties, citrus rootstocks, floral biology, transgenic

1. By Kunzang Lamo

2. CITRUS
Family Rutaceae
Subfamily Aurantoideae
Tribe Citreaea
Genus Citrus
Chromosome No. 2n=18
Origin South East Asia
 Citrus is the most cultivated fruits in the world with the highest acrage concentrated in the
tropical and subtropical regions of the world.
 In world, Brazil, USA, China, Mexico, and Spain are the five largest citrus producing
countries.
 In India, citrus occupies third position in fruit production after banana and mango. Citrus is
being cultivated on 1024’000 Hectare with an annual production of 11581’000 MT and the
productivity is 11.30 MT/Hectare (Anonymous, 2016).
 Citrus fruits are not only refreshing and delicious but are also very rich in Vitamin C and
contain 25-60 mg of vitamin C per 100 g of juice. Apart from this fruits are good source of
minerals like calcium, phosphorous and iron.

3. Domestication
 The domestication and distribution of edible citrus types began several thousand
years ago in Southeast Asia and spread globally following ancientl and sea routes.
 The Citrus species which are undoubtedly indigenous or those domesticated for a
long time are listed below in table (Bhattacharya and Dutta, 1956; Singh, 1977).
Species Common name Distribution
Citrus medica L. Bira-jora, Soh-manong, Mitha-
jora, Yema-kaipuli, Citron
All over the country
Citrus limon Burn Asssam lemon, Pati-lebu, Jora-
tenga, Naya Changney
North-Eastern India
Citrus limon var. Decumana Soh-long, Hill lemon, Galgal or
Kil-Kil
North Eastern region and foot hills
of Western India
Citrus limon var. Limonum (citron
hybrids)
Gandhraj lemon, Godhapti-lebu All over India

4. Contd....
Citrus jambhiri Lush Soh-sarkar, Nibu, Karna Khatta, Karna North-eastern region and western India
Citrus aurantifolia Swingle Abhayapuri lime, Karimganj lime,
Kagzi lime
Widely distributed in the country
Citrus reticulate var. Crenatifolia
Blanco
Nagpur mandarin, Khasi mandarin,
Coorg mandarin, Soh-niamtara, Lahore
local, Deshi, Darjeeling mandarin
All over country
Citrus reshni Tan. Kodakithuli, Kokni, Soh-seim All over the country
Citrus indica Tan. Indian wild orange North-eastern India
Citrus nobilis Jeneru-tenga North-eastern India
Citrus sinensis Osbeck Soh-niangriang North-eastern India
Citrus pennivesiculata Tan. Gajanimmia, Baduvapulli South India
Citrus pennivesiculata var. assamensis Ada-jamir North-eastern India (greening)
Citrus grandis Osbeck Dowli, Nowgong Burni, Gagar All over the country
Citrus megaloxycarpa Lush. Bor-tenga, Hukma-tenga, Holong-
tenga, Jamir-tenga, Amilbed
North-East and Several other parts of
the country
Citrus limonia Osbeck Rangpur lime, Marmalade orange,
Kusai lime, Sylhet lime
North-Eastern & Central India
Citrus macroptera Moni. Tih-Kara, Sat-kara North-Eastern India
Citrus madurensis Lour Calomondin North-East region

5. GENETIC RESOURCES
There is an in situ gene sanctuary for citrus in the Garo Hills in the Northeast , state of
Assam, which is a field genebank with 627 accessions (Singh, 1981). Ex situ
collections consist of 451 or 521 accessions at eight sites:Chetalli, Bangalore,
Rahuri, Tirupati, Abohar, Bhatinda, Yercaud, and Delhi)
Important Citrus rootstocks and their characteristics used in breeding
Rough Lemon (C. Jambhiri Lush) Good tolerance to tristeza virus, poor tolerance to Phytophthora root
rot. Susceptible to all citrus nematode. Fair tolerant to saline and
good tolerance to calcareous soils.
Rangpur Lime (Citius limonia) A vigorous hardy rootstock with good adaptability to wide wide range
of soil, particularly for heavy soils. Tolerant to tristeza virus and also
to salt. It is very susceptible to Phytophthora. Compatible with orange,
grapefruit and tangelo scions.
Sour orange (C. aurantium L.) Susceptible to tristeza virus, with orange, grapefruit and mandarin
tree shows quick decline and death within few years after infection.
Excellent tolerant to Phytophthora foot rot. Susceptible to all citrus
nematode. Moderately cold hardy. Good tolerance to soil salinity
and calcareous soils.
‘Cleopatra’Mandarin
(C. reshmi)
It is commonly used rootstock. Cleoptera is most salt tolerant
rootstock with ability to exclude sodium and chloride taken up by the
roots. It is good tolerant to tristiza virus. Susceptible to gummosis and
root rot and citrus nematode.

6. Contd..
Trifoliate orange
(Poincirus trifoliatea
(L.) Raf.)
A deciduous relative of citrus. This is tolerant to tristeza virus. Resistant to citrus
nematode and Phytophthora foot rot, and frost. Trees on trifoliate gave high yield
with excellent fruit quality.
Citrange C 35
(Hybrids: Ruby orange
x Webber- Fawcett
trifoliate)
Good tolerance to citrus tristeza virus. Excellent resistant to Phytophthora root rot.
Resistant to all citrus nematode. Well adapted to sandy soils. Poor tolerance to
salinity (chloriodes) and to calceareous soils. Compatible with all oranges,
grapefruit and Lisbon lemons.
Carrizo Troyer
Citranges:(Washington
navel orange x
Trifoliate orange)
Poor tolerance to salinity and to highly calcareous soils. Good tolerance to tristeza.
Resistance to Phytophthora. Resistance to some nematode. Good freeze tolerance.
Good compatible with all oranges, grapefruit and Lisbon lemon.
Swingle Citrumelo
(Hybrids; Duncan
grapefruit x Trifoliate)
Performs poorly on heavy soils. Poor tolerance to salinity and to calcareous soils.
Overall fruit quality is good. Excellent resistant to Phytophthora root rot. Resistant
to some citrus nematode races, susceptible to others.freeze tolerant.
Volkameriana: Natural
hybrid occurs in India.
Also known as Volk
lemon
Often performs poorly on heavy soils. Fair tolerance to salinity and good tolerance
to calcareous soils. Good tolerance to tristeza, moderate tolerance to Phytophthora
root rot. Susceptible to all citrus nematode. Poor freeze tolerance.
Macrophylla Good Tolerance to Phytophthora root rot. Susceptible to all citrus nematode races.
Susceptible to tristeza. Good freeze tolerance. Good tolerance to salinity and
calcareous soils.
Sweet orange (Citrus
sinensis)
Can be used as a rootstock but it is susceptible to Phytophthora and citrus
nematode often limit the productivity and life span of trees.

7. FLOWER BIOLOGY
Inflorescence in citrus species is cymose type. Generally anthesis takes place in
the morning hours between 9.00 am to 12.00 noon. In citrus both staminate and
hermaphrodite flowers are produced on the same plant. The flowers are white or
pinkish purple in colour.
CHEMOTAXONOMY
Citrus species are commonly known to contain phytochemicals.
Species Flavonoids Coumarins and psoraiens Carotenoids
C.
aurantifolia
Hesperidin, naringin, tangeretin,
naringenin, erioctrin
Bergapten, bergamottin, beragaptol,
byakangelicin, citropten (limettin), imperatorin,
isoimperatorin, isopimpinellin, phellopterin,
prangol, scopoletin
C. aurantium Naringin, neoeriocitrin, hesperidin,
auranetin, neohesperidin, rutin
rhoifolin, tangeretin, nobiletin,
Aurapten, auraptenol, bergapten, meranzin,
meranzin hydrate, seselin, isomeranzin, osthol,
scoparon, umibelliferone, xanthyletin
C. bergamia 5-Hydroxy-7,8,3,4-tetramethoxyflavone Bergapten, bergaptol, bergamottin, citropten
C. paradisci Naringin, kaempferol glycoside,
isosakuranetin, hesperidin, poncirin
Aurapten, bergapten, bergamottin, bergaptol,
byakangelicin, citropten, marmin, meranzin
Phytoene, phytofluene, β-
carotene, cryptoxanthin,

8. Contd...
C. reticulate Hesperidin, neohesperidin,
xanthomicrol, nobiletin,
auranetin, ponkanetin
Aurapten, bergapten,
bergaptol, citropten, osthol,
auroptenol
β-carotene, cryptoxanthin, violaxanthin,
zeaxanthin, antheraxanthin,
luteoxanthins mutatoxanthins, lutein,
C. sinensis Rutin, limocitrin, sinensetin,
isosinensetin, tangeretin,
nobiletin, ponkanetin
Auraptenol, scoparon,
xanthyletin
Phytoene, phytofluene, β -carotene,
lutein, cryptoxanthin, luteoxanthins,
antheraxanthin, violaxanthin,
mutatoxanthins, auroxanthins
C. limon Naringin, eriocitrin, hesperitin,
hesperidin, neohesperidin,
diosmin, apigenin, chrysoeriol,
limocitrin, rutin, phlorin,
isorhoifolin, neoponcitrin,
nobeletin,limocitrol, isolimocitrol,
quercetin, isovitexin, eryodictyol
Bergapten, bergamottin,
byakangelicin, citropten,
imperatorin,isoimperatorin,
isopimpinellin, prangol,
phellopterin, scoparon,
scopoletin, umbelliferone,
umbelliprenin, xanthyletin
Phytofluene, β-carotene,
cryptoxanthin,violaxanthin,
auroxanthin

9. CYTOTAXONOMY
 Family: Rutaceae. Swingle (1948) recognised only 16 species under genus Citrus whereas
Tanaka (1954) described as many as 144 species.
 Subgenera: Swingle (Eucitrus and Papeda) and Tanaka (Archicitrus and Metacitrus).
Taxonomic work on Indian Citrus was started as early as in 1874 by Roxburg.
Citrus species and their hybrids
Common name Botanical name
Grapefruit Citrus× paradisci (C. maxima and c. sinensis)
Mandarin C. reticulate
Pummelo C. maxima
Sweet orange C. sinensis (an introgression of C. maxima and C. reticulata)
Sour orange C. aurantium (an introgression of C. maxima and C. reticulata)
Calamodin Citrus.× Citrofortunella (Hybrid between C. reticulate and Fortunella)
Tahitilime Citrus latifolia (C. aurantifolia × C. medica)
Tangelo C. × tangelo (C. paradisci × C. reticulata)
Tangor C. × nobilis (C. reticulate × C. sinensis)
Citrange Poincirus trifoliate and sweet orange
Citrangor Citrange × C. sinensis Source: Janick (2006)

10. Other citrus species
Common name Botanical name
Assam lemon Citrus longilimon
Cleoptera mandarin Citrus reshmi
Ichang lime Citrus ichangensis
Indian sweet lime Citrus limettioides
Indian wild orange Citrus indica
King mandarin Citrus nobilis
Rough lemon Citrus jambhiri
Volkamer lemon Citrus volkameriana
Yuzu Citrus junos
Macrophylla rootstocks Citrus macrophylla
Sour mandarin Citrus sunki
Khasi papeda Citrus latipes
Adajamir Citrus pennivesiculata var. assamensis
Source: Janick (2006)

11. Citrus varieties
 Mandarin
 Kinnow: It is originated between King and Willow Leaf mandarin (Citrus
noblis × Citrus deliciosa) at Riverside, California. This hybrid was developed
by Dr. H.B.Frost in 1915 and was released for cultivation in U.S.A in 1935.
Kinnow was introduced in Abohar. It matures in January- February, and have
12 to 25 seeds.
 Nagpur santra: it is most important mandarin cultivar of India. Fruits are
medium to large, having prominent oil glands. It has 10-11 segments, easily
seperable peel and contain 4 -10 seeds.
 Coorg mandarin: It is commercial variety of Coorg. Fruits are oblate to
globose, golden yellow coloured, with depressed base and abundant juice
content.
 Khasi mandarin: the cultivar is commercially grown in North-eastern region
of India. It is polyembryonic in nature and keeping quality is good.
 PAU Kinnow -1: It was produced through mutation breeding of Kinnow. Its
fruits are medium globose to oblate, skin golden orange when fully mature. It
is mid season variety matures in January. The fruits are low seeded as
compare to Kinnow with 0-9 seeds per fruit.
 Daisy : Daisy is a cross between Fortune and Fremont varieties of mandarin

12. Contd…
 W. Murcott: The fruit is mostly medium sized, sort of flat shaped with a
thin, smooth easy to peel skin. Low seeded fruit. It is mid season variety,
maturing in January.
 Local: Local mandarin is grown in some pockets of Gurdaspur,
Hoshiarpur and Ropar districts of Punjab. Only 3-7 seeds are present
per fruit. Fruit matures in December –January.
 Sweet orange
 Mosambi: this is a very distinctive variety with its fruits being small to
m,edium in size and broadly sub-globose. The flesh is pale yellow or
whitish with 20-25 seeds. It is early maturing and ripens in November.
 Sathgudi: medium to large sized fruits are spherical to globose, colour
light yellow to pale orange, difficult to peel, flesh firm and juicy.
 Hamlin: medium size oval with smooth surface, orange red in colour. It
contain 2-5 seeds per fruit.
 Jaffa: The colour of this fruit is orange red under favourable conditions. It
has around 8-10 seeds. It is mid-season variety with maturity in
December. Trees of this variety are vigrous, upright with dense foliage.

13.  Blood Red: it is prized variety of Punjab. It has 8-10 seeds per fruit, fruit ripe
in the month of December- January. The development of blood colouration is
variable and is best in the sub-montane districts.
 Valencia Late: its fruits are slightly oval, medium in size and deep golden
orange coloured at maturity. Fruit contain few seeds, usually 5 to 6. This is
late maturing variety and matures in February –March.
 Grapefruit: Marsh Seedless: this variety is known for its almost absence of
seeds, if present are few (0-6) and usually rudimentary. The colour is pale
yellow at maturity. Fruits ripen in December- January.
 Duncan: its fruit are large, with about 30-50 seeds per fruit. Flesh is tendet,
acidic and the sweetness is medium, very juicy. Fruit ripen in Decmber-
January.
 Red Blush: this variety is liked for the attractive appearance provided by the
pigmentation of the rind and its deep flesh colouration. It contain seeds (0-8),
mostly aborted. Peel is smooth, glossy, deep yellow in colour. Ripen during
lat week of November.
 Foster: fruit are medium to large, oblate to spherical, having large numbet of
seeds with 40-50 seeds per fruit. Primary colour is pale to light yellow, but
sometimes rind blushed with pink, extending into the albedo. Fruit ripens in
November-December.

14.  Limes and Lemons
 Punjab Baramasi Lemon: fruits are medium to large in size with a smooth rind.
Fruits is very juicy and seedless. Average yield is 84 kg per tree and the juice has 5.6
percent acidity.
 PAU Baramasi Lemon-1: the fruit is lemon yellow, round, tapering towards the base
with round apex. Seedless and contains about 7 percent acidity.
 Eureka: the fruits are medium in size, oblong in shape. Skin colour is lemon yellow at
maturity. The fruits ripen in the August- September. Tree is medium in vigour, spread
and size and open in growth habit and virtually thornless.
 Punjab Galgal: its trees are vigorous, fruits is medium in size, oval in shape, glossy,
medium thick and yellow at maturity. 5-8 seeds per fruit. It matures in the last week of
November and gives 80-100 kg fruit per tree.
 Kagzi: tree is medium sized, hardy and semi-vigorous. The fruit is small, round and
thin skinnede, pulp greenish white and the juice is strongly acidic.
 Local: the fruit is medium, globose to ellipsoid, the rind is smooth with distinctive
aroma. The juice is abundant, lacking in acidity, insipid, seeds 5-6 and ripens in the
beginning of September.
 Vikram and Pramilini (Acid lime): these cultivars have been developed through
clonal selection. Fruits borne in cluster.
 Tahiti lime: this is believed to have been introduced from Tahiti to California, Florida.
Fruits are medium, obovate-oblong, base and apex roundish, rind thin, tight, smooth ,
light yellow in colour. It is seedless in nature.

15. INHERITANCE OF TRAITS
 Characters that show inheritance are like Trifoliate leaf
characters in trifoliate orange show essentially complete
dominance over monofoliate.
 Purple colour in young leaves of lemon is governed by one or
two dominant gene and nucellar embryo in citrus is controlled
by one or two dominant gene.
 Small size of the fruits, seediness and paleness of colour
appeared to be dominant. However, characters like presence of
thorns, pubescence and oil glands are dominant characters.
 Dwarfness is a recessive character.
INCOMPATIBILITY SYSTEMS IN CITRUS: Soost (1969)
reported gametophytic incompatibility systems in Citrus. When
pollen tube starts growing through the style, but could not
progress more than 50% of the style length.

16. METHODS OF IMPROVEMENT
 INTRODUCTION
 SELECTION
 HYBRIDIZATION
 MUTATION
 POLYPLOIDY BREEDING
 BIOTECHNOLOGY
BOTTLENECKS
 Long juvenile period
 Heterozygous nature
 Pollen and ovule sterility
 Self and cross Incompatibility
 Polyembryony

17. OBJECTIVES
Scion improvement
 Seedlessness, high TSS/acid ratio, thin loose and coloured rind in
sweet orange and mandarin.
 Prolonged shelf life, easy to peel and resistance to greening
Rootstock improvement
 To develop rootstock with dwarf stature, imparting high yield and good
quality to the fruits on scion variety.
 Develop a rootstock resistant to biotic (Phytophthora, citrus nematode,
tristeza and exocortis) and abiotic stresses (salinity and alkalinity)..

18. INTRODUCTION
Cultivar Country Characteristics
Pummelo
US -145
USA It is white fleshed and fruit weights about 700-800 gm. It has sweet taste
like Mosambi. The fruit has 15 segments which are very juicy. The
productivity is about 45-50 tonns/ Ha. Fruit mature in August and can be
harvested up to October. This is an early fruit of citrus group since
Nagpur mandarin comes to market in October.
Cutter
Valencia
USA This is a tight jacket orange of sweet orange group and is considered best
fruit world over for processing. Fruit matures in last week of November
and can be harvested up to January. It can be used for table and juice
purpose. The yield is 21 tonnes at 6×6 m spacing and can increase up to
30 tonne at 5×5 m
Flame
grapefruit
seedling
USA The grapefruit is seedless, red fleshed and has good blend of acid and
sweet and bitter taste. The productivity is about 25 tonne per hectare (6×6
m distance) which increases to double in high density (4×4m) cultivation.
Recent achievements
Recently, in 2016 ICAR-CCRI, Nagpur released five citrus
varieties

19. Contd...
Cultivar State Characteristics
NRCC
Pummelo – 5
Jungles of North-East
on Mizoram-Tripura
border
Fruit has excellent sweet taste and has a little tinge of
guava flavor. It is red fleshed and juicy, fruit weight is
about 800g.
The yield is high and a grown-up plant bears over 400
fruits. The yield could be 32 tonnes per hectare at 55 m
spacing.
NRCC
Grapefruit – 6
North-east region This variety also yields around 400 fruits per plant,
which is about 152 kg per plant, or 42 tonnes per
hectare.
Two varieties developed through indigenous material

20. ` SELECTION
In India, over the last ten years clonal selection has been done in sweet orange and in
acid lime at IARI New Delhi.
 Sweet orange:
 Pusa Round: It is a promising selection having dense foliage and attractive round
fruit. It has uniform bigger sized fruits (268.68 g), high juice (119.00 ml/fruit;
48.26%) and high TSS (10.140B) content with moderate acidity (0.92%).
Furthermore, the plants are moderately vigorous and about 400 plants of this
selection may be accommodated in a hectare (5 m x 5 m). On per plant basis, it
yields about 3.5-fold higher than Jaffa and 2.4-fold higher than Valencia. Therefore,
it will give higher productivity per unit area. Its fruits are free from granulation.
 Pusa Sharad: It is a selection having medium vigorous trees, leaves unifoliolate,
petiole wing absent, more or less plane, thorn present, dense canopy, suitable for
planting at a distance of 5 m x 5 m. Average fruit weight 227.56 g, round in shape,
juice recovery 50.12%, medium thick peel, higher TSS (9.20 ºBrix) and moderate
acidity (0.77%).It yields 2.6-fold higher than Jaffa and 1.8-fold higher than Valencia.
Therefore, it will give higher productivity per unit area. Its fruits are free from
granulation.
www.iari.res.in

21.  Acid lime:
 Pusa Abhinav: It is a promising clonal selection having medium
vigorous trees, dense foliage, and attractive bright yellow round
shaped fruits. It has round the year fruiting with peak harvesting
during summer months (March-April and August-September) and is
moderately susceptible to citrus canker.
 Pusa Udit: It is a clonal selection has dense canopy, heavy yielder,
fruits round in shape, medium in size. It produces round the year
crop with peak harvesting from August-September and February-
March. It is moderately susceptible to citrus canker. Round the year
fruiting with two season harvesting will make this selection highly
suitable for commercial cultivation as well in kitchen garden.
 Recently, in 2014-15 two new superior varieties of acid lime has
been developed using clonal selection at National Research Centre
for Citrus (NRCC) or CCRI, Nagpur. These varieties yield five
times more than the normal yield, are more juicy, have thinner skin
and are bigger in size. NRCC acid lime-7 bears single fruit whereas
NRCC acid lime-8 is bunch bearer. (www.ccringp.org.in)

22. Achievements: In china
TYPES VARIETY
RELEASED
RELEASED YEAR
Mandarin Huagan 2005
Mandarin Shiyuechu 2005
Pummelo Feicuiyou 2002
Pummelo Taoxizaoyou 2003
Source: Liu (2007)

23. SELECTION
‘DaisySL’ Mandarin
Roose and Williams (2010)U.S.A.

24.  ‘DaisySL’ is a mandarin selection developed by the University
of California Riverside from an budwood irradiation of the
diploid Daisy mandarin.
 Mid-season maturing variety , low seeded (2.2 seeds/fruit)
 Fruit size is moderately large (68 mm) averaging 135
grams/fruit. Fruit are slightly obconate in shape with a very
deep orange rind colour and an extremely smooth rind texture.
Flesh color is very deep orange and finely textured; fruit are
very juicy (47%) with a rich, sweet and very distinctive
flavour when mature.
 Tree growth habit is spreading with excellent production
commencing in the third year after planting. Thornless variety.
 Release and Distribution: ‘DaisySL’ was released for
propagation in California in September 2009.

25. HYBRIDIZATION
HYBRID CHARACTERISTICS
Gold Nugget
(Wilking  Kincy)
Diploid hybrid released in 1999. Seedless late maturing hybrid
(February – March), on tree storage life for 4 -6 months, high brix
(15-18°Brix), excellent storage abilities, alternate bearing is the
problem.
TDE 2 (Shasta Gold) Patented, triploid hybrid, late season maturing (February – March),
Seedless, production is very high, average 800-900 cartons/acre.
TDE 3 (Tahoe Gold) Patented, Seedless, mid late season maturing (January – February),
very deep rind colour, on tree storage life of fruits (1-2months).
Production is excellent 900-1000 cartons/acre.
TDE 4 mandarin
(Yosemite Gold )
Patented, Seedless, Large alternative fruit, late maturing (February–
March) on tree storage life of fruit (2-3 months). Production is very
good of 700-900–cartons/acre.
Source: Roose and Williams (2000)
Improvement of citrus rootstocks was initiated in 1972 at Chethalli and IIHR Banglore.
Achievements
In india, CRH-3, CRH-5 and CRH-47: Rangpur lime x Trifoliate orange, an inter-generic hybrid.
These rootstocks are resistant to citrus nematode, released from, IIHR Bangalore.
Rasraj: Rasraj is an inter-generic polyembryonic hybrid developed for resistance to bacterial canker disease.
Nepali Round lemon and Acid lime.
Mandarin hybrids from University of California, Riverside

26. Contd...
Rootstock, In Spain
Rootstock hybrid Parentage Important traits
Forner- Alcaide 5 Cleopatra mandarin
x trifoliate orange
Semidwarfing, Resistant to tristeza, citrus nematode,
Tolerant to lime induced chlorosis, Cl- excluder,
excellent tolerance to wet and flooded soils, good
fruit quality (can replace Carrizo in Spain).
Forner- Alcaide 13 Cleopatra mandarin
 trifoliate orange
Trees deciduous, CTV resistant, phytophthora
resistance, susceptible to citrus nematode but salinity
tolerant.
Rootstock hybrid Parentage Important traits
US-812 Sunki  Benecke
Trifoliate orange
It is highly productive of good quality fruit on a
moderate sized tree and exhibits tolerance or
resistance to citrus tristeza virus. Released in May
2001 from U.S.A.
US-897 Mandarin  trifoliate
orange
Resistant to Huanglongbing (HLB)
Source: Bowman and Rouse (2006)
Source: Forner et al (2003)
In USA

27. Mutation
TYPES VARIETY
RELEASED
RELEASED
YEAR
REGION BREEDING
METHOD
Citrus reticulata Ganpon 1997 Jiangxi Bud sport
Qianyang seedless
Pongan
1998 Hunan Bud sport
Mingliu Tianchu 2006 Guangdong Bud sport
C. sinensis Fengjie wancheng 2005 Chongqing Bud sport
Xinjing No.101 2005 Hubei Bud sport
C. unshiu Egan 2004 Hubei Bud sport
Hongroumiyou 2004 Fujian Bud sport
Recent achievements in china
Source: Liu (2007)

28. SPARSE SEEDED MUTANT OF KINNOW (Citrus reticulata Blanco)
The mV1 plants treated with 20 Gy dose was used for successive vegetative
propagation upto mV5 as sparse seeded character was retained upto mV5 . A
sparse seeded (5 ± 3 seeds/fruit) mutant was evolved.
Flowering stage: After 3 to 4 years all the plants entered the flowering stage
fruits from these plants were analyzed for the number of seeds per fruit.
Mutagen treatment: Radiation (gamma) doses of 20, 40, 60, 80 and 120 Gy
were applied.
Propagation: The irradiated bud scions were grafted onto Rough Lemon
rootstock, using the side-graft technique.
Explant: Dormant bud scions of Kinnow were subjected to gamma
irradiation.
Khalil et al (2011)
Induced mutation

29. Table 1. Physical characteristics of kinnow and its seedless mutant (range and mean)
crop year fruit diameter (mm) seeds/ fruit
Non-irradiated (mV0)
1987/1988
2003/2004
65-80 (74.3)
64-81 (73.6)
20-30 (23.50)
19-28 (23.00)
Mutant (mV1)
1987/1988
2003/2004
63-78 (70.2)
65-79 (72.4)
3-7 (4.67 )
2-8(4.39)
Mutant (mV2)
1992/1993
2003/2004
65-79 (71.79)
61-82 (70.78)
3-7 (5.00)
3-7 (4.89)
Mutant (mV3)
1996/1997
2003/2004
65-82 (72.78)
63-81 (79.5)
3-8 (5.11)
3-7 (4.95)
Mutant (mV4)
2000/2001
2003/2004
60-78 (76.2)
62-79 (76.5)
2-8 (5.04)
3-7 (4.72
Mutant (mV5)
2003/2004
60-80 (75.7) 2 – 7 (4.91)

30. Table 2 Biochemical constituents of juice of Kinnow and its seedless mutant (range and
mean)
crop year TSS(%) Acidity (%)
Non- irradiated (mV0)
1987/1988
2003/2004
10.5-11.2 (11.0)
10.4-11.5 (11.2)
0.58-0.62 (0.59)
0.60-0.62 (0.60)
Mutant (mV1)
1987/1988
2003/2004
9.95-10.7 (10.41)
10.3-10.8 (10.62)
0.61-0.62 (0.62)
0.60-0.62 (0.62)
Mutant (mV2)
1992/1993
2003/2004
10.6-11.1 (10.69)
10.5-11.4 (10.75)
0.60-0.63 (0.62)
0.62-0.62(0.62)
Mutant (mV3)
1996/1997
2003/2004
10.3-11.9 (10.48)
9.8-11.5 (10.70)
0.59-0.62 (0.61)
0.61-0.62 (0.61)
Mutant (mV4)
2000/2001
2003/2004
10.5-11.7 (11.0)
10.7-11.6(11.2)
0.62-0.65 (0.63)
0.60-0.63(0.60)
Mutant (mV5)
2003/2004
10.4-10.7 (10.5) 0.62-0.66 (0.63)
Reduction of seed number is caused by the abortion of both ovules and seeds presence of
certain chemical factors leads to discontinuation of the seed development process. Ovule +
seed abortion in parent Kinnow = 20%; ovule + seed abortion in the seedless Kinnow
mutant = 80%.

31. Fig 1. Mutant kinnow plant and its sparse seeded fruit

32. Production of Bingtang sweet orange somaclone (DG-2)
tolerant to citrus canker disease by in vitro mutagenesis with EMS
 Causal organism of citrus canker is Xanthomonas citri subsp. citri
(Xcc)
 Somaclones are the individuals that undergo genetic variation.
 In vitro culture combined with induced mutation had been proven
speed up the breeding programme (Ahloowalia et al., 2004).
 In vitro cultured explant provide a wider choice of controlled selection
following mutagenic treatment. It required less space to handle large
population within short time.
China Ge et al (2015)

33. Fig 1. a The formation of embryogenic callus from undeveloped ovules of sweet orange. b
The embryonic callus grew well sub cultured on MT medium without any plant growth
regulator. c The established cell suspension culture of embryogenic callus in liquid MT
medium. These cell suspension was treated with EMS and washed properly with LMT
medium and survival rates, lethal concentration was assessed with the help of
spectrophotometer. Cell aggregates were recovered and cultured on LMT med. for 1 week.
The resulted cell aggregates were transferred to LMT med. containing 10% Xcc- crude
extract for selection.
(a) (b) (c)

34. Fig 3. Test of the sensitivity of ‘Bingtang’
sweet orange cell suspensions to EMS
treatment
Fig 4. Determination of lethal EMS
treatment concentration for ‘Bingtang’
sweet orange cell suspension. Thus,
1.5% for 1h is the lethal concentration.
Determination of lethal concentration of EMS for cell
suspension culture

35. Determination of duration of Xcc-crude extract
treatment
Fig 5. Influences of Xcc-crude extract treatment time on the survival rate of the ‘Bingtang’
sweet orange cells aggregates. The ‘Bingtang’ sweet orange cell suspension cultured in the
LMT medium containing 10 % Xcc-crude extract solution was observed in comparison
with the same suspension in the LMT medium containing 10 % LB liquid medium (control)

36. Fig 6. selection of Xcc-tolerent mutants and regeneration of somaclone plant. A cell
suspension under selection were planted on solid medium for embryogenesis tolerent callus
colonies were further grew up and embryo were formed. B Less than 20 callus colonies
grew out in petridish from 2000 original cells aggregates, the embryo were germinated into
shoots, which were rooted and plantlets were regenerated. C in total 271 putative plantlets
were obtained. D normal growth of control plantlets. E 271 plants were grown in 10% Xcc-
crude and those grown in water were served as control at the end 19 out of 271 survived. F
after inoculation leaves shows symptoms (yellow, wilting and necrotic lessions) and leaves of
control plants remain normal.

37. Fig. g In vitro selected plants by inoculation of the detached leaves with Xcc crude extract .
Lastly seven Xcc-crude extract tolerent plants were identified and transplanted in the
greenhouse. h Shoots (from Xcc tolerent plants) were grafted on the rootstock of sour
orange. i The potted plants growing in greenhouse. j–m In vivo assay of the somaclones for
the tolerance to the pathogen. The symptoms of ‘Bingtang’ sweet orange plants on 3d (j)
and on 7d (k); No symptoms on the leaves of the tolerant somaclone (l) and on (m). Among
all grafted plants, a somaclone named DG-2 did not show any symptom of citrus canker.

38. Improvement through
Biotechnology

39. In vitro micrografting for production of Indian citrus ring spot virus (ICRSV)-
free plants of kinnow mandarin
Fig. 1 a Etiolated seedlings (2wk old) of rough lemon (C. jambhiri) prepared for micrografting. b Rootstock
after 12 days of micrografting. c Rootstock with scion showing two expanded leaves after 30 days of
micrografting. d Micrografted plantlet after acclimatization. e Micrografted plantlets growing in screen
house. First reported in India by Byadgi et al. (1993) . Almost all citrus sp. Are susceptible, it is
graft transmissible disease. 98% yield loss
Singh et al (2008)Attari, Punjab

40. Fig. 2 Agarose gel electrophoresis of RT-PCR products from in vitro micro grafted plants to
detect presence or absence of ICRSV (Lane M: 100 bp ladder as marker; Lane 1: virus-infected
mother plant as control; Lane 2: 0.2-mm scion micro grafted plant; Lane 3: 0.3-mm scion
micrografted plant; Lane 4: 0.4-mm scion micrografted plant; Lane 5: 0.5-mm scion micro
grafted plant, Lane 6: negative control)

41. Table 1. Effect of shoot apex size on micro grafts survival in MS liquid
medium containing 5% sucrose and on elimination of ICRSV

42. Table 2 Effect of different concentration of sucrose in MS liquid medium
on micrografting success using shoot apex of 0.2 mm size

43. Early age propagation of three commercial citrus species
through micro budding technique
Cultivar Bud
wood
Size
(mm)
Rootstock
Budding
height
(cm)
Micro
budding
Success %
No. Of
Leaves
Leaf
Length
(cm)
Leaf
width
(cm)
Scion stem
diameter
(mm)
Plant
height
(cm)
No. of
days to
form first
leaf
Grapefruit
Shambar
3 10 36.00ab 4.80bc 7.92ab 4.08ab 2.64de 12.5a 16.35 b
14 68.00ab 7.20a 6.64c 3.50bc 2.45def 9.08e
4 10 28.00d 2.60d 8.44a 4.90c 2.35ef 9.00e
14 36.00cd 7.20a 6.76ac 3.86bc 3.80a 19.76a
Mandarin
Feutrell’s
Early
3 10 28.00d 6.80a 6.36c 3.56bc 3.00bc 8.76e 17.05 b
14 24.00d 2.40d 6.68bc 3.82bc 2.30f 13.56c
4 10 24.00d 3.60cd 5.76c 3.02c 2.52def 17.28b
14 44.00c 4.00bc 6.76bc 3.60bc 2.52def 11.44cd
Sweet lime 3 10 44.00c 4.20bc 8.68a 4.48a 2.22f 16.22a 19.8 a
14 60.00b 6.80a 6.02c 3.24bc 2.7cd 11.92cd
4 10 76.00a 5.00a 5.76c 3.08c 3.23a 12.58c
14 36.00cd 2.60a 8.76a 4.90a 3.18b 10.22de
Alam et al (2006)Faisalabad

44. TRANSGENIC APPROACHES
 Dutt et al (2011) develop transgenic sweet orange cultivars
‘Hamlin’ and ‘Valencia’ resistance to HLB, expressing an
Arabidopsis thaliana NPR1 gene under the control of a
constitutive CaMV 35S promoter or a phloem specific
Arabidopsis SUC2 (AtSUC2) promoter were produced.
Overexpression of AtNPR1 resulted in trees with normal
phenotypes that exhibited enhanced resistance to HLB.
 Guo et al (2005) first successful transfer of a target fruit-
quality gene by protoplast transformation with recovery of
transgenic plants in Valencia sweet orange. There is
reduction in activity of thermostable pectin methylesterase
(TSPME) in juice and overexpression of CsPME4 to verify
the thermostability of the protein product and/or to
downregulate the gene.

45. THANK YOU