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Advances In Citrus Breeding
Introduction & Importance
 Citrus is the most cultivated fruits in the world with the highest acreage 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 10.24 Lac Hectare with an annual production of 1.15
Crore 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.
 The domestication and distribution of edible citrus types began several thousand
years ago in South -East Asia and spread globally following ancient and sea routes.
 The Citrus species are undoubtedly indigenous or domesticated for a long time.
(Bhattacharya and Dutta, 1956; Singh, 1977).
Origin and Distribution
■ Center of origin is South-East Asia bordered by North Eastern India, Myanmar
(Burma) and Yunnan province of China.
■ From China, India and West Indies, it dispersed all over the world and now, it is
grown almost throughout world.
■ Most scion and rootstock cultivars that are widely grown in the main commercial
producing areas of the world are originated as either chance seedling selections or
bud sport mutations (Hodgson, 1967).
Species Origin
Mandarin Indo-China and South China
Sweet orange South China
Lemon Unknown
Lime East India and Archipelago
Pummelo Malaysia and Indian Archipelago
Grape fruit West Indies
Kingdom Plantae
Division Phanerogams
Sub division Angiosperm
Class Dicotyledonae
Order Geraniales (1 of 21 orders)
Sub order Geraniinea (1 of 12 sub orders)
Family Rutaceae (1 of 12 families)
Sub family Aurantoideae (1 of 7 sub families)
Tribe Citrae (1 of 2 tribes)
Sub tribe Citrinae (1 of 3 groups)
Group C True citrus fruit trees (1 of 6 generas)
1. Poncirus - 1 species
2. Fortunella (Kumquat) – 4 species
3. Eremocitrus
4. Clymenia
5. Microcitrus
6. Citrus – 2 subgenera
* Generally, 3 related genera Poncirus,
Fortunella and Citrus are included in the
study of citrus fruit/ Citriculture.
Genetic
Classification
*Source: Swingle & Reece, 1967.
Taxonomy
Sub-genus : Eucitrus (10 species)
1. C. medica (Citron)
2. C. limon (Lemon)
3. C. aurantifolia (Acid/sour lime)
4. C. aurantinum (Sour/bitter orange)
5. C. sinensis (Sweet orange)
6. C. reticulata (Mandarin)
7. C. grandis (Pummelo)
8. C. paradisi (Grapefruit)
9. C. indica (Indian wild orange)
10. C. tachibana (Tachibana orange)
Fruits are edible and widely cultivated.
Sub-genus : Papeda (6 species)
1. C. ichangenesis
2. C. latipes
3. C. micrantha
4. C. celebica
5. C. macroptera
6. C. hystrix
Fruits are inedible and presence of
numerous droplets and oil in juice sacs.
Genus Citrus
* Source: Janick 2006
WT Swingle (1948)
Genus Citrus
■ Citrus word is originated from Greek word – ‘Kedros’ originally meant ‘Cedar’ or
‘Cedarcone’ which means – Fruit of the godly tree in Palestine.
■ Gk. Word ‘Kedros’ is latinized to ‘Cedrus’ which later changed to ‘Citrus’.
■ Genus is commercially important as many species are cultivated for fruit, which is
eaten fresh, pressed for juice, preserved in marmalades and pickles.
■ In most of species, both bud and seed mutations are known to occur giving rise to
‘Sports’.
■ The species of Fortunella, Poncirus and Citrus hybridize freely producing natural
hybrids. Similarly, budding and grafting are easily done reciprocally among the
species and genera.
Genetic variability
■ Citrus has wide variability with respect to fruit morphology & quality.
■ Limes are smallest scarcely exceeds 3 cm in size, while pummelo may attain
diameter of 30 cm.
■ Fruit rind colour ranges from yellow green of lime to red orange of some
mandarins.
■ Shape varies from oblate to pyriform.
■ At maturity, some cultivars are high in acid while others have almost none.
■ All species are evergreen but related genus Poncirus is deciduous.
■ Altogether, there is much variability within genus with which breeder can work
and closely related genera provide an even wider range of characters.
Genus Fortunella
■ Kumquat is the popular name of the Fortunella.
■ The plants are evergreen shrub & most cold hardy.
■ Fruits are orange to golden yellow colour, palatable with spicy aromatic ring.
■ The four species of Fortunella are F. margarita (Oval kumquat), F. japonica
(Round kumquat), F. crassifolia & F. hindsii (Hongkong wild Kumquat).
Genus Poncirus
■ Monotypic genus: has only one species, popularly known as trifoliate orange.
■ Plants deciduous with trifoliate leaves, highly thorny & fruits are inedible.
■ Dark green twig with strong and stout thorns make the species an excellent
hedge in ornamental gardens.
■ Plants are cold hardy and the species is principally used as rootstock.
Cytogenetics.
Studies of cellular aspects of heredity (chromosomes)
■ Most of the citrus species are diploid (2n=2x=18).
■ One natural triploid is Tahiti lime (Citrus latifolia).
■ Fortunella hindsii is tetraploid species of Kumquat.
■ Genome size of Citrus sinensis is 360 Mb.
Chemotaxonomy
Species Flavonoids Coumarins and Psoraiens Carotenoids
C.
aurantifolia
Hesperidin, Naringin,
Naringenin, Erioctrin
Tangeretin
Bergapten, Bergamottin, Beragaptol,
Phellopterin, Prangol, Scopoletin
-
C. aurantium Naringin, Neoeriocitrin,
auranetin, neohesperidin,
tangeretin, hesperidin
Aurapten, auraptenol, bergapten, meranzin,
meranzin hydrate, seselin, isomeranzin, osthol,
scoparon, umibelliferone, xanthyletin
-
C. paradisi Naringin, hesperidin Aurapten, bergapten, bergamottin, bergaptol,
citropten, marmin, meranzin
Phytoene, Phytofluene,
Carotene.
C. reticulate Hesperidin, Neohesperidin,
Xanthomicrol, Nobiletin
Aurapten, Bergapten, Bergaptol, Citropten,
Osthol, Auroptenol
β-carotene, Cryptoxanthin,
Violaxanthin,
C. sinensis Rutin, limocitrin, sinensetin Auraptenol, Scoparon Phytoene, β- carotene
C. limon Naringin, Eriocitrin,
Hesperitin, Hesperidin
Bergapten, Bergamottin,
Byakangelicin, Citropten, Imperatorin,
Phytofluene, β-carotene,
Cryptoxanthin
Centre of diversity
■ There are three major centers of diversity in India.
■ The first in the North-East including Assam and adjoining areas. It includes Papedas, pummelos and their
hybrids, citron, lemons and mandarins and other interesting types like jenera-tenga, soh synteng, a sour
fruit similar to the sweet lime and soh siem, a mandarin type.
■ The second diversity in south India, indigenous types include Gajanima, kichili and some wild mandarin
types.
■ The third in North-West region at the foot of Himalayas where the hill lemon (galgal) is common.
■ The various types of mandarins, hybrids of pummelo, citron, lemons, karna khatta and rough lemon are
found all over the country.
■ In general, the wild types are more common in the foot hills. Many of the progenitors of citrus fruits are
believed to have originated in India. These include C. latipes, C. limonia, C.kama, C.pennevesiculata,
C.maderaspatana, many of these are wild types.
■ Presence of Sah- Niangriang, a wild sweet orange and a wild mandarin (C.indica) furnishes strong
evidence that Eastern India might be the centre of orgin for many citrus fruits (Tanaka, 1981).
Germplasm resources
 Exotic collection of citrus germplasm was started in 1940.
 Kinnow mandarin was one of the collections which is now a leading cultivar in North – Western India.
 Besides, other exotic collections were Valencia Late, Washington Navel, Jaffa, Malta Blood Red, Pineapple,
Ruby orange, Satsuma, Dancy Tangerine, Climentime, and Cleoptera wilking ,Temple, Duncan, Marsh seedless,
Lisbon lemon, Trifoliate orange, Dancy, Lisbon lemon, Trifoliate orange (Dutta,1958).
 More than 650 accessions are being maintained at CHES, Chethali, Bangalore, CHES, Ranchi, RFRS, Abhor, NRC
on citrus, Nagpur, Horticultural Experiment Station, Bathinda, IARI, New Delhi, MPKV, Rahuri, Citrus
Improvement Project, Tirupati, Citrus Experiment station, Nagpur, HC&RI, Periyakulam, and Citrus Experiment
Station, Tinsukia, Assam.
 Attempt has been made during 1978 by NBPGR to preserve the C. indica which is progenitor of C. reticulata
(Singh, 1981).
 For establishment of gene sanctuary, National Park, the natural genetic diversity of C. indica was observed in
the forest of Garo hills in Megalaya which exhibited plant characters varying from bush to climber with high
frequency of distribution in dense forest and showing resistance to biotic stresses.
 Genetic material of citrus is conserved in field gene bank or repository.
National Status
■ In India, Citrus genetic resources are being managed by multiple agencies such as research
institutes of Indian Council of Agricultural Research (ICAR), State Agricultural Universities
and also by the State Horticultural Farms and Fruit Research Stations.
■ Besides these several private nurseries, orchards, gardens, parks etc. which are owned by
progressive farmers and public enterprises are also maintaining Citrus germplasm as field
collections
■ In India, National Research Centre for Citrus (NRCC), of ICAR, located at Nagpur in Central
India is the only dedicated institute for Citrus research, development and extension. This
Centre has also been designated as National Active Germplasm Site (NAGS) for Citrus by
NBPGR.
■ All India Coordinated Research Project (AICRP) on Tropical Fruits (Citrus) of ICAR with its
Network at nine centres at SAU's and ICAR institutes in various agro-ecological regions of
India has been taking up work on genetic resources management, crop improvement, crop
production and crop protection. Since last several decades crop specific responsibilities for
multi-location evaluation and improvement work has been assigned to these centres.
Inheritance of characters in citrus
Character Inheritance
Leaf character Two principal genes
Anthocyanin pigment in leaf Dominant gene controlling this reddish colour
Anthocyanin pigment in fruit More than one recessive genes are involved
Tristeza virus Single allele
Texture Polygenic control
Polyembryony More than one single gene
Seedless character A recessive gene
Nucellar embryony One or two dominant gene
Tolerance for biotic stress
Biotic stress Cultivar
Root rot Rangpur lime, Marmalade orange, Troyer citrange, Carrizo citrange.
Nematode Marmalade orange, Sweet lime, trifoliate orange, Troyer citrange, Carrizo
citrange, Karna khatta, Nasnaran, Survernia disticha.
Tristeza virus Rangpur lime, Marmalade orange, Rough lemon, Cleopatra mandarin, Trifoliate
orange, Troyer citrange, Carrizo citrange, Nasnaran, Sweet orange.
Exocortis Rough lemon, Sweet orange, Sour orange, Cleopatra mandarin, Grapefruit.
Gummosis Trifoliate orange, Sour orange, Rangpur lime, Cleopatra mandarin, Grapefruit.
Greening Poncirus, Gajanima, Florida rough, Karna khatta.
Genetic resource resistant/tolerance for abiotic stress
Abiotic stress Cultivars
Salt Rangpur lime, Marmalade orange, Nasnaran, Cleopatra mandarin.
Drought Rangpur lime, Marmalade orange, Rough lemon, Sour orange.
Genetic resource for yield and quality traits
Trait Cultivars
Good yield Rangpur lime, Rough lemon, Cleopatra mandarin, Sour orange, Karna khatta.
Good quality fruits Sour orange, Carrizo citrange, Troyer citrange , Sweet orange, Nasnaran.
Varieties resistance to abiotic stress
Resistance Cultivar
Cold hardiness Duncan grapefruit
Cold hardiness Valencia sweet orange
Cold hardiness Carrizo citrange
Salt tolerance C. jambhiri and C. macrophylla
Salt tolerance ‘Cleopatra’ mandarin for ‘Star Ruby’
Main Cultivated Species
Mandarin Citrus reticulata Blanco
Sweet orange Citrus sinensis (L.) Osbeck
Lemon Citrus limon Burmann
Lime Citrus aurantifolia Swingle
Pummelo Citrus maxima (L.) Osbeck
Grape fruit Citrus paradisi Macfadyen
Citron Citrus medica L.
Kumquat Fortunella species
Main Cultivated Cultivars
Type Cultivar
Sweet orange
i. Common or round orange Hamlin, Valencia, Netal, Pera, Shamouti, Jaffa, Pineapple, Mosambi, Delta,
Campbell, Frost, Premier, Sathgudi, Ambersweet, Delicious, Malta.
ii. Navel orange Washington Navel, Thompson, Dream, Autumn Gold Late, Atwood, Fisher,
Newhall, Navelina, Navelate, Summerfield, Marrs, Bajaninha, Palmer, Cara .
iii. Pigmented oranges Malta Blood Red, Ruby, Moro, Tarocco.
iv. Acidless or sugar orange Lima, Succari, Piralima, Oro , Sicily, Sanguinello.
Limes
i. Acid lime Kagzi, Mexican, Palmetto.
ii. Persian lime (Tahiti lime) Tahiti, Pond, Bearss lime.
iii. Mandarin lime Rangpur lime.
iv. Sweet/acidless lime Sweet lime, Local, Palestine.
Lemons Lisbon, Eureka, Lucknow Seedless, Kagazi Kalan, Baramasi, Villafranca, Pant
Lemon -1, Meyer Lemon, Femminello, Malta Lemon, Nepali, Oblong.
Main Cultivated Cultivars
Type Cultivar
Mandarins
i. Satsuma group (C. unshiu Marcvitch) Ovari, Kara, Brown’s Select, Miho Wase, Miyagawa Wase.
ii. Mediterranean group (C. deliciosa Tenore) Willow Leaf mandarin, Wilking, Kinnow.
iii. Common group (C. reticulata) Nagpur Santra, Coorg, Khasi, Local, Darjeeling, Dancy, Daisy,
Kinnow, Encore, Murcott.
iv. King mandarin group (C. nobilis Loureiro) Jeneru Tenga.
v. Tangerine group Dancy, Beauty, Naartja.
vi. Mitis group Cleopatra, Calamondin.
Mandarin - Like Tangors (Hybrid between Tangerine × Sweet orange)
Temple, Clementine.
Mandarin - Like Tangelos (Hybrid between Tangerine × Grapefruit)
Orlando, Minneola.
Type Cultivar
Grapefruit
i. White fleshed Duncan, Marsh, Walters.
ii. Pink fleshed Ruby (syn. Ruby Red, Red Blush), Foster, Ray Ruby, Ruben SL, Henderson 13.
iii. Red fleshed Star Ruby, Rio Red, Flame, Nelruby, Oran Red (syn. Rouge la toma), Henderson.
Pomelos
i. Thai group Chander (Pink Fleshed), Kao Panne and Kao Phuang (White fleshed).
ii. Chinese group Goliath, Mato, Shatinyu.
iii. Indonesian group Banpeiyu (White-fleshed), Djeroek deleema, Kopjar (Pink-fleshed).
iv. Indian group Galgal (White or Pink-fleshed).
Kumquat (Fortunella spp.) Nagami, Metwa, Marumi, Calamondin.
Main Cultivated Cultivars
Cultivars grown in India
Sweet orange Mosambi, Satgudi, Valencia Late, Jaffa, Pineapple, Shamouti, Washington Navel.
Mandarins Nagpur Sangtra, Khasi mandarin, Darjeeling mandarin, Coorg Kinnow.
Lemon Eureka, Lisbon, Villafranca, Assam lemon, Punjab Galgal.
Grapefruit Duncan, Triumph, Marsh Seedless, Star Ruby, Foster.
Floral Biology of Citrus
■ Flowers are hermaphrodite produced on current
season growth in cymes, both at axillary and
terminal position.
■ Flower opening starts from morning and extend
upto evening, but maximum anthesis is between
11.00 a.m. to 12.00 noon.
■ The viability of pollen grains varies from 45-80%
depending upon the season.
■ The dehiscence of anthers takes place 45 minutes
before anthesis or within 45 minutes after
anthesis. It varies up to 5 hours after anthesis.
■ The receptivity of stigma starts either 15 minutes
to 2 hours before anthesis or within 35 minutes to
5 hours after anthesis depending upon weather.
The receptivity lasts for 4-8 days after anthesis.
Flowering, fruit set and maturity of citrus
Country Flowering time Fruit set Ripening
N. India March (Feb.-April) April-May January ( Mandarins)
Nov.-Jan. (Sweet Oranges)
S. India Dec. –April,
Sept-Dec.
Sept.-Dec. March –April,
Sept-Oct.
Central India March-April,
Sept. - Oct.
June,
Oct. – Feb.
Aug.-Oct. (Sweet Oranges)
Feb-March
* Lemon and lime throughout the year.
USA Feb-March April-May Dec.-Feb.
Australia April-May May-June Jan.-March
Japan April-May May-June Dec.-Feb.
Brazil March-April April-May Dec.-Jan.
Problems in conventional breeding
■ Male or female sterility
■ Self and cross-incompatibility
■ Long juvenile period
■ Nucellar embryony
Kinds of male sterility occurring in citrus
* Source: Iwamasa, 1966
Objective of improvement for Scion
■ Seedlessness.
■ High productivity.
■ Sensory fruit quality (loose peel, pigments).
■ Quality (TSS/ acid ratio, Juice content, Flavour and colour).
■ Wide season of ripening & good post harvest life.
■ Resistance to adverse climatic and soil conditions.
Objective of improvement for rootstock
■ Better stock-scion compatibility, reduction of tree size without affecting yield.
■ Resistance to pests and diseases or hardiness to adverse climactic conditions.
■ Resistance to nematode species.
■ Must produce many seeds & be highly nucellar in order to provide uniformity in mass
multiplication.
■ Cold, salinity & drought tolerance.
CCRI Nagpur Vision 2050
Genetic Resources and Crop Improvement
■ Functional genomics and phenomics based gene identification for specific trait and gene pyramiding to have cultivar of
multiple resistance on one hand and identifying genes for some negative traits like genes leading rootstock
vulnerability to Phytophthora, drought tolerance, nutrient deficiency etc. on the other.
■ Characterization and cataloguing of citrus germplasms using microsatellite molecular markers.
■ Marker-aided-selection of citrus germplasm for useful traits e.g. less seeded, suitable for processing, tolerance to
drought, floods, salinity and diseases.
■ Rootstock improvement through protoplast fusion.
■ Anti-oxidant and neutraceutical profiling of citrus germplasm could be new objectives in citrus breeding programme.
■ Clonal selection for better yield and quality and ploidy manipulation for seedlessness.
■ Evaluation and barcoding of citrus germplasm.
■ Hybridization for scion and rootstock improvement.
■ An engineered rootstock capable of withstanding both biotic as well as abiotic stresses, besides fitting well into high
density planting will improve factor productivity of citrus orchards.
■ Identification of germplasm tolerant to water stress (drought and flood).
Breeding Approaches & Achievements
Selection of new plants
Hybrids, mutants, cybrids, somaclones, transformants etc.
Identification of desirable promising plants
Replicated field trials
Evaluation for horticultural characteristics
Identification of new cultivar
Multiplication & release of the cultivar to the growers
Selection of parents
Breeding procedure
Hybridization, Mutation, Somatic hybridization & other in vitro techniques, Genectic transformation
General outline of breeding methods
Breeding in USA
(University of California, University of Florida and Texas University)
Scion breeding
■ For Sweet oranges, cultivar which holds well late in the season is desired.
■ For Mandarins, important traits are seedlessness, easy peeling, good flavor, high rind color and low tendency to
alternate bearing.
■ For grapefruit, a deep pigmented with low naringin cultivar is breeding objective.
Stock breeding
■ Rootstocks resistant to problems like Phytophthora, citrus tristeza virus, citrus nematodes, citrus blight, high pH,
salinity.
Scientists
■ Kim D Bowman – USDA, ARS, Horticultural research aboratory, Florida.
■ JW Grosser, JL Chandler and FG Gmitter Jr – CREC, University of Florida, lake Alfred, USA.
■ Mikeal Roose, Timothy E Williams, Department of Botany and Plant Sciences, University of California, Riverside.
Breeding in China and Spain
■ China
The major breeding objectives are to develop seedless cultivar better than Ponkan mandarin w. r. t. fruit
size, variation in maturity.
■ Scientists
XX Deng: National Key laboratory of Crop genetic Improvement and citrus research Institute, Huazhong
agricultural university, Wuhan, Hubei.
■ Spain
To breed rootstocks resistant to citrus tristeza virus, Phytophthora, adapted to Spanish conditions especially
saline and calcareous soils and give good quality fruits.
■ Scientists
JB Forner, MA Forner, AA AClaide- Instituto Valenciano de investtigaciones Agrarias, Moncanda, Valencia,
Spain.
Breeding in India
Scion breeding:
■ For mandarins- Low juvenility, high yield, reddish orange color fruits without neck or collar, seedless
or with few seeds with proper sugar acid blend extended period of maturation, low bittering
substances in Kinnow.
■ In sweet orange- Fruits free from granulation, seedless or low seed content with TSS and acidity,
and juice content should not be less than 35 %.
■ In Acid lime- resistance to canker and scab, large fruit size with high juice content, seedless or low
seed content.
Stock breeding:
■ Rootstocks resistant to Phytophthora, citrus tristeza virus, exocortis, greening, salinity, drought,
water logging and good yield and quality of fruit in scion cultivars.
Introduction
Introduction
■ The mandarin variety ‘Santra’ is known to have been grown in India for many centuries. It was
introduced into the Central Provinces (now Maharashtra) by Ranghojee Bhonsal II from
Aurangabad in eighteenth century.
■ Sweet orange varieties including Washington Navel, Valencia introduced from America.
■ Grapefruits were introduced from California and Florida.
■ Lemons from China.
■ Malta from USA and Italy.
■ ‘Mosambi’ seems to have been introduced in Nagpur during the beginning of the 20th century.
■ The introduction of ‘Kinnow’ mandarin (King x Willow leaf) in 1947 showed great promise in
North India. It was introduced in South India in 1958 and Punjab in 1959 and has performed
extremely well in Punjab.
Citrus Cultivation in Punjab
Mandarins PAU Kinnow 1, Daisy (2013), W. Murcott (2013), Kinnow (1968), Local (1968).
Sweet oranges Valencia (1968), Mosambi (1962), Jaffa (1962), Blood Red (1962).
Grapefruit Star Ruby (2009), Red Blush (1995) Marsh Seedless (1968), Duncan (1968),
Foster (1968).
Lemon Punjab Baramasi Lemon (2008), Punjab Galgal (1994), PAU Baramasi Lemon-
1 (1990), Eureka (1968).
Lime (Nimboo) Kagzi (1968), Sweet Lime (Mitha), Local (1968).
Rootstock Rough Lemon, Carrizo, Cleopatra, Pectinifera.
* Source: Citrus cultivation by Rattanpal et al., 2017
Mosambi Jaffa
PineappleHamlin
Valencia Late
Vaninglia SanguignoFewtrell’s Early
Blood Red
Cambell ValenciaOlinda Valencia
Queen Frost Valencia
Star Ruby
Red Blush
Marsh Seedless
Duncan
Three exotic varieties introduced
viz. ‘Pummelo US -145’, ‘Cutter
Valencia’ and ‘Flame grapefruit’ by
Institute.
Two varieties developed through
indigenous material viz NRCC
Pummelo – 5 and NRCC Grapefruit-
6 have been released by Institute.
ICAR-CCRI
*Source: Information for ICAR-CCRI Website (November, 2016 to June, 2017)
Selection
Selection at IARI, New Delhi
■ 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 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 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.
■ Acid lime
■ Pusa Abhinav: It is a promising clonal selection and is moderately susceptible to citrus canker.
■ Pusa Udit: It is a clonal selection has round the year fruiting with two season harvesting will make this
selection highly suitable for commercial cultivation as well in kitchen garden.
Hybridization
Hybridization
■ The mating or crossing of two plants of dissimilar
genotype is known as hybridization.
■ In plants, crossing is done by placing pollen grains from
one genotype (male parent) onto the stigma of flowers
of other genotype (female parents).
■ The seeds as well as progeny resulting from hybridization
are known as hybrid or F₁. The progeny of F₁, obtained by
selfing of F₁ plants, & the subsequent generations are
termed as segregating generations.
■ Genus Citrus is its vast array of forms and cultivars has
great genetic diversity. Intergeneric hybridization is the
key process by which genetic diversity of citrus and its
relatives can be added to genetic diversity of citrus.
* Riccardo & Licciardello: 2014 (Nature)
Hybrids released by
University of California, Riverside
■ Gold Nugget
■ TDE 1 (Triploid hybrid)
■ TDE 2 (Shasta Gold)
■ TDE 3 (Tahoe Gold)
■ TDE 4 (Yosemite Gold)
■ USDA 88-2 (Lee × Nova)
* Source: Roose and Williams (2000); Nir Carmi 2014
■ USDA 88-3 (Robinson × Lee)
■ Valentine
■ Flamingo- Red Pomelo
■ Hanna
■ Einat
■ Aliza
Wilking
(Willow leaf × King)
Kincy
King × Dancy
Gold Nugget
Wilking × Kincy
Gold Nugget
■ (Wilking X Kincy)
■ Diploid hybrid released in 1999
■ Seedless
■ Late maturing hybrid (February - March)
■ On tree storage life is 4-6 months
* Source: Roose and Williams (2000)
TDE 1 (Triploid hybrid)
(Temple x Dancy) x Encore mandarin
(4x X 2x cross)
Temple
TDE 1 (Trioloid hybrid
EncoreDancy
* Source: Roose and Williams (2000)
TDE 2 (Shasta Gold)
• Triploid hybrid
• Late season maturity
• Seedless
TDE 3 (Tahoe Gold)
• Mid late season
• Very deep rind color
• 1-2 months on tree storage life
TDE 4 (Yosemite Gold)
• Seedless
• Late maturity (Feb. - March)
• 2-3 months on tree storage life.
* Source: Roose and Williams (2000)
USDA 88-3
• Diploid hybrid of Robinson x Lee
• Seedless in isolation
• Can cause seeds in other mandarins
• Early maturity (Nov.)
• Deep orange rind colour
• Poor fruit holding ability on the tree
Oden
• Cross of Orah and Shani
* Source: https://news.ifas.ufl.edu 2009
Flamingo- Red Pomelo
• Chandler pomelo x (Shamouti orange x Clementine)
• Ripens October to December
• Maintains its red color when the temperature drops
• Sweet (14% sugar)
• Seedless
• Presence of lycopene
Hanna
• Tahitian pomelo x Chandler pomelos
• Ripens from October to December
• Especially sweet taste (12.6% sugar)
• Seedless
* Source: Nir Carmi 2014
Sugar Belle
• University of Florida created 1st citrus variety
intended for commercial production.
• Clementine x Minneola
• “Many old-timers in citrus have said this is
the best-tasting citrus they’ve ever had,”
Gmitter
Valentine
• Siamese Sweet (Acidless pummelo) x (Dancy x
Ruby blood)
• Valentine’ combines large size and low acidity
from its pummelo parent
• It combines complex, floral taste from ‘Dancy’
& juicy red pulp from ‘Ruby’
* Source: https://news.ifas.ufl.edu 2009
Einnat
• Hudson x Pomelo
• Ripens: October to January
• Maintains its inner pink color
(lycopene) throughout all the season.
• Sugar (11.5%)
• Seedless
Aliza
• 'Chandler' pomelo x "Ora”
• Ripens during Nov. to February
• Sugar (16%)
• Seedless
• Dominates the global market
Citrus Intrageneric (interspecific) &
Intergeneric Hybrids
(Grapefruit)
(Mandarin)
(Orange)
Grapefruit Tangelo
Tangeloes Minneola, Orlando, Pearl, Sampson
Tangelo
Tangor
Intrageneric or Interspecific hybrids
Hybrid Percentage Examples
Tangor Mandarin × Sweet orange Eg. Temple, Monreal, Umatilla
Tangelo Mandarin × Grapefruit Eg. Sampson, Orlando, Minneola
Lemonime Lime × Lemon Eg. Perrine
Intergeneric hybrids
Citrange Sweet orange × Trifoliate orange Eg. Troyer, Carrrizo, Willits.
Citrangedin Calamondin × Willits citrange Eg. Glen
Limequat Lime × Kumquat Eg. Eustis, Lakeland
Citrangor Citrange × Sweet orange
Cicitrange Citrange × Trifoliate orange
Citrumelo Grapefruit × Trifoliate orange Eg. Swingle citrumelo
Citrandrin Mandarin × Trifoliate orange
Citremon Lemon × Trifoliate orange
Artificial hybrids
Citrus Hybrids for Ornamental Use
■ 04-K5 (Chinotto × ‘Buddha’s Hand’ Citron)
■ FH-2 (F. hindsii spp. Open Pollinated)
■ LM X CH-3 (‘Meyer’ Lemon × Chinotto Small Leaf
■ LM X CH-13 (‘Meyer’ Lemon × Chinotto Small Leaf
■ LM X LR-14 (‘Meyer’ Lemon × Red Lime)
■ ARCOBAL (‘Meyer’ lemon × ‘Doppio Sanguigno’ Orange)
■ 04-F5 (‘Oval’ Kumquat × ‘Buddha’s hand’)
* Source: Recupero et al., 2015
04- K5 FH-2 LM × CH-3
LM X CH-13 LM X LR-14 ARCOBAL
Rootstock hybrid
(Release year)
Parentage Important traits
US-1516 (2015) African Pummelo (C. grandis) × Flying Dragon (P.
trifoliata)
Tolerant to HLB
US-1283 (2014) Ninkat mandarin x P. trifoliata Tolerant to HLB
US-897 (2011) Citrus reticulata x P. trifoliata Tolerant to HLB
US- 942 (2010) Sunki mandarin (C. reticulata) × Flying Dragon (P.
trifoliata)
Resistant to nematode, phytophthora,
induction of good quality fruits, easy to
propagate
US 812 (2006) Sunki mandarin (C. reticulata) × Flying Dragon (P.
trifoliata)
Highly productive, resistance to citrus
blight
US-815 (2001) Sunki mandarin (C. reticulata) × Benecke
trifoliate orange
Resistance to phytophthora, citrus
nematode, Tristeza and citrus blight.
US 8-52 (2000) Changsha Mandarin × Trifoliate orange Cold hardy, resistance to phytophthora,
citrus nematode, Tristeza and citrus blight.
Rootstock developed in USA
* Source: Forner 2010
Rootstock developed in India
Country Rootstock
hybrid
Parentage Important traits
IIHR, Bangalore CRH-12 Rangpur lime x Trifoliate orange Highly resistant to phytophthora and
citrus nematode and moderately
tolerant to salinity and tolerant to
drought
CRH-57 Rough lemon x Trifoliate orange Highly resistant to phytophthora,
citrus nematode and tolerant to
salinity and drought
CRH 47 Cleopatra Mandarin xTrifoliate
orange
Moderately tolerant to salinity
CRC, Nagpur NRCC 1 to NRCC 10
Mutation Breeding
Mutation Breeding
■ Mutation can be defined as the changing of the structure of a gene,
resulting in a variant form that may be transmitted to subsequent
generations, caused by the alteration of single base units in DNA, or
the deletion, insertion, or rearrangement of larger sections of genes
or chromosomes.
■ Two types of mutation
■ Spontaneous mutation: It occurs naturally i.e. without human intervention
and at a low rate (one in a population of ten lacs). Eg. Natural budsports.
■ Induced mutation: It can be induced artificially by a treatment with certain
physical or chemical mutagens.
■ The first important cultivar from a mutation breeding programme was
the ‘Star Ruby grapefruit that resulted from the irradiation of seeds
of Hudson grapefruit. Star Ruby is having very deep flesh colour and
is commercially seedless (Hensz, 1977).
■ Liu et al., (2007) reported a spontaneous bud mutation in sweet
orange (Citrus sinensis L. Osbeck) „Hong Anliu‟, which results in fruits
with lycopene accumulation, low citric acid, and high sucrose was
reported in a previous study.
Crop Parent variety Mutant cultivar Nature of mutation
Navel orange Bahia Bahianinha Limb sport
Washington Navelina, Navelate Marrs,
Autumn Gold, Powell Sum
mer, Weller Gold
Limb sport
Mandarin Owari Clausellina Bud sport
Pongan Pongan 86-1 -
Grapefruit Walter (White fleshed) Foster (Pink fleshed) Limb sport
Foster (Pink fleshed) Hodoun (Red fleshed) Bud sport
Thompson (light pink flesh) Red Blush (deep red flesh) Limb sport
Henderson Flame Bud sport
Pummelo Local cultivar Wuheputaoyou Bud sport
Spontaneous mutation in citrus
* Source: Ray, 2000
Star Ruby Red Blush Foster
Fruit Crop Parent
Veriety
Mutant Improved Trait Mutagen
used
Reference
Citrus deliciosa Comun EC31/1 Low seed content and
cold tolerant
Gamma rays Anderson
2000
Citrus reticulata Pongan Fuyu 28 Resistant to canker &
drought
Gamma rays Huang et
al. 2000
Citrus reticulata Daisy,
Encore,
Fairchild,
Fremont,
Kinnow,
Murcott
and Nova
Daisy IR,
Encore IR 1,
Fairchild IR 2,
Fremont IR 2,
Kinnow IR
1,Tango and
Nova IR 10
Seedless or less seed
content, Tango released
as a variety- late
maturing, fruit
moderately
Gamma rays Williams
2008
Induced mutation in citrus
Fruit crop Parent variety Mutant Improved trait Mutagen used References
Mandarin Shatangju Wuzishat
angju
Fruits are small
& seedless
Gamma rays ZiXing et al., 2006
Mandarin Qianyang
Seedless
Qianyang
Seedless
Seedlessness Gamma rays Bing Hong 2003
Penggan
mandarin 1
Penggan 2 - - -
Tangelo Tangelo
cultivar
Jianyang
Tangelo
Seedlesness &
171 g weight
Gamma rays Liu-Tao and
Wu-Rui Dong 2007
Kumquat Meiwa
kumquat
Yubeni Large fruits &
low seeded
Colchicine
treatment
Yasuda et al., 2008
Contd….
Studies on mutation breeding in mandarin cv. Kinnow
• Rattanpal et al. 2019 studied variability in
Kinnow (Citrus nobilis Lour × C. deliciosa Tenora)
using gamma radiation.
• Four hundred buds of Kinnow were irradiated at
30 Gy and budded on rough lemon rootstock in
February 2007.
• The resulting 188 MV1 plants were planted in
the field in October 2009 and observations in
the present trial were recorded from 2015 to
2018.
• Among the 188 MV1 plants, eleven Kinnow
mutants (seven solid mutant trees and four
mutant branches) having average seed number
less than eight were identified and were
compared in detail with parent variety Kinnow.
Gray (Gy): Unit of absorbed dose of energy per unit mass of
tissue. 1 gray = 1 Joule/kilogram and also equals 100 rad.
One of the low seeded mutant was released as a new variety named
‘PAU Kinnow-1’ for cultivation in Punjab.
Polyploidy Breeding
Polyploidy Breeding
■ Polyploids are organisms with multiple sets of
chromosomes in excess of the diploid number
(Acquaah, 2007) & implications of polyploidy in plant
breeding is known as polyploidy breeding.
■ The Hongkong wild kumquats, Fortunella hindsii may
have been the first reported tetraploid.
■ The seedless lime (C. latifolia) a triploid. Triploids have
favorable characteristics and yield well but they are
sterile.
■ Polyploidy manipulation by crossing of tetraploids with
diploids yielded some valuable triploid varieties like
‘Oroblanco’ and ‘Melogold’.
Triploid frequency of sexual hybridization and
pollen and ovary development in mandarins
■ Costa et al. (2019) aimed to ascertain the triploid frequency of sexual hybridization and the
pollen and ovary development in Citrus.
■ The ‘Fortune’ mandarin (Citrus clementina Hort. ex Tan. × C. tangerina Hort. ex Tan.) accession
was selected as female parent. The male parents used were the ‘Cravo’ (C. reticulata Blanco)
and ‘Dancy’ (C. reticulata) mandarins.
■ The pollinations were always manually performed in the morning, and the pollinated flowers
were identified for control. During the flowering period, flower samples at different stages of
development of the ‘Fortune’ variety were collected for analysis.
■ Microspore development occurred regularly in the 4–5 mm (length) phase of the floral buds,
and a bicellular micro-gametophyte was formed in the floral buds with 5–6 mm in length.
■ The formation of the female gametophyte occurred later, in flower buds with approximately 10
mm in length.
*Source : Costa et al., 2019
The hybridizations resulted in 19 triploid plants: five when the ‘Cravo’ mandarin was used as the male
parent and 14 when ‘Dancy’ mandarin was used as the male parent, evidencing the importance of the
male genitor also in this process.
Extensive citrus triploid hybrid production by 2x X 4x sexual
hybridizations and parent-effect on the length of the juvenile phase
a Monoembryonic undeveloped seed.
b Polyembryonic undeveloped seed.
c Germination of embryo from
monoembryonic undeveloped seed.
d Germination of multiple embryos from
polyembryonic undeveloped seed
Aleza et al. 2012 reported effective methodology to recover citrus triploid plants
from 2x X 4x sexual hybridizations and the parental effect on the length of the
juvenile phase.
The juvenile phase length of the triploid hybrids obtained with ‘Fortune’
mandarin as female parent and tetraploid ‘Orlando’ tangelo as male parent was
shorter than the juvenile phase obtained with a clementine as female parent and
tetraploids of ‘Nova’, ‘W. Leaf’ and ‘Pineapple’ male parents. *Source:https://www.researchgate
.net/publication/286730366
Alkantara
‘Oroval’ clementine
× ‘Tarocco’ orange 4x
Mandared
Nules’ clementine ×
‘Tarocco’ orange 4x
Early Sicily
‘Oroval clementine ×
‘Tarocco’ orange 4x
Sweet Sicily
‘Comune’clementine
× ‘Tarocco’ orange 4x
* Source: Giuseppe Russo et al., 2015; Recupero et al., 2005.
Triploids
* Source: Giuseppe Russo et al., 2015; Cuenca et al., 2010.
Garbi
‘Fortune’ mandarin x
diploid tangor ‘Murcott’ (C.
reticulata x C. sinensis)
Safor
Diploid ‘Fortune’ mandarin
and diploid ‘Kara’mandarin
(C. unshiu x C. nobilis)
Albir
‘Fortune’ and Kara’mandarin
(C. unshiu × C. nobilis)
Dawn
Diploid Acidless pummelo
x 4n ‘Ruby’ blood
Triploids
Citrus Biotechnology
In vitro Plant Regeneration in Cleopatra (Citrus reshni Hort. ex Tan.)
by Direct Organogenesis
Objectives
To develop in vitro regeneration system for citrus
rootstock Cleopatra.
Direct induction of adventitious shoot buds and
plant regeneration via culture of different explants
from in vitro grown seedlings in Cleopatra.
(A) Initiation of shoot formation.
(B) Emergence of shoot buds from the longitudinal cut of
epicotyl segment.
(C) Shoot development from epicotyl segment on MT +
BAP (2 mg/l) + NAA (0.2 mg/l).
(D) High frequency of regeneration of multiple shoots
from epicotyl segment.
(E) Rooted plants.
(F) In vitro hardened plantlet.
* Source: Devi and Rattanpal 2018
Valenfresh (N7-3)
• Somaclone (protoclone)
of Valencia sweet orange
• Late-maturing
Valquarious
• Early maturing Jan-Feb
• Selection with typical Valencia
quality
OLL 8
• Low seeded
Somaclonal variations
■ Somaclonal variation is defined as genetic variation observed among progeny of plants
regenerated from somatic cells cultured in vitro.
■ It can be used in the development of crops with novel traits.
Production of sweet orange somaclones tolerant to
citrus canker disease by in vitro mutagenesis with EMS
■ Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease
worldwide.
■ Ge et al. (2015) conducted study, aimed at the establishment of an efficient protocol for in vitro
ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange
somaclones tolerant to citrus canker disease.
■ The results suggested that the established protocol for somaclones variation by treating sweet
orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the
pathogen crude extract was effective.
■ The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar
selection procedures.
*Source: https://www.researchgate.net/publication/281608821
Micro grafting
■ Micro grafting is an in vitro grafting
technique which involves the placement
of a meristem or shoot tip explant onto a
decapitated rootstock that has been
grown aseptically from seed or micro
propagated cultures (Hussian et al.,
2014).
■ Shoot tips having size (0.1-0.2 mm) is
very efficient for elimination of all citrus
graft-transmissible pathogens from local
or imported varieties.
■ STG was used to regenerate the
‘Clemenules’ haploid plant that has been
used by the International Citrus Genome
Consortium to sequence the whole citrus
genome (Jaurez et al., 2015).
Standard STG in vitro technique description.
a-c. Rootstock preparation.
d-f. Scion preparation.
g-m. Grafting and culture in vitro of grafted plants.
n. Plant recovered by STG.
* Source : Jaurez et al., 2015
Citrus shoot-tip grafting in vitro
Applications of Citrus STG in vitro
■ The main application of STG is to control graft transmissible pathogens.
■ According to the studies conducted by Navarro et al. (1988) and Navarro (1992) the elimination of
viruses in citrus by STG is as follows:
■ Regeneration of somatic hybrids from embryos difficult to germinate.
■ Regeneration of plants from irradiated shoots to produce seedless varieties.
■ Regeneration of plants from haploid embryos that are very difficult to germinate.
■ Production of stable tetraploid plants of non-apomictic genotypes, which are very useful for triploid
breeding.
■ Regeneration of transgenic plants from shoots that are very difficult to root in vitro. STG has become a
routine application in citrus genetic transformation.
Name of the viruses Ease of elimination Percentage of germination
Exocortis infection, Tristeza Very easy 100
Tristeza Easy 80
Psorosis Difficult <25
* Source : Jaurez et al., 2015
In vitro shoot tip grafting
(a) Vertical incision on rootstock
(b) Scion insertion at vertical
incision (cleft grafting)
(c) Inverted T incision on
rootstock
(d) Scion insertion at inverted T
cut
In vitro micrografting using three diverse indigenous rootstocks for the
production of Citrus tristeza virus-free plants of Khasi mandarin
• Singh et al. 2018 reported the prospective applicability of three indigenous rootstocks belonging to
different species viz. Nemutenga, Tayum and Tasi cultivars of north eastern Himalayan region for producing
Citrus tristeza virus (CTV; a viral species of the Closterovirus genus) free quality planting materials of Khasi
mandarin (Citrus reticulata Blanco) through micrografting or shoot-tip grafting (STG).
• Viral assessment with enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase
chain reaction (RT-PCR) revealed negative results for all plants from 0.3 to 0.5 mm scion while 0.7-mm
scion-raised micrografts showed 60% negative with RT-PCR for CTV.
*Source: https://doi.org/10.1007/s11627-018-9946-6
In vitro shoot tip grafting
(e) In vitro graft culture after kinetin pre-treatment
(f) Effect of kinetin (1.0 mg L−1) pre-treatment for Nemutenga rootstock with scion showing
new expanded leaves of micrograft for inverted T and cleft grafting
(g) Effect of kinetin (1.0 mg L−1) pre-treatment for Tayum rootstock with scion showing new
expanded leaves of micrograft for inverted T and cleft grafting
(h) Effect of kinetin (1.0 mg L−1) pre-treatment for Tasi rootstock with scion showing new
expanded leaves of micrograft for inverted T and cleft grafting
Conti..
In vitro shoot tip grafting
(i), (j) Proliferation of micrografts in MS supplemented with 5% sucrose along with 0.5
mg L−1 BAP and 0.1 mg L−1 IAA
(k) In vitro root induction in MS supplemented with 1.0 mg L−1 NAA and 1.0mg L−1 IBA;
(l) Pre-hardened micrografted plants
(m) micrografted plantlet after hardening
Conti..
Somatic hybridization
■ Somatic embryogenesis is a powerful biotechnological
tool for the mass production of economically important
cultivars.
■ It can be defined as the process by which somatic cells or
tissues develop into differential embryo and each fully
developed embryo is capable of developing into a new
plantlet (Iqbal 2015).
Somatic hybrids Protoplast material Method
of fusion
Objective Reference
Mandarin + pummelo
hybrids
Suspension cultures PEG Sting nematode
resistance
Grosser et al.,
2007
Hamlin sweet orange +
Montenegrina mandarin
Suspension cultures and
leaf
- Tolerance to citrus
canker and xylella
fasidiosa
Pavan et al.,
2007
Somatic embryogenesis through in vitro anther
culture of Citrus sinensis L. Osbeck ‘Moro’
■ In many crops, anther culture is the most used method to induce gametic embryogenesis,
aimed to regenerate homozygous plants. However, also somatic embryogenesis can be
obtained by this method, when somatic tissue is involved in the regeneration process.
■ Lacuzzi et al. 2019 collected anthers of Citrus sinensis L. Osbeck 'Moro', at the vacuolate
stage, and after a chilling (4°C) pre-treatment of 7 days, were placed on the same medium,
evaluating different temperature stresses applied after placing them in culture.
■ In this study, the effect of three thermal treatments, compared with direct in vitro culture of
the anthers (after the pre-treatment of the floral buds at 4°C for 7 days), was observed in
cultivar 'Moro'.
■ Embryo regeneration has been obtained and their characterization, through ploidy and
molecular analyses, showed that they were heterozygous tetraploids.
*Source: https://www.researchgate.net/publication/330445894_
Anther culture
■ Anther culture is a technique by which the developing
anthers at a precise and critical stage are excised aseptically
from unopened flower bud and are cultured on a nutrient
medium where the microspores within the cultured anther
develop into callus tissue or embryoids that give rise to
haploid plantlets either through organogenesis or
embryogenesis.
■ Anther culture is very important to plant breeding, due to
their numerous applications in mutation research, selection,
genome sequencing, genetic analysis, and transformation
(Lacuzzi et al. 2019).
*Source: https://www.researchgate.net/publication/262572007
In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck)
genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid
* Source: https://www.researchgate.net/publication/262572007
Anther culture of sweet orange (Citrus sinensis Osbeck.) genotypes
a Bud flower size for anther culture
b Transverse section of a cultured anther showing swollen tissue, the
parietal layers (pl) and microspores (mi) during in vitro culture;
c Non-responsive (nr), swollen (sw), cream-colored callus (cc) and white-
greenish callus (gc); Embryogenic callus induction
d Proliferation
e & f From anther culture of C. clementine × C. sinensis ‘Hamlin’
Cardoso (2014) reported that anther cultures of the hybrid C.
clementina × C. sinensis produced tri-haploid (3n) embryogenic
calli and the embryos obtained were homozygous when analyzed
by molecular markers (sample sequence repeats), confirming the
more responsive characteristic of clementine to microspore
embryogenesis through anther culture.
Embryo rescue
The difficulty of recovering triploid hybrids
from interploid hybridization or diploid ×
diploid hybridization (due to an unbalanced
ploidy ratio between embryo and endosperm
as well as polyembryony) should be
overcome by in vitro embryo rescue.
In vitro culture of excised fully developed
embryos was described 40 years ago to
obtain zygotic plantlets from polyembryonic
cultivars.
*Source: Maheshwari and Ranga Swamy, 1958
Recovery of citrus triploid hybrids by embryo rescue and
flow cytometry from 2x X 2x sexual hybridisation and its
application to extensive breeding programs.
• Citrus triploid plants can be recovered by 2x X 2x sexual hybridisations by in vitro embryo
rescue and ploidy level determination by means of flow cytometry.
• The influence of parents and environmental conditions on obtaining triploid hybrids has
been analysed (Aleza et al. 2010).
• The strongest effect was associated with the genotype of the female parent while a strong
interaction was found between the male parent genotype and environmental conditions.
The effect of the female parent genotype on the length of the juvenile phase was also
demonstrated by observing a large number of progenies over the last 10 years.
• The methodology helped to obtain over 4,000 triploid hybrids & these triploid hybrids have
been analysed with simple sequence repeats markers to differentiate all the new triploid
varieties and their parents.
* Source: https://www.ncbi.nlm.nih.gov/pubmed/20607244
Production of three new grapefruit cybrids with
potential for improved citrus canker resistance
■ Omar et al. 2017 by using a cybridization approach, several putative cybrids were created by
protoplast fusion of embryogenic suspension culture-derived protoplasts of canker resistant
‘Meiwa’ kumquat (Citrus japonica Thunb), with mesophyll-derived protoplasts of three grapefruit
(Citrus paradisi Macfad.) cultivars ‘Marsh,’ ‘Flame,’ and ‘N11-11’ somaclone of ‘Ruby Red.’
■ In an effort to generate new grapefruit cultivars with enhanced canker resistance, putative cybrid
grapefruit plants morphologically equivalent to standard grapefruit from all three combinations
were produced.
■ EST-SSR marker analysis confirmed that the nuclear genome in all the generated cybrids came
from the grapefruit parent.
■ All the cybrid clones have been propagated and are undergoing extensive canker assays to identify
any clones that have improved canker tolerance/resistance.
■ These cybrid populations provide a valuable tool for investigating the contribution of cytoplasmic
organelles to plant disease resistance.
*Source: https://link.springer.com/article/10.1007/s11627-017-9816-7
Cryopreservation of Citrus anthers in the
National Crop Gene bank of China
■ Genebank conservation of pollen is valuable because it makes genetic resources immediately
available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen
can be easily transported and used to develop new varieties with pathogen resistance and
desirable quality and yield traits.
■ Zhang et al. 2017 conducted study to develop and improve air-desiccation cryopreservation
protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study,
warming, rehydration, and in vitro germination conditions were optimized to achieve high levels
of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure.
■ The highest viability of 93% was obtained for C. cavaleriei. The methods identified in the current
study could be used to cryopreserve C. cavaleriei and C. maxima anthers.
*Source: https://link.springer.com/article/10.1007/s11627-017-9848-z
Mapping &
Marker-assisted Selection
Linkage maps
■ Genetic linkage maps are a statistical description of the organization of DNA segments into
chromosomes and the order and recombination frequency of DNA segments within chromosomes.
■ Maps can include both DNA markers and trait genes that are detected by affecting the phenotype
of an individual.
■ A good map should include one linkage group that corresponds to each chromosome of the
genome (nine in Citrus and closely related genera).
■ Each linkage group will be comprised of a linearly ordered set of markers and/or trait genes, with
distances between adjacent markers determined by the proportion of gametes that have a
crossover event between the markers.
■ For citrus breeders, the objective of mapping is to identify markers that can be used in marker-
assisted selection (MAS).
■ MAS involves identification of DNA markers that are near a variable trait gene, and selection of
progeny carrying markers that predict which progeny will carry the desired trait gene allele.
*Source: Citrus by I Khan
Characteristics of some common types of DNA markers
■ AFLP Amplified fragment length polymorphism- PCR based, gel analysis, multilocus, sequence anonymous.
■ CAPS Cleaved amplified polymorphic sequence – PCR followed by restriction enzyme digestion, gel
analysis, single locus per gel, sequence known, can target genes.
■ RAPD Random amplified polymorphic DNA – PCR based, gel analysis, single to a few loci per gel, sequence
anonymous.
■ RFLP Restriction fragment length polymorphism – Restriction enzyme and gel based, single to a few loci
per gel, can target genes.
■ ISSR Inter-simple sequence repeat – PCR based, gel analysis, multilocus, sequence anonymous.
■ SNP Single nucleotide polymorphism – PCR based, gel, plate or chip analysis; single locus, but multiplexing
possible, sequence known, can target genes.
■ STS Sequence-tagged site – PCR based, gel analysis, single locus, sequence known.
■ SSR Simple sequence repeat – PCR based, gel analysis, single locus, sequence known, can target some
genes.
■ The method of detection of the DNA sequence (e.g. PCR) is listed, the method for distinguishing alleles (e.g. gel
analysis), whether the method generally detects one or several to many marker loci in a single analysis, whether the
DNA sequences detected are known or anonymous, and whether the method can target specific genes.
Relationship between DNA sequence differences between two alleles and
banding patterns observed for five types of gel-based marker systems
DNA Marker types
Examples of marker variation for five types of DNA marker systems
• The RFLP and RAPD markers were separated on agarose gels.
• The SSR and AFLP patterns were analysed on an automated DNA analyser.
• The ISSRs were analysed on a silver-stained polyacrylamide gel and show
patterns in seedlings from different source trees, not a single segregating
population.
*Source: Citrus by I Khan
Summery of linkage maps of citrus
*Source: Modified from Roose et al. 2000
eQTL analysis
■ A SNP is an eQTL if it is associated with an allelic difference in mRNA levels of a gene.
■ For an eQTL gene under investigation, a corresponding eQTL SNP occurs on the top of a peak in a
regional association plot, i.e. has a low association p-value compared to other SNPs nearby.
The goal of eQTL is to identify genomic locations where genotype significantly affects gene expression.
QTLs and eQTLs mapping related to citrandarins’
resistance to citrus gummosis disease
Map, linkage group (LG), flanking markers, position of the QTL in
centiMorgans (cM position), LOD score, proportion of
phenotypic variation (R2) explained by QTL in % and additive
effect by the QTLs mapping
Distribution of the number of eQTLs detected
for each candidate gene in relation to the
Poncirus trifoliate & Citrus sunki maps
eQTL are genomic loci that explain all or a fraction of variation in expression levels of mRNAs.
* Source: https://www.researchgate.net/publication/326162981
Lima et al. (2018) conducted first study to use eQTLs mapping in the Phytophthora-citrus interaction & concluded that
the resistance of some citrandarins to the infection by P. parasitica is due to a favorable combination of QTLs and
eQTLs transmitted by both parents.
 Heatmap of the gene expression profile by
clustering analyses between 19 evaluated
target genes with 55 selected genotypes (51
hybrids, their two parents, Rangpur lime and
Swingle citrumelo), allowed the grouping of
genes and related genotypes, using the
Pearson correlation as metric distance to
obtain the best intra and intervariable
grouping possible.
 The genotypes were separated into five
secondary clusters distributed in two main
clusters, while the genes in three secondary
clusters arranged in two main clusters.
 The color scale representing the log2Fold
change values showed that the gene
expression data ranged from − 8.29 to 5.53.
Conti..
Gene tagging
Gene tagging is one of the most useful methods to identify and
isolate specific genetic loci of an organism.
In vitro selection of resistant/tolerant mutants lines of Citrus jambhiri
Lush. using crude culture filtrate of Phytophthora parasitica and their
randomly amplified polymorphic DNA analysis
■ Pati et al. (2017) reported in vitro -induced mutagenesis and selection of Phytophthora tolerant
lines of Citrus jambhiri and their regeneration.
■ In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in
comparison with the control plant, which confirms the presence of variations at genetic level.
* Source: https://www.researchgate.net/publication/319407543
Identification and Parentage Analysis of Citrus
Cultivars Developed in Japan by CAPS Markers
■ Nonaka et al. (2016) developed a method of cultivar identification based on cleaved amplified
polymorphic sequence (CAPS) markers, to protect the rights of breeders of the major citrus cultivars
developed under breeding programs by the national institute of Japan and to evaluate identity and
parentage of cultivars.
■ They selected 19 CAPS markers that had a single-locus origin and moderate polymorphism, and used
them to construct genotyping data for 59 citrus cultivars (including American accessions), local
varieties, and selections. Of the 19 CAPS markers, 8 were sufficient to discriminate among all accessions
except ‘Mato’ buntan (Citrus grandis Osbeck) and ‘Hirado’ buntan (Citrus grandis Osbeck).
■ Among the 33 Japanese cultivars, the parentage of 30 agreed with that reported, but ‘Setoka’,
‘Southern Red’, and ‘Reikou’ had discrepancies at one or more loci.
■ Using 15 to 18 CAPS markers to validate the putative parentage revealed that the seed parent of
‘Setoka’ was ‘KyEn No. 4’, not ‘Tsunonozomi’, and the pollen parent of ‘Southern Red’ was ‘Osceola’, not
ponkan (C. reticulate Blanco). The seed parent of ‘Reikou’ remains unknown.
*Source: https://www.jstage.jst.go.jp/article/hortj/86/2/86_OKD-026/_html/-char/en
Genetic diversity and DNA fingerprinting of indigenous and
exotic mandarin genotypes in India using SSR markers
■ Singh et al. (2016) determined genetic variability and fingerprint profiles of 19 indigenous and exotic mandarin
genotypes introduced from different parts of India and USA using 60 SSR markers.
■ Of the 57 SSR markers amplified, a total of 96 alleles were detected by 39 polymorphic SSR loci and maximum 5
alleles were amplified with an average of 2.46 alleles per primer pair.
■ The CAT01 was the highly informative marker as it revealed maximum number of alleles (5), PIC value (0.75) and
genetic diversity (0.79).
■ The higher average expected heterozygosity (35.6%) with in a mandarin group as compared to the average
observed heterozygosity (27.2%) may be explained by selfing, which reduced the proportion of heterozygotes.
■ The genotypes were classified in three clusters i.e. cluster- I, cluster- -II and cluster -III. All the indigenous genotypes
(selections) were grouped in cluster -I and it had maximum genetic similarity coefficient. However, the exotic
genotypes (hybrids) were grouped in cluster- II and cluster- II. Clustering was according to the breeding history of
genotypes but independent to their geographic origin. The low observed heterozygosity frequency, PIC value, and
number of alleles explained the narrow genetic base in the present set of mandarin genotypes.
*Source: https://www.researchgate.net/publication/317717144
Dendrogram illustrating genetic relationship
among 19 mandarin genotypes generated by
UPGMA tree analysis.
The cluster I was sub-divided into two sub cluster IA
with eleven genotypes (N-38, Khasi, Clone 11, N-34,
N-43,N-4, N-28,CRS-4,N-51,Nagpur Seedless and
Nagpur) and IB with three genotypes (Coorg,
Darjiling, and Mudhkher) respectively.
In cluster II, three genotypes (Nova, Kinnow and
Fremont) were clustered and cluster III comprised
of two genotype (W. Murcutt and Daisy).
Genetic Diversity and Population Structure Analysis of Citrus
Germplasm with Single Nucleotide Polymorphism Markers
■ Yu et al. 2017 conducted research trial to determine their SNP fingerprints and to assess genetic
diversity, population structure, and phylogenetic relationships, and thereby to test the efficiency of
using the single genotype-derived SNP chip with relatively low cost for these analyses, on a collection
of 80 citrus accessions.
■ A model-based clustering program detected five basic groups and revealed that C. maxima
introgressions varied among mandarin cultivars and segregated in mandarin F1 progeny.
■ In addition, reciprocal differences in C. maxima contributions were observed among citranges and
may be caused by the influence of cytoplasmic DNA and its effect on selection of cultivars.
■ The relatively inexpensive SNP array used in this study generated informative genotyping data and
led to good consensus and correlations with previously published observations based on whole
genome sequencing (WGS) data.
■ The genotyping data and the phylogenetic results may facilitate further exploitation of interesting
genotypes in the collection and additional understanding of phylogenetic relationships in citrus.
*Source: https://www.researchgate.net/publication/329333504
Dendrogram of 80 Citrus and
Citrus relatives based on identity
by state (IBS) calculated from the
data of 1366 genome-wide single
nucleotide polymorphism markers.
IBS is the allelic similarity between
two individuals at the given loci
regardless of their common
ancestry, via computing the
probability of two individuals
sharing the same copy of an allele.
The cluster analysis is performed
using a matrix of IBS pairwise
distances, and groups are
determined by a permutation test.
Genetic Transformation
Genetic
Transformation
■ Genetic transformation involves the
integration of gene into genome by
means other than fusion of
gametes or somatic cells. The
foreign gene (termed the
"transgene") is incorporated into
the host plant genome and stably
inherited through future
generations.
■ This plant transformation approach
is being used to generate plant
processing trails, unachievable by
conventional plant breeding,
especially in case where there is no
source of the desired trait in the
gene pool.
The Genetic Transformation of Sweet Orange (Citrus sinensis L. Osbeck)
for Enhanced Resistance to Citrus Canker: Methods and Protocols
■ Developing disease resistance is one of the most important components of any plant breeding
program.
■ Citrus traditional breeding methods (bud sport selection, crossbreeding, and other breeding
channels) are a laborious task and often hampered by long juvenility, a high degree of
heterozygosity, polyembryony, self-incompatibility, and abortion of reproductive organs.
■ An interesting alternative to the classical breeding approach is the use of genetic
transformation, which provides the means for adding a single agronomic trait to a plant without
otherwise altering its phenotype.
■ Sendin and Filippone (2019) carried out Agrobacterium tumefaciens-mediated transformation
with numerous hybrids and citrus species & introduced the Bs2 gene in Citrus, as well as to
increase citrus canker resistance in transgenic Bs2 gene-expressing lines.
*Source: https://www.researchgate.net/publication/328874799
Efficient genetic transformation of sour orange, Citrus aurantium L.
using Agrobacterium tumefaciens containing the coat protein
gene of Citrus tristeza virus
■ Ghaderi et al. 2018 conducted research, in which hypocotyl, and epicotyls parts of C. aurantium
plant were inoculated with A. tumefaciens strain EHA- 105.
■ Then, explants were transferred into the regeneration medium and shoot growth containing
various concentrations of kinetin and IAA growth regulators. In order to detect transgenic shoots,
herbicide Basta (ammonium glufosinate) was added at a rate of 1 mg/L to the regeneration
medium. Production buds happened in explants which Marker BAR gene resistant to Basta
herbicides was received. After PCR, to approve the final transgenic plants, transformation
efficiency was evaluated by 9.16%.
*Source: https://doi.org/10.1016/j.plgene.2018.02.00
Regeneration of explants treated with hormones showed a
significant difference at 1% compared with control explants. The
Maximum regeneration was showed in 0.5 mg/L kinetin
treatment. These results indicated that the combination
between kinetin and IAA could be effective in increasing
regeneration. Regeneration of transgenic explants
Novel Plastid-Nuclear Genome Combinations Enhance
Resistance to Citrus Canker in Cybrid Grapefruit
■ Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram-
negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been
identified.
■ Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from
different species, was used to create cybrids between citrus canker resistant ‘Meiwa’ kumquat (Fortunella
crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars.
■ Murata et al. 2019 reported that, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat
chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were
generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts
had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the
grapefruit controls.
■ The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and ‘Marsh’
grapefruit had different gene expression profiles.
■ These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They
also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the
question of whether other alien chloroplast genotypes would condition similar results.
*Source: https://www.researchgate.net/publication/329541592
Gene expression overview of
‘Marsh’ grapefruit, M-102
cybrid (Cybrid-G), M-9 cybrid
(Cybrid-K) and ‘Meiwa’ kumquat
in response to Xanthomonas
citri subsp. citri infection. Green
arrows indicate upregulation
and orange arrows indicate
downregulation.
Conti…
Enhanced resistance to citrus canker in transgenic
mandarin expressing Xa21 from rice
■ Genetic engineering approaches offer an alternative method to the conventional
breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown
pollen parent) is one of the most commercially important cultivars grown in many
regions around the world.
■ Omar et al. 2018 made transformation of ‘W. Murcott’ mandarin was achieved by
direct DNA uptake using a protoplast transformation system. DNA construct (pAO3),
encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas
resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin.
■ Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast
transformation from embryogenic callus with the Xa21 gene from rice are being
evaluated under field conditions to validate the level of resistance.
*Source: https://www.researchgate.net/publication/323180663
Sweet orange genetic transformation with the attacin A gene
under the control of phloem-specific promoters and
inoculation with Candidatus liberibacter asiaticus
■ An alternative approach to control Huanglongbing disease (Citrus degreening: Causal organisms is
Candidatus liberibacter asiaticus) in Citrus is the development of transgenic plants expressing genes
that may influence pathogen development.
■ Tavano et al. 2018 reported production of sweet orange transgenic plants bearing gene constructs
containing the antimicrobial gene attacin A (attA) under the control of phloem-specific promoters.
■ The non-transgenic inoculated plants showed a significant reduction in shoot development comparing
to transgenic inoculated plants indicating that the expression of attA gene may influence the plant
tolerance to HLB disease.
* Source : https://www.researchgate.net/publication/327002860
Gene Editing
CRISPR-mediated technologies suitable for breeding
non-transgenic HLB resistance citrus
*Source: Gmitter et al. 2015
CRISPR, or Clustered
Regularly Interspaced
Short Palindromic
Repeats, is an integral
part of a bacterial
defense system. It is
also the basis of the
CRISPR-Cas9 system
that uses site-directed
nucleases to target
and modify DNA with
great accuracy.
Approach to create Non-transgenic
HLB Resistance Citrus Cultivars
Albino mutants of
citrus with CRISPR
Genome Sequencing
Complete Cholorplast Genome Sequence of Omani Lime (Citrus
aurantifolia) & Comparative Analysis within the Rosids
* Source: Huei-Jiun Su et al. 2014
Resistance to Abiotic Stress
Resistance to Abiotic Stress
Citrus
genotypes
Vector/method Gene Result/remarks References
Rough lemon A. tumefaciens HAL2 Salt tolerance Ali et al.,2012
Swingle citrumelo A. tumefaciens P5CS F129A Drought resistant Campos et al., 2011
Carrizo citrange A. tumefaciens P5CS Drought resistant Molinari et al., 2004
Carrizo citrange A. tumefaciens HAL 2 Salt tolerance Cervera et al., 2000
Disease Resistance
Disease Resistance
Citrus genotype Gene Remarks Reference
Citrus jambhiri Beta-1-3-
glucanase
Phytophtora Sandhu et al., 2016
Hamlin SABP2 Citrus canker Attílio et al., 2014
‘Anliucheng’ sweet
orange
Xa21 Citrus canker Ding et al., 2013
Hamlin, Natal, Pera and
Valencia
Xa21 Citrus canker Mendes et al., 2010
Duncan and Hamlin NPR1 Citrus canker Zhang et al., 2010
Hamlin Rice Xa21 Citrus canker Ahmad 2006
Hamlin Attacin A gene Citrus canker Boscariol et al., 2006
Carrizo citrange Expansins related
P12
Citrus blight Kayim et al., 2004
Future Goals
■ Collection & evaluation of germplasm from indigenous & exotic sources for desired
horticultural traits & processing qualities.
■ Introduction & evaluation of indigenous & exotic rootstocks.
■ Improvement of acid lime & lemon by interspecific hybridization.
■ Linkage mapping.
■ Establishment of in vitro gene banks.
Thank You

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Citrus

  • 2. Introduction & Importance  Citrus is the most cultivated fruits in the world with the highest acreage 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 10.24 Lac Hectare with an annual production of 1.15 Crore 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.  The domestication and distribution of edible citrus types began several thousand years ago in South -East Asia and spread globally following ancient and sea routes.  The Citrus species are undoubtedly indigenous or domesticated for a long time. (Bhattacharya and Dutta, 1956; Singh, 1977).
  • 3. Origin and Distribution ■ Center of origin is South-East Asia bordered by North Eastern India, Myanmar (Burma) and Yunnan province of China. ■ From China, India and West Indies, it dispersed all over the world and now, it is grown almost throughout world. ■ Most scion and rootstock cultivars that are widely grown in the main commercial producing areas of the world are originated as either chance seedling selections or bud sport mutations (Hodgson, 1967). Species Origin Mandarin Indo-China and South China Sweet orange South China Lemon Unknown Lime East India and Archipelago Pummelo Malaysia and Indian Archipelago Grape fruit West Indies
  • 4. Kingdom Plantae Division Phanerogams Sub division Angiosperm Class Dicotyledonae Order Geraniales (1 of 21 orders) Sub order Geraniinea (1 of 12 sub orders) Family Rutaceae (1 of 12 families) Sub family Aurantoideae (1 of 7 sub families) Tribe Citrae (1 of 2 tribes) Sub tribe Citrinae (1 of 3 groups) Group C True citrus fruit trees (1 of 6 generas) 1. Poncirus - 1 species 2. Fortunella (Kumquat) – 4 species 3. Eremocitrus 4. Clymenia 5. Microcitrus 6. Citrus – 2 subgenera * Generally, 3 related genera Poncirus, Fortunella and Citrus are included in the study of citrus fruit/ Citriculture. Genetic Classification *Source: Swingle & Reece, 1967.
  • 5. Taxonomy Sub-genus : Eucitrus (10 species) 1. C. medica (Citron) 2. C. limon (Lemon) 3. C. aurantifolia (Acid/sour lime) 4. C. aurantinum (Sour/bitter orange) 5. C. sinensis (Sweet orange) 6. C. reticulata (Mandarin) 7. C. grandis (Pummelo) 8. C. paradisi (Grapefruit) 9. C. indica (Indian wild orange) 10. C. tachibana (Tachibana orange) Fruits are edible and widely cultivated. Sub-genus : Papeda (6 species) 1. C. ichangenesis 2. C. latipes 3. C. micrantha 4. C. celebica 5. C. macroptera 6. C. hystrix Fruits are inedible and presence of numerous droplets and oil in juice sacs. Genus Citrus * Source: Janick 2006 WT Swingle (1948)
  • 6. Genus Citrus ■ Citrus word is originated from Greek word – ‘Kedros’ originally meant ‘Cedar’ or ‘Cedarcone’ which means – Fruit of the godly tree in Palestine. ■ Gk. Word ‘Kedros’ is latinized to ‘Cedrus’ which later changed to ‘Citrus’. ■ Genus is commercially important as many species are cultivated for fruit, which is eaten fresh, pressed for juice, preserved in marmalades and pickles. ■ In most of species, both bud and seed mutations are known to occur giving rise to ‘Sports’. ■ The species of Fortunella, Poncirus and Citrus hybridize freely producing natural hybrids. Similarly, budding and grafting are easily done reciprocally among the species and genera.
  • 7. Genetic variability ■ Citrus has wide variability with respect to fruit morphology & quality. ■ Limes are smallest scarcely exceeds 3 cm in size, while pummelo may attain diameter of 30 cm. ■ Fruit rind colour ranges from yellow green of lime to red orange of some mandarins. ■ Shape varies from oblate to pyriform. ■ At maturity, some cultivars are high in acid while others have almost none. ■ All species are evergreen but related genus Poncirus is deciduous. ■ Altogether, there is much variability within genus with which breeder can work and closely related genera provide an even wider range of characters.
  • 8. Genus Fortunella ■ Kumquat is the popular name of the Fortunella. ■ The plants are evergreen shrub & most cold hardy. ■ Fruits are orange to golden yellow colour, palatable with spicy aromatic ring. ■ The four species of Fortunella are F. margarita (Oval kumquat), F. japonica (Round kumquat), F. crassifolia & F. hindsii (Hongkong wild Kumquat). Genus Poncirus ■ Monotypic genus: has only one species, popularly known as trifoliate orange. ■ Plants deciduous with trifoliate leaves, highly thorny & fruits are inedible. ■ Dark green twig with strong and stout thorns make the species an excellent hedge in ornamental gardens. ■ Plants are cold hardy and the species is principally used as rootstock.
  • 9. Cytogenetics. Studies of cellular aspects of heredity (chromosomes) ■ Most of the citrus species are diploid (2n=2x=18). ■ One natural triploid is Tahiti lime (Citrus latifolia). ■ Fortunella hindsii is tetraploid species of Kumquat. ■ Genome size of Citrus sinensis is 360 Mb.
  • 10. Chemotaxonomy Species Flavonoids Coumarins and Psoraiens Carotenoids C. aurantifolia Hesperidin, Naringin, Naringenin, Erioctrin Tangeretin Bergapten, Bergamottin, Beragaptol, Phellopterin, Prangol, Scopoletin - C. aurantium Naringin, Neoeriocitrin, auranetin, neohesperidin, tangeretin, hesperidin Aurapten, auraptenol, bergapten, meranzin, meranzin hydrate, seselin, isomeranzin, osthol, scoparon, umibelliferone, xanthyletin - C. paradisi Naringin, hesperidin Aurapten, bergapten, bergamottin, bergaptol, citropten, marmin, meranzin Phytoene, Phytofluene, Carotene. C. reticulate Hesperidin, Neohesperidin, Xanthomicrol, Nobiletin Aurapten, Bergapten, Bergaptol, Citropten, Osthol, Auroptenol β-carotene, Cryptoxanthin, Violaxanthin, C. sinensis Rutin, limocitrin, sinensetin Auraptenol, Scoparon Phytoene, β- carotene C. limon Naringin, Eriocitrin, Hesperitin, Hesperidin Bergapten, Bergamottin, Byakangelicin, Citropten, Imperatorin, Phytofluene, β-carotene, Cryptoxanthin
  • 11. Centre of diversity ■ There are three major centers of diversity in India. ■ The first in the North-East including Assam and adjoining areas. It includes Papedas, pummelos and their hybrids, citron, lemons and mandarins and other interesting types like jenera-tenga, soh synteng, a sour fruit similar to the sweet lime and soh siem, a mandarin type. ■ The second diversity in south India, indigenous types include Gajanima, kichili and some wild mandarin types. ■ The third in North-West region at the foot of Himalayas where the hill lemon (galgal) is common. ■ The various types of mandarins, hybrids of pummelo, citron, lemons, karna khatta and rough lemon are found all over the country. ■ In general, the wild types are more common in the foot hills. Many of the progenitors of citrus fruits are believed to have originated in India. These include C. latipes, C. limonia, C.kama, C.pennevesiculata, C.maderaspatana, many of these are wild types. ■ Presence of Sah- Niangriang, a wild sweet orange and a wild mandarin (C.indica) furnishes strong evidence that Eastern India might be the centre of orgin for many citrus fruits (Tanaka, 1981).
  • 12. Germplasm resources  Exotic collection of citrus germplasm was started in 1940.  Kinnow mandarin was one of the collections which is now a leading cultivar in North – Western India.  Besides, other exotic collections were Valencia Late, Washington Navel, Jaffa, Malta Blood Red, Pineapple, Ruby orange, Satsuma, Dancy Tangerine, Climentime, and Cleoptera wilking ,Temple, Duncan, Marsh seedless, Lisbon lemon, Trifoliate orange, Dancy, Lisbon lemon, Trifoliate orange (Dutta,1958).  More than 650 accessions are being maintained at CHES, Chethali, Bangalore, CHES, Ranchi, RFRS, Abhor, NRC on citrus, Nagpur, Horticultural Experiment Station, Bathinda, IARI, New Delhi, MPKV, Rahuri, Citrus Improvement Project, Tirupati, Citrus Experiment station, Nagpur, HC&RI, Periyakulam, and Citrus Experiment Station, Tinsukia, Assam.  Attempt has been made during 1978 by NBPGR to preserve the C. indica which is progenitor of C. reticulata (Singh, 1981).  For establishment of gene sanctuary, National Park, the natural genetic diversity of C. indica was observed in the forest of Garo hills in Megalaya which exhibited plant characters varying from bush to climber with high frequency of distribution in dense forest and showing resistance to biotic stresses.  Genetic material of citrus is conserved in field gene bank or repository.
  • 13. National Status ■ In India, Citrus genetic resources are being managed by multiple agencies such as research institutes of Indian Council of Agricultural Research (ICAR), State Agricultural Universities and also by the State Horticultural Farms and Fruit Research Stations. ■ Besides these several private nurseries, orchards, gardens, parks etc. which are owned by progressive farmers and public enterprises are also maintaining Citrus germplasm as field collections ■ In India, National Research Centre for Citrus (NRCC), of ICAR, located at Nagpur in Central India is the only dedicated institute for Citrus research, development and extension. This Centre has also been designated as National Active Germplasm Site (NAGS) for Citrus by NBPGR. ■ All India Coordinated Research Project (AICRP) on Tropical Fruits (Citrus) of ICAR with its Network at nine centres at SAU's and ICAR institutes in various agro-ecological regions of India has been taking up work on genetic resources management, crop improvement, crop production and crop protection. Since last several decades crop specific responsibilities for multi-location evaluation and improvement work has been assigned to these centres.
  • 14. Inheritance of characters in citrus Character Inheritance Leaf character Two principal genes Anthocyanin pigment in leaf Dominant gene controlling this reddish colour Anthocyanin pigment in fruit More than one recessive genes are involved Tristeza virus Single allele Texture Polygenic control Polyembryony More than one single gene Seedless character A recessive gene Nucellar embryony One or two dominant gene
  • 15. Tolerance for biotic stress Biotic stress Cultivar Root rot Rangpur lime, Marmalade orange, Troyer citrange, Carrizo citrange. Nematode Marmalade orange, Sweet lime, trifoliate orange, Troyer citrange, Carrizo citrange, Karna khatta, Nasnaran, Survernia disticha. Tristeza virus Rangpur lime, Marmalade orange, Rough lemon, Cleopatra mandarin, Trifoliate orange, Troyer citrange, Carrizo citrange, Nasnaran, Sweet orange. Exocortis Rough lemon, Sweet orange, Sour orange, Cleopatra mandarin, Grapefruit. Gummosis Trifoliate orange, Sour orange, Rangpur lime, Cleopatra mandarin, Grapefruit. Greening Poncirus, Gajanima, Florida rough, Karna khatta.
  • 16. Genetic resource resistant/tolerance for abiotic stress Abiotic stress Cultivars Salt Rangpur lime, Marmalade orange, Nasnaran, Cleopatra mandarin. Drought Rangpur lime, Marmalade orange, Rough lemon, Sour orange. Genetic resource for yield and quality traits Trait Cultivars Good yield Rangpur lime, Rough lemon, Cleopatra mandarin, Sour orange, Karna khatta. Good quality fruits Sour orange, Carrizo citrange, Troyer citrange , Sweet orange, Nasnaran.
  • 17. Varieties resistance to abiotic stress Resistance Cultivar Cold hardiness Duncan grapefruit Cold hardiness Valencia sweet orange Cold hardiness Carrizo citrange Salt tolerance C. jambhiri and C. macrophylla Salt tolerance ‘Cleopatra’ mandarin for ‘Star Ruby’
  • 18. Main Cultivated Species Mandarin Citrus reticulata Blanco Sweet orange Citrus sinensis (L.) Osbeck Lemon Citrus limon Burmann Lime Citrus aurantifolia Swingle Pummelo Citrus maxima (L.) Osbeck Grape fruit Citrus paradisi Macfadyen Citron Citrus medica L. Kumquat Fortunella species
  • 19. Main Cultivated Cultivars Type Cultivar Sweet orange i. Common or round orange Hamlin, Valencia, Netal, Pera, Shamouti, Jaffa, Pineapple, Mosambi, Delta, Campbell, Frost, Premier, Sathgudi, Ambersweet, Delicious, Malta. ii. Navel orange Washington Navel, Thompson, Dream, Autumn Gold Late, Atwood, Fisher, Newhall, Navelina, Navelate, Summerfield, Marrs, Bajaninha, Palmer, Cara . iii. Pigmented oranges Malta Blood Red, Ruby, Moro, Tarocco. iv. Acidless or sugar orange Lima, Succari, Piralima, Oro , Sicily, Sanguinello. Limes i. Acid lime Kagzi, Mexican, Palmetto. ii. Persian lime (Tahiti lime) Tahiti, Pond, Bearss lime. iii. Mandarin lime Rangpur lime. iv. Sweet/acidless lime Sweet lime, Local, Palestine. Lemons Lisbon, Eureka, Lucknow Seedless, Kagazi Kalan, Baramasi, Villafranca, Pant Lemon -1, Meyer Lemon, Femminello, Malta Lemon, Nepali, Oblong.
  • 20. Main Cultivated Cultivars Type Cultivar Mandarins i. Satsuma group (C. unshiu Marcvitch) Ovari, Kara, Brown’s Select, Miho Wase, Miyagawa Wase. ii. Mediterranean group (C. deliciosa Tenore) Willow Leaf mandarin, Wilking, Kinnow. iii. Common group (C. reticulata) Nagpur Santra, Coorg, Khasi, Local, Darjeeling, Dancy, Daisy, Kinnow, Encore, Murcott. iv. King mandarin group (C. nobilis Loureiro) Jeneru Tenga. v. Tangerine group Dancy, Beauty, Naartja. vi. Mitis group Cleopatra, Calamondin. Mandarin - Like Tangors (Hybrid between Tangerine × Sweet orange) Temple, Clementine. Mandarin - Like Tangelos (Hybrid between Tangerine × Grapefruit) Orlando, Minneola.
  • 21. Type Cultivar Grapefruit i. White fleshed Duncan, Marsh, Walters. ii. Pink fleshed Ruby (syn. Ruby Red, Red Blush), Foster, Ray Ruby, Ruben SL, Henderson 13. iii. Red fleshed Star Ruby, Rio Red, Flame, Nelruby, Oran Red (syn. Rouge la toma), Henderson. Pomelos i. Thai group Chander (Pink Fleshed), Kao Panne and Kao Phuang (White fleshed). ii. Chinese group Goliath, Mato, Shatinyu. iii. Indonesian group Banpeiyu (White-fleshed), Djeroek deleema, Kopjar (Pink-fleshed). iv. Indian group Galgal (White or Pink-fleshed). Kumquat (Fortunella spp.) Nagami, Metwa, Marumi, Calamondin. Main Cultivated Cultivars
  • 22. Cultivars grown in India Sweet orange Mosambi, Satgudi, Valencia Late, Jaffa, Pineapple, Shamouti, Washington Navel. Mandarins Nagpur Sangtra, Khasi mandarin, Darjeeling mandarin, Coorg Kinnow. Lemon Eureka, Lisbon, Villafranca, Assam lemon, Punjab Galgal. Grapefruit Duncan, Triumph, Marsh Seedless, Star Ruby, Foster.
  • 23. Floral Biology of Citrus ■ Flowers are hermaphrodite produced on current season growth in cymes, both at axillary and terminal position. ■ Flower opening starts from morning and extend upto evening, but maximum anthesis is between 11.00 a.m. to 12.00 noon. ■ The viability of pollen grains varies from 45-80% depending upon the season. ■ The dehiscence of anthers takes place 45 minutes before anthesis or within 45 minutes after anthesis. It varies up to 5 hours after anthesis. ■ The receptivity of stigma starts either 15 minutes to 2 hours before anthesis or within 35 minutes to 5 hours after anthesis depending upon weather. The receptivity lasts for 4-8 days after anthesis.
  • 24. Flowering, fruit set and maturity of citrus Country Flowering time Fruit set Ripening N. India March (Feb.-April) April-May January ( Mandarins) Nov.-Jan. (Sweet Oranges) S. India Dec. –April, Sept-Dec. Sept.-Dec. March –April, Sept-Oct. Central India March-April, Sept. - Oct. June, Oct. – Feb. Aug.-Oct. (Sweet Oranges) Feb-March * Lemon and lime throughout the year. USA Feb-March April-May Dec.-Feb. Australia April-May May-June Jan.-March Japan April-May May-June Dec.-Feb. Brazil March-April April-May Dec.-Jan.
  • 25. Problems in conventional breeding ■ Male or female sterility ■ Self and cross-incompatibility ■ Long juvenile period ■ Nucellar embryony
  • 26. Kinds of male sterility occurring in citrus * Source: Iwamasa, 1966
  • 27. Objective of improvement for Scion ■ Seedlessness. ■ High productivity. ■ Sensory fruit quality (loose peel, pigments). ■ Quality (TSS/ acid ratio, Juice content, Flavour and colour). ■ Wide season of ripening & good post harvest life. ■ Resistance to adverse climatic and soil conditions.
  • 28. Objective of improvement for rootstock ■ Better stock-scion compatibility, reduction of tree size without affecting yield. ■ Resistance to pests and diseases or hardiness to adverse climactic conditions. ■ Resistance to nematode species. ■ Must produce many seeds & be highly nucellar in order to provide uniformity in mass multiplication. ■ Cold, salinity & drought tolerance.
  • 29. CCRI Nagpur Vision 2050 Genetic Resources and Crop Improvement ■ Functional genomics and phenomics based gene identification for specific trait and gene pyramiding to have cultivar of multiple resistance on one hand and identifying genes for some negative traits like genes leading rootstock vulnerability to Phytophthora, drought tolerance, nutrient deficiency etc. on the other. ■ Characterization and cataloguing of citrus germplasms using microsatellite molecular markers. ■ Marker-aided-selection of citrus germplasm for useful traits e.g. less seeded, suitable for processing, tolerance to drought, floods, salinity and diseases. ■ Rootstock improvement through protoplast fusion. ■ Anti-oxidant and neutraceutical profiling of citrus germplasm could be new objectives in citrus breeding programme. ■ Clonal selection for better yield and quality and ploidy manipulation for seedlessness. ■ Evaluation and barcoding of citrus germplasm. ■ Hybridization for scion and rootstock improvement. ■ An engineered rootstock capable of withstanding both biotic as well as abiotic stresses, besides fitting well into high density planting will improve factor productivity of citrus orchards. ■ Identification of germplasm tolerant to water stress (drought and flood).
  • 30. Breeding Approaches & Achievements
  • 31. Selection of new plants Hybrids, mutants, cybrids, somaclones, transformants etc. Identification of desirable promising plants Replicated field trials Evaluation for horticultural characteristics Identification of new cultivar Multiplication & release of the cultivar to the growers Selection of parents Breeding procedure Hybridization, Mutation, Somatic hybridization & other in vitro techniques, Genectic transformation General outline of breeding methods
  • 32. Breeding in USA (University of California, University of Florida and Texas University) Scion breeding ■ For Sweet oranges, cultivar which holds well late in the season is desired. ■ For Mandarins, important traits are seedlessness, easy peeling, good flavor, high rind color and low tendency to alternate bearing. ■ For grapefruit, a deep pigmented with low naringin cultivar is breeding objective. Stock breeding ■ Rootstocks resistant to problems like Phytophthora, citrus tristeza virus, citrus nematodes, citrus blight, high pH, salinity. Scientists ■ Kim D Bowman – USDA, ARS, Horticultural research aboratory, Florida. ■ JW Grosser, JL Chandler and FG Gmitter Jr – CREC, University of Florida, lake Alfred, USA. ■ Mikeal Roose, Timothy E Williams, Department of Botany and Plant Sciences, University of California, Riverside.
  • 33. Breeding in China and Spain ■ China The major breeding objectives are to develop seedless cultivar better than Ponkan mandarin w. r. t. fruit size, variation in maturity. ■ Scientists XX Deng: National Key laboratory of Crop genetic Improvement and citrus research Institute, Huazhong agricultural university, Wuhan, Hubei. ■ Spain To breed rootstocks resistant to citrus tristeza virus, Phytophthora, adapted to Spanish conditions especially saline and calcareous soils and give good quality fruits. ■ Scientists JB Forner, MA Forner, AA AClaide- Instituto Valenciano de investtigaciones Agrarias, Moncanda, Valencia, Spain.
  • 34. Breeding in India Scion breeding: ■ For mandarins- Low juvenility, high yield, reddish orange color fruits without neck or collar, seedless or with few seeds with proper sugar acid blend extended period of maturation, low bittering substances in Kinnow. ■ In sweet orange- Fruits free from granulation, seedless or low seed content with TSS and acidity, and juice content should not be less than 35 %. ■ In Acid lime- resistance to canker and scab, large fruit size with high juice content, seedless or low seed content. Stock breeding: ■ Rootstocks resistant to Phytophthora, citrus tristeza virus, exocortis, greening, salinity, drought, water logging and good yield and quality of fruit in scion cultivars.
  • 36. Introduction ■ The mandarin variety ‘Santra’ is known to have been grown in India for many centuries. It was introduced into the Central Provinces (now Maharashtra) by Ranghojee Bhonsal II from Aurangabad in eighteenth century. ■ Sweet orange varieties including Washington Navel, Valencia introduced from America. ■ Grapefruits were introduced from California and Florida. ■ Lemons from China. ■ Malta from USA and Italy. ■ ‘Mosambi’ seems to have been introduced in Nagpur during the beginning of the 20th century. ■ The introduction of ‘Kinnow’ mandarin (King x Willow leaf) in 1947 showed great promise in North India. It was introduced in South India in 1958 and Punjab in 1959 and has performed extremely well in Punjab.
  • 37. Citrus Cultivation in Punjab Mandarins PAU Kinnow 1, Daisy (2013), W. Murcott (2013), Kinnow (1968), Local (1968). Sweet oranges Valencia (1968), Mosambi (1962), Jaffa (1962), Blood Red (1962). Grapefruit Star Ruby (2009), Red Blush (1995) Marsh Seedless (1968), Duncan (1968), Foster (1968). Lemon Punjab Baramasi Lemon (2008), Punjab Galgal (1994), PAU Baramasi Lemon- 1 (1990), Eureka (1968). Lime (Nimboo) Kagzi (1968), Sweet Lime (Mitha), Local (1968). Rootstock Rough Lemon, Carrizo, Cleopatra, Pectinifera. * Source: Citrus cultivation by Rattanpal et al., 2017
  • 41. Star Ruby Red Blush Marsh Seedless Duncan
  • 42. Three exotic varieties introduced viz. ‘Pummelo US -145’, ‘Cutter Valencia’ and ‘Flame grapefruit’ by Institute. Two varieties developed through indigenous material viz NRCC Pummelo – 5 and NRCC Grapefruit- 6 have been released by Institute. ICAR-CCRI *Source: Information for ICAR-CCRI Website (November, 2016 to June, 2017)
  • 44. Selection at IARI, New Delhi ■ 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 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 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. ■ Acid lime ■ Pusa Abhinav: It is a promising clonal selection and is moderately susceptible to citrus canker. ■ Pusa Udit: It is a clonal selection has round the year fruiting with two season harvesting will make this selection highly suitable for commercial cultivation as well in kitchen garden.
  • 46. Hybridization ■ The mating or crossing of two plants of dissimilar genotype is known as hybridization. ■ In plants, crossing is done by placing pollen grains from one genotype (male parent) onto the stigma of flowers of other genotype (female parents). ■ The seeds as well as progeny resulting from hybridization are known as hybrid or F₁. The progeny of F₁, obtained by selfing of F₁ plants, & the subsequent generations are termed as segregating generations. ■ Genus Citrus is its vast array of forms and cultivars has great genetic diversity. Intergeneric hybridization is the key process by which genetic diversity of citrus and its relatives can be added to genetic diversity of citrus.
  • 47. * Riccardo & Licciardello: 2014 (Nature)
  • 48. Hybrids released by University of California, Riverside ■ Gold Nugget ■ TDE 1 (Triploid hybrid) ■ TDE 2 (Shasta Gold) ■ TDE 3 (Tahoe Gold) ■ TDE 4 (Yosemite Gold) ■ USDA 88-2 (Lee × Nova) * Source: Roose and Williams (2000); Nir Carmi 2014 ■ USDA 88-3 (Robinson × Lee) ■ Valentine ■ Flamingo- Red Pomelo ■ Hanna ■ Einat ■ Aliza
  • 49. Wilking (Willow leaf × King) Kincy King × Dancy Gold Nugget Wilking × Kincy Gold Nugget ■ (Wilking X Kincy) ■ Diploid hybrid released in 1999 ■ Seedless ■ Late maturing hybrid (February - March) ■ On tree storage life is 4-6 months * Source: Roose and Williams (2000)
  • 50. TDE 1 (Triploid hybrid) (Temple x Dancy) x Encore mandarin (4x X 2x cross) Temple TDE 1 (Trioloid hybrid EncoreDancy * Source: Roose and Williams (2000)
  • 51. TDE 2 (Shasta Gold) • Triploid hybrid • Late season maturity • Seedless TDE 3 (Tahoe Gold) • Mid late season • Very deep rind color • 1-2 months on tree storage life TDE 4 (Yosemite Gold) • Seedless • Late maturity (Feb. - March) • 2-3 months on tree storage life. * Source: Roose and Williams (2000)
  • 52. USDA 88-3 • Diploid hybrid of Robinson x Lee • Seedless in isolation • Can cause seeds in other mandarins • Early maturity (Nov.) • Deep orange rind colour • Poor fruit holding ability on the tree Oden • Cross of Orah and Shani * Source: https://news.ifas.ufl.edu 2009
  • 53. Flamingo- Red Pomelo • Chandler pomelo x (Shamouti orange x Clementine) • Ripens October to December • Maintains its red color when the temperature drops • Sweet (14% sugar) • Seedless • Presence of lycopene Hanna • Tahitian pomelo x Chandler pomelos • Ripens from October to December • Especially sweet taste (12.6% sugar) • Seedless * Source: Nir Carmi 2014
  • 54. Sugar Belle • University of Florida created 1st citrus variety intended for commercial production. • Clementine x Minneola • “Many old-timers in citrus have said this is the best-tasting citrus they’ve ever had,” Gmitter Valentine • Siamese Sweet (Acidless pummelo) x (Dancy x Ruby blood) • Valentine’ combines large size and low acidity from its pummelo parent • It combines complex, floral taste from ‘Dancy’ & juicy red pulp from ‘Ruby’ * Source: https://news.ifas.ufl.edu 2009
  • 55. Einnat • Hudson x Pomelo • Ripens: October to January • Maintains its inner pink color (lycopene) throughout all the season. • Sugar (11.5%) • Seedless Aliza • 'Chandler' pomelo x "Ora” • Ripens during Nov. to February • Sugar (16%) • Seedless • Dominates the global market
  • 56. Citrus Intrageneric (interspecific) & Intergeneric Hybrids (Grapefruit) (Mandarin) (Orange)
  • 57. Grapefruit Tangelo Tangeloes Minneola, Orlando, Pearl, Sampson Tangelo
  • 59. Intrageneric or Interspecific hybrids Hybrid Percentage Examples Tangor Mandarin × Sweet orange Eg. Temple, Monreal, Umatilla Tangelo Mandarin × Grapefruit Eg. Sampson, Orlando, Minneola Lemonime Lime × Lemon Eg. Perrine Intergeneric hybrids Citrange Sweet orange × Trifoliate orange Eg. Troyer, Carrrizo, Willits. Citrangedin Calamondin × Willits citrange Eg. Glen Limequat Lime × Kumquat Eg. Eustis, Lakeland Citrangor Citrange × Sweet orange Cicitrange Citrange × Trifoliate orange Citrumelo Grapefruit × Trifoliate orange Eg. Swingle citrumelo Citrandrin Mandarin × Trifoliate orange Citremon Lemon × Trifoliate orange Artificial hybrids
  • 60. Citrus Hybrids for Ornamental Use ■ 04-K5 (Chinotto × ‘Buddha’s Hand’ Citron) ■ FH-2 (F. hindsii spp. Open Pollinated) ■ LM X CH-3 (‘Meyer’ Lemon × Chinotto Small Leaf ■ LM X CH-13 (‘Meyer’ Lemon × Chinotto Small Leaf ■ LM X LR-14 (‘Meyer’ Lemon × Red Lime) ■ ARCOBAL (‘Meyer’ lemon × ‘Doppio Sanguigno’ Orange) ■ 04-F5 (‘Oval’ Kumquat × ‘Buddha’s hand’) * Source: Recupero et al., 2015
  • 61. 04- K5 FH-2 LM × CH-3
  • 62. LM X CH-13 LM X LR-14 ARCOBAL
  • 63. Rootstock hybrid (Release year) Parentage Important traits US-1516 (2015) African Pummelo (C. grandis) × Flying Dragon (P. trifoliata) Tolerant to HLB US-1283 (2014) Ninkat mandarin x P. trifoliata Tolerant to HLB US-897 (2011) Citrus reticulata x P. trifoliata Tolerant to HLB US- 942 (2010) Sunki mandarin (C. reticulata) × Flying Dragon (P. trifoliata) Resistant to nematode, phytophthora, induction of good quality fruits, easy to propagate US 812 (2006) Sunki mandarin (C. reticulata) × Flying Dragon (P. trifoliata) Highly productive, resistance to citrus blight US-815 (2001) Sunki mandarin (C. reticulata) × Benecke trifoliate orange Resistance to phytophthora, citrus nematode, Tristeza and citrus blight. US 8-52 (2000) Changsha Mandarin × Trifoliate orange Cold hardy, resistance to phytophthora, citrus nematode, Tristeza and citrus blight. Rootstock developed in USA * Source: Forner 2010
  • 64. Rootstock developed in India Country Rootstock hybrid Parentage Important traits IIHR, Bangalore CRH-12 Rangpur lime x Trifoliate orange Highly resistant to phytophthora and citrus nematode and moderately tolerant to salinity and tolerant to drought CRH-57 Rough lemon x Trifoliate orange Highly resistant to phytophthora, citrus nematode and tolerant to salinity and drought CRH 47 Cleopatra Mandarin xTrifoliate orange Moderately tolerant to salinity CRC, Nagpur NRCC 1 to NRCC 10
  • 66. Mutation Breeding ■ Mutation can be defined as the changing of the structure of a gene, resulting in a variant form that may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. ■ Two types of mutation ■ Spontaneous mutation: It occurs naturally i.e. without human intervention and at a low rate (one in a population of ten lacs). Eg. Natural budsports. ■ Induced mutation: It can be induced artificially by a treatment with certain physical or chemical mutagens. ■ The first important cultivar from a mutation breeding programme was the ‘Star Ruby grapefruit that resulted from the irradiation of seeds of Hudson grapefruit. Star Ruby is having very deep flesh colour and is commercially seedless (Hensz, 1977). ■ Liu et al., (2007) reported a spontaneous bud mutation in sweet orange (Citrus sinensis L. Osbeck) „Hong Anliu‟, which results in fruits with lycopene accumulation, low citric acid, and high sucrose was reported in a previous study.
  • 67. Crop Parent variety Mutant cultivar Nature of mutation Navel orange Bahia Bahianinha Limb sport Washington Navelina, Navelate Marrs, Autumn Gold, Powell Sum mer, Weller Gold Limb sport Mandarin Owari Clausellina Bud sport Pongan Pongan 86-1 - Grapefruit Walter (White fleshed) Foster (Pink fleshed) Limb sport Foster (Pink fleshed) Hodoun (Red fleshed) Bud sport Thompson (light pink flesh) Red Blush (deep red flesh) Limb sport Henderson Flame Bud sport Pummelo Local cultivar Wuheputaoyou Bud sport Spontaneous mutation in citrus * Source: Ray, 2000
  • 68.
  • 69. Star Ruby Red Blush Foster
  • 70. Fruit Crop Parent Veriety Mutant Improved Trait Mutagen used Reference Citrus deliciosa Comun EC31/1 Low seed content and cold tolerant Gamma rays Anderson 2000 Citrus reticulata Pongan Fuyu 28 Resistant to canker & drought Gamma rays Huang et al. 2000 Citrus reticulata Daisy, Encore, Fairchild, Fremont, Kinnow, Murcott and Nova Daisy IR, Encore IR 1, Fairchild IR 2, Fremont IR 2, Kinnow IR 1,Tango and Nova IR 10 Seedless or less seed content, Tango released as a variety- late maturing, fruit moderately Gamma rays Williams 2008 Induced mutation in citrus
  • 71. Fruit crop Parent variety Mutant Improved trait Mutagen used References Mandarin Shatangju Wuzishat angju Fruits are small & seedless Gamma rays ZiXing et al., 2006 Mandarin Qianyang Seedless Qianyang Seedless Seedlessness Gamma rays Bing Hong 2003 Penggan mandarin 1 Penggan 2 - - - Tangelo Tangelo cultivar Jianyang Tangelo Seedlesness & 171 g weight Gamma rays Liu-Tao and Wu-Rui Dong 2007 Kumquat Meiwa kumquat Yubeni Large fruits & low seeded Colchicine treatment Yasuda et al., 2008 Contd….
  • 72. Studies on mutation breeding in mandarin cv. Kinnow • Rattanpal et al. 2019 studied variability in Kinnow (Citrus nobilis Lour × C. deliciosa Tenora) using gamma radiation. • Four hundred buds of Kinnow were irradiated at 30 Gy and budded on rough lemon rootstock in February 2007. • The resulting 188 MV1 plants were planted in the field in October 2009 and observations in the present trial were recorded from 2015 to 2018. • Among the 188 MV1 plants, eleven Kinnow mutants (seven solid mutant trees and four mutant branches) having average seed number less than eight were identified and were compared in detail with parent variety Kinnow. Gray (Gy): Unit of absorbed dose of energy per unit mass of tissue. 1 gray = 1 Joule/kilogram and also equals 100 rad.
  • 73. One of the low seeded mutant was released as a new variety named ‘PAU Kinnow-1’ for cultivation in Punjab.
  • 75. Polyploidy Breeding ■ Polyploids are organisms with multiple sets of chromosomes in excess of the diploid number (Acquaah, 2007) & implications of polyploidy in plant breeding is known as polyploidy breeding. ■ The Hongkong wild kumquats, Fortunella hindsii may have been the first reported tetraploid. ■ The seedless lime (C. latifolia) a triploid. Triploids have favorable characteristics and yield well but they are sterile. ■ Polyploidy manipulation by crossing of tetraploids with diploids yielded some valuable triploid varieties like ‘Oroblanco’ and ‘Melogold’.
  • 76. Triploid frequency of sexual hybridization and pollen and ovary development in mandarins ■ Costa et al. (2019) aimed to ascertain the triploid frequency of sexual hybridization and the pollen and ovary development in Citrus. ■ The ‘Fortune’ mandarin (Citrus clementina Hort. ex Tan. × C. tangerina Hort. ex Tan.) accession was selected as female parent. The male parents used were the ‘Cravo’ (C. reticulata Blanco) and ‘Dancy’ (C. reticulata) mandarins. ■ The pollinations were always manually performed in the morning, and the pollinated flowers were identified for control. During the flowering period, flower samples at different stages of development of the ‘Fortune’ variety were collected for analysis. ■ Microspore development occurred regularly in the 4–5 mm (length) phase of the floral buds, and a bicellular micro-gametophyte was formed in the floral buds with 5–6 mm in length. ■ The formation of the female gametophyte occurred later, in flower buds with approximately 10 mm in length. *Source : Costa et al., 2019
  • 77. The hybridizations resulted in 19 triploid plants: five when the ‘Cravo’ mandarin was used as the male parent and 14 when ‘Dancy’ mandarin was used as the male parent, evidencing the importance of the male genitor also in this process.
  • 78. Extensive citrus triploid hybrid production by 2x X 4x sexual hybridizations and parent-effect on the length of the juvenile phase a Monoembryonic undeveloped seed. b Polyembryonic undeveloped seed. c Germination of embryo from monoembryonic undeveloped seed. d Germination of multiple embryos from polyembryonic undeveloped seed Aleza et al. 2012 reported effective methodology to recover citrus triploid plants from 2x X 4x sexual hybridizations and the parental effect on the length of the juvenile phase. The juvenile phase length of the triploid hybrids obtained with ‘Fortune’ mandarin as female parent and tetraploid ‘Orlando’ tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of ‘Nova’, ‘W. Leaf’ and ‘Pineapple’ male parents. *Source:https://www.researchgate .net/publication/286730366
  • 79. Alkantara ‘Oroval’ clementine × ‘Tarocco’ orange 4x Mandared Nules’ clementine × ‘Tarocco’ orange 4x Early Sicily ‘Oroval clementine × ‘Tarocco’ orange 4x Sweet Sicily ‘Comune’clementine × ‘Tarocco’ orange 4x * Source: Giuseppe Russo et al., 2015; Recupero et al., 2005. Triploids
  • 80. * Source: Giuseppe Russo et al., 2015; Cuenca et al., 2010. Garbi ‘Fortune’ mandarin x diploid tangor ‘Murcott’ (C. reticulata x C. sinensis) Safor Diploid ‘Fortune’ mandarin and diploid ‘Kara’mandarin (C. unshiu x C. nobilis) Albir ‘Fortune’ and Kara’mandarin (C. unshiu × C. nobilis) Dawn Diploid Acidless pummelo x 4n ‘Ruby’ blood Triploids
  • 82. In vitro Plant Regeneration in Cleopatra (Citrus reshni Hort. ex Tan.) by Direct Organogenesis Objectives To develop in vitro regeneration system for citrus rootstock Cleopatra. Direct induction of adventitious shoot buds and plant regeneration via culture of different explants from in vitro grown seedlings in Cleopatra. (A) Initiation of shoot formation. (B) Emergence of shoot buds from the longitudinal cut of epicotyl segment. (C) Shoot development from epicotyl segment on MT + BAP (2 mg/l) + NAA (0.2 mg/l). (D) High frequency of regeneration of multiple shoots from epicotyl segment. (E) Rooted plants. (F) In vitro hardened plantlet. * Source: Devi and Rattanpal 2018
  • 83. Valenfresh (N7-3) • Somaclone (protoclone) of Valencia sweet orange • Late-maturing Valquarious • Early maturing Jan-Feb • Selection with typical Valencia quality OLL 8 • Low seeded Somaclonal variations ■ Somaclonal variation is defined as genetic variation observed among progeny of plants regenerated from somatic cells cultured in vitro. ■ It can be used in the development of crops with novel traits.
  • 84. Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS ■ Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. ■ Ge et al. (2015) conducted study, aimed at the establishment of an efficient protocol for in vitro ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange somaclones tolerant to citrus canker disease. ■ The results suggested that the established protocol for somaclones variation by treating sweet orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the pathogen crude extract was effective. ■ The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar selection procedures. *Source: https://www.researchgate.net/publication/281608821
  • 85. Micro grafting ■ Micro grafting is an in vitro grafting technique which involves the placement of a meristem or shoot tip explant onto a decapitated rootstock that has been grown aseptically from seed or micro propagated cultures (Hussian et al., 2014). ■ Shoot tips having size (0.1-0.2 mm) is very efficient for elimination of all citrus graft-transmissible pathogens from local or imported varieties. ■ STG was used to regenerate the ‘Clemenules’ haploid plant that has been used by the International Citrus Genome Consortium to sequence the whole citrus genome (Jaurez et al., 2015).
  • 86. Standard STG in vitro technique description. a-c. Rootstock preparation. d-f. Scion preparation. g-m. Grafting and culture in vitro of grafted plants. n. Plant recovered by STG. * Source : Jaurez et al., 2015 Citrus shoot-tip grafting in vitro
  • 87. Applications of Citrus STG in vitro ■ The main application of STG is to control graft transmissible pathogens. ■ According to the studies conducted by Navarro et al. (1988) and Navarro (1992) the elimination of viruses in citrus by STG is as follows: ■ Regeneration of somatic hybrids from embryos difficult to germinate. ■ Regeneration of plants from irradiated shoots to produce seedless varieties. ■ Regeneration of plants from haploid embryos that are very difficult to germinate. ■ Production of stable tetraploid plants of non-apomictic genotypes, which are very useful for triploid breeding. ■ Regeneration of transgenic plants from shoots that are very difficult to root in vitro. STG has become a routine application in citrus genetic transformation. Name of the viruses Ease of elimination Percentage of germination Exocortis infection, Tristeza Very easy 100 Tristeza Easy 80 Psorosis Difficult <25 * Source : Jaurez et al., 2015
  • 88. In vitro shoot tip grafting (a) Vertical incision on rootstock (b) Scion insertion at vertical incision (cleft grafting) (c) Inverted T incision on rootstock (d) Scion insertion at inverted T cut In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus-free plants of Khasi mandarin • Singh et al. 2018 reported the prospective applicability of three indigenous rootstocks belonging to different species viz. Nemutenga, Tayum and Tasi cultivars of north eastern Himalayan region for producing Citrus tristeza virus (CTV; a viral species of the Closterovirus genus) free quality planting materials of Khasi mandarin (Citrus reticulata Blanco) through micrografting or shoot-tip grafting (STG). • Viral assessment with enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) revealed negative results for all plants from 0.3 to 0.5 mm scion while 0.7-mm scion-raised micrografts showed 60% negative with RT-PCR for CTV. *Source: https://doi.org/10.1007/s11627-018-9946-6
  • 89. In vitro shoot tip grafting (e) In vitro graft culture after kinetin pre-treatment (f) Effect of kinetin (1.0 mg L−1) pre-treatment for Nemutenga rootstock with scion showing new expanded leaves of micrograft for inverted T and cleft grafting (g) Effect of kinetin (1.0 mg L−1) pre-treatment for Tayum rootstock with scion showing new expanded leaves of micrograft for inverted T and cleft grafting (h) Effect of kinetin (1.0 mg L−1) pre-treatment for Tasi rootstock with scion showing new expanded leaves of micrograft for inverted T and cleft grafting Conti..
  • 90. In vitro shoot tip grafting (i), (j) Proliferation of micrografts in MS supplemented with 5% sucrose along with 0.5 mg L−1 BAP and 0.1 mg L−1 IAA (k) In vitro root induction in MS supplemented with 1.0 mg L−1 NAA and 1.0mg L−1 IBA; (l) Pre-hardened micrografted plants (m) micrografted plantlet after hardening Conti..
  • 91. Somatic hybridization ■ Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. ■ It can be defined as the process by which somatic cells or tissues develop into differential embryo and each fully developed embryo is capable of developing into a new plantlet (Iqbal 2015). Somatic hybrids Protoplast material Method of fusion Objective Reference Mandarin + pummelo hybrids Suspension cultures PEG Sting nematode resistance Grosser et al., 2007 Hamlin sweet orange + Montenegrina mandarin Suspension cultures and leaf - Tolerance to citrus canker and xylella fasidiosa Pavan et al., 2007
  • 92. Somatic embryogenesis through in vitro anther culture of Citrus sinensis L. Osbeck ‘Moro’ ■ In many crops, anther culture is the most used method to induce gametic embryogenesis, aimed to regenerate homozygous plants. However, also somatic embryogenesis can be obtained by this method, when somatic tissue is involved in the regeneration process. ■ Lacuzzi et al. 2019 collected anthers of Citrus sinensis L. Osbeck 'Moro', at the vacuolate stage, and after a chilling (4°C) pre-treatment of 7 days, were placed on the same medium, evaluating different temperature stresses applied after placing them in culture. ■ In this study, the effect of three thermal treatments, compared with direct in vitro culture of the anthers (after the pre-treatment of the floral buds at 4°C for 7 days), was observed in cultivar 'Moro'. ■ Embryo regeneration has been obtained and their characterization, through ploidy and molecular analyses, showed that they were heterozygous tetraploids. *Source: https://www.researchgate.net/publication/330445894_
  • 93. Anther culture ■ Anther culture is a technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids that give rise to haploid plantlets either through organogenesis or embryogenesis. ■ Anther culture is very important to plant breeding, due to their numerous applications in mutation research, selection, genome sequencing, genetic analysis, and transformation (Lacuzzi et al. 2019). *Source: https://www.researchgate.net/publication/262572007
  • 94. In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid * Source: https://www.researchgate.net/publication/262572007 Anther culture of sweet orange (Citrus sinensis Osbeck.) genotypes a Bud flower size for anther culture b Transverse section of a cultured anther showing swollen tissue, the parietal layers (pl) and microspores (mi) during in vitro culture; c Non-responsive (nr), swollen (sw), cream-colored callus (cc) and white- greenish callus (gc); Embryogenic callus induction d Proliferation e & f From anther culture of C. clementine × C. sinensis ‘Hamlin’ Cardoso (2014) reported that anther cultures of the hybrid C. clementina × C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture.
  • 95. Embryo rescue The difficulty of recovering triploid hybrids from interploid hybridization or diploid × diploid hybridization (due to an unbalanced ploidy ratio between embryo and endosperm as well as polyembryony) should be overcome by in vitro embryo rescue. In vitro culture of excised fully developed embryos was described 40 years ago to obtain zygotic plantlets from polyembryonic cultivars. *Source: Maheshwari and Ranga Swamy, 1958
  • 96. Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x X 2x sexual hybridisation and its application to extensive breeding programs. • Citrus triploid plants can be recovered by 2x X 2x sexual hybridisations by in vitro embryo rescue and ploidy level determination by means of flow cytometry. • The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed (Aleza et al. 2010). • The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. • The methodology helped to obtain over 4,000 triploid hybrids & these triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents. * Source: https://www.ncbi.nlm.nih.gov/pubmed/20607244
  • 97. Production of three new grapefruit cybrids with potential for improved citrus canker resistance ■ Omar et al. 2017 by using a cybridization approach, several putative cybrids were created by protoplast fusion of embryogenic suspension culture-derived protoplasts of canker resistant ‘Meiwa’ kumquat (Citrus japonica Thunb), with mesophyll-derived protoplasts of three grapefruit (Citrus paradisi Macfad.) cultivars ‘Marsh,’ ‘Flame,’ and ‘N11-11’ somaclone of ‘Ruby Red.’ ■ In an effort to generate new grapefruit cultivars with enhanced canker resistance, putative cybrid grapefruit plants morphologically equivalent to standard grapefruit from all three combinations were produced. ■ EST-SSR marker analysis confirmed that the nuclear genome in all the generated cybrids came from the grapefruit parent. ■ All the cybrid clones have been propagated and are undergoing extensive canker assays to identify any clones that have improved canker tolerance/resistance. ■ These cybrid populations provide a valuable tool for investigating the contribution of cytoplasmic organelles to plant disease resistance. *Source: https://link.springer.com/article/10.1007/s11627-017-9816-7
  • 98. Cryopreservation of Citrus anthers in the National Crop Gene bank of China ■ Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. ■ Zhang et al. 2017 conducted study to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. ■ The highest viability of 93% was obtained for C. cavaleriei. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers. *Source: https://link.springer.com/article/10.1007/s11627-017-9848-z
  • 100. Linkage maps ■ Genetic linkage maps are a statistical description of the organization of DNA segments into chromosomes and the order and recombination frequency of DNA segments within chromosomes. ■ Maps can include both DNA markers and trait genes that are detected by affecting the phenotype of an individual. ■ A good map should include one linkage group that corresponds to each chromosome of the genome (nine in Citrus and closely related genera). ■ Each linkage group will be comprised of a linearly ordered set of markers and/or trait genes, with distances between adjacent markers determined by the proportion of gametes that have a crossover event between the markers. ■ For citrus breeders, the objective of mapping is to identify markers that can be used in marker- assisted selection (MAS). ■ MAS involves identification of DNA markers that are near a variable trait gene, and selection of progeny carrying markers that predict which progeny will carry the desired trait gene allele. *Source: Citrus by I Khan
  • 101. Characteristics of some common types of DNA markers ■ AFLP Amplified fragment length polymorphism- PCR based, gel analysis, multilocus, sequence anonymous. ■ CAPS Cleaved amplified polymorphic sequence – PCR followed by restriction enzyme digestion, gel analysis, single locus per gel, sequence known, can target genes. ■ RAPD Random amplified polymorphic DNA – PCR based, gel analysis, single to a few loci per gel, sequence anonymous. ■ RFLP Restriction fragment length polymorphism – Restriction enzyme and gel based, single to a few loci per gel, can target genes. ■ ISSR Inter-simple sequence repeat – PCR based, gel analysis, multilocus, sequence anonymous. ■ SNP Single nucleotide polymorphism – PCR based, gel, plate or chip analysis; single locus, but multiplexing possible, sequence known, can target genes. ■ STS Sequence-tagged site – PCR based, gel analysis, single locus, sequence known. ■ SSR Simple sequence repeat – PCR based, gel analysis, single locus, sequence known, can target some genes. ■ The method of detection of the DNA sequence (e.g. PCR) is listed, the method for distinguishing alleles (e.g. gel analysis), whether the method generally detects one or several to many marker loci in a single analysis, whether the DNA sequences detected are known or anonymous, and whether the method can target specific genes.
  • 102. Relationship between DNA sequence differences between two alleles and banding patterns observed for five types of gel-based marker systems DNA Marker types
  • 103. Examples of marker variation for five types of DNA marker systems • The RFLP and RAPD markers were separated on agarose gels. • The SSR and AFLP patterns were analysed on an automated DNA analyser. • The ISSRs were analysed on a silver-stained polyacrylamide gel and show patterns in seedlings from different source trees, not a single segregating population. *Source: Citrus by I Khan
  • 104. Summery of linkage maps of citrus *Source: Modified from Roose et al. 2000
  • 105. eQTL analysis ■ A SNP is an eQTL if it is associated with an allelic difference in mRNA levels of a gene. ■ For an eQTL gene under investigation, a corresponding eQTL SNP occurs on the top of a peak in a regional association plot, i.e. has a low association p-value compared to other SNPs nearby. The goal of eQTL is to identify genomic locations where genotype significantly affects gene expression.
  • 106. QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease Map, linkage group (LG), flanking markers, position of the QTL in centiMorgans (cM position), LOD score, proportion of phenotypic variation (R2) explained by QTL in % and additive effect by the QTLs mapping Distribution of the number of eQTLs detected for each candidate gene in relation to the Poncirus trifoliate & Citrus sunki maps eQTL are genomic loci that explain all or a fraction of variation in expression levels of mRNAs. * Source: https://www.researchgate.net/publication/326162981 Lima et al. (2018) conducted first study to use eQTLs mapping in the Phytophthora-citrus interaction & concluded that the resistance of some citrandarins to the infection by P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both parents.
  • 107.  Heatmap of the gene expression profile by clustering analyses between 19 evaluated target genes with 55 selected genotypes (51 hybrids, their two parents, Rangpur lime and Swingle citrumelo), allowed the grouping of genes and related genotypes, using the Pearson correlation as metric distance to obtain the best intra and intervariable grouping possible.  The genotypes were separated into five secondary clusters distributed in two main clusters, while the genes in three secondary clusters arranged in two main clusters.  The color scale representing the log2Fold change values showed that the gene expression data ranged from − 8.29 to 5.53. Conti..
  • 108. Gene tagging Gene tagging is one of the most useful methods to identify and isolate specific genetic loci of an organism.
  • 109. In vitro selection of resistant/tolerant mutants lines of Citrus jambhiri Lush. using crude culture filtrate of Phytophthora parasitica and their randomly amplified polymorphic DNA analysis ■ Pati et al. (2017) reported in vitro -induced mutagenesis and selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. ■ In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in comparison with the control plant, which confirms the presence of variations at genetic level. * Source: https://www.researchgate.net/publication/319407543
  • 110. Identification and Parentage Analysis of Citrus Cultivars Developed in Japan by CAPS Markers ■ Nonaka et al. (2016) developed a method of cultivar identification based on cleaved amplified polymorphic sequence (CAPS) markers, to protect the rights of breeders of the major citrus cultivars developed under breeding programs by the national institute of Japan and to evaluate identity and parentage of cultivars. ■ They selected 19 CAPS markers that had a single-locus origin and moderate polymorphism, and used them to construct genotyping data for 59 citrus cultivars (including American accessions), local varieties, and selections. Of the 19 CAPS markers, 8 were sufficient to discriminate among all accessions except ‘Mato’ buntan (Citrus grandis Osbeck) and ‘Hirado’ buntan (Citrus grandis Osbeck). ■ Among the 33 Japanese cultivars, the parentage of 30 agreed with that reported, but ‘Setoka’, ‘Southern Red’, and ‘Reikou’ had discrepancies at one or more loci. ■ Using 15 to 18 CAPS markers to validate the putative parentage revealed that the seed parent of ‘Setoka’ was ‘KyEn No. 4’, not ‘Tsunonozomi’, and the pollen parent of ‘Southern Red’ was ‘Osceola’, not ponkan (C. reticulate Blanco). The seed parent of ‘Reikou’ remains unknown. *Source: https://www.jstage.jst.go.jp/article/hortj/86/2/86_OKD-026/_html/-char/en
  • 111.
  • 112. Genetic diversity and DNA fingerprinting of indigenous and exotic mandarin genotypes in India using SSR markers ■ Singh et al. (2016) determined genetic variability and fingerprint profiles of 19 indigenous and exotic mandarin genotypes introduced from different parts of India and USA using 60 SSR markers. ■ Of the 57 SSR markers amplified, a total of 96 alleles were detected by 39 polymorphic SSR loci and maximum 5 alleles were amplified with an average of 2.46 alleles per primer pair. ■ The CAT01 was the highly informative marker as it revealed maximum number of alleles (5), PIC value (0.75) and genetic diversity (0.79). ■ The higher average expected heterozygosity (35.6%) with in a mandarin group as compared to the average observed heterozygosity (27.2%) may be explained by selfing, which reduced the proportion of heterozygotes. ■ The genotypes were classified in three clusters i.e. cluster- I, cluster- -II and cluster -III. All the indigenous genotypes (selections) were grouped in cluster -I and it had maximum genetic similarity coefficient. However, the exotic genotypes (hybrids) were grouped in cluster- II and cluster- II. Clustering was according to the breeding history of genotypes but independent to their geographic origin. The low observed heterozygosity frequency, PIC value, and number of alleles explained the narrow genetic base in the present set of mandarin genotypes. *Source: https://www.researchgate.net/publication/317717144
  • 113. Dendrogram illustrating genetic relationship among 19 mandarin genotypes generated by UPGMA tree analysis. The cluster I was sub-divided into two sub cluster IA with eleven genotypes (N-38, Khasi, Clone 11, N-34, N-43,N-4, N-28,CRS-4,N-51,Nagpur Seedless and Nagpur) and IB with three genotypes (Coorg, Darjiling, and Mudhkher) respectively. In cluster II, three genotypes (Nova, Kinnow and Fremont) were clustered and cluster III comprised of two genotype (W. Murcutt and Daisy).
  • 114. Genetic Diversity and Population Structure Analysis of Citrus Germplasm with Single Nucleotide Polymorphism Markers ■ Yu et al. 2017 conducted research trial to determine their SNP fingerprints and to assess genetic diversity, population structure, and phylogenetic relationships, and thereby to test the efficiency of using the single genotype-derived SNP chip with relatively low cost for these analyses, on a collection of 80 citrus accessions. ■ A model-based clustering program detected five basic groups and revealed that C. maxima introgressions varied among mandarin cultivars and segregated in mandarin F1 progeny. ■ In addition, reciprocal differences in C. maxima contributions were observed among citranges and may be caused by the influence of cytoplasmic DNA and its effect on selection of cultivars. ■ The relatively inexpensive SNP array used in this study generated informative genotyping data and led to good consensus and correlations with previously published observations based on whole genome sequencing (WGS) data. ■ The genotyping data and the phylogenetic results may facilitate further exploitation of interesting genotypes in the collection and additional understanding of phylogenetic relationships in citrus. *Source: https://www.researchgate.net/publication/329333504
  • 115. Dendrogram of 80 Citrus and Citrus relatives based on identity by state (IBS) calculated from the data of 1366 genome-wide single nucleotide polymorphism markers. IBS is the allelic similarity between two individuals at the given loci regardless of their common ancestry, via computing the probability of two individuals sharing the same copy of an allele. The cluster analysis is performed using a matrix of IBS pairwise distances, and groups are determined by a permutation test.
  • 117. Genetic Transformation ■ Genetic transformation involves the integration of gene into genome by means other than fusion of gametes or somatic cells. The foreign gene (termed the "transgene") is incorporated into the host plant genome and stably inherited through future generations. ■ This plant transformation approach is being used to generate plant processing trails, unachievable by conventional plant breeding, especially in case where there is no source of the desired trait in the gene pool.
  • 118. The Genetic Transformation of Sweet Orange (Citrus sinensis L. Osbeck) for Enhanced Resistance to Citrus Canker: Methods and Protocols ■ Developing disease resistance is one of the most important components of any plant breeding program. ■ Citrus traditional breeding methods (bud sport selection, crossbreeding, and other breeding channels) are a laborious task and often hampered by long juvenility, a high degree of heterozygosity, polyembryony, self-incompatibility, and abortion of reproductive organs. ■ An interesting alternative to the classical breeding approach is the use of genetic transformation, which provides the means for adding a single agronomic trait to a plant without otherwise altering its phenotype. ■ Sendin and Filippone (2019) carried out Agrobacterium tumefaciens-mediated transformation with numerous hybrids and citrus species & introduced the Bs2 gene in Citrus, as well as to increase citrus canker resistance in transgenic Bs2 gene-expressing lines. *Source: https://www.researchgate.net/publication/328874799
  • 119. Efficient genetic transformation of sour orange, Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus ■ Ghaderi et al. 2018 conducted research, in which hypocotyl, and epicotyls parts of C. aurantium plant were inoculated with A. tumefaciens strain EHA- 105. ■ Then, explants were transferred into the regeneration medium and shoot growth containing various concentrations of kinetin and IAA growth regulators. In order to detect transgenic shoots, herbicide Basta (ammonium glufosinate) was added at a rate of 1 mg/L to the regeneration medium. Production buds happened in explants which Marker BAR gene resistant to Basta herbicides was received. After PCR, to approve the final transgenic plants, transformation efficiency was evaluated by 9.16%. *Source: https://doi.org/10.1016/j.plgene.2018.02.00 Regeneration of explants treated with hormones showed a significant difference at 1% compared with control explants. The Maximum regeneration was showed in 0.5 mg/L kinetin treatment. These results indicated that the combination between kinetin and IAA could be effective in increasing regeneration. Regeneration of transgenic explants
  • 120. Novel Plastid-Nuclear Genome Combinations Enhance Resistance to Citrus Canker in Cybrid Grapefruit ■ Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram- negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been identified. ■ Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from different species, was used to create cybrids between citrus canker resistant ‘Meiwa’ kumquat (Fortunella crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars. ■ Murata et al. 2019 reported that, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the grapefruit controls. ■ The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and ‘Marsh’ grapefruit had different gene expression profiles. ■ These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the question of whether other alien chloroplast genotypes would condition similar results. *Source: https://www.researchgate.net/publication/329541592
  • 121. Gene expression overview of ‘Marsh’ grapefruit, M-102 cybrid (Cybrid-G), M-9 cybrid (Cybrid-K) and ‘Meiwa’ kumquat in response to Xanthomonas citri subsp. citri infection. Green arrows indicate upregulation and orange arrows indicate downregulation. Conti…
  • 122. Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice ■ Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. ■ Omar et al. 2018 made transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. ■ Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance. *Source: https://www.researchgate.net/publication/323180663
  • 123. Sweet orange genetic transformation with the attacin A gene under the control of phloem-specific promoters and inoculation with Candidatus liberibacter asiaticus ■ An alternative approach to control Huanglongbing disease (Citrus degreening: Causal organisms is Candidatus liberibacter asiaticus) in Citrus is the development of transgenic plants expressing genes that may influence pathogen development. ■ Tavano et al. 2018 reported production of sweet orange transgenic plants bearing gene constructs containing the antimicrobial gene attacin A (attA) under the control of phloem-specific promoters. ■ The non-transgenic inoculated plants showed a significant reduction in shoot development comparing to transgenic inoculated plants indicating that the expression of attA gene may influence the plant tolerance to HLB disease. * Source : https://www.researchgate.net/publication/327002860
  • 125. CRISPR-mediated technologies suitable for breeding non-transgenic HLB resistance citrus *Source: Gmitter et al. 2015 CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is an integral part of a bacterial defense system. It is also the basis of the CRISPR-Cas9 system that uses site-directed nucleases to target and modify DNA with great accuracy.
  • 126. Approach to create Non-transgenic HLB Resistance Citrus Cultivars Albino mutants of citrus with CRISPR
  • 128. Complete Cholorplast Genome Sequence of Omani Lime (Citrus aurantifolia) & Comparative Analysis within the Rosids * Source: Huei-Jiun Su et al. 2014
  • 130. Resistance to Abiotic Stress Citrus genotypes Vector/method Gene Result/remarks References Rough lemon A. tumefaciens HAL2 Salt tolerance Ali et al.,2012 Swingle citrumelo A. tumefaciens P5CS F129A Drought resistant Campos et al., 2011 Carrizo citrange A. tumefaciens P5CS Drought resistant Molinari et al., 2004 Carrizo citrange A. tumefaciens HAL 2 Salt tolerance Cervera et al., 2000
  • 132. Disease Resistance Citrus genotype Gene Remarks Reference Citrus jambhiri Beta-1-3- glucanase Phytophtora Sandhu et al., 2016 Hamlin SABP2 Citrus canker Attílio et al., 2014 ‘Anliucheng’ sweet orange Xa21 Citrus canker Ding et al., 2013 Hamlin, Natal, Pera and Valencia Xa21 Citrus canker Mendes et al., 2010 Duncan and Hamlin NPR1 Citrus canker Zhang et al., 2010 Hamlin Rice Xa21 Citrus canker Ahmad 2006 Hamlin Attacin A gene Citrus canker Boscariol et al., 2006 Carrizo citrange Expansins related P12 Citrus blight Kayim et al., 2004
  • 133. Future Goals ■ Collection & evaluation of germplasm from indigenous & exotic sources for desired horticultural traits & processing qualities. ■ Introduction & evaluation of indigenous & exotic rootstocks. ■ Improvement of acid lime & lemon by interspecific hybridization. ■ Linkage mapping. ■ Establishment of in vitro gene banks.