Citrus constitutes a major group of fruits comprising of mandarins, oranges, lemon, pummelo, grape fruit, tangelo, trifoliate orange, citron, citranges etc. Citrus fruits have important position among other popular fruits of the world. It is one of the popular fruit crops of both tropical and subtropical region of the world due to its wide adaptability to different climatic conditions. Of all Citrus species, there are numerous cultivars and hybrids. A wide range of variability exist in citrus with respect to fruit morphology and quality. The smallest fruits are the limes, which scarcely exceeds 30 cm, while pummelo may attain a diameter of 30 cm. Fruit and rind colour ranges from the yellow-green of the limes to the red orange of some mandarins and shape varies from oblate to pyriform. At maturity, fruits of some cultivars are high in acid while others have almost none. Tree size also exhibits tremendous range. Altogether there is a tremendous amount of variability within the genus with which the Breeder can work and closely related genera provide even a wider selection of characters. Several man-made and natural hybrids are now available for use in breeding programmes as parents. Problems in citrus breeding: 1. Incompatibility 2. Nucellar embryony 3. Juvenility Breeding objectives:  Producing early maturing citrus fruits with high yield and fruit quality.  To breed varieties suitable for different agro-ecological region.  To develop varieties having less tendency of granulation.  To breed varieties suitable for export.  In rootstock breeding, the main emphasis has been given on the development of root stock resistant to tristeza virus, Phytophthora, nematodes, etc.  Most of the breeding programmes make use of Poncirus, which is a carrier of resistance to tristeza, Phytophthora and nematodes besides cold hardiness.  Salt tolerant rootstocks have also been found possible in some progenies involving Cleopatra and Sunki mandarin and Rangpur lime. Breeding Methods: 1. Introduction 2. Clonal Selection 3. Hybridization 4. Mutation breeding 5. Polyploidy breeding Shoot tip Grafting : Shoot Tip Grafting (STG) or Micrografting is an in vitro technique which involves the placement of a meristem or shoot tip explants on to a decapitated rootstock that has been grown aseptically from seed or micropropagated cultures. (Hartmann et al., 2002). In Citrus, STG was first developed by Murashige et al. (1972) and later improved by Navarro et al. in 1975. This technique was further improved in 1975 by Navarro, Roistacher and Murashige where they tested various media formulas, diffèrent ways of placement of scion tip, different rootstocks, light intensities and size of scion on graft success. STG potentially can combine the advantages of rapid in vitro multiplication with increased productivity that results from grafting, superior rootstock and scion combinations (Zilka et al., 2002).

1. By
Mr. Sanjay Chetry
Ph.D. Scholar
Fruit Science
ID: 2020632001
Department of Fruit Science
Horticultural College and Research Institute
Tamil Nadu Agricultural University,
Coimbatore

2. ADVANCES IN
CITRUS BREEDING
TERM PAPER
ON
Course: FSC-601-Advances in breeding of
fruit crops (2+1)

3. 3
Common Name: Citrus/Micro-nutrient loving plant/Fruit of the
Godly tree/Buddha’s hand fruit
Scientific Name: Citrus spp.
Chromosome Number: 2n=2x=18
Origin: South-East Asia
Order: Sapindales
Family: Rutaceae
Sub family: Aurantioideae
Inflorescence: Cymose
Fruit type: Hesperidium
Edible part: Juicy placental hairs
Botanical classification of Citrus

4. Common Name Scientific Name
Mandarin Citrus reticulata
Sweet Orange Citrus sinensis
Acid lime Citrus aurantifolia
Lemon Citrus limon
Pummelo Citrus grandis
Grapefruit Citrus paradisi
Rangpur lime Citrus limonia
Tahiti lime Citrus latifolia
Sweet lime Citrus limettoides
Sour orange Citrus aurantium
Citron Citrus medica
Indian wild orange Citrus indica
Different species of Citrus

5. Origin and Distribution
 Centre of origin is South East Asia bordered by North Eastern India, Myanmar
(Burma) and Yunnan province of China.
 It is believed that about 78 species under the family rutaceae have their origin in
India.
 North Eastern region is considered as natural home and place of origin of a
number of species.
 Different strains of Citrus medica, Citrus aurantium, C. indica, C. ichangensis,
C. macroptera and C. latipes are found to be grown wild in NE region.
 From China, India and West indies, it dispersed all over the world and now, it is
grown almost throughout the world.
 Introduction and spread of Citrus species to new world started during beginning
of Christian era.
 Voyagers, Prortuguese and Spanish traders, travellers and missionaries
spreaded Citrus to different places.
 By the end of 15th century, many Citrus spp. had reached almost all tropical and
sub-tropical countries in the world.

6. Citrus Growing Countries
Major Citrus Producing Countries are
⁕ Spain
⁕ USA
⁕ Israel
⁕ Morocco
⁕ South Africa
⁕ India
⁕ Japan
⁕ Brazil
⁕ Turkey
⁕ Cuba

7. Citrus growing states in India
1.Andhra Pradesh
2.Maharashtra
3.Punjab
4.Madhya Pradesh
5.Gujarat
6.Rajasthan
7.Karnataka
8.Orissa
9.Bihar
10.Assam

8. Country Citrus species
USA C. paradisi, C. grandis
China C. reticulata
Brazil C. sinensis
India C. aurantifolia
Italy C. limon
Largest producer of Citrus spp. in the world
Mandarin Sweet orange Grapefruit Pummelo
Lemon Acid lime Citron

9. Origin Citrus species
China C. reticulata, C. sinensis, Poncirus and Fortunella
India C. aurantifolia, C. limon, C. medica, C. indica
South-East Asia C. limon, C. grandis
West Indies C. paradisi

10. Closely related genera are: Citrus, Fortunella and Poncirus.
TAXONOMY
 Family: Rutaceae (7 subfamilies have been recognized).
 The orange subfamily Aurantioideae is of main concern, because it
includes citrus and its nearer relatives represented by 28 genera in the
tribe Citreae.
 6 of these genera (Citrus, Eremocitrus, Poncirus, Fortunella, Clymenia
and Microcitrus) comprise the true citrus fruits.
Poncirus, a monotype genus occurs naturally in Central and Northern
China, deciduous and highly resistant to cold.
Fortunella (from Southern China) is evergreen and hardiness to a
prolonged winter dormancy.
Eremocitrus (from Australia) is highly drought tolerant.

11. Classification of Citrus given by Swingle and Tanaka is noteworthy
Classification of Citrus is quite complicated and confused.
This is because the large number of cultivars and hybrids, polyploids,
mutations and polyembryony exist in nature with wide variability.
Classification by Swingle and Tanaka
Swingle recognized 16 species
under genus Citrus with 2 sub
genera i.e., Eucitrus (10 species)
and Papeda (6 species).
Tanaka described as many as
144 species with 2 sub genera
i.e., Archicitrus (98 species) and
Metacitrus (46 species)

12. Classification of Swingle (2 subgenera; 16 species
Eucitrus (10 spp)
1. C. medica
2. C. limon
3. C. aurantifolia
4. C. aurantium
5. C. sinensis
6. C. reticulata
7. C. grandis
8. C. paradisi
9. C. indica
10. C. tachibana
Fruits are edible and widely
cultivated
Papeda (6 spp)
1. C. ichangensis
2. C. latipes
3. C. micrantha
4. C. celebica
5. C. macroptera
6. C. hystrix
Fruits are inedible and presence
of numerous droplets and aerid
oil in juice sacs.

13. Classification of Tanaka (2 subgenera; 144 species)
Archicitrus (5 sections, 98 spp)
1. Papeda (12 spp)
2. Limonellus (16 spp)
3. Citrophorum (21 spp)
4. Aruntium (28 spp)
5. Cephalocitrus (21 spp)
Metacitrus (3 sections, 46 spp)
1. Osmocitrus (9 spp)
2. Acrumen (36 spp)
3. Pseudofortunella (1 spp)

14. EVOLUTIONARY MECHANISM
Wu et al. 2018

15. TBM (Tachibana Mandarin)
SCM (Sun Chu Sha Kat)
CLP (Cleopatra Mandarin)
SNK (Sunki Mandarin)
HLM (Huanglingmiao Mandarin)
CSM (Changsha Mandarin)
DNC (Dancy Mandarin)
WLM (Willowleaf Mandarin)
PKM (Ponkan Mandarin)
CLM (Clementine Mandarin)
WMM (W. Murcott Mandarin)
UNS (Satsuma Mandarin)
KNG (King Mandarin)
SO5 (Ambersweet orange)
SWO (Sweet Orange)
SSO (Seville sour orange)
BO2 (Chinese Sour Orange)
BO3 (Chinese Sour Orange)
GF0 (Cocktail Grapefruit)
PAR (Marsh Grapefruit)
LIM (Eureka Lemon)
LMA (Rangpur Lime)
RRL (Red rough Lemon)
MXL (Mexican Lime)
CHP (Chandler Pummelo)
STP (Shatian Pummelo)
LAP (Low-acid Pummelo)
GXP (Guanxi Pummelo)
BUD (Buddha’s Hand Citron)
COR (Corsica Citron)
HUM (Humpang Citron)
VEU (Mac Veu Mountain citron)
CAL (Calamondin)
FOR (Kumquat)
MIC (Micrantha)
M Series (Chinese Mandarin)

16. 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.

17.  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.
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

18. Cytogenetics
 The branch of genetics which deals with the study of cell and structure and
function of the chromosomes is called cytogenetics
 Studies of cellular aspects of heredity (chromosomes)

19. Chemotaxonomy

20. Segregation of character in citrus progenies indicate the action of one or
few genes (Soost and Cameron, 1975)
Inheritance Pattern
Character Inheritance
Leaf character Two principal genes
Anthocyanin pigment (Leaf) Dominant gene controlling reddish colour
Anthocyanin pigment (Fruit) More than one recessive genes
Resistance to Tristeza virus Dominant allele at a single locus
Texture Polygenic control
Polyembryony (recessive character) More than one single gene
Seedless character A recessive gene
Nucellar embryony One or two dominant gene
Small fruit size, seediness and paleness
of colour (undesirable characters)
Dominant gene

21. Centre of diversity
North-
East
South India
North-
Western
region

22. During 1988 as a result of systematic exploration by NBPGR in
North-Eastern region, C. Indica and many endangered species were
collected for conservation.
Germplasm resources
Exotic collection of Citrus germplasm was started in 1940.
Kinnow mandarin is now a leading cultivar in North-Western India.
Besides, other exotic collections: Valencia Late, Washington Navel,
Jaffa, Malta Blood Red, Pineapple, Ruby orange, Satsuma, Dancy
Tangerine, Clementine and Cleopatra, Wilking ,Temple, Duncan,
Marsh seedless, Lisbon lemon, Trifoliate orange, Dancy,
(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.

23. •North-Eastern region is a hunting
ground of biodiversity of Citrus species.
•Chakrawar et.al., (1988) identified two
promising clones of acid lime Vikram and
Pramalini in Maharashtra.
•At Nagpur, seedless Santra has been
selected which gives high yield and
quality fruits (Anon., 1989)
•Attempt has been made during 1978 by
NBPGR to preserve the C. indica which is
progenitor of C.reticulata (Singh, 1981).
•Natural genetic diversity of C .indica was observed in the forest of Garo Hills in
Meghalaya which exhibited plant characters varying from bush to climber with high
frequency of distribution in dense forest and showing resistance to biotic stresses.
•The gene sanctuary for conservation of Indian Wild Orange (Citrus indica) was
established by NBPGR at Garo Hills, Meghalaya in 1978.

24. NOKREK BIOSPHERE RESERVE, GARO HILLS,
MEGHALAYA

25. Indian wild orange (Citrus indica)

26. Diversity of Citrus indica

27. Problems in Citrus
Breeding

28. JUVENILITY
Citrus being perennial in nature takes more time for
bearing.
The long period from seed to fruiting remains a major barrier to
rapid progress in Citrus breeding
However, this period can be reduced to a maximum of half by top
working the seedling on an old tree.
Eg: When grapefruit (C. paradisi) or pummelo (C. grandis) is used as a parent,
the resultant offsprings (hybrids) appear to be precocious.

29. Monoembryonic spp: C. grandis, C. medica, C.
reticulata var Nagpur
Santara, C. latifolia
When cross is made, the breeder gets seeds
possessing both zygotic as well as nucellar (true to
type) embryos.
Nucellar
Embryony
Nucellar embryony has been a major obstacle in
Citrus breeding.
Cultivars that produce nucellar seedlings also
occasionally produce zygotic twins, but typical
monoembryonic cvs. have not been shown to produce
nucellar seedlings under neutral conditions. (Soost and
Cameron 1975)

30. Self
incompatibility
Both self and cross incompatibility widely occur in Citrus
All tested cvs. of C. grandis have been self incompatible.
Some cvs. of C.limon, C. sinensis and C. reticulata exhibit self
incompatibility
Satsuma Mandarin, Washington Navel orange are mostly pollen
sterile and if not cross pollinated with viable pollen, they yield
parthenocarpic fruit.
Many of the commercial cultivars are practically seedless (2 to 4
seeds) due to low pollen viability and/or to high degree of ovule
sterility.
If the breeder is utilizing SI parents, he/she needs to be aware that lack of
fruiting in some individuals may result.
Self incompatibility refers to the failure of pollen from a flower to
fertilize the same flower or other flowers on the same plants.

31. Citrus fruits are usually grown by budding scions on rootstocks.
Rootstocks greatly influences the performance of scion by modifying its tree
morphology and imparting resistance to biotic and abiotic stresses.
Thus, breeding programmes have been carried on for both scions and
rootstocks.
BREEDING
OBJECTIVES

32. OBJECTIV
ES
SCION ROOTSOCK
Seedlessness
High productivity
Sensory fruit quality (loose peel,
pigments)
Quality (TSS/acid ratio, juice
content, flavour and colour)
Wide season of ripening and good
post harvest life
Resistance to adverse climatic and
soil conditions.
Better stock-scion compatibility,
reduction of tree size without
affecting yield.
Resistance to pests and diseases or
hardiness to adverse climatic
conditions.
Resistance to nematodes.
Must produce many seeds and be
highly nucellar in order to provide
uniformity in mass multiplication.
Cold, salinity and drought
tolerance.

33. Floral biology
Flowering in citrus takes place during
February-April.
In North India, sweet orange and mandarins
bloom only once in March.
However, it is reported that sweet oranges
bloom twice in a year under Bihar conditions
i.e., February-March and June-July.
Inflorescence in citrus species is of cymose
type
Generally anthesis takes place in the morning
between 9.00 am to 12.00 noon.

34. Flowers on shaded side of the tree have been observed to
open later than those exposed to sunshine.
Dehiscence of anther take place at different time viz. in
lemon before anthesis and in sweet orange, sweet lime etc.
after anthesis.
Oozing of gummy substances on the stigmatic surface is
indication of stigma receptivity.
In various citrus varieties receptivity starts 2-3 days earlier
to anthesis and last four days after anthesis with maximum
receptivity on the day of anthesis.
In citrus both staminate and hermaphrodite flowers are
produced on the same plant.

35. Breeding Approaches and
Achievements

36. General Outline of Breeding
Methods

37. BREEDING METHODS
1. Introduction
2. Clonal Selection
3. Hybridization
4. Mutation
breeding
5. Polyploidy
breeding

38. Introduction has been one of the most potent improvement
methods.
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 18th century.
Tangerines, St. Michael Blood Orange and Large White Orange
were imported and cultivated at Goojranwallah in Punjab
during 1880.
Introduction
Plant introduction pertains to taking a genotype or a group of genotypes into a
new environment, where they were not being grown before.

39.  Lemons from China
 Malta from USA and Italy
 Grapefruits were introduced from California and Florida
 Washington Navel and Valencia were introduced from America
 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.

40. ICAR-CCRI

41. ICAR-CCRI

42. ⁂Exploitation of natural variability existing in a variety has resulted in the
isolation of some promising clones in Citrus.
⁂PKM 1 lime is a clonal selection from seedling progenies of Kadayam
type of Tirunelveli district of Tamil Nadu.
⁂Yuvaraj Blood Red is a seedless and early maturing clonal selection
from `Blood Red' orange.
⁂Pramalini and `Vikaram, the two kagzi lime varieties were developed
through clonal selection at Marathwada University.
⁂Chakradhar is a thorn less and seedless selection from Kagzi lime.
Clonal selection
A clone is a group of plants exclusively from a single individual plant
through asexual reproduction.
Clonal selection involves:
i. Collection of clonal variability.
ii. Evaluation of clone w.r.t yield, quality and biotic stress resistance.

43. TAHITI LIME (Citrus latifolia)
Variety: VRM 1
Released by: Agricultural Research Station, Virinjipuram, Tamil Nadu
Year of release : 2016
Suitable for home garden
Suitable for preparation of juice and pickles
High Vitamin ‘C’ (96 mg/100 ml)
More ascorbic acid and less acidity than local lime.
Yield potential of 25-30 tonnes per hectare.
Resistant to Leaf miner and Citrus canker

44. HYBRIDIZATION
When crossing is done between different varieties of same species
is known as inter-varietal cross or intra-specific cross.
When a crossing is done between two parents within the same
variety, it is known as intra-varietal cross.
When crossing is done between two different genus, it is known as
inter-generic cross
Crossing of any two genetically unlike individuals
In plants, crossing is done by placing pollen grains from one genotype (male parent)
onto the stigma of flowers of other genotype (female parent)

45. •The mature flower buds on the female parent are emasculated
early in the morning on the day of opening and are bagged.
•The flowers to be used as male parent are bagged the previous
evening.
•The next morning as the day warms up, the anthers dehisce
releasing the pollen grains when these flowers can be plucked to
pollinate the receptive stigmas of emasculated flowers.
•The pollinated flowers are bagged, opened after about a week
and allowed to mature into ripe fruits.
Hybridization Technique

46. Note: When Trifoliate orange is used as male
parent, difficulties are encountered as its
flowering is over before other citrus
varieties flower. Therefore, pollen has to
be stored at low humidity and
temperature.

48. Evaluation for rootstock and scion
Rootstock Scion
 Rootstock hybrids should have
desirable attributes like high
percentage of nucellar embryony,
resistance to different diseases and
nematodes.
 The selected hybrids are then tested
with different scion varieties and
compared with the commercial
rootstock.
 Various plant and fruit characters,
yield and yield contributing characters
are recorded.
 In the first round of evaluation, the
zygotic seedlings are raised on
suitable rootstock and observations on
different vegetative and fruit
characters are recorded.
 Meanwhile, the resistance to different
diseases is also confirmed.
 Selected hybrids are tested on
different rootstocks at different
locations and compared with the
commercial varieties.

49. Citrus intergeneric and intrageneric hybrids
Hybrids Parents
Intrageneric (same genus different species)
Tangor C. reticulata x C. sinensis
Tangelo C. reticulata x C. paradisi
Lemonine C. limon x C. aurantifolia
Lemonnage C. limon x C. sinensis
Lemandarin C. limon x C. reticulata
Intergeneric ( different genus)
Citrange P. trifoliata x C. sinensis
Citrangequat Poncirus x Citrus x Fortunella (Trigeneric)
Citrangedin Citrange x Calamondin
Citrangor Citrange x C. sinensis
Cicitrange Citrange x P. trifoliata
Citrumelo P. trifoliata x C. paradisi
Citremon P. trifoliata x C. limon
Citrandarin P. Trifoliata x C. reticulata
Citradia P. Trifoliata x C. aurantium
Citrumquat P. Trifoliata x F. japonica

51. WILKING
KINCY
GOLDEN NUGGET
Diploid hybrid released in 1999
Seedless, late maturing hybrid
(February-March), on tree
storage life for 4-6 months

58. King Willow leaf
Kinnow
Kinnow Mandarin was developed by H.B. Frost [University of California]
Kinnow was developed in 1915 and released for commercial cultivation in
USA during 1935 and introduced in India during the year 1959

59. Spontaneous mutation occurs frequently in Citrus.
Mutations are often observed as limb sport or sectors on fruits; they are also detected
occasionally in nucellar seedlings or their budded progeny (Soost and Cameron 1975)
Washington Navel (Orange), Marsh and Thompson (Grapefruit) are few examples of
spontaneous mutation.
Budwood and seeds are treated for short period with gamma irradiation to induce
mutations without killing the cells.
Seeds are more tolerant to gamma irradiation than buds.
Star Ruby (Grapefruit)- developed through seed irradiation. Selected from radiated seeds
of Hudson in 1970 for its extremely deep red flesh colour.
Mutation Breeding
Sudden heritable change in characteristic of the plants is termed as mutation. It
may be spontaneous (without any treatment by man) or induced (artificially
induced by a treatment with certain physical or chemical agents) in plant
population

60. Cultivars developed due to mutation in Citrus
Crop Original variety Mutant cultivar Nature of
mutation
Navel Orange Bahia Baianinha Limb sport
Washington
Navelina, Navelate, Marrs,
Leng, Autumn Gold,
Powell Summer,
Weller Red
Limb sport
Mandarin Owari Clausellina Bud sport
Pongan Pongan 86-1 Bud sport
Grapefruit Walters (White fleshed) Foster (pink fleshed) Limb sport
Foster (Pink fleshed) Hudson (deep red fleshed) Bud sport
Hudson (deep red flesh) Star ruby (deepest red peel) Limb sport
Thompson (light pink flesh
and peel)
Red blush (deep red flesh) Limb sport
Henderson Flame Bud sport
Pummelo A local cv. Wuheputaoyou Bud sport

62. Diploidy is general rule. Occurrence of polyploidy has been reported in
many cvs.
The Hongkong wild kumquats, Fortunella hindsii may have been the
first reported tetraploid.
Polyploidy breeding: large sized fruit with dwarf plant types.
4x 2x 3x (Seedless varieties)
C. latifolia is a triploid. Triploids have favorable characteristics and yield
well but they are sterile.
Polyploidy breeding
It is the variation in chromosome number of individual plant, different
from the normal diploid chromosome number

63. Pummelo Grapefruit
Orablanco
and
Melogold
Orablanco and Melogold are
seedless, triploid varieties of Citrus
developed in 1982 and 1985
respectively

64. TRIPLOIDS

65. Shoot Tip Grafting (STG) or Micrografting is an in vitro
technique which involves the placement of a meristem or
shoot tip explants on to a decapitated rootstock that has
been grown aseptically from seed or micropropagated
cultures. ( Hartmann et al., 2002).
In Citrus, STG was first developed by Murashige et al.
(1972) and later improved by Navarro et al. in 1975.
SHOOT TIP GRAFTING

67. STAGES OF SHOOT TIP GRAFTING
Navarro et al., 1975

68. Seed coats
are
removed
Surfaced
sterilised with
0.25-0.7%
NaOCl+ 0.1%
Tween-20
Washed 4-5
time with
sterilized
distilled water
Sown in culture
tubes containing
20-25 ml MS
Media solidified
with 0.8% agar
PREPARATION OF THE ROOTSTOCK

69. Fig: A. Troyer seeds with and without integuments. B. Freshly sown seed and seedling after 2
weeks. C. Sown seeds incubated in dark at 27±2°C. D. 2 week old seedling and decapitated seedling
ready for grafting (Juarez et al., 2015)
C
D

70. LOREM IPSUM
SOURCES OF SCION
Direct source
Seasonal flush
Lower rate of
elimination of
pathogens
 Induced whenever
necessary
 Can be subjected to
warm pre-treatment to
improve efficiency of
pathogen elimination
Faster scion
source and
gives better
grafting success
Field grown plants Greenhouse Plants Bud wood cultured in vitro
Navarro et al., 1980 Navarro et al., 2002
Juarez et al., 2015
SOURCE & PREPARATION OF THE SCION

71. Young flush & shoot prepared for surface disinfection
Inducing vegetative flushes by bud wood
cultured in vitro
Inducing vegetative flushes with greenhouse
plant

72. MICROGRAFTING

73. Flushes of 3 cm long are collected from the desired source
Stripped off of the larger leaves. Smaller leaves can be removed under
microscope with the help of dissecting instruments
Stripped off of the larger leaves and cut to 1 cm long
Shoot tips composed of the apical meristem and three leaf primordia (0.1-0.2 mm in
length) are then excised with a razor blade. The shoot tip can then be placed inside
the incision of the rootstock with its cut surface in contact with the cortex exposed by
the horizontal cut of the incision made at the top of the decapitated epicotyl.
Sterilised by immersion in 0.25% NaOCl solution

75. Different type of Cuts
THE INVERTED ‘T’ CUT
THE WEDGE/CLEFT
GRAFT
THE LATERAL
INCISION
TOP SURFACE
PLACEMENT GRAFT
Citrange seedlings (Navarro and Juárez, 1977)

76.  Micrografted plants are
cultured in a liquid nutrient
medium composed of the plant
cell culture salt solution of
Murashige and Skoog (1962) with
a modification in the sucrose
concentration, i.e., 75 g/l
(Navarro et al., 1975).
A folded filter paper perforated
at the centre is used as a platform
to hold the grafted plant in place
and to prevent it from collapsing.
CULTURE OF THE GRAFTED PLANTS (in vitro)

77. Scions of successful grafts having at least 2 expanded leaves (4-6 weeks after
grafting) can be transplanted to soil. Sterilized garden soil, vermiculite and
sand can be used for planting the micro-grafted plants

78. The pots are then enclosed in polythene bags that are closed with rubber bands
and placed in a shaded area at 18-25°C for 8-10 days.
After 8-10 days the bags are opened and after another 8-10 days the bags are
removed and the plants are grown under normal greenhouse condition.

79. Picture source: Zamora-Rodríguez et al., 2015
Plants recovered by STG do not
have juvenile characters, if the
shoot tips are excised from adult
plants.
Thus, they usually flower & set
fruit within 2 years from grafting
(Navarro & Juárez, 1977).

80.  Murashige, T.; Bitters, W.P.; Rangan, E.M.; Naue, E.M.; Roistacher, C.N.
and Holiday, P.B. 1972. A technique of shoot apex grafting & its utilization
towards recovering virus free clones. HortScience. 7:118-119 .
 Navarro, L., Roistacher, C.N. and Murashige, T. 1975. Improvement of
shoot tip grafting in vitro for virusfree citrus. J. Amer. Soc. Hort. Sci. 100:
471-79.
 Ray, P.K. (2002). Breeding Tropical and Subtropical Fruits. Narosa
Publishing House, 84-101.
 Velasco, R., & Licciardello, C. (2014). A genealogy of the citrus family.
Nature biotechnology, 32(7), 640- 642.
 Wu, G. A., Terol, J., Ibanez, V., López-García, A., Pérez-Román, E.,
Borredá, C., ... & Talon, M. (2018). Genomics of the origin and evolution of
Citrus. Nature, 554(7692), 311-316
References