Role of tissue culture techniques in overcoming major breeding constraints in fruit crops

1. M K Saini
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Presented by:
Mandeep Kaur
L-2018-A-35-D
PhD Fruit Science
Role of tissue culture techniques in
overcoming major breeding constraints in
fruit crops
Presented to:
Dr H S Dhaliwal
Dr Monika Gupta
Dr Rachna Arora

2. M K Saini
CONTENT
 Introduction to Fruit Breeding
 Fruit Breeding Constraints
 Tissue Culture Techniques
a. Embryo Rescue
b. Production of Haploid Plants
c. Somatic Hybridization
d. Somaclonal Variations
e. Cryopreservation & In- vitro Germplasm Storage
f. Quality Improvement
 Conclusion

3. M K Saini
Fruit Breeding
• Breeding objectives for rootstock:
– Resistant to biotic and abiotic stress
– Dwarf growth stature – without affecting productivity of scion.
– Wide geographical adaptability
– Easily propagated through asexual means
• Breeding objectives for scion cultivars:
– High productivity
– Resistant to biotic and abiotic stress
– Dwarf growth stature
– Regular, precocious and prolific bearer per unit canopy area
– Longer shelf life and transport quality
– Good quality fruits:
 Appearance –> Size, shape, color
 Eating quality –> Seedlessness (few or soft seeds), flesh color, texture, flavor

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Fruit Breeding Constraints
• Long juvenile phase
• Heterozygous nature
• Wide/distant hybridization – Interspecific & Intergeneric crosses.
• Sexual incompatibilities in both self and cross combination
• Susceptibility to biotic & abiotic stress
• Polyploidy – Banana, Strawberry
• Polyembryony – Citrus, Mango
• Parthenocarpy and seedlessness – Banana, Pineapple
• Loss of natural germplasm
• Lack of knowledge on inheritance pattern
• Excessive fruit drop – Mango, Citrus
• Presence of single seed in single fruit – Mango, Litchi
• Seed dormancy
• Low seed viability

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1. Embryo Rescue

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1. Embryo Rescue
• Post - zygotic embryo abortion – Major breeding constraint.
• Embryo rescue refers to in vitro technology whose purpose is to promote
the development of an immature or weak embryo into a viable plant.
• Embryo rescue procedures – Ovule culture, Ovary culture, Embryo culture.
• It has been successfully applied in grapes, citrus, mango, stone fruits.
• Grapes variety developed by IARI, New Delhi using embryo rescue :-
• Pusa Purple Seedless: Cross between ‘Pearl of Casaba’ and ‘Beauty
Seedless’, raised through embryo rescue.
• It is an extra-early berry ripening variety (in the 3rd week of May) under
sub-tropical region.

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Applications of embryo rescue techniques
1. Shortening the breeding cycle
2. Developing interspecific and intergeneric hybrids
3. Seedlessness
4. Overcoming embryo abortion
5. Determining of seed viability
6. Overcoming dormancy – Malus and Prunus spp.

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In vitro efficiency of embryo rescue of interspecific
hybrids of sweet cherry and Chinese cherry cultivars
Effects of different low temperature treatments & GA₃ on cherry embryo germination
through in vitro rescue.
Wu et al (2021) China
 Chaoyang-1 (CY-1) – Sweet cherry
 Duangbing (DB) – Chinese cherry
• Failure to obtain hybrid cherry seedlings during outbreeding.

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In vitro embryo rescue for the production of
hypotetraploids after cross between hypotetraploid
and tetraploid grape cultivars
Effect of the time of ovule culture and basal medium on development of the embryos and in
vitro growth of embryos excised after ovule culture
Kim et al (2020) Korea
• Hypotetraploid (2n = 4x-1 = 75) - To develop seedless grapes with large berries.
• (4x-1=75) => Hanareum => female parent
• (4x=76) => Honey Black and Kyoho => male parents

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Effect of different sampling times on embryo recovery
rate in different seedless grapes crosses
Li et al (2020) China
Plant
Development
(%)
Embryo
Recovery
(%)
• Stenospermocarpy leads to embryo abortion during development.

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Plant material Remarks Reference
Vitis vinifera L. Ovule culture of seedless grapes (Vitis vinifera L.) Singh et al (1991)
Peach Ovule rescue in peaches: incubation period Raseira and Einhardt (2010)
Citrus Regenerating triploid plants by embryo rescue
technique
Liu et al (2010),
Aleza et al (2010)
Grape Embryo rescue of seedless grape with disease and
cold resistance
Niu et al (2012)
Musa species Embryo culture and embryo rescue studies in wild
Musa spp. (Musa ornata)
Dayarani et al (2014)
Citrus Production of common sour orange × Carrizo
citrange hybrids using embryo rescue
Kurt and Ulger (2014)
Grapevine Immature embryo rescue and in vitro evaluation of
intraspecific hybrids from Brazilian seedless
grapevine ‘Superior × Thompson’ clones
Menezes et al (2014)
Embryo rescue in fruit crop improvement

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2. In Vitro Production
of Haploids

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2. In Vitro Production of Haploids
• Haploid plants are obtained from pollen grains by culturing them.
• Two methods - Anther culture & pollen culture.
• Double haploid - Homozygous pure plant.
• Applications of haploid plants:
– Production of genetically pureline by selfing is impractical because of their
long generation cycle & frequent self incompatibility.
– To solve of problem of high heterozygosity – No segregation in meiosis.
– Shorten the time for generation of homozygous lines.
– QTL mapping.
– Genome sequencing.
– DH: To study heterozygous maternal parent.
Haploid
1 n
Diploid
2 n
Colchicine

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In vitro regeneration of haploids using isolated
microspore culture of banana cv. Bimkol
• Musa balbisiana cv. Bimkol => BB genomic group (2n= 2x= 22).
• Long crop cycle (740 days) => Breeding problem.
• 30 days old immature male flowers => Explants.
• 58.33 % haploid plants
• 41.66 % diploid plants
Gogoi et al (2020) India
Haploid plant Diploid (Control)

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3. Somatic Hybridization

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3. Somatic Hybridization
• Somatic hybridization is an alternative to sexual reproduction in order to
combine the genomes from otherwise incompatible parents.
• It involves fusing protoplasts of two different genomes followed by the
selection of desired somatic hybrid cells and subsequent regeneration of
hybrid plant.
• It involves four steps:
 Protoplast isolation
 Protoplast fusion
 Selection of hybrid cells
 Regeneration of hybrid plants
• Two types of hybrids - symmetric hybrid and asymmetric hybrids

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Applications of somatic hybridization
• Production of interspecific and intergeneric hybrids – by combining
nuclear and cytoplasmic genomes belonging to different species & genus.
• Production of fertile diploids and polypoids – by crossing sexually sterile
haploids, triploids and aneuploids
• Production of heterozygous lines in the asexually propagated species
• Transfer of cytoplasmic information (mtDNA) for the production of male
sterile plants and seedless triploids.
• To create novel germplasm as a source of elite breeding parents.
• To create novel hybrids with increased yield and resistance to biotic and
abiotic stress.

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Triploid Mandarin:
Sugar Belle X Nova + Succari
Triploid Lime:
‘Todo del Ano’ lemon X ‘Mexican’ lime + ‘Valencia’
Triploid Lemon:
‘Todo del Ano’ lemon X ‘Hamlin’ + Femminello’
Fig: Seedless fruits from triploid hybrids produced by somatic hybridization
Grosser and Gmitter (2011) USA

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Response of four allotetraploid somatic hybrids to
Citrus tristeza virus induced infections
Abbate et al (2018) Italy
Fig: Detection of mild (CTV-DS1) and severe (CTV-DS2) strains in the
investigated genotypes by using DAS-ELISA and qRT-PCR assays.
Biological barriers - sexual incompatibility, polyembryony, male/female sterility, long
juvenility and different bloom phases

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Production of diploid and tetraploid somatic
cybrid plants between male sterile Satsuma mandarin
and seedy sweet orange cultivars
• Polyembryony: Problem to obtain sexual hybrids
• Protoplast fusion - Satsuma mandarin cv. Guoqing No. 1 (G1) and three seedy sweet
oranges i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’.
• mtDNA of G1 SM introduced into SO cultivars for potential seedlessness.
Xiao et al (2014) China

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Somatic hybridization in fruit crop improvement
Somatic hybrids Characters References
Musa acuminata cv. Mas + M. silk cv. Guoshanxiang Disease resistance (Fusarium) Xiao et al (2009)
Citrus reticulata cv. Red Tangerine + Poncirus trifoliata Tolerant to CTV (citrus tristeza
virus) and CEV ( citrus exocortis
virus )
Guo et al (2002)
Citrus reticulata + C. limon Carotenoid compounds Bassene et al (2009)
Murcott tangor (Citrus reticulata Blanco × C. sinensis
(L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C.
grandis (L.) Osbeck)
Seedless Cai et al (2010)
Citrus sinensis Osbeck cv. Yoshida navel orange +
Citrus unshiu Marc cv. Okitsu satsuma mandarin
Seedless An et al (2008)
Bingtang orange (Citrus sinensis (L.) Osbeck) +
Calamondin (C. microcarpa Bunge)
Asiatic citrus canker-tolerant and
ornamental citrus breeding
Cai et al (2010)

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4. Somaclonal variations &
In vitro mutagenesis

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4.Somaclonal variations & In vitro mutagenesis
• Somaclonal variation is defined as genetic variation observed among
progeny of plants regenerated from somatic cells cultured in vitro.
• Somaclonal variants in vitro selected with or without induced
mutagenesis to obtain tolerant plants to a variety of stresses.
Grosser and Gmitter (2011) USA

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Production of sweet orange somaclones tolerant to
citrus canker disease by in vitro mutagenesis with EMS
• Mutation was introduced by treating the cell suspension of embryogenic
callus with 1.5 % of EMS for 1 hour.
• The survival plants were tested by in vitro and in vivo inoculation with Xcc.
• One somaclone (DG-2) was identified to be resistant to the canker disease.
Ge et al (2015) China
The symptoms of wild type plants
on 3 dpi (j) and on 7 dpi (k);
No symptoms of tolerant somaclone
on 3 dpi (l) and on 7 dpi (m)

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In vitro mutagenesis for improvement of Banana
• In vitro mutagenesis was applied to banana cv. “Giant Cavendish” to develop
dwarf, early maturing mutants with increased fruit yield.
• Multiple shoots of banana were gamma ray irradiated (10 Gy).
• Later, irradiated plants and control plants were multiplied by tissue culture.
Ganapathi and Badigannavar (2020) BARC, Mumbai
TBM-2

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5. Cryopreservation & in
vitro germplasm storage

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5. Cryopreservation & in vitro germplasm storage
• Genetic resources are not only pre-requsite but key ingredient for genetic
improvement.
• Loss of genetic variability –> Genetic erosion.
• Field gene bank –> Damage by pests, pathogens & climatic disorders.
• In vitro germplasm conservation is the only solution.
• Cryopreservation –
– Storage at ultra cool temperature (-196 ᵒC).
– Plants cells, Shoot tips, Somatic & Zygotic embryos.
– 4 Steps –> Freezing, Storage, Thawing & Reculturing.
• In vitro germplasm storage
– INIBAP –> Banana
– NBPGR –> Pomegranate, Banana, Phalsa, Bael & Jackfruit.

28. M K Saini
Conclusion
• Biotechnology has brought great challenges, opportunities
and prospects for conventional breeding.
• However, biotechnology as transgenic breeding or genetic
manipulation cannot replace conventional breeding but is and
only is a supplementary addition to conventional breeding.
• Therefore, integration of biotechnology into conventional
breeding programs will be an optimistic strategy for fruit crop
improvement in the future.

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