Wide hybridization in vegetable crops

Horti. Stat, 2018
13-Jun-21 Veg.Dept 2

ICAR-NBPGR, 2019

University of horticultural sciences , bagalkot
k. R. C. college of horticulture , arabhavi
department of vegetable science
WIDE HYBRIDIZATION IN Vegetable crops
Basavaraj S Panjagal
Ph.D in Vegetable Science
13-Jun-21 Veg.Dept
Seminar – I
5

Introduction
History of wide hybridization
Types and features
Barriers associated with wide Hybridization
Role of Wide crossing in crop improvement
Techniques for production of DistantHybrid
Case studies
Limitations of Wide Hybridization
Achievements Of Wide Hybridization In Vegetable Crops
Conclusion
Topic Division

Why Wide hybridization?
Disease
resistance
Insect pest
resistance
Stress
environments
Quality
attributes
http://cwcdiversity.org

Wide hybridization ?
Genus
Species x Species
F1
Family
Genus x Genus
F1
Interspecific Hybridization Intergeneric Hybridization
Wide/Distant Cross
Sharma, 2009

How Wide Hybridization differs?
Wide hybridization differ from normal hybridization because individuals used for
hybridization in such cases are taxonomicallymore distantly related than different
variety from thesame species.
13-Jun-21 Veg.Dept
Crossing between two genetically dissimilar parent is called
hybridization.
Variety
Species
Genus
Family
9
Sharma, 2009

History
1st interspecific hybridization was done between Carnation
(Dianthus caryophyllus) and Sweet William (Dianthus
barbatus) by Thomas Fairchild in 1717
 Rimpu (1890):
Produce the first intergeneric hybrid triticale (Wheat x Rye)
which have greaterpotential than raphanobrassica.
 Karpechenko (1928): An interesting intergeneric hybrid,
Raphanobrassica (Radish x cabbage) was produced.
Singh, 2013

Features of Interspecific hybridization
Desirable trait is not
found
Transferring desirable
gene
Introgression
3 types of crosses
a) fully fertile,
b) Partiallyfertile
c) Fully sterile
a) Fully fertile:
1.L. esculentum (24) x L. peruvianum(24)
2. C. frutescence (24) x C. annum (24)
3. P. sativum (14) x P. fulvum (14)
,
b) Partiallyfertile
Wheat, Cotton, Tobacco
T. aestivum x T. durum
(AABBDD) (AABB)
( 2n=6x=42) (2n=4x=28)
(7 univalent) 14 bivalents
F1 (AABBD) partially fertile
Occasionally seed set
c) Fully sterile
A. esculentus x A. manihot ssp.
manihot
(n=65) (n=97)
F1 sterile (2n=162)
Colchicine treatment
2n= 324 fertile F1
Singh, 2013

8
Fig 1.Brassica U’ s triangle Ram, 2012

Features of Intergeneric hybridization
Desirable trait/gene is
not found
Rarely used in crop
improvement
F1 hybrids are always
sterile
Develop new crop
Singh, 2013

2n=28
2n=14
3n=21 Colchicine
6n=42
Zillinsky, 1974

Table 1: Differences between Interspecific and
Intergeneric hybridization
13-Jun-21 Veg.Dept
Particulars Interspecific Hybridization Intergeneric
hybridization
Parents involved Involve two different species of the
same genus
Involve two different species
of the same family
Fertility Such hybrids vary from completely
fertile to completely sterile
Such hybrids always sterile
Seed setting More than intergeneric crosses Low
Use in crop
improvement
More than intergeneric crosses less than interspecific
crosses
Release of Hybrid
Varieties
Possible in some crops Not Possible
Evolution of new
crops
Not possible, but evolution of new
species is some times possible
Sometimes Possible,
eg: Triticale
15
Ram, 2012

Application of wide hybridization in crop improvement
 Generating variability
 Wider adaptation
 Yield
 Quality
 Disease resistance
 Transfer of small chromosomal segment
 Mode of reproduction
 Transfer of cytoplasm
 Utilization as new varieties
 Development of new crop species
 Root nodulation

Role of wide crosses in crop improvement
a) Disease and insect resistance
Crop Character
transferred
Wild Species Species Source
Cotton Jassid
resistance
G.tomentosum
G.arboreum
G.hirsutum
G.barbadense
Singh,
1996
Okra Resistance to
YVMV
Abelmoschus
manihot
A. esculenta Ram, 2012
Brinjal Bacterial wilt S. stenotomum S. melongena
Wheat Rust resistance Agropyron T. aestivum Singh, 2013
Tobacco Resistant to
mosaic virus
N.repanda N. tabaccum
Potato Late blight, leaf
roll, virus-x
S. demissum S. tuberosum Peter, 2012
French
bean
Rust resistant P. flavescens P. vulgaris
Cucumber Fruit fly resistance C. callosus C. sativus

b) Improvement in quality
Crop Charactertransferred Species transferred
from
Species transferred
to
Cotton Fibre length
Male sterility
G. thurberi &
G. raimondii
G. harkenssii
G. hirsutum
G. hirsutum
Potato Starch content
Frost resistance
Solanum acaule S. tuberosum
Tomato Carotenoid content Lycopersicon
hirsutum
L. esculentum
Chilli High capsaicin, more
pungency
C. frutescence C. annum
c) Improvement in yield:
This also been achieved through the use of wild Spp. in some crops eg. Oat,
Vigna, Potato,Tobacco.
13-Jun-21 Veg.Dept Continue..
Singh, 1996

d) Improvement in adaptation:
Eg: Tolerance to cold in Onion ( A. porrum), Potato (S.commersonii) etc
Tolerance to high temperatureTomato (L. cheesmani),
Drought tolerance in Peas, Tomato
e) Mode of Reproduction:
Male sterility -alteration in the mode of reproduction.
CMS has been reported in wheat , cotton, potato.
Transfer of Radish cytoplasm to B. napus to produce CMS lines.
f) Other characters :
Eg: Transfer dark green colour and excellent leaf texture in lettuce
bright red thin flesh in red peppers.
G) Development of hybrid varieties
Eg: Okra
Singh, 1996

Problems associated with wide
crosses
Barriers
Fortified
Foods
13-Jun-21 Veg.Dept
3.Hybrid
Sterility
1.Cross
incompatibility
2.Hybrid
Inviability
• 4.Hybrid
Breakdown
20
Tonosaki et al., 2016
Zygote arrested
Less interaction
Lethel genes
Disharmony
Embry abortion

Fig. 2: Schematic representations of multi stage controlled pollen
tube guidance and pre-zygotic barriers
Tonosaki et al., 2016

Selection of Plants
Reciprocal Crosses
Manipulation of
Ploidy Level
Use of Growth
regulators
Manipulation of Pistil Use of pollen mixture
TECHNIQUES TO MAKE WIDE CROSSES SUCCESSFUL
Van and Jeu, 2000

Techniques to overcome crossability barriers
Wide
hybridization
Bridging
species
Embryo
rescue
Somatic
hybridization
Chromosome
doubling
Backcrossing
Van and Jeu, 2000

1.Bridge species technique
Potato :
a. S. demissum x S. tuberosum
2 n = 72 ↓ 2 n = 48
F1 sterile (univalents)
S. demissum x S. rybinii
(+LB,PVA,PVY) (Bridge species)
↓
F1 (Fertile) x S. tuberosum
↓
Fertile progenies
Potato :
b. S. acuale x S. tuberosum
↓
F1 sterile (univalents)
S. acuale x S. simplicifolium
(PVX,PLV ) ↓ (Bridge species)
F1 (fertile) x S. tuberosum
↓
Fertile progenies
Selvakumar, 2006

2.Embryo rescue

Development of interspecific Solanum lycopersicum
and screening for Tospovirus resistance
Sohrab et al., 2015

Materials and methods
Screening and selection of tomato plants resistance to Tospovirus
Hybrid nature of F1 , F2, and F3 plants evaluated by visual
morphological observation of parents and their hybrid plants
Embryo rescue and development of interspecific F1 hybridtomato
Collection of fertilized tomato fruits and isolation of immature
embryos
Plant material: S.lycopersicum (492-BC) × L. peruvianum (921-
L00671)
Sohrab et al., 2015

Fig. 5: various stages of interspecific F1 tomato plant development (A)
germinating immature embryos (B) shoot induction (C) shoot
elongation and rooting (D) regenerated plants in pots
Sohrab et al., 2015

Table 2: Total no. of interspecific F1 tomato plants developed
Tomato fruits (days
after pollination)
Total number of
fruits harvested
Total number of
plants developed
28 135 8
29 142 12
31 171 24
33 109 5
Total 557 49
Sohrab et al., 2015

Fig. 6: morphological variations of interspecific plants (A) Leaf
(wild). (B) Leaf (cultivated). (C) F1 fruits. (D) F1 seeds. (E) F2
seeds (F) F3 seeds
Sohrab et al., 2015

Fig. 7: Inoculation and symptoms of PBNV on cowpea (A)
naturally infected tomato (B) inoculated cowpea
Maintenance of pure virus
Sohrab et al., 2015

Table 3: Screening and selection of interspecific F1 tomato
plants resistance to Tospovirus
Sohrab et al., 2015

Wide hybridization and embryo rescue in chilli
Debbarama et al., 2015

3.Somatic hybridization

Interspecific potato somatic hybrids between
Solanum tuberosum and Solanum cardiophyllum
Chandel et al., 2015

Materials and methods
Plant material: Solanum tuberosum dihaploid
“C-13” and diploid wild species Solanum
cardiophyllum (PI 341233)
Protoplast isolation, fusion and regeneration
DNA isolation and analysis using RAPD, ISSR,
SSR and AFLP markers
Cluster analysis was done with program
NTSYS-PC 2.21
Phenotypic characterization, male fertility and
hybridization and lastly late blight resistance
assay was performed

Table 4: Summary of markers analyses in potato
somatic hybrids between C-13 and S. cardiophyllum
SN Marker type No. of markers
used
No. of markers
confirmed
hybridity
1. RAPD 35 24
2. ISSR 24 13
3. SSR 26 10
4. AFLP 30 30
Fig. 10:Somatic hybrids between C-13 and S.cardiophyllum confirmed with
possessing parental bands by a RAPD marker OPACO6, and b. ISSR marker
ISSR10. P1= C-13, P2= S. cardiophyllum, P1+P2=pooled parental DNA before
PCR

Fig. 11: SSR marker STI0012 confirmed hybridity of somatic hybrids C-13(+) S.
cardiophyllum possessing parental bands as indicated by arrow a. C-13, b. S.
cardiophyllum, and c. somatic hybrid analysed on “3500 GeneticAnalyzer”(ABI)

Fig. 12: Cluster analysis based on the Jaccard’s coefficient of
molecular profiles generated by RAPD, ISSR, AFLP and
cytoplasmic markers showing genetic distinctness among somatic
hybrid clones and their parents

Table 5: Late blight assay of potato somatic hybrids and their parents by
inoculation of Phytophthora infestans
S. No. Genotype Late blight infection
(AUDPC)
Class
1. Crd-6 0.00 0.00 HR
2. Crd-10 0.00 0.00 HR
3. Crd-16 0.00 0.00 HR
4. Crd-23 0.00 0.00 HR
5. S.Cardiophyllum 0.00 0.00 HR
6. C-13 155.00 160.00 S
7. Kufri Jyoti 180.00 190.00 S
8. Kufri Girdhari 25.00 30.00 HR
AUDPC- Area under disease progress curve value: HR (<50), R (50-100), MR
(100-150), and S (>150)
(HR highly resistant, R resistant, MR moderately resistant and S susceptible)

Fig. 13: i) somatic hybrids confirmed by phenotypes (a) leaf,
(b) flower and (c) tuber traits possessing their parental
characters; ii) Late blight resistance test of somatic hybrids
confirmed by challenge inoculation of Phytophthora infestans

Interspecific somatic hybrids produced by protoplast electrofusion of the cells
of potato cv. Delikat (S. tuberosum) and S. tarnii. Selected somatic hybrids
backcrossed with cv. Delikat. Furthur hybrids and BC1 progeny were
assessed for resistance to PVY. Somatic hybrids shows resistance (Ramona
et al., 2008).
Somatic hybrids between the Japanese radish and cauliflower (Brassica
oleracea) were produced by protoplast electrofusion in order to introduce
clubroot disease resistance in the Japanese radish (Raphanus sativus) into
Brassica crops (Hagimori et al., 1992).

4. Chromosome doubling
Crossability behaviour and fertility restoration
through Colchiploidy in interspecific hybrids of Okra
Reddy, 2015

Fig .14: Flow chart for overcoming sterility through colchiploidy in F1 hybrid 44
Reddy, 2015

Cross combination No. of cross
made
No. of fruit
set
No. of fruits
with seeds
Fruit set
(%)
Crossabil
ity (%)
A. esculentum (RNOYR-19) x
A. manihot subsp.tetraphyllus
10 9 9 90 90
Table 7: Germinability and dormancy in interspecific F1 hybrid
Cross combination No. of seed
sown
No. of seeds
germinated
Germination
(%)
Dormancy
100 94 94 Absent
Table 8: Hybrid lethality and hybrid breakdown in interspecific F1 hybrid
Cross
combination
No. of seed
sown
No. of seeds
germinated
No .of
plants
died
No. of viable
plants
No. of
plants
reached
maturity
Hybrid
lethality (%)
Hybrid
breakd
own
(%)
(RNOYR-19) x
A. manihot
subsp.tetraphyllus
10 10 0 10 10 0 0
Table 6: Crossability behaviour in interspecific F1 hybrid
Reddy, 2015

Table 9: Sterility in interspecific F1 hybrid
Cross combination No. of plants
grown
No. of plants
set fruits
No. of plants set
mature seeds
No. of mature
seeds/fruit
10 10 0 0
Table 10: Colchiploidy as a means of fertility restoration in interspecific F1hybrid
Cross
combination
No. of Plants Avg. no. of seeds per fruit
Treated Survived Set
seed
Total no.
of seeds
No. of
mature seeds
No.of abortive
seeds
Seed
set (%)
(RNOYR-19) x
A. m subsp.
tetraphyllus
75 75 52 32 17 15 53.12
Table 11 : Restoring complete fertility in colchiploids through selfing
Cross
combination
No. of Plants Avg. no. of seeds per fruit
Selfed Set seed Total no.
of seeds
No. of mature
seeds
No.of abortive
seeds
Seed set
(%)
(RNOYR-19) x
A. m subsp.
tetraphyllus
10 10 32 32 0 100
Reddy, 2015

Colchicine (0.1%) treatment on apical buds at two leaf stage of the seedlings for
65h in the interspecific hybrids of A. esculentus cv. Phule Utkarsha x A.
manihot subsp. manihot was effective in inducing amphidiploids with fertility
(Jatkar et al., 2007).
Interspecific hybrid between Cucumis sativus (2n=14) and C. hystrix (2n=24)were
first produced C. hytivus (2n= 38) was obtained through chromosome doubling
of the interspecific hybrid ( Chen and Kirkbride, 2000).
Amphidiploid Raphanofortii was synthesized by colchicinization of the F1 hybrid
Brassica tournefortii (2n = 20)×Raphanus caudatus (2n = 18). The hybrid
plants were intermediate for most of the morphological attributes and showed
very low pollen fertility compared to the parents. The newly synthesized
Raphanofortii has great potential as a new commercial crop (Choudhary et al.,
2000)

5.Backcrossing
Transfer of CMS from B. juncea and B. napus to
cauliflower through interspecific hybridization and
embryo culture
Chamola et al., 2015

Transfer of CMS from B. juncea and B. napus to
cauliflower through interspecific hybridization and
embryo culture

Material and methods
 CMS B. juncea cv. RLM 198 (M. arvensis) – Black Mustard
 CMS B. napus (E. canariense) – Rape seed
 B. Oleracea var. botrytis cv. Pusa Meghna – Cauliflower
B. juncea cv. RLM 198 x B. oleracea var. botrytis cv. Pusa Meghna
ovaries for invitro culture – ovule culture- shoot-root growth- hardening
F1 plants x B. oleracea var. botrytis cv. Pusa Meghna
BC1 (Emasculation, pollination, ovary/embryo culture
were repeated during successive backcross)
BC1 x B. oleracea var. botrytis cv. Pusa Meghna
BC2 x B. oleracea var. botrytis cv. Pusa Meghna
BC3

Table 12: Summary of results of embryo rescue on the recovery of
interspecific hybrids between B. juncea or B. napus and B. oleracea var.
botrytis
Cross Time of
sampling of
ovaries
No. of
ovaries
cultured
No. of
surviving
ovaries
No. of
ovules
obtained
and
cultured
No. of
plants
recorded
CMS (M.
arvensis)
B. juncea x
B. oleracea
5 DAP 60 12 11 -
9 DAP 60 47 73 14
14 DAP 60 19 15 2
CMS (E.
canariense)
B. napus x
B. oleracea
5 DAP 60 0 0 0
9 DAP 60 28 23 2
14 DAP 60 32 47 5

Fig. 15 Sequential ovary and embryo culture to obtain interspecific hybrids
between B. juncea / B. napus and B. oleracea.
a. Ovary culture b. dissected embryos from 20-day-old ovary culture
c. Germinated embryos d. Young seedling e. Hybrid plants at hardening stage

Fig. 16 Interspecific
hybridization between B.
juncea x B.oleracea
a. F1 plant
b. BC1 plant
c. BC2 plant
d. Crooked and fused
pistils of BC1 flower
e. leaves of B. juncea
(L), BC3 (c), B.
oleracea (R)
f. Male sterile flowers of
BC3 plant
g. Meiotic preparation of
BC2 plant at the late
metaphase I stage
showing2 multivalents,
7 bivalents and 4 pairs
of univalents migrating
towards poles

Fig. 17 Interspecific
hybridization between B.
napus x B.oleracea
a. F1 plant
b. leaves of B. napus (L),
BC1 (c), B. oleracea
(R) BC2 plant
c. curd formation in BC1
d. curd formation in BC2
e. curd formation in BC3
f. leaves of B. napus (L),
BC3 (c), B. oleracea
(R).
g. Inflorescence in Bc3 of
CMS B. oleracea
h. Meiotic preparation of
BC2 plant at the late
metaphase I stage
showing 9II

Developing stable progenies of Brassicoraphanus through
induced mutation using microspore culture
Lee et al., 2011
10 progeny lines
BB-1
BB-4
Mutagen N-methyl –N-
nitrose-urethane (NMU)
0.1µm

Table 13: Number of seeds per siliqua (seed fertility) on artificial self pollination of Mi1 plants
and Mi2 derived from microspore culture with or without NMU treatment in Brassicoraphanus
Plant code NMU
treat.(0.01 µm)
No. of seeds per siliqua
Mi1 Plants Mi2 Plants
Mean Best stalk Mean Best stalk
548 Yes 0.1 0.8 1.2 7.5
550 Yes 0.4 0.6 0.6 3.8
551 Yes 0.2 0.4 1.2 6.8
552 Yes 0.1 0.9 0.2 1.0
554 No 0.1 0.3 - -
556 No 0.3 1.6 0.0 0.3
558 Yes 0.3 4.9 0.5 1.6
559 Yes 0.3 1.0 0.5 2.6
560 Yes 0.5 3.0 1.4 8.6
562 Yes 0.2 0.5 1.8 6.6
289 No (wild) 0.2 0.7 0.5 1.4
Lee et al., 2011

Number of seeds per siliqua was counted from 100 siliqua.
Table 14: No. of seeds per siliqua and seed yield on open pollination of four mutant lines in Mi3
generation of Brassicoraphanus
Line code No. of
plants
Number of
seeds per
siliqua
Seed yield
Harvested
(g)
Per plant(g) Per ha (kg)
548 30 7.6 1.253 41.8 835
551 40 8.8 2.296 57.4 1148
560 40 7.6 1.967 49.2 983
562 40 8.0 1.498 37.5 749
BB-1 (check A) 39 2.3 559 20.4 410
BB-2 (check B) 21 2.1 441 21.0 421
Lee et al., 2011

Line code Uniformity Plant wt
(kg)
Leaf wt
(kg)
No. of
leaves
Leaf length
(cm)
Flower
stock
length (cm)
Check A Fair 1.5 1.4 17.0 46.7 Uneven
Check B Fair 1.4 1.3 14.7 47.0 23
548 Excellent 1.5 1.4 17.7 40.0 13
551 Excellent 1.3 1.1 14.0 40.3 17
560 Excellent 1.6 1.5 19.0 39.7 14
Table 15 : Characteristics of high fertile mutants compared to check lines in spring growing
Lee et al., 2011

Fig 18 a)Morphology of roots of four mutant lines
b) representative plants of the wild line and a mutant
c) ovules matured and degenerated in wild and a mutant
Lee et al., 2011

ACHIEVEMENTS

Inter specific hybrids in different vegetables
Okra
Pusa A4 A. esculentus x A. manihot ssp.
manihot
Dhankhar et al.,
1999
Tolerant to
jassids, Fruit
and shoot
borer
Punjab-7 A. esculentus (Pusa Sawani) x A.
manihot ssp. manihot
(Ghana)(2n=194)
Thakur, 1976
Resistance to
YVMV
Punjab Padmini A. esculentus (Rashmi) x A.
manihot ssp. manihot (Ghana)
Sharma, 1982
Parbhani Kranti A. esculentus (Pusa Sawani) x A.
manihot
Jambhale and
Nerkar, 1986
Arka Anamika A. esculentus(2n=130) x A.
tetraphyllus (2n=138)
Dutta, 1984
Arka Abhay A. esculentus(2n=72) x A.
tetraphyllus (2n=130)
Dutta, 1984 Resistance to
YVMV
And tolerant to
fruit borer
Anjitha A. esculentus cv. Kiran x A.
manihot
Manju and
Gopimani, 2009
Resistance to
YVMV

Inter specific hybrids in different vegetables
Tomato
Pusa Red Plum S. lycopersicum x S. pimpinellifolium Rich in Vit-C
HissarAnmol HissarArun x S. hirsutum f. glabratum Resistant to TLCV
Potato
Kufri Kuber (Solanum curtilobum × S. tuberosum) × S.
andigenum
High tuber yield
Amaranthus
Pusa Kiran A. tricolour x A. tristis Rainy season
Cucumber
C. hytivus Cucumis sativus x C. hystrix Resistant to downy
mildew
Selvakumar, 2006

Intergeneric hybridization
New Crop Parents Special feature
Hakurana Cabbage x Chinese
cabbage
(Developed by
Embryo culture)
New leafy vegetable in
Japan
Resistant to soft rot, drought
and heat
Selvakumar, 2006
Nabicol Kale x Turnip
Caulicob Cabbage x
Cauliflower
Swede Turnip x Cabbage Root vegetable
Raphanobrassica Radish x cabbage Fodder crop Naughton et al.,
1973
Baemoochae
(Brassicoraphan
us) (2n=38)
B. rapa ssp.
pekinensis (Big head
Chinese cabbage) x
R. sativus (big root
radish)
New leafy vegetable Lee et al., 2011

Limitations of Wide hybridization
1. Incompatible crosses
2. F1 sterility
3. Problems in creating new species
4. Lack of homoeology between chromosome of the parental species
5. Undesirable linkages
6. Problems in the transfer of recessive oligogenes and quantitative
traits
7. Lack of flowering in F1
8. Problems in using improved varieties in distant hybridization
9. Dormancy

Future thrust
• Reduce time in breeding programme
• Exploration of wild relatives
• Quality improvement needs special attention.
• Multiple disease resistance is the need of future.

Diversity
Food
security
Global plant
hub
Pre-breeding
Stable
production

Wide hybridization in vegetable crops

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