ADVANCES IN HYBRID SEED ADVANCES IN HYBRID SEED PRODUCTION TECHNOLOGY OF PRODUCTION TECHNOLOGY OF

TOMATOTOMATOSpeaker:

Akshay ChittoraPh.D. Horticulture

CONTENTSCONTENTS• Introduction• Hand emasculation and pollination• Use of male sterility• Biotechnological tools in hybrid seed

production• Major constraints in utilization of F1

hybrids• Conclusion

TOMATOTOMATOCommon Name :Tomato

Botanical Name :Solanum lycopersicum formerly Lycopersicon esculentum

Introduced in India :19th century by Britishers

Chromosome No. :2n = 24

Family :Solanaceae (nightshade)

Centre of origin :Peru (South America)

AREA, PRODUCTION AND PRODUCTIVITY OF TOMATO

AREA(000' ha)

PRODUCTION(000’Mt)

PRODUCTIVITY(Mt/ha)

World 4815.71 163029.746 33.9

China 1000 50000 50

India 882.03 18735.9 21.2

NHB (2014)

Tomato shares 9.4% of total vegetable area and 11.5% of total vegetable production in India

Historical BackgroundHistorical Background Among the vegetable crops, first commercial F1

hybrid of brinjal was released during 1924 in Japan In tomato first F1 hybrid was developed in 1940 in

Japan

At national level first hybrid vigor was reported in chilli during 1933 in by IARI, New Delhi

First public sector hybrid developed was Pusa Meghdoot in bottle gourd in 1971

The first hybrid vegetable seeds (Karnataka of tomato and Bharat of bell pepper) were marketed in India by IAHS in 1973

a) Single cross hybrid (AXB)

b) Three way cross hybrid (AXB) X C

c) Double cross hybrid (AXB) X (CXD)

d) Modified single cross (AXA’) X B

e) Double modified single cross (AXA’) X (BXB’)

f) Modified three-way hybrid (AXB) X (CXC’)

g) Top cross hybrid (Inbred X Variety or family etc.)

Types of HybridsTypes of Hybrids

Advantages of Hybrids Increased yield Early maturity Heat & cold tolerance Disease and pest resistance Better uniformity Better fruit quality Better transportability Better keeping quality

STEPS OF HYBRID SEEDS PRODUCTION

Production of inbred linesIn Self-Pollinated : Pure lineIn Cross-Pollinated : Inbred (by selfing)

Testing of combining ability

GCA for additive Gene ActionsSCA for dominant Gene ActionPredictive Information from SCA by

(Single Cross, Double Cross, Three way Cross, Top Cross Poly Cross and Diallele)

Improvement of inbred lines / varietiesFor disease and quality trait

Production of hybrid seedTypes of Hybrids and their Seed ProductionHybridization

The commonly utilized mechanisms for developing

commercial hybrids in vegetablesMechanism Commercially exploited crops

Hand emasculation + MP Tomato, Eggplant, Okra

Pinching of staminate flowers + MP/NP Cucurbits

Male sterility + MP Tomato, Hot pepper, Sweet pepper

Male sterility + NP Onion, Cabbage, Cauliflower, Carrot, Radish, Muskmelon, Chilli

Self incompatibility + NP Cauliflower, Cabbage

Gynoecism + NP Cucumber, Muskmelon

PGR & pinching of staminate flowers + NP Summer squash, Winter squash etc.

Hazra and Som (2009)MP: Manual pollinationNP: Natural pollination

FLOWERING BEHAVIOURANTHESIS DEHISCENCE RECEPTIVITY

OF STIGMAStarts at 6 AM and maximum flower opening till late morning.

8 AM – 11 AM. 16 hrs before and 5 days after anthesis.

Hand emasculation and Hand emasculation and pollination as hybridization pollination as hybridization

processprocess Plant male and female plants in a separate plots.

Plant male seedlings 2-3 weeks earlier to obtain adequate

amount of pollen

Ratio of female to male is generally 4:1

Spacing

Female plant : 75-90 X 60-75 cm

Male plant : 60 X 45-60 cm

Roguing at before flowering, early flowering and fruit setting

stage and fruiting stage

Emasculation procedure

Collection of pollen

Pollination

Hybrid fruits

Treatments No. of flowers crossed per

plant

No. of crossed fruit per

plant

Fruit set (%)

Crossed fruit yield per plant

(g)

Seed yield / plant (g)

Stage of bud (S)S1 76.52 20.73 27.12 1549.71 6.19

S2 76.84 40.65 52.92 2558.29 7.75

SEm± 2.15 0.61 1.21 61.08 0.17

CD @ 5% NS 1.83 3.67 183.87 NS

No. of pollinations (P)P1 77.31 29.26 38.07 1899.80 6.50

P2 76.10 29.69 39.00 1989.04 6.84

P3 76.62 33.11 43.00 2533.16 7.23

SEm± 1.98 0.50 1.05 49.21 0.15

CD@ 5% NS 1.52 3.15 146.15 0.44

(Jolli et al., 2009)

Effect of stage of bud and no. of pollination on different parameters of tomato hybrid (DTH-1)

S1: <50% bud open, S2: >50% bud openP1: One time pollination (morning), P2: Two time pollination (morning + evening), P3: Three time pollination (morning, evening and next day morning)

USE OF MALE STERILE LINES

Types of male sterility found in tomato and there inheritance pattern(Kaul, 1988:

TYPES OF STERILITY

DESCRIPTION INHERITANCE

Pollen Pollen abortive Monogenic recessive (ms series)

Positional Sigma exerted Monogenic recessive (ps)

Functional Anthers do not dehisce Monogenic recessive (ps 2)

Staminal Stamens absent Monogenic recessive (sl)

Positional Male Sterility

sl (Stamenless mutant) in tomato

Natural population

Artificially induced through • Mutagenesis •Distant hybridization• Protoplast fusion • Genetic engineering

PROCEDURE FOR DEVELOPMENT HYBRID USING GENEIC MALE STERILE LINES

Parents msms X MsMs

(male sterile) (male fertile)

Msms

(male fertile)

F2 1 MsMs : 2Msms : 1 msms Ratio 3:1

(male fertile) (male sterile)

F1

x

Study done at IARI using male sterile & male fertile lines in Tomato

• Time saved by male sterile line was 63%.

• Average fruit set by using male sterile line was 79.35%.

• Average fruit set using male fertile line was 65.40%.

• Hybrid seed cost by using male sterile line per kg was Rs. 466/-

• Hybrid seed cost by using male ferile line per kg was Rs. 3691/-

Tewari (1997)

Dhaliwal and Cheema (2008)

Time required for crossing 50 flower buds on male fertile (Ms33 IPA) and genetic male sterile (ms33 IPA) plants in tomato.

Maintenance of male Maintenance of male sterilitysterility

• Genic or pollen male sterilitymsms (male sterile) x Msms (male fertile)

1 Msms : 1 msms (rogued out)

Drawbacks in male sterilityDrawbacks in male sterility

• Maintenance (Identification and roguing) is difficult in pollen abortive (ms) sterility

• ps 2 sterility has variable expressivity and there is necessity of staminal emasculation

• Outcrossing leading to poor seed set (0.3-1.5 kg/acre seed as a result of natural outcrossing)

(Yardanov, 1983)

Linkage of ms gene with the marker gene in tomato

Marker geneMarker gene ReferencesReferences• Potato leaf shape & green stem colour•Parthenocarpic fruit•Enzyme markers• Purple coloured hypocotyle•Anthocyanin absent

•Kaul,1988

•Soressi & Salamini,1975•Tanksley et al.,1984

•Georgiev, 1991

The ms-1035 gene is linked with a recessive marker gene aa (absence of anthocyanin). Hence, ms-1035 sterile plant can be identified at seedling stage and fertile plant can be rouged out in the nursery itself. Moreover, no effect of genes ms1035aa on plant and fruit characteristics was established.

(Atanassova and Georgiev, 2006)

For utilizing functional sterility anther emasculation could be made easier if ps 2 was combined with short style. Such flowers can be emasculated without using forceps (Georgiev and Atanassova 1981)

USE OF CHEMICAL HYBRIDIZING AGENTS (CHA) Applied chemicals Remark(s) Reference

GA3 Treated plants produced separatestamens and split pistils

Chandra Sekharand Sawhney,1990

Gibberlin synthesisCCC inhibitors

Selectively inhibited the development of stamen or msuppressed pollen

Rastogi and Singh,1988

ABA (Abscisic acid) Selectively inhibited the development of stamen or suppressed pollen

Rastogi and Singh,1988

FW-450 (Mendok) Showed promise for commercialutilization

Moore, 1959

Dalapon Male sterility was induced Brauer, 1959TIBA (TriiodobenzoicAcid)

Some degree of male sterility wasinducing

Rehm, 1952

NAA Induced male sterility McRae, 1985

Biotechnological tools useful in Hybrid Biotechnological tools useful in Hybrid seed productionseed production

Tissue culture Clonal multiplication via in vitro fertilisation Haploid culture Protoplast fusion

Transgenic approaches for male sterility for developing resistant line terminator seed technology

Molecular Markers

Application of haploid Application of haploid cultureculture

CROP FINDINGS REFERENCE

Tomato Haploidy has been successfully used for developing male sterile pure line

Zamit et al., 1980Schereva et.al., 1990

42% MS plant+34% normal plant obtained by culturing cv.Roma & MS line with ms 1035

Oankh et al., 1986

Application of Protoplast fusion

Through protoplast fusion of Solanum esculentum with Solanum acuale & S. tuberosum, cytoplasmic male sterile cybrid plant with different flower morphology have been isolated (Melchers et al., 1992)

Male sterile cytoplasm has been transferred into S. pennelli & then CMS pennelli has been successfully crossed with esculentum (Petrova et al., 1999)

Use of molecular markers Marker assisted transfer of specific genes/ QTLs

controlling heterosis for desirable traits

Assessment of genetic diversity Establishment of heterotic pools

Prediction of hybrid performance

Testing of genetic purity of parental lines and hybrid seeds

Analysis of Genetic Diversity in 11 Tomato Varieties using RAPD Markers

Tabassum et al. (2013)

Twenty arbitrary oligonucleotide primers used in the RAPD‐PCR produced a total of 584 different marker bands with an average of 29.2 bands per primer.

Based on the banding pattern 94.168% polymorphism observed among the tomato varieties

The values of pair‐wise genetic distances ranged from 0.1838 - 0.9049, indicating the presence of wide genetic diversity.

Determination of Genetic Purity of Hybrid Seed in Watermelon and Tomato Using RAPD

markers

Hashizume et al. (1993)

Mechanism Remark(s) Reference

Nuclear malesterility

Monogenic recessive mutant was utilized todevelop cost effective experimental crosses.

Sawhney, 1997;Kumar et al., 2001

Auxotrophy A very attractive and feasible model wasproposed utilizing monogenic recessivenutritional mutants e.g. thiamin dependent.

Barabas, 1991

Incongruity

Models to transfer barrier (incompatibilitybetween pollen and stigma) genes andcorresponding penetration genes weredemonstrated.

Hogenboom et al.,1978

Commercially unexploited mechanisms for the development of hybrids in tomato

HYBRID INSTITUTE

Arka Abhijit IIHR, Bangalore

Arka Shreshtha IIHR, Bangalore

Arka Vardan IIHR, Bangalore

Arka Vishal IIHR, Bangalore

Arka Rakshak IIHR, Bangalore

Arka Ananya IIHR, Bangalore

Arka Samrat IIHR, Bangalore

Pusa Divya IARI, New Delhi

Pusa Hybrid-1 IARI, New Delhi

Pusa Hybrid-2 IARI, New Delhi

Pusa Hybrid-4 IARI, New Delhi

TH-802 PAU, Ludhiana

TH-2312 PAU, Ludhiana

RHRTH-57 MPKV, Rahuri

RHRTH-92 MPKV, Rahuri

COTH-1 TNAU, Coimbatore.

VRTH-1 IIVR,Varanasi

VRTH-2 IIVR,Varanasi

LIST OF F1 HYBRIDS IN TOMATOHYBRID INSTITUTE

Rupali IAHS

Vaishali IAHS

Naveen IAHS

Rashmi IAHS

Megha Beejo Sheetal Seeds

Madhuri Beejo Sheetal Seeds

Manisha Beejo Sheetal Seeds

Meenakshi Beejo Sheetal Seeds

ARTH-3 Ankur Seeds

ARTH-4 Ankur Seeds

Arka Rakshak

• First public triple disease resistant tomato F1 hybrid in India• High yielding F1 hybrid giving yield of 90-100 tons per hectare in 140-150

days• Triple disease resistance to tomato leaf curl virus, bacterial wilt and early

blight• Suitable for summer, kharif and rabi seasons• Crossing an advanced breeding line bred at IIHR with another breeding line

bred at AVRDC, Taiwan

MAJOR CONSTRAINTS

High cost of F1 hybrid seeds Lack of awareness among the growers about

hybrid crop production techniques Unorganized marketing system for vegetables Lack of postharvest management techniques Non availability of quality seeds Non availability of other inputs at proper time Non availability of biotic stress resistant hybrids

CONCLUSICONCLUSI

ONSONS