Anther culture Rice final
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Anther Culture Response of some Salt Resistant and Susceptible Indica Rice Varieties
Presented By:
M.C.Mohamed Zakeel
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H.M.P.S. Kumari, U.N.D. Silva, W.M. Abayarathne, D.S. De Z. Abeysiriwardena,
I.W.M.K.N.K. Yatawara and D.N. Sirisena
Annals of Sri Lanka Department of Agriculture 2006
8:137-145
Rice Research and Development Institute
BatalagodaIbbagamuwa
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Introduction
Important biotech tool in rice breeding Genetically diverse haploid, Homozygous diploid within short time Early expression of recessive gene Increased selection efficiency
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Cont…
First successful haploid Datura Innoxia (1960)
Applied to rice Present promising approach China, Taiwan, South Korea, Japan, the
USA, India Efficient callusing & regeneration in
Indica – still challenging field
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Cont…
Cold treatment induces calli formation Delayed senescence & entry of nutrients Various auxins & cytokinins
combinations Regeneration by water stress Spontaneous or induced doubling
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Cont…
Spontaneous in tetep & tulsiEndomitosis Irregular meiosisSpindle fusionNuclear fusion
Callus induction ability inherited as recessive trait
8 QTLs for rice anther culturability
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Objectives
Tremendous improvement in rice conventional breeding
Creation of genetic variability & application of advanced technologies develop varietal resistance for biotic & abiotic stress- LIMITED
To investigate anther culture response of selected rice varieties showing varying response to salt stress
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Materials and Methods
8 popular rice varieties Grown under natural condition
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Variety Reaction to salinity
Traditional Pokkali Tolerant
Nonabokra Tolerant
Improved At 354 Tolerant
At 401 Tolerant
Bw 400 Moderately tolerant
Bg 94-1 Susceptible
Bg 300 Susceptible
Bg 750 ***
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Cont…
Panicle collected at late uninucleate microspore stage
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Pretreatment and sterilization of plant material
Cold treatment (10 oC) Panicle sterilization – 70% ethanol Opened panicle –
20% Clorex for 20 min 70% ethanol Sterilized distilled water
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Culture media for callus initiation & plant regeneration
N6 medium Different combination & concentration of
growth regulators Anthers removed from spikelets 100 anthers per each petridish 25 petridish per each medium Incubated under dark at 25oC
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Treatment Sugar type Growth regulators (mg/l)
Sucrose % Maltose % 2,4 D KN NAA BAP
Callus induction
N6-1 6 2 0.5 - -
N6-2 6 - 0.5 2 -
N6-3 6 2 1 - -
N6-4 6 - 1 2 -
N6-5 6 2 0.5 2 -
N6-6 6 2 1 2 -
Plant regeneration
MS1 3 - 0.5 1 1
MS2 3 - 0.5 1 2
MS3 3 - 2 1 0.5
MS4 3 - 2 1 2
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Partial desiccation of calli & plantlet regeneration
Primary calli (1-5 mm) subjected to 24 hr water stress
Petridish & Watmann No. 1 filter paper Desiccated calli transferred to modified
MS medium (regeneration) Incubated at 250C (16hr/8hr – light/dark)
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Identification of ploidy level & plantlet establishment in soil
Root tip with aceto carmine stain Plantlet transferred to sterilized soil Kept in green house Seeds screened under field condition
Uniform growthSeed settingAgainst disease & salt stress
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Result and Discussion
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Callus induction
7 days cold trt - highest response to callusing & regeneration
Variety x medium interaction not significant Fast calli proliferation eventually turned necrotic Higher con. of auxins suppresses callus initiation
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Variety N6-1 N6-2 N6-3
# of planted anthers
# of calli produced
# of planted anthers
# of calli produced
# of planted anthers
# of calli produced
Pokkali 484 3 465 6 430 7
BW 400 352 10 314 5 374 9
Bg 300 267 1 275 1 284 -
Big 750 306 17 244 22 269 18
Bg 94-1 310 3 291 - 285 4
At 354 281 - 290 - 206 -
At 401 395 6 282 11 397 2
Nonabokra 294 40 175 45 244 40
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Variety N6-4 N6-5 N6-6
# of planted anthers
# of calli produced
# of planted anthers
# of calli produced
# of planted anthers
# of calli produced
Pokkali 494 27 295 - 312 -
BW 400 387 15 325 - 316 -
Bg 300 304 2 245 - 302 -
Bg 750 202 - 224 - 220 -
Bg 94-1 215 - 205 - 196 -
At 354 260 - 254 - 276 -
At 401 123 - 208 8 156 27
Nonabokra
106 44 170 - 165 -
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Plant regeneration
Variety x medium interaction not significant Pokkali, Bg 94-1, At 401, Bw 400 produced
plants on MS2 Frequency of regeneration 82.7%, 20.8%,
46.7%, 26.7% MS2 BEST among all others High frequency of green (82,7%) & albino (15%)
in Pokkali
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Variety MS1 MS2
# of calli cultured
Regenerated plants
# of calli cultured
Regenerated plants
Pokkali 17 2 (11.7%) 29 24 (82.7%)
Bg 750 39 - 26 -
Bg 300 7 - 5 -
Bg 94-1 7 - 24 5 (20.8)
At 401 17 - 15 7 (46.7%)
Bw 400 10 - 15 4 (16.7%)
Nonabokra 12 - 11 -
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Variety MS3 MS4
# of calli cultured
Regenerated plants
# of calli cultured
Regenerated plants
Pokkali 16 2 (12.5%) 41 16 (39%)
Bg 750 41 - 10 -
Bg 300 3 - 7 -
Bg 94-1 17 - 10 -
At 401 36 - 33 -
Bw 400 15 - 8 2 (25%)
Nonabokra 9 - 9 -
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Cont…
16 Pokkali & 2 Bw 400 plants were established in soil
Somatic metaphase analysis – Haploid & diploid chromosome number
All Pokkali plants showed homogeneity, uniformity, seed setting & shorter plant height
Bw 400 – homogeneity, uniformity, 100% fertility
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Conclusion
Variety x medium interaction not significant in calli initiation & plant regeneration
Only Pokkali & Bw 400 successfully established in soil
Breeding lines obtained fro Pokkali with improved chars
Anther culture tech can be used in rice improvement
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Cont…
Creating genetic variants Producing double haploidEarly expression of recessive genes Shortened breeding cycle
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Thank you