Anther Culture Response of some Salt Resistant and Susceptible Indica Rice Varieties Presented By: M.C.Mohamed Zakeel
Anther Culture Response of some Salt Resistant and Susceptible Indica Rice Varieties
Presented By:
M.C.Mohamed Zakeel
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
Introduction
Important biotech tool in rice breeding Genetically diverse haploid, Homozygous diploid within short time Early expression of recessive gene Increased selection efficiency
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
Cont…
Cold treatment induces calli formation Delayed senescence & entry of nutrients Various auxins & cytokinins
combinations Regeneration by water stress Spontaneous or induced doubling
Cont…
Spontaneous in tetep & tulsiEndomitosis Irregular meiosisSpindle fusionNuclear fusion
Callus induction ability inherited as recessive trait
8 QTLs for rice anther culturability
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
Materials and Methods
8 popular rice varieties Grown under natural condition
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 ***
Cont…
Panicle collected at late uninucleate microspore stage
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
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
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
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)
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
Result and Discussion
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
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
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 -
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
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 -
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 -
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
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
Cont…
Creating genetic variants Producing double haploidEarly expression of recessive genes Shortened breeding cycle
Thank you