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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
Batalagoda
Ibbagamuwa
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 & tulsi


 Endomitosis
 Irregularmeiosis
 Spindle fusion
 Nuclear 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 growth
 Seed setting
 Against 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 # of calli # of # of calli # of # of calli
plante produ plan produc plan produc
d ced ted ed ted ed
anthe anth anth
rs ers ers
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 # of calli # of # of calli # of # of calli
plant produc plant produc plant produc
ed ed ed ed ed ed
anth anth anth
ers ers ers
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
Nonabok 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 Regenerated # of calli Regenerated


culture plants culture plants
d d
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 Regenerated # of calli Regenerated


cultured plants cultured 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 haploid
 Early expression of recessive genes
 Shortened breeding cycle
Thank you