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