in vitro callus induction and plantlet regeneration...

152Current Trends in Biotechnology and PharmacyVol. 8 (2) 152-159 April 2014, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractEffect of organic adjuvants, synthetic plant

growth regulators and diverse carbon sources ininfluencing anther culture response in two indicarice genotypes (GR 11 and Gurjari) wereassessed due to genotypic difference.Androgenic callus induction as well as greenplantlet regeneration was more when callus wasinduced on N6 fortified with 100 mgl-1 yeastextract (YE). However, casein hydrolysate (CH)at 50, 250 and 500 mgl-1 concentrationsencouraged green plantlet regeneration. Highcallus induction was observed with syntheticgrowth regulators in comparison with the controlset. However, green plantlet regeneration wasobserved when callus induction medium (CIM)was supplemented with 2, 4-D, NAA and Kn inboth the varieties. Between two varieties, Gurjarishowed better callus induction on N6 mediumsupplemented with 6% sucrose. Maximum callusinduction was observed in Gurjari when 6%sucrose was used as carbon source. Plantletregeneration in the present study was found tobe very low. This study elucidates that thegenotypic differences to in vitro response andthe probability of the exploitation of androclonalvariation in indica rice.

Keywords: Anther culture, doubled haploids, 2,4-D, NAA, Yeast extract, Casein hydrolysate, Oryzasativa.

IntroductionIn the recent past anther culture in rice

has been improved substantially. However,detailed study on various factors governingculture response of anthers under in vitroconditions especially in indica rice is extremelylimited. Generally green plant regeneration fromandrogenic calli is very low irrespective of therace (1). Low anther culture response, highpercent of albino plantlet regeneration andabundance of haploids are the principalconstraints in establishing successful antherculture in rice.

Plantlets developed directly frommicrospores provide ample scope in developinghomozygous doubled haploids (DH) withoutinterference of heterozygosity. Anther cultureprovides an easy to handle in vitro selection forgenetic improvisation of characters (2) forsuperior performance. Androclonal variationseems to have immense prospect albeit it wasquantified in a very few cases in rice (3). DHlines produced from microspore culture and/oranther culture were proved to be highly potentialin genetic improvement by broadening thegenetic diversity through production ofhomozygous lines within short time (4,5), incontrast to numerous cycles of inbreeding orback crossing as conventionally practiced enroute plant breeding. Owing to the importanceof anther culture in speeding up the breeding

In Vitro Callus Induction and Plantlet RegenerationStudies through Anther Culture in Two Indica Rice

(Oryza Sativa L.) Varieties

J. K. Patel1*, N. Subhash2 and R. S. Fougat2

Department of Agricultural Botany, B. A. College of Agriculture, Anand, 388 110, India1Agricultural Research Station, Anand Agricultural University, Sansoli 387 130, India

2Plant Tissue Culture Laboratory, Department of Biotechnology, Anand Agricultural University,Anand 388 110, India.

*For Correspondence - [email protected]

Anther Culture Studies In Rice


process, plants with special agronomiccharacters such as development of earliness,increased grain weight, superior grain quality,disease resistance (6,7), dwarf plant type andabiotic stress tolerances have been developed(8). Unlike somaclonal variation, utilization ofandroclonal variation (AV) in rice geneticimprovement is reported to be exceedingly less.Upon culturing anthers from an inbred line or fromF

1 recombinants, one might generate

androclones with altered characters differentfrom the parent due to culture induced AV. Thus,anther culture prospects amply in rice geneticimprovement. Considering the above aspects ofanther culture, the present study was aimed atincreasing the green plantlet regeneration byoptimizing plant growth regulators (PGR), organicadjuvants and carbon sources and to asses therange and magnitude of variation in pollenderived plants of traditional cultivars, GR 11 andGurjari which were widely grown in the state ofGujarat and are popular for its high demand indomestic and international markets owing to highconsumer preference.

Materials and methodsPlant materials and callus induction: Thevarieties, GR 11 and Gurjari were employed forthis study. The panicles with boot leaf sheathwere wiped with 70% ethanol. They were coveredwith moist tissue paper, kept in polyethylene bagand cold shocked at 8ºC for eight days in arefrigerator prior to anther plating. On the day ofculture, selected spikelets were surface sterilizedin Erlenmeyer flask with 0.1% freshly preparedHgCl

2 solution for 10 min. The HgCl

2 was drained

off and the panicles were washed three times insterile distilled water. Fifty to sixty spikelets werecut at a time on sterile petri dishes under laminarairflow (LAF) bench. Individual spikelets were cutat the base to free the anthers from the filaments.They were plated aseptically onto autoclavedErlenmeyer flasks (100 ml capacity, Borosilicateglass) containing callus induction medium (CIM)- N6 (9) supplemented with differentconcentrations of plant growth regulators (2,4-Dat 1.0 and 2.0 mgl-1; NAA at 1.0 and 2.0 mgl-1

and Kinetin at 0.5 and 1.0 mgl-1), organicadjuvants (Yeast extract at 100, 200 and 500 mgl-1 and casein hydrolysate at 50, 250 and 500 mgl-1) and carbon sources (sucrose, maltose andglucose at 4, 6 and 8% singly) in liquid form. Thecultures were kept in dark at 25 + 2ºC. The flaskswere examined periodically at weekly intervalsto observe the progress in respect of callusformation. Embryogenic calli of at least ~2 mmdiameter were transferred to 25 x 150 mm culturetubes (Borosil) containing 14 ml regenerationmedium consisting of MS (10) supplemented withdifferent concentrations (2.0, 3.0 and 4.0 mgl-l)of Kinetin, (1.0 and 2.0 mgl-l) GA

3, 3% (w/v)

sucrose and 0.8% (w/v) agar. The pH of themedium was adjusted to 5.8 with 0.1N HCl or0.1N NaOH before adding agar and autoclavedat 121ºC, 15 lb pressure for 20 minutes. Theculture tubes were plugged with non-absorbentcotton wrapped in one layer of cheesecloth.Inoculated cultures were kept for four weeksunder 16/8 h light (~130 μE m-2 s-1) /dark at 25 +2ºC.

Results and DiscussionEffect of plant growth regulators: Higher orderinteraction of mean values for the effect of 2,4-D, NAA, Kn and variety on liquid medium forcallus induction per cent and days to callusinduction (Table 1) showed non-significantvalues. Analysis of variance revealed that therewas significant differences for 2,4-D, NAA, Kn,variety, 2,4-D and NAA interaction, NAA andvariety interaction, Kn and variety interaction and2,4-D, NAA and Kn interaction, 2,4-D, NAA andvariety interaction and NAA, Kn and varietyinteraction.

Present investigation showed that 2.0mgl-1 2,4-D incorporated in N6 media gave highcallus induction per cent (2.07%). Overall, N6medium supplied with 1.0 mgl-1 concentration ofNAA showed significantly high callus inductionper cent (2.13%) among NAA levels. At the sametime when N6 medium was supplemented with0.5 mgl-1 concentration of Kn showed overall bestcallus induction per cent (2.33%) as well as earlycallus induction (46.50 days) when only Kn levels

Patel et al


Tab

le 1

. E

ffect

of

2,4-

D,

NA

A,

Kn

and

varie

ty o

n m

ean

valu

es f

or p

erce

ntag

e of

cal

lus

initi

atio

n.

Trea

tmen

t

V

arie

ty (D

)2,

4-D

NAA

KnVa

riety

A x

BB

x C

C x

DAx

BxC

AxBx

DBx

CxD

2,4-

D (A

)N

AA (B

)Kn

(C)

GR

11

Gur

jari

Mea

nM

ean

Mea

nM

ean

Mea

nM

ean

Mea

nM

ean

Mea

nM

ean

1.0

1.0

0.5

0.77

2.71

1.77

2.13

2.33

GR

11

1.70

1.65

2.42

1.84

1.74

1.16

1.74

1.0

1.55

1.55

1.55

2.13

3.10

2.0

0.5

2.33

2.71

1.89

1.84

2.81

2.52

1.84

2.13

1.0

1.35

1.16

1.26

1.94

1.55

2.0

1.0

0.5

2.71

3.48

2.07

1.70

1.51

Gur

jari

2.13

2.62

2.23

1.57

3.10

2.71

1.94

1.0

2.71

1.55

2.13

2.52

2.52

2.0

0.5

1.55

2.33

1.52

1.18

1.45

1.94

1.10

1.00

1.0

0.65

1.55

1.10

1.94

1.36

A x

B x

C x

D

S. E

m. +

0.22

0.08

0.11

0.16

C. D

. 0.0

5N

S0.

230.

320.

45C

. V. %

20.1

3

S.Em

.: St

anda

rd e

rror o

f mea

n, C

.D. 0

.05:

Crit

ical

diff

eren

ce a

t 5.0

0% p

roba

bilit

y, C

.V. %

: Coe

ffici

ent o

f var

ianc

e pe

r cen

t

Tab

le 2

. Effe

ct o

f org

anic

adj

uvan

ts a

nd th

eir

conc

entr

atio

ns o

n ca

llus

initi

atio

n pe

r ce

ntA.

Tre

atm

ent

Var

iety

(C

)A

vera

geA

vera

ge o

fA

x B

of o

rgan

icco

ncen

trat

ion

Ave

rage

Org

anic

Con

cent

ratio

nG

R 1

1G

urja

riad

juva

ntA

djuv

ant

(A)

mgl

-1(B

)

Yea

st E

xtra

ct10

0 (L

ow)

1.54

(2.3

7)1.

54(2

.38)

1.25

(1.

57)

1.34

(1.

79)

1.54

(2.

37)

200

(Med

ium

)1.

31(1

.73)

1.31

(1.7

3)1.

31 (

1.72

)50

0 (H

igh)

0.92

(0.8

7)0.

91(0

.85)

1.31

(1.

72)

0.91

(0.

84)

Cas

ein

Hyd

roly

sate

50 (

Low

)1.

14(1

.30)

1.14

(1.3

0)1.

12 (

1.26

)1.

14 (

1.29

)25

0 (M

ediu

m)

1.31

(1.7

3)1.

31(1

.73)

0.91

(0.

84)

1.31

(1.

72)

500

(Hig

h)0.

92(0

.87)

0.91

(0.8

5)0.

91 (

0.84

)

S.

Em

. +

0.08

870.

0362

0.04

440.

0627

C.

D.

0.05

NS

0.11

0.13

0.18

C. V

. %12

.94

A S

quar

e ro

ot t

rans

form

atio

n, r

e-tr

ansf

orm

ed v

alue

s in

par

enth

esis

. S

.Em

.: S

tand

ard

erro

r of

mea

n, C

.D.

0.05

:C

ritic

al d

iffer

ence

at 5

.00%

pro

babi

lity,

C.V

. %: C

oeffi

cien

t of v

aria

nce

per

cent



were compared and these values were foundsignificant. Between two varieties, Gurjarishowed significantly high callus induction per cent(2.13%) than variety GR 11.

Medium N6 supplemented with growthregulator combination of 2.0 mgl-1 2,4-D+ 1.0 mgl-1 NAA showed significantly high callus inductionper cent (2.62%) than any other medium overvariety and concentrations of Kn. Liquid N6medium supplemented with 1.0 mgl-1 NAA + 1.0mgl-1 Kn showed highest callus induction per cent(2.42%) and the value was at par with 2.0 mgl-1

NAA + 1.0 mgl-1 Kn medium. Variety Gurjarishowed highest callus induction per cent on liquidN6 medium supplied with 1.0 mgl-1 Kn (2.81%).Liquid medium N6 supplied with 2.0mgl-1 2,4-D +1.0 mgl-1 NAA + 0.5 mgl-1 Kn showed high callusinduction per cent (3.10%) which was foundsignificant. Variety GR 11 showed highest callusinduction per cent (2.71%) on medium N6supplied with 2.0 mgl-1 2,4-D + 1.0 mgl-1 NAA andat the same concentration of growth regulatoron variety Gurjari the value was at par in theexperiment. Variety Gurjari showed significantlyhigh callus induction per cent (3.10%) whenanthers were cultured on liquid N6 mediumsupplemented with 1.0 mgl-1 NAA + 0.5 mgl-1 Kn.

Compact embryogenic calli of ~2 mmdiameter were transferred to regenerationmedium (RM) for further plant regeneration.Albino plantlets were also obtained in both thevarieties.

Effect of organic adjuvants: Two organicadjuvants viz., Yeast Extract and CaseinHydrolysate at three different concentrationswere studied. Mean values for organic adjuvants,their concentration and variety interaction (Table2) showed non significant differences for callusinduction per cent and days to callus induction.Analysis of variance showed significantdifferences for type of organic adjuvants,concentration of organic adjuvants andinteraction between organic adjuvant and itsconcentration for callus induction per cent. Varietyshowed non-significant effect for callus induction

per cent as well as days to callus induction. Daysto callus induction showed non-significantdifferences for organic adjuvants, itsconcentration and interaction effects.

Both the varieties produced androgenic calliin N6 supplemented with three differentconcentrations (100, 200 and 500 mgl-1) of YEand (50, 250 and 500 mgl-1) CH each. High callusinduction (1.79%) was observed at lowconcentration (100 mgl-1) of YE in both antherculture responsive varieties. This showed thatlower concentration of YE facilitated androgeniccallus initiation. The results also indicatesubstantial variation in callus induction percentfor type of organic adjuvants as well as amongdifferent concentrations.

Between two organic adjuvants tried, yeastextract gave significantly higher callus inductionper cent (1.25) than casein hydrolysate (1.12).Low concentration of organic adjuvant showedsignificant difference for callus induction per cent(1.34) and was at par with medium concentration(1.31) for the same.

When N6 medium was supplementedwith yeast extract at low concentration (100 mgl-1) showed significantly high callus induction percent (1.54). At the same time mediumconcentration of casein hydrolysate (250 mgl-1)showed better callus induction per cent amongthe different concentrations of caseinhydrolysate.

Effect of carbon sources: Diverse carbonsources viz. sucrose, glucose and maltose wereused to pinpoint the appropriate compound forprolific callus induction and facile green plantletregeneration in anther culture system. Maximumcallus induction (3.34%) was observed in Gurjarion N6 with 6% sucrose. Callus induction wasextremely low in GR 11. However, callus inductionwas observed in all the treatments for both thevarieties.

Mean values for type of sugars, theirconcentrations and variety showed non-significant interactions (Table 3). Analysis of

Patel et al


Table 3. Effect of sugars and their concentrations on callus initiation per cent.

Treatments Variety (C) Average Average of Average of A x B B x Cof type concentra- variety

Sugar (A) Concentration (B) GR 11 Gurjari of sugar tion Interaction Interaction

Sucrose 4% (Low) 1.61 1.81 2.20 1.94 GR 112.05 1.71 1.886% (Medium) 3.01 3.34 3.18 2.018% (High) 1.61 1.81 1.71

Glucose 4% (Low) 1.81 1.81 1.95 2.79 1.81 2.686% (Medium) 1.81 2.34 Gurjari2.39 2.08 2.908% (High) 1.61 2.34 1.98

Maltose 4% (Low) 2.21 2.41 2.51 1.93 2.31 1.616% (Medium) 3.21 3.01 3.11 2.258% (High) 1.61 2.61 2.11

S. Em. + 0.17 0.0703 0.0574 0.12 0.0994C. D. 0.05 NS 0.20 0.16 0.35 0.16C. V. % 13.42

S.Em.: Standard error of mean, C.D. 0.05: Critical difference at 5.00% probability, C.V. %: Coefficientof variance per cent

Table 4. Effect of Kn and GA3 on percentage of plantlet regeneration for variety GR 11 andGurjari

Treatments Green Plantlet % A Albino Plantlet % A Total Plantlet % B

Kn GA3 GR 11 Gurjari GR 11 Gurjari GR 11 Gurjari

2.0 1.0 4.21 (17.24) 2.99 (8.45) 2.73 (6.97) 2.49 (5.68) 30.12 (25.18) 24.34 (16.98)2.0 4.16 (16.82) 2.99 (8.45) 2.73 (6.97) 2.23 (4.46) 31.45 (27.22) 23.01 (15.28)

3.0 1.0 4.01 (15.60) 3.50 (11.74) 3.76 (13.60) 2.23 (4.46) 33.07 (29.78) 24.63 (17.37)2.0 4.67 (21.29) 2.99 (8.45) 2.99 (8.45) 2.73 (6.97) 34.40 (31.92) 24.63 (17.37)

4.0 1.0 4.01 (15.60) 3.25 (9.42) 3.24 (10.00) 2.23 (4.46) 30.54 (25.82) 24.34 (16.98)2.0 3.95 (15.13) 3.76 (13.60) 3.50 (11.74) 2.73 (6.97) 31.74 (27.68) 25.96 (19.16)

Variety 4.17 (16.89) 3.25 (10.04) 3.16 (9.48) 2.44 (5.45) 31.89 (27.90) 24.48 (17.18)Average

S. Em. + 0.22 0.37 1.18C. D. 0.05 0.62 0.16 3.35

Kn x GA3 x Variety interaction

S. Em. + 0.53 0.53 2.89C. D. 0.05 NS NS NSC. V. % 32.07 42.01 22.92

A Square root X + 0.5 Transformation, B ARC sin transformation, Re-transformed value in parenthesisS.Em.: Standard error of mean, C.D. 0.05: Critical difference at 5.00% probability, C.V. %: Coefficient ofvariance per cent



variance showed significant difference for themean values of type of sugar, concentration ofsugar, variety, interaction between sugars andits concentration and interaction between sugarconcentration and variety for callus induction percent. Variety showed significant difference fordays to callus induction. Interaction betweensugar, its concentration and variety did not showsignificant difference for callus induction and daysto callus induction.

In this experiment N6 mediumsupplemented with maltose showed significantlyhigher callus induction per cent (2.51%) followedby sucrose (2.20). Over all, mediumconcentration of sugar (6%) showed significantlyhigher callus induction per cent (2.79%) thanother concentrations of sugar. Variety Gurjari

Plate 1. A callus initiation of Liquid N6 + 2.0 mgl-12,4 - D+1.0 mgl-1 NAA+0.5 mgl-1 kn for variety 1)GR 11 and 2) Gujari,B1 - 4 Embryo development of variety GR 11C1 - 4 Embryo development of variety GurjariD Embryo germination on semi-sold medium1) MS+3.0 mgl-1Kn+2.0 mgl-1 GA, +3%Sucrose+0.8% Agar-agar, variety GR II2) MS+4.0mgl-1 Kn+2.0 mgl-1 GA3 + 3%sucrose+0.8 Agar-agar, variety gurjari

showed significantly higher callus induction percent (2.39%) but induced later callusing (50 days)than variety GR 11 (2.05% and 48 days) in theexperiment.

Interaction of sugar and itsconcentrations on callus induction per centshowed significant differences. Sucrose atmedium concentration (6%) showed highestcallus induction per cent (3.18%) and was at parwith the same concentration of maltose (3.11%).Gurjari variety showed significantly higher callusinduction per cent (2.90) at mediumconcentration (6%) of sugar.

The embryogenic calli were transferredto RM for regeneration of plantlets. High percentof albino plantlets were observed from all the in

Plate II : Primary Hardened Plantlets variety a) GR IIGurjari Hardened plants established in earthen potsvariety C) GR II D) Gurjari

Patel et al


vitro culture responsive cultivars. Mean valuesof green, albino and total plantlet regenerationfor the effect of Kn, GA

3 and variety interaction

(Table 4) showed non-significance differences.Analysis of variance showed that variety hadsignificant effect for the number of green, albinoand total plantlet regeneration. Kn and GA

3 did

not show any effect on plantlet regenerationindividually as well as interaction.

Variety GR 11 showed significantly highmean values for number of green (4.17), albino(3.16) and total (31.89) plantlet regeneration thanvariety Gurjari.

Manipulation of chemical and physicalculture environments is essential to provokemicrospore to switch on to an embyogenic ratherthan a gametophytic pattern of development (11).The effect of different treatments and varietiesplay an important role for callus induction andplantlet regeneration. It was also found that YE(100 mgl-1), 2,4-D (2 mgl-1) and 6% glucose inCIM promoted green plantlet regeneration. N6supplemented with organic adjuvants like YE andCH showed enhanced androgenic callusinduction in indica rice to a considerable extent.Null relationship was discernable between callusinduction/plantlet regeneration and differentconcentrations of medium/organic adjuvants,plant growth regulators, carbon sources andgenotypes. This indicates that the callus inductionand subsequent green plantlet regeneration werestrictly genotype specific. However, applicationof 6% maltose as carbon source in the mediuminflated plantlet regeneration substantially. Greenplantlet regeneration was found to be very low. Itis mentionable that the success of plantletregeneration under in vitro culture systemdepends on the type of medium used in eachphase of culture starting from callus induction,proliferation to plantlet regeneration and the typeand dose of different growth regulators especiallyauxins, and cytokinins used in cereal antherculture. Among the auxins, 2,4-D was found tobe useful for callus induction and subsequentlyin green plantlet regeneration. Interestingly,number of responding calli was high in GR 11.

Very low culture response was observed inGurjari. Among the treatments, more cultureresponse was observed when medium wasadded with organic adjuvants like CH and YE,plant growth regulators (2, 4-D, NAA and Kn) andmaltose as carbon source. Both the varietiesshowed green plantlet regeneration (Plate: 1 and2). The regenerated plantlets were rooted in thesame medium. After two weeks on RM, profusetillers were also observed. The rooted plantletswere shifted to experimental glass house forhardening. After two weeks of hardening, plantswere finally transferred to earthen pots. Theandrogenic plantlets obtained from GR 11showed flowering on time, however, producedcent percent spikelet sterility owing to theirhaploid nature. The plants developed in Gurjarialso flowered and produced filled grains and withhigh sterility perhaps due to their genesis anddevelopment on relatively harsh in vitroenvironment.

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6. Zhang, Z.H. (1989). In: Mujeeb Kazi A. andSitch L. A. (Eds.) Review of PlantBiotechnology 1985-88: The practicabilityof anther culture breeding in rice. CIMMYT,Mexico and IRRI, Philippines p. 31-42

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