ANDROGENIC DOUBLED HAPLOIDS IN INDICA RICE : IN VITRO INDUCTION, PLANT REGENERATION

AND EVALUATION

A Thesis Submitted for the Degree of

DOCTOR OF PHILOSOPHY

By

Rabindra Nath Bhuyan

NORTH·EASTERN HILL UNIVERSITY

SHILLONG

1996

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We certi~y that the thesis entitled II Androgenic

doubled haploids in rice : in vitro induction~ plant regeneration

and evaluation" submitted by Mr. Rabindra Nath Bhuyan ~or the

degree o~ Doctor o~ Philosophy in Botany o~ the North Eastet·n

Hill University, Shillong embodies the t·ecm·d 0~ original

investigation carried out by him under our supervision. He has been

duly registered and the thesis presented is worthy ~or being

considered ~or the award o~ Ph.D. degree. The work has not been

submitted ~or any degree o~ any other university.

(Prcunod Tandon)

Department o~ Botany

North-Eastern Hill University

Shillong 793 022

Date: June 5, 1996

Place: Shillong

, ' J \,1 \".{ 0 5 I \ q q -b (H.S.Gupta)

Division o~ Plant Breeding

ICAR Research Complex ~or

North Eastern Hill Region

Barapani - 793103

ACKNOWLEDGEMENT

I express my deep sense o-f gratitude to Dr. H. S. Gupta,

Principal Scientist and Head, Division o-f Plant Breeding, ICAR

Research Complex -for North Eastern Hi 11 Region, Barapani and

Dr. PrQtnod Tandon, Department o-f Botany,

North - Eastern Hill University~ Shillong -for their constant

guidance, valuable suggestion, const t-uct i ve criticism and

g~nerous attitude throughout the course o-f this investigation and

instrumentalization o-f this dissertation.

I am deeply thank-ful to Dr. B. K. Sarma, and

Mr. A. Pattanayak, o-f the Division o-f Plant Breeding ICAR

Research

ways.

Compex -for NEH Region -for helping me in all possible

I am highly indebted to the Di t-ectm- o-f ICAR Research

Complex -for North Eastern Hill Region ·Fm- pt-oviding -ficilities

during the course o-f this investigation.

I -further express thanks to Drs. D . K • Pandey,

M. Singh , U. K. Hazarika • S. Banerjee and Mr. A • Khound -for

their generous help in computer works.

I also sincerely thank Miss. B. Bhattacharjee, Mr. Ram Murti

and Mr. Hari Govind -for their valuable assistance in laboratory

works.

I am indebted to Miss. Verginia Kalita and Ms Suman Kumaria

o-f Department o-f Botany Nor-th-- E"'stet-n Hill University

~or their kind cooperation at the time o-f seminars.

Sincere thanks are also due to Mt- • A. Ahmed and

Mr. Nandalal Chetri -for helping the author in matters related to

-field trials.

I wish to thank Bishnu Sita Sharma and

Jotshna Bora -for their sel-fless service to the author in

routine works o-f the laboratory~

Sincere thanks are also due to the sta-f-f memebers o-f Centre

o-f In-formation Technology, New Kenche's Trace, Shillong -for

technical help in preparing the manuscript.

Last but not the least, I great-fully acknowledge the

constant inspiration and help received -from the members o-f my

-fami 1 y without which it would have been impossible to camp lete

the present investigation.

~~si~l=~k ( RABINDRA NATH BHUYAN )

Chapters

1.

2.

2.1.

2.2.

2.2.1.

2.2.2.

2.2.2.1.

2.2.2.2.

2.2.2.3.

2.2.3.

2.2.3.1.

2.2.3.2.

CONTENTS

Title

Introduction

Materials and methods

Materials

Methods

Time or collection or panicles

Preparation of staining solutions

and staining procedure.

Acetocarmine ~tain

Feulgen stain

Iron alum - haematoxylin stain.

Anther culture

Methodology

List or media and their composition

used for anther callusing

pp. No.

1 14

15 - 35

15

16

16

16

16

17

17

18

18

18

2.2.3.2.1. Callus prolire~ation and regeneration 22

2.2.3.2.2. Ploidy status 24

2.2.3.3 Production or promising double haploids 25,

2.2.3.3.1. Selection and hybridiza~ion 25

2.2.3.3.2. Culturing or a~thers from F1 plants,

regeneration a~d production or double

haploid lines 25

2.2.4.

2.2.4.1.

2.2.4.2.

2.2.4.3.

2.2.4.4.

2.2.4.5.

2.2.4.6.

2.2.4.7.

2.2.4.8.

3.

3.1.

3.1.1.

3 .. 1.1.1.

3.1.1.2.

Field evaluation

Data collectjon

Data analysis

Analysis o~ variance

Coe~~icient o~ variation

Her it ab i 1 it y

Genetic: advance

Correlation

Path coe~~ic~ent analysis

Results

Anther culture callus induction and

plant regeneration ~rom local c:old

tolerant cultivars

Standardization o~ conditions ~br

c:allusing'o~ mic:rospores

Time o~ collection or panicles

Environmental conditions at the time or

collection or panicles

36 -

27

27

28

28

30

31

31

32

33

60

36

37

37

37

3.1.1.3. Length or cold pretreatment 38

3.1.1.4. Staging o~ microspores 39

3.1.1.4.1. Staging with acetocarmine stain 39

3.1.1.4.2. Staging with ~eulgen stain 39

3.1.1.4.3. Staging with iron alum-haematoxylin stain 40

3.1.2.

3.1.3.

3.2.

3.2.1.

3.2.2.

3.2.3.

3.2.3.1.

3.2.3.2.

3.2.3.3.

3.3.

3.3.1.

3.2.

3.3.3.

3.3.4.

3.3.5.

E~~ect o~ di~~erent culture media on

callusing ~requency and callus

proli-Feration

Regeneration and con-Firmation o~ ploidy

status

Production o~ double haploids

Selection of= parents and hybridization

Raising o~ F 1 and their comparision

with parents

Culture o~ anthers derived ~rom F 1 plants

Callusing

Regeneration

Evaluation o~ double haploid ( DH )

lines

Field evaluation and per~ormance o~ double

haploid ( DH 2 ) lines

Means and ranges ~or di~~erent characters

Genetic variability

Analysis o~ variance

Simple correlation coe~~icience among some

major yield contributing characters.

Direct and indirect e~~ects o~ di~f=erent

characters on grain yield

40

45

51

~51

51

52

52

53

55

57

57

58

59

59

60

4.

4.1.

4.2.

4.3.

4.4.

4.:S.

5.

Discussion 61 - 79

Standardization o~ conditions ~or

callusing o~ microspores in local cultivar 62

Anther /microspore culture

Ploidy status o~ plants derived through

anther culture

Production o~ promising doubled haploid

lines and their evaluation

Field evaluation and per~ormance o~

doubled haploid ( DH ) lines

Summary

Bibiliography

80

84

64

69

70

74

83

99

INTRODUCTION

Rice, the major source o-f calories -for 407. o-f the

global population, is ranked as the world's most important -food

crop. Consequently, it is one o-f the most intensively

investigated crop plant. The accumulated knowledge-base has been

o-f considet-able value in enhancing t-ice productivity and, in

turn, in the betterment o-f millions o-f people who depend on it as

a source o-f -food and income.

As in the case o~ other crop plants~ rice breeders have

mainly pursued the exploitation o~ natural variation ~or

tailoring cultivars suitable ~or the demands o~ agriculture. The

process o~ hybridization and selection have been the main

vehicles o~ this delivery system. The achievements in this regard

have been impressive. Taichung Native! (TN 1) and IR series

starting ~rom IRS to IR74 lines/varieties released by the

International Rice Linstitute, Manila, the Philippines and L Institution o~ National Agriculture Research System are known ~or

their impact on enhancing productivity per unit area and time.

The increase in rice production has helped

alleviate widespread hunger- and -famine by keeping pace with

ever gt·owing human population. I:Jith the rice-consuming

population increasing at an alarming rate of= hundred

million per

would exceed

year, by 2020 A.D. the .. --~

760 million ~,s_~) The

rice requirement o-f Asia

situation in India, the

J.:n·gest rice gtowing countt-y, is also not stable. Although we

are sel~ su~~icient at pn~sent, ot.n- rice n,·qu i rement is

estimated to increase at a rate o~ 25 to 30 million tones every

decade. This means /~- "'

producing 100 million ~s

tonnes) by the year 2000 A.D.

we should achieve the target o-f

pr~r (present 1 y 75 mi 11 ion

This requirement would increase

2

to 160 million tonr1es by 2020 A.D. Accordingly, the production o~

rice has to be doubled in next 25 years. It has to be recognised

here that plant breeding

has severe biological

is a slow and painstaking process that d_e--q....~-Ad

1 imitations, and th<'\t such d[stimatea-,,

increase in yield and productivity r:annot be sustained

inde~initely. It is in this context that rapidly emerging

techniques o~ plant cell and molecular biology have attracted

much attention -for they pt-ovide a powet-~ul and novel means to

supplement and complement the traditional methods n~ plant

improvement.

Since 1930's, plant tissue culture has progressed mostly

on dicotyledonous plants, and studies on monocotyledonous plants

lagged behind. The in vitro culture o~ rice started with the

organ culture, when Fujiwara and Ojima (1955) cultured excised

roots. The culture conditions ~or vat-ious stages o~ immature

embryos were examined by Amemi ya et al. ( 1956) and obtained

seedlings ~t-om even -five day old cultLu-ed embryos. F1 hybrids

~rom otherwise unsuccess~ul rice crosses were· subsequently

obtained by Bourhamont (1961)~"-Iyer and Govila (1964). Cultut-e o~

nodes and young seedlings on Heller's medium containing vitamins

and auxins was reported by Furuhashi and Yatazawa in 1964. This

was -followed by plant t-egenet-ation ~rom seed derived callus

(Tamura,1968; Nishi et. al.~ 1968 and M~eda(~-;<-~.,1968). Plants "---··--"""·""' ! "·

were also regenerated -from root callus (Kawata and

Ishihara,1969)as well as pollen derived callus (Niizeki and

Oono,1968; Cho and Zapata, 1988).

The demonstt-at ion ~ Guha and 1'1aheshwar i ( 1964, 1966,

1967)/ that immature anthet-s of D,:;tura .i. n.nox.i a when cui tured

in an appropriate nutrient medium can give rise to plantlets with

a haploid numbet- o-f chromosomesy opened up a new approach to the

induction o-f haploidy in plants genet-ally known as androgenesis.

Soon it was -found that this technique can be e-f-fectively employed

in Orvza (Niizeki and Oono, 1968; Guha et. a1,.1970), .Nicotianet

(Bourgin and Nitsch, 1967; Nitsch et. al.,1969; ~kata and Tanaka

,1968; Nitsch and Nitsch,1969; Sunderland and Wricks, 1969;

Helchers and Labib, 11J70), B:·-A":;t';_ic,::~ O<ameya and Hinata, 1970) and

1-fordeum and Lol i um (Clapham, 1971). In Or·l;za sat i \-'a, varietal

g were-reported by Guha etj al.(1970).

The underlying principle o-f androgenesis is to direct the

...v=--~ . ./ development o-f the microspores whose -fate is~ to become~

gamete~ and to -force its development dit-ectly into a plant or

callus. This abnormal pathway is possible i-f the pollen cell is

provided with certain speci-fic conditions.

Utilization o-f anther culture technique in the breeding

programmes has been in practice, mostly in China, -for more than

4

20 years. Since the release for cultivation of Hua-Yu No. 1 and

Hua-Yu No. 2, the rice vat- iet i es developed through anther

culture breeding in 1976, Chinese t-esearchers ~e: • rwpur.t;ea M3

have developed over 100 varieties and lines with objectives like

earliness, yield, resistance to disease and grain quality

(Shen et al.~ 1983). Reseat-chet-s in China, have also successfully

used anther culture for several other purposes like, solving

sterility problems in indica-japonica hybrids. Outside China,

utilization of anther culture in rice breeding programme has been

comparatively of a lesser magnitude. In Korea, the technique has

been mainly used for developing blast resistan~ and cold

tolerant varieties (Gun~ 198~). In laiwan, application of anther

culture was mainly to produce inter-subspecific and inter­

specific Fl hybt-ids (Woo and Chen,. 1982). International Rice

Research Institute, Manila's programme of anther culture has been

focussed qn production of varieties fOt- cold tolerance using Fl

hybrids of japonica-indica and indica-japonica cresses.

Researchet-s in Nepal (Agrawal et al., 1994) have also t-egenerated

plants from anthers of cold tolerant rice. The biggest programme

outside China is at CIAT (F'ulvet- and Jennings, 1986) as for

example, 20,000 diploid R2 lines are reported to have been raised

from approximately 100 triple crosses for obtaining Rl lines for

blast resistance, Fe-tolerance and white belly determination.

5

Factor-s a-f-fecting andr-ogenesis in rice have been

r-eviewed by Chen et al. ( 1991). Cold m- heat shock -for shor-t

period is known to enhance the yield o~ pollen embr-yos and gr-een

plants r-emad~ably. This bene~icial e~-fect o-f cold pr-etr-eatment

was -first repor-ted by Nitsch and Norreel ( 1973) in Datura

innoxia. It has also been shown to increase the r-atio o-f

responding anther-s in many species, such as N."ic:otiana tabac:um

(Nitsch, 1974), Datur.:.; :innoxic:t., llvo.c::.c.,·i.imus n.i.gc::-r. fkn-deum "'Lilgare

(Sunder-land and Roberts, 1977) and i}rvzc.· sBt:i-.,'a (Chale-f-f et.

al., 1975). In t-he_~ t·ir.e anthet- culture, this

tr-eatment has been an essential step and broadly adopted by many

r-esQat-cher-s. Callus induct ion -ft-equency 0~ Keng ( a~-vza StElt .i ,,,a

Subsp. japonica) or Hsien ((Jryz,::i .c::. •. :~tiva Subsp. indica) rice can

be r-aised -from two to more than tm1 times (Ying, 1986) via an

appropr-iate cold tr-eatment.

The time needed -fot- pt-etreatment depends on the

temper-atur-e used. In general, lower the temper-atur-e (as low

as 3°- 5°C) shorter- the time needed,

( as high as 10°- 15°C longer

and

the

higher the temper-atur-e

period o-f treatment

r-equir-ed. Through the experiment by Wang and his associates

( 1974) in indica rice, 10 day pretreatment at 3° - 5°C, 10 to

15 day at 6° - soc, or 15 to 20 day at 9° - 10°C was optimum -for

6

the enhancement o~ callus induction ~requency. ~hen the period o~

cold pretreatment is too long, the ~requency o~

di~~erentiated ~rom callus decreased dramatically

green plantlet (U-1'

while Lo~ albinoJ

increased greatly. The appropriate cold pretreatment bene~its the

di~~erentiation o~ green plantlets and raises the di~~erentiation

~requency and ~ormation o~ green 0

(!ti} 1983; Go and Chen , 1 983a) •

7?5f'

r ·~

plantlets (Zhou and(Chang? 1981; '-.. ---· ~~--- .. ~ ?

Physiological investigation has revealed that cold

pretreatment could reduce the respiration intensity o~ anther and

the consumption o~ materials, and could prolong the li~espan o~

anther. In addition, it was ~ound that the weight o~ anther also

increased during cold prett·eatment. All the!Se e~-fects would be

advantageous to the initiation and development o~ pollen grains

inoculated (~, 1983). Qu and Chen, ( 1983 1;:1) demonstrated that 'S the cold pretreatment supported the sut-vi val o~ most o~ the

grains and completion o-f the induction process -for androgenesis

at the same time, and thet·e-fore lef'd to a mLICh higher induction "<..

~requency o-f callus than that o-f the control.

Other treatments such as heat shock (Kellet- and

Armstrong 1978, 1979), placing the detached tillers in watet· at

room temperature (Wilson et al. 1978 ) and putting the excised

anthers in a watet· satLu·ated atmosphet·e (DLmwell, 1981 ) have

7

also been ~ound bene~icial to production o~ embryos and plants.

Formation o~ embryo or callus depends greatly on the

appropriate nutrients in the culture medium (Nitsch, 1969; Wang

et al., 1974). Clapham ( 1973) discovered that high concentration

o~ ammonium ions in LS medium (Linsmaier and Skoog, 1965) was

disadvantageous ~or callus ~ormation in barley microspores.

Subsequently, Chu et al.(1975) developed N6 medium which is

characterised by having a low concentration o~ (NH4 >2so4 and high

KN03 • This medium proved to be very e~~icient ~or rice anther

culture (Chu, 1978; Genovesi and Maqill, 1979; Chen et al. 1982;

Tsay et al., 1982). Specially i~1

·'iraminae, a high sucrose

concentration has been ~ound to be bene~icial -fot- plant

production through anther culture (Clapham,1973; Ouyang et al.,

1973; Dono and Lat-tet-, 1976; Miao et al., 1978). Chen (1978)

reported that in t- ic:e 6% sucrose in callusing medium and 3'Y.

sucrose in regenerating medium resulted in highest -frequency o-f

callus and green plant regeneration @~pability in rice. But some

investigators have n~pm-ted that 6% sucrose is too high and 5%

was recommended (Huang et al., 1978. Chu,1978; Chale~~ and 1-

Stolarz, 1981). It has been observed that high concentration o-f

sucrose is responsible -for maintaining osmotic pt-essure o-f the

nutrient medium (Wang et. al., 1974; Ch;de~-f and Stolarz,1981).

Plant growth regulatot-s like au:dns and cyf:okinins play a crucial

8

role in the process o~ androgenesis. In earlier days, 2,4-

dichlordphenoxyacetic ac:in (2.4--D) ,.,as thought to be the only ._.,.

hormone essential ~or androgenesis and it was always added to the

culture medium with or without cytokinin (usually kinetin)

and/or other auxins (Niizeki and Oono, 1968; Harn, 1969; Nishi

and t'1itsuoka, 1969; Guha- t1ukhfr- jee, 1973; Wang et al., 1974).

The inhibitory e~~ect o~ 2, 1l-D, and that it can be

replaced by <X - naphthylacetic acid (NAA), was ~it-st reported by

Niizeki and Oono,(1971). A~ter~anJs it was also -found out that

~or obtaining callus o~ high morphogenic potential cytokinin is

also needed (Chen, 1977; Chalef~ and Stolarz, 1981; Lee and Chen,

1982). However, this conclusion o~ hormonal requirement may not

be applicable to all cultivars as genotypic di-f~erences in

hormone requirement has been reported (Liang, 1978; Cornejo

-Martin and Primo- Millo, 1981).

Light and temper-atw-e play an impm-tant role in

androgenesis (Maheshwari et al., 1980). The light conditions

varied ~t-om complete dar-kness (Niizeki and Dono, 1968; Hat-n,

1969; Woo and Tung, 1972; Guha -- t-1ukhfr jee, 1973) to continuous

illumination (Nishi and Mitsuoka, 1969; Niizeki and Dono, 1971).

Darkness is ~avourable ~or initiation o~ callus (Cornejo - Martin

and Primo- Millo,1981). For plant regeneration high intensity o~

light is essential.

9

Other -factors that influence anther response are

composition o-f the gas mixture in the culture vessel (Dunwell,

1979; Johansson et al.,1982) and the density o-f anther inoculated

in the culture medium (Bajaj et al., 1977; Sunderland et al.,

1981).

The genotype o-f the donm- plantj has a strong e-f-fect on

pollen plant -formation. There at-e several reports on genotypic

variation in anther response o-f rice (Guha et al., 1970; Iyer and

Raina, 1972; Wang et al-~1974; Raina and Iyer,1974; Tsai and Lin,

1977; Chen and Lin,1976; Chu, 1982; Rush and Shao 1982; Ding

et al 1983; Zapata, 1985; Hu, 1985; Miah et al.,1985; Karim et

al., 1985; Boyajiev and Kuong, 1986; Guidet-doni et al., 1986;

Mikami and Kinoshita,1988). In the initial study o-f lyer and

Raina (1972), out o-f 15 cultivars only 5 showed callusing and o-f

these only one (IARI 5788) t-egenerated roots and shoots.

Genotypic variation is now seen mostly in the extent o-f response,

rather than in the existence o-f some responsive genotypes and

some unresponsive , genotypes (Raina, 1989). In general,

japonica rice varieties are known to respond better than indicas

(Chen and Lin, 1976; Hu, 1978; ~:w::h and Shao, 1982; Chu 19B2;Woof

and Chen,1982; Gun, 1982; Zapata 1985). Hu (1984. 1985) t-eported

10

that the induction ~requency o~ green plants was more than lOX in

japonica cultivars, whereas it was only 1% in indica rice.

The reason that this technique oF androgenesis has

attracted such wide attention is the possibility o~ obtaining

haploid cells, tissues and complete ~-~anism~, which has a single

dose o~ gametic in~ormation. Instant homozygous lines could then

be obtained by doubling the chromosome complement in these

haploids without having to resort to a lengthy and time consuming

programme o~ inbreeding, which is indispensable ~or obtaining

homozygosity by conventional methods. In addition, recessive

mutants can be readily discovered because o~ presence o~ only one

set o~ chromosomes, and hence only one copy o~ each gene.

The other attt·act ion o~ anthet· cui ture is the

possibility o~ early ~ixation o~ recombinants. Pollen plant

population derived ~rom anther culture o~ Fl hybrids will express

segregation patterns otherwise expressed in the F2 or F3

generation. Diploidization oF haploids will render them

homozygous, resulting in the t·apid establishment o~ pure lines

that otherwise would require several generations o~ sel~ing. The

saving o~ time and resources would be ~ar greater in long

duration compan~d to dut-at ion cultivars, in which

homozygosity can be achieved rathet- speedily through conventional

11

methods such as single seed descent, by growing two or three

crops per year. In rice, there~ore, anther culture breeding would

be more advantageous in situations involving temperate

environment, photosensitivity ot- environmental stresses because

in these cases only one generation can be raised in a year.

Increased selection e~~iciency is the other advantage

o~ anther culture breeding, especially when dominance variation

is signi~icant. In conventional breeding, early generation lines

(F2 - F4) show phenotypic di~rerences to which both additive and

dominance e~~ects contribute. In contrast, double haploid lines

derived ~rom F1 anther culture will show only additive variance

and, there~ore, high heritability due to elimination o~ dominance

e~~ects. Thus, compared to the F2 population, ~ewer double

haploid plants will be required -for the purposes or selection or

the desired recombinant(s). To elabm-ate this point, Baenziger

and Schi\e~-fer-,(1983) cited the hypothetical example o-f a cross

segregating ~or three recessive genes and ror three dominant

genes. In the case o-f three recessive genes, 1/8 o-f the double

haploids derived -from such a cross would be selected as compared

to 1/64 or the F2. In the case of three dominant genes, wherea~

1/8 o~ the double haploids, ~~~ of which would breed true, 27/64

o-f F2 plants would be selected, on the basis or phenotype, but

only 1/64 would be true bn.:>edino. Chinese scientists have

12

estimated that about 150 pollen plants det-ived -from Fl anthers

would be enouqh instead o~ 4 - 5.000 F2 plants. ~or the purpose

o-f selection o~ desirable genotypes (Shen et. al., 1983). Double

haploid breeding would be particulat-ly use-ful in situation in

which extensive recombination is not impm-tant (Chale-f-f, 1979;

Chu, 1982). In rice, e-f~orts to combine the desirable traits o~

indica and japonica cultivars have o-ften been ~rustrated by the

high degree o~ sterility o-f the hybrids and vigorous segregation

toward the parental types in F2 and subsequent generations.

Double haploids produced through anther culture o-f the Fl hybrid,

or o-f a promising segt-egant, ~·muld ensure stabi 1 ization and

thereby produce and arrest segregation in successive generations.

Use o-f anther culture ~or solving the pt-oblems arising -from

inter-subspeci~ic and intet--spec i ~ic hybt- idi zat ion can be

regarded as the most important area o-f its application in rice

improvement.

Another area o~ use o-f double haploid is the selection

o-f the races o-f pathogen o-f insect biotypes (Rush and Shao,

1982). Use o~ 'pure line' varieties or breeding lines may cause

considerable experimental error due to residual heterozygosity o-f

the genotypes investigated. Anther culture could be used to

obtain homozygous lines Fm- utilization in such situations, as

well as in sct-eeninq -rm- t-esistr.1nce to herbicides, physiological

13

studies, and in puri~ication o~ A~ B and R lines ~or hybrid rice

progt·ammes.

The present investigation is aimed at standardization

o~ conditions ~ot- anthet- cul tw-e-det· i ved callus induct ion and

plant regenet·ation -from cold tolet-ant t-ice vat·ieties qrown in

high altitude areas o-f Nm-th Easten1 Hill of= India. All these

varieties are red kerneled, possess cold tolerance and they are

poor yielders. Having standar-dized the conditions -for

androgenesis, we produced sevet-a I double haploid 1 ines tht-ouqh

anther culture o-f Fl derived hybrids between IR 70, an elite IRRI

bt·eeding line and Khonm-ullo. a local cold tolerant line. The

double~ haploid 1 ines wet-e evaluated .fot· agronomic charactet·s

undet· medium altitude ( 950 m. msl Q.

~~2_~-~!:nd condition .:md

promising ones with high yield and \l'lhite ket·nel colour wet·e

identi-fied -for -further evaluation.

14

MATERIALS AND METHODS

2.1 MATERIALS

The -following locally adapted varieties of= rice (Oryza

satival L). were used initially to assess the response to callus

induction during anther culture:

1. Khonorullo

2. RCPL 1 - 1C (Meqha Rice 2)

-~. RCPL 1 - 2C

4. RCPL 1 - 3C

I:' ..Jo DR 92 and

6. Pus a 33

15

They were qrown in the experimental ~ields o~ Plant

Breeding Division, Indian Council o~ Agricultural Research l~or

North Eastern Hills Region, Barapani~ Meghalaya, with normal

~ertilizer and crop management.

In addition, IR 65, IR 68, IR 70~ Khonorullo, Yamuk, Pusa

33 and DR 92 (Subhadra) were grown in pots, with normal

~ertilizers ~or crossing. One cross i.e., IR70 X Khonorullo was

made and attempted ~or daub le hap laid pt·oduct ion.

2.2 METHODS

2.2.1 Tim• of collection of panicle•

Panicles were collected while still inside the sheath,

mostly in the mornings (between 8.30 AM - 10.30 AM) between

middle or August to the end o~ September i.e, in the rlowerinq

period. They were wrapped in polythene bags with a little water

and stored at 8 - 10°C ~or 1 to 16 days.

2.2.2 Preparation o~ staining solutions and staining procedure

2.2.2.1 Acetocarmine stain:

2 gm o~ carmine was dissolved in 100 ml of 45X acetic

acid and was kept ovet·night. Ne:::t day the stain was ~iltered and

used ~or staging o~ microspores. A ~ew anthers were squashed in a

drop o~ stain and observed under a research microscope.

16

2.2.2.2 Feulgen stain•

Feulgen reagent was prepared by adding 1 gm basic

fuschin, 2 gm potassium metabisulphite, 5 ml 1N HCl, 85 ml

water and 1 gm activated charcoal (Nitsch 1981). The solution was ,--~.

allowed to stand overnight in fudg~. , ___ /

The anthers were dissected out from the spikelets and

fixed in acetic alcohol (1 : 3) for a period ranqinq between

overnight to 24 hour. They were brought to water level through

alcohol grades and hydrolyzed with 1 N HCl at 60°C for 5 to 7

minutes. After the hydrolysis the vials were immediately put

into ice cold water to stop further action of HCl. After repeated

washing with water the anthers were squashed in a drop of the

staining solution and observed under the microscope after 5

minutes.

2.2.2.3 Iran Alum Haemataxylin staina

A stock solution of 4/. haematoxylin in 45/. acetic acid

was prepared and 1 gm of ir-on alum was added to it. The solution

was allowed to stand -for 24 houri.:_ To 10 ml of this stock ~ solution, 4 qm of chloral hydrate was added and after complete

dissolution)it was used (Chang et al.~ 1978)

Anthers were squashed in a drop of the staining

solution under a cover glass. After 2 to 5 minutes, the slides

17

were observed under a research microscope.

2.2.3 ANTHER CULTURE

2.2.3.1 Methodology

sterilized.

Panicles o~ the varieties mentioned above were sur~ace

Since individual panicles contain tfiicrospo~~~~---,at "-··~ . -.- . ..- - ~-

A stages o~ development at di~~et-ent places, the sheath di~-ferent

was opened at -five place~and ~~loret was taken out -from

opening. Anthers o-f these ~lorets were examined under

each

the

microscope

containing

-for ascertaining the nuclear stage.

microspores at mid-uninucleate stage were

Lfortions

taken out

and anthers were removed -from it. The anthers were then passed

through 70/. ethanol -for a -few seconds, and then "-sterilized in

O.l'l. mercuric chloride -for 7 to 8 minutes and rinsed 3 times 0

with sterile distilled "Jatet-. They wet-e then inoculated jn the

-following semisolid/liquid medium.

2.2.3.2 List OT media and their composition used TOr anther callusing

Medium No. 1 65 (Semisolid)

Medium No. 2 E24 (Semisolid)

Medium No. 3 Minimal (Semisolid)

Medium No. 4 LB (Semisolid)

18

Medium No. 5

Medium No. 6

Medium No. 7

Medium No. 8

MP2 (Semisolid)

E10G (Liquid)

Modi~ied Nb (Liquid)

E10 YK (Liquid)

Medium No. 1 G5 (Samisolid)a

Blaydes medium/.._ NaH2P04 (150 mg/1), (NH4 >2 so4 (134

mg/1), KN03 (2500 mq/1), MqS04 • 7H2 0 (250 mg/1), CaC1 2 • 2H2 0 (150

mg/1), KI (0.75 mg/1), Na2 Mo04 (0.25 mg/1), CuS04 .5H2o (0.025

mg/1), CoC1 2 (0.025 mg/1), [ Na2EDTA (745 mg), FeS04 .7H2 0 (557

mg) dissolved in 100 ml o~ water and 5 ml o~ this solution is

used ~or 1 litre], Sucrose (50 gm/1), Agar (8 gm/1), ~orti~ied

with 2,4-D (2 mg/1), Nicotinic acid (0.5 mg/1), Pyridoxin HCl

(0.5 mg/1), Thiamine HC1 (1.0 mg/1), Glycine (2.0 mg/1) and

Inositol (100 mg/1).

Medium No. 2 E24 (semisolid)•

G5 basic salts~orti~ied with 2,4-D (1 mg/1), BAP (0.5

mq/1), IAA (0.5 mg/1) and Inositol ( 160 mg/1).

Medium No. 3 Minimal (semisolid);

Med i urn No. 12 o+ Snnq ct . a I ( 1978) :

19

mg/lJ, KN03 ( 100 mg/1) !' KH2Po4 (300 mq/1), MqS04 • 7H2D (200 mg/1),

CaC1 2 • 2H20 (110 mg/1), MnS04 .4H2D (10 mq/1), [Na2EDTA (745 mg)~

FeS04 .7H20 (557 mg) dissolved in 100 ml o-F water and 5 m1 o-F

this solution was used -For 1 litre], Sucrose (30 qm/1) Agar

(0.8%) -Forti-Fied with 2,4-D (2 mg/1), Nicotinic acid (0.5 mg/1),

Pyridoxin HCl (0.1 mg/1), Thiamine HCl (0.1 mg/1) and Glycine (2

mg/1).

Medium No.4 LS C Semisolid ):

KN03 (3000 mg/1), l"lqSD4 .7H20 (185 mq/1), CaC1 2 .2H20

(150 mg/1), KH2Po4 (540 mq/1), [FeS04 .7H20 (557 mg), Na2EDTA (745

mq/1) dissolved in 100 m1 o-F water and 5 ml o-F this solution was

used -For 1 litre], Mnso4 .4H2 0 (22.30 mg/1), Znso4 .7H2 o (10 mq/1),

KI (1.0mg/l), H3 Bo3 (6.0 mq/1), Nicotinic acid (3.0 mg/1),

Thiamine HCl (2.5 mq/1), Pyridoxin HCl (5.0 mg/1), Inositol (100

mq/1), Glycine (2.0 mg/1) and Lactalbumin hydolysate or Ca~ein

hydrolysate (300 mg/1), Kinetin (1.0 mg/1), Sucrose (50 g/1) and

Agar (6 g/1).

- ~~---~-- --------.......

HRdium No. 5 ~( Liqu~-~ Potato extract 10% aqueous (-For 1 litre o-F medium, 100

gm o-F -Fresh potato tubers were weighed out and cleaned with

water, the sprouts on the tubers i-F any were.removed. The tubers

20

were cut into small pieces, boiled in a given volume o~ distilled

water -for 20-30 minutes and -fi 1 ter-ed tht-ouqh two layers o~ cheese.

cloth. The residual potato pieces were extracted once aqain in

th~ same manner. Finally, the two -filtered liquids were mixed ~or

immediate use), KN03 (100 mq/1), (NH4 >2so4 (100 mg/1), Ca(N03 >2

( 100 mg/1), MgS04 • 7H20 ( 125 mq/1), t<H2Po4 (200 mg/1), KCl (35

""'"'""A-~~~ mg/1), Sucrose (100 gm/1) and Agar (8 gm/1). ~- !~- Q

Medium No. 6 E10G ( Liquid ):

G5 medium without agar and -forti-fied with 2, 4-D (1

mg/1), BAP (0.5 mg/1), IAA (0.5 mg/1), Glucose (5 gm/1),

Nicotinic acid (0.5 mg/1), Pyridoxin HCl (0.5 mg/1, Thiamine HCl

( 1 mg/ 1), Glycine ( 2 mg/1), Inosi to1 ( 100 mg/ 10) and Glutamine

(47 mg/1).

Medium No. 7 Modi~iad N6 (Liquid)& J

CNH4)2S04 (463 mg/1), KN03 (2830 mg/1), KH2Po4 (400

mg/1), MgS04.?H20 (185 mg/1), CaC1 2 • 2H2 o (166 mg/1), Mnso4 .4H20

(4.4 mg/1), ZnS04 .7H2 0 (1.5 mg/1), H3 Bo3 (1.6 mq/1), KI (0.8

mg/1), [Na2EDTA (745 mg), Feso4 .7H2D (557 mg) -dissolved in 100

ml o-f water and 5 ml o-f this solution was taken ~er 1 litre or

medium], -fortiFied with 2,4-D (4 mg/1), Nicotinic acid (0.5

mg/1), Pyridoxin HCI (0.5 mg/1), Thiamine HCl (1.0 mg/1) and

21

Glycine (2 mg/1) Sucrose (60 g/1).

Medium No. 8 E10YK ( liquid )I

G5 salts without agar and ~orti~ied with 2,4-D (1.0

mg/1), BAP (0.5 mg/1), IAA (0.5 mg/1), Glucose (5 mg/1),

Nicotinic acid (0.5 mg/1), Pyridoxin HC1 (0.5mg/l), Thiamine HCl

(1.0 mg/1), Glycine (2.0 mg/1), Myo-inositol (100 mg/1), Yeast

extract ( 3g/l ) and Kinetin ( 1.0 mg/1 ).

The media pH was adjusted to 5.8 in all cases and they ':)

were autoclaved at 1.06 kg/cmL ~or 20 minuites.

2.2.3.2.1 Callus proliTeration and regeneration•

Small calli were trans~erred to proli~eration media

viz. N-19 and J-19 on a paper boat. The composition o~ J-19 and

N-19 media are given below.

J - 19 (Liquid ) 1

B5 basic sal tsri thout agat- and ~orti~ied with sucrose

(20 gm/1), Inositol (160 mg/1), Kinetin (1mg/l) and NAA (1 or 2

mg/1).

N - 19 (Liquid ) 1

MS (Murashige,--J:_t-97-4) basic salts without aqar and

~orti~ied with sucrose (30 gm/1), NAA (1 or 2 mg/1), Kinetin

22

(1 mg/1) and Inositol (100 mq/1).

Calli thus obtained were trans~erred to di~~erent

regenerating media. Ten media i.e., MSA, MSB, MSI, MSBT, MSC,

MSD, MSBS, RGA, RG2 and N6 without hormones were tried ~or

regeneration. The composition o~ the media are given in Table 1:

Table 11 Di~~arent regenerating media with their composition•

Media ----~ ~~tokinins,.)

------ ----- -·

MSA MS+

MSB MS+

MSI MS ..

MSBT MS+

MSC MS+

MSD MS+

MSBS MS+

RGA MS+

RG2 MS+

and N6 without

NAA (mg/1)

1.0

0.5

2.0

0.5

1.0

1.0

0.5

1.0

0.5

hormone.

Concentration or /

Vitamins and Sucrose.

BAP Kinetin (mg/1) (mq/1)

Thiamine HCL (mg/1)

1.0 0.5

3.0

1.0 2.0

3.0 10.0 10.0

0.5 0.6

0.1 3.0

3.0

'2.0 0.6

5.0

23

Auxins,

Sucrose (gm/1)

30

30

30

30

30

30

60

60

30

2.2.3.2.2 Ploidy Status

Excised roots o-f about 1 em long were pretreated with

2 mH (h d ~. L y roxyqu1J-1ne at room temperature -for 2-3 hours to

arrest/accumulate cells at metaphase. These were -fixed in

Carney's -fixative (6:3:1 absolute ethanol : chloro-form . . glacial acetic acid ; with two changes). The roots were stored at

4°C -for at least 2 days which can be extended upto 60 days.

The -fixed roots were washed in distilled water -for 2-3 times (5-6 minutes each) and then in ice cold O.OlH citrate

bu-f-fer (0.01M citric acid monohydrate in 0.01M sodium citrate, pH

4.6) 2-3 times (2-3 minutes each). Root tips (2-3 mm) were cut

and trans-ferred to an ependro-f-f tube containing an enzyme

solution [2-6'X Cellulase (Onozuka R-10) and 1-27. Pectolyase

(Yakult Y-23) in citrate bu-f-fer] held at 37°C in a water bath.

The rootf tips were digested -for 45-75 minutes and washed in

0.01H citrate bu-f-fer 2-3 times (10 minutes each) and then in

sterile distilled water twice (5-10 minutes each) to remove the

enzyme solution (Islam and Stich, 1989).

Root tips were trans-ferred singly to an acid cleaned

and ethanol washed slide. E>=:cess water was removed without

disturbing the slide. A drop o~ 3 : 1 absolute ethanol: glacial

acetic acid was placed onto the root tip and squashed immediately

with a needle. Another drop o~ 3 : 1 -fixative was added to and

24

the ~aterial was spread~ over the slide. Slides were air dried

~ and stored in a des~icator overniqht.

~ Slides were examined under phase contrast microscope.

For better contrast the chromosomes were stained by placing a

drop o~ tX acetocarmine or 1/. propionic carmine over the air

dried cells and covered with a cover slip and heated brie~ly over

a spirit lamp. Excess stain was removed be~ore applying liqht

pressure to the cover slip.

2.2.3.3 Production of promising double haploids• I

2.2.3.3.1 Selection and hybridization

A~ter standardizing the media and environmental

conditions ~or callus induction and plant regeneration, crosses

were made between IR70 and Khonorullo. The crossings were carried

out by ~allowing standard procedures o~ emasculation in the

evening and pollination next morninq.

2.2.3.3.2 Culturing of anthers from Fl, regeneration and

production o~ double haploid lines

In the next season, Fl derived seeds obtained from the

crosses were grown sep~-ately. The anthers obtained ~rom them ~

were cultured for callLisinq in di~~erent media as mentioned

earlier. The resultant calli were transFerred to regeneration

25

""' medium.

Regenerated plantlets were transferred singly to pots

where they matured. The data on agrobotanic characters of Fl's

were recorded and seeds were harvested from fertile plants.

During the ne}~t year, seeds of

--------

di-f-ferent double / I

haploid~~--1 ) 'lines were multiplied in upland, lowland and

pots and double haploid ~~--Y 1 ines thus obtained

in

were

evaluated for yield and yield contributing characters under

medium altitude ( 950 m. msl ) lowland condition.

26

2.2.4 Field evaluation

The experiment was conducted on the ~arm o~ the ICAR

Research Comple;, ~or NEH Region, Barapani in the wet season o~

1994. The test entries, 9 DH lines (line no. 1, 6, 7, 10, 13, 15,

16, 19 and 21) and 2 parents (IR70 and l<honot-u 11 o) were evaluated ~

design; in a 3m X 1m plot in }._randomized block with -five

rt=;::>lications. Each plot contained ~ive rows with 30 plants/row.

Single seelding was transplanted/hill at 20cm X 10cm spacing.

Recommended dose o~ NPK ~ertilizer was applied.

2.2.4.1 Data collection

Observations were recorded on plants in the three

middle rows (excluding border plants) ~or ~allowing characters:

Panicle length: Average length ~rom ~ive plants/plot measured in

em at maturity.

Panicle weight: Average weight ~rom ~ive plants /plot weighed as

gm at maturity.

Percent sound grain: Calculated as percentage o~ ~illed grains

~rom total number o~ spikelets/panicle o~ ~ive plants/plot.

Plant height: In em, ~rom the ground to the tip o~ the main

panicle at maturity.

E5'ar bearing t i llet-s: • Average number o~ ear bearing tillers -from ~

~ive plants/plot at matlu-ity.

27

Grain weight/panicle: Average weight o~ grain ~rom ~ive

panicles/plot at maturity.

Thousand grain weight: Recorded ~rom evenly oven dried seeds o~

~ive plants/plot.

Number o~ spikelet/panicle: Average number o~ spikelets ~rom

~ive panicles/plot.

Grain yield: Weight o~ clean and dry grains harvested ~rom the

whole plot (excluding border plants) computed at 14X moisture

content in t/ha.

2.2.4.2 Data analysis

In order to have the basic in~ormation on the extent o~ ~

genetic variability~ statistical analvsjs were carried out on

analysis o~ variance~ coe~~icient of variation and heritability.

2.2.4.3 Analysis of variance

The analysis o~ variance was carried out to test the

genetic di~~erence between the genotypes under investigation

~allowing the method suggested by Gomez and Gomez (1984).

The analysis o~ var1ance was based on the assumptions

underlying linear model. A simple additive model is qiven below:

28

r :; ( v t

where i = j = 1 •....• n

k = 1 •••••••••• r-

Y i jk= the phenotypic value o-f genotype i j gt-own in Kth

block.

u= the qeneral mean

qij= the generatio~ e-f-fect o-f the ijth genotype

bk= the kth e-f-fect

eijk= the environmental e-f-fect associated with each

observation.

The u,qij and bk are assumed to be -fixed unknown parameters. The

eijk's are assumed to be normally and independently distributed

with mean zero and variance~ The analysis o-f variance table is

qiven below:

Source o.f d.f Mean sum o-f E~<pected mean variation squ.:u-e squat-e

,.... ..., Block r-1 1 "'- x"-,, .

j g j t_g

'"") x2 Genotype g-1 1 .-"'-A

i (f2 ? + r- (f~ g

r- i f"g

Error (r-1) (g-1) By subtraction

29

~ J-, 0 /~': .J-.., ._2 ""i

Total rg-1 ;-:: I ' A ){...:. <_:

ij i rg

Various mean squares were tested against error variance by using

F test. The standard error o~ mean and di-f-ference o-f

means were calculated as ( EMS/t-) 11 2 and ( 2 EMS &/r) 1/2

respectively where EMS is the errot· mean square.

2.2.4.4 Coefficient of variation

For comparing the extent o-f variability between di-f-ferent

characters, coe-f-ficient o-f variation was calculated at phenotypic

and genotypic levels using the methodology o-f Burton ( 1952 ).

Phenotypic coe-f-ficient o-f vat·iation ( PCV ) :

PCV= X 1 (H)

X

Genotypic coe~-ficient o-f variation ( GCV )

~~ -l ' q.'

GCV= '..___./. X 1 00

30

Where p and are phenotypic and genotypic variances

~ obtained ~rom the expectation o~ mean squares in the analysis o~

variance tables andVs the qenet-al mean o~ the populations.

2.2.4.5 Heritability /:::::\

Heritability <tH )) in a broad sense was calculated by the L./

method described by Allard ( 1960 ) and was de~ined in percentage

where, -? ~~ h2 = Heritability is percentage

u 2 p = Phenotypic variance

u2g = Genotypic variance.

2.2.4.6 Genetic advance

It is calculated as

GA = vg :X ' K

~ vP

Where K is the selection di-f-ferential based on phenotypic

deviation ( K = 2.06 at 5/. selection intensity level )

Genetic advance as percentage o~ mean is calculated as

GA GAl. = X 100

mean

31

2.2.4.7 Correlation

Correlation coef~icient ( ,-) between various characters

studied were worked out according to the method suggested by

Robinson et al. ( 1951 ). The ~orm o~ analysis o~ covariance is

given below:

~ Analysis of covariance

Source d~ Mean sum 0~ E}~pectat ion o-f mean Products Sum of products

Replication r-1 r12 ee12 + ger12 J

) ~l Genotype q-1 t12 ee12 + reg12

Error ( r-1) ( g-1) e12 ee12

Also, di~ferent simple correlation coefficient among different

characters were worked out:

Phenotypic coe-fficient o~ correlation ( rp12 ):

-11 2 2

rp 12=u -l u-- >~ u--P12 Pt P2

Where,

rP12 = Correlation between phenotypic values o-f character 1 and 2.

2 u--- = Variation o~ phenotypic values o~ character 1

Pt

32

'") . ~ u--- = Variation o~ phenotypic values o~ character 2

P2

2 u--- = Covariance between 1 and 2

Pt2

The signi~icance o~ phenotypic coe~~icient o~ correlation was

tested against "r" values and error degrees o~ ~reedom at p=0.05

and p= 0.01 levels.

2.2.4.<6 Path coe~~icient analysis

Path coe~~icient is simply a standardised partial regression

coe~~icient and as such measut-es the direct in~luence o~ ~ one

variable upon another. Path coe~~icient analysis was done to

partition the total correlation into direct and indirect e~~ects

by usinq the concepts o~ Wriqht 1921 ). The phenotypic

correlation among di~~erent character combinations were utilized

~or this purpose. The path coe~ficient analysis was per~ormed in

accordance with the method given by Dewey and Lu ( 1959 ). The

basic theory for the calculation of path coe~ficient is as

~allows:

33

Where, ·

ryi is the total correlation between x 1 ·s (independent

variable) and Y ( dependent variable )

Pyi is the direct e~~ect o~ variance x 1 on Y

Pvn ezn is the indirect e~~ect a~ variable xi on Y through vat- iab le xn

Then the equation in matrix ~arm are as under:

=

Hence = }:: n .-, r yi

Where,

( R >,. x n = Correlation matrix

Pyi = Column vector o~ path coe~~icient

ryi = Column vector correlation coe~~icient o~ individual

trait with dependent variable

34

It ( R )-l r x n = ( C ) r R n 'the path coe~~icient are

calculated as under :

Direct e~~ects:

Ply = C11 rly + c12t-2y + c 1 -,t-"7 + . . . . . . . . . . ._:. ·-'Y

p2y = C21 rly + C22r-2y + c23r-3y + . . . . . . . . . . Pny = cnl t•l y + Cn--:.t-'1,. + Cn3t-3y + . . . . . . . . . .

.c. ..:..y

worked out as Piyrni·

P 1yr 12 is the indirect e~~ect o~ x1 on Y via .X2 :

P2yr23 is the indirect e~~ect o~ X2 on Y via X3 :

and similarly all the indirect e~~ects were calculated.

Residual e-f-fect:

The variation in Y may be due to some other variables

not studied, e~rect or pseudo variables which have no

correlation with any other independent variable etc. It is termed

as residual e-f~ect.

35

3.RESULTB

3.1 Anther culture- callus induction and plant reqeneration

from local cold tolerant cultivars

Microspores o~ rice during anther culture are known

to give riset:ingle or multiple calli which can be regenerated

into plants. In order to apply anther culture technique in rice

breeding ~or compressing the breeding cycle~ anther culturability

o~ the local varieties was ~irst tested. Apart ~rom in vitro

responsiveness~various physical parameters and suitable culture

conditions are also needed to be standardized. There~ore,

36

experiments were ~irst undertaken to work out suitable conditions

~or induction o~ callusing in the anthers.

3.1.1 Standardization OT conditions ~or callusino OT microspar•

3.1.1.1 Time DT collection DT panicles

The panicles collected in the morning hours gave better

callusing response compared to the panicles collected at

di~~erent times though the di~~erence was not signi~icant. The

experiments were done with panicles collected in the Torenoon

(between 8 AM to 10.30 AM ).

3.1.1.2 Environmental conditions at the time a~ collection a~

panicles

A signi~icant di~~erence in ~requency o~ callusinq was

observed in the panicles collected on di~~erent days. In order to

~ind out optimum conditions~ panicles were collected between 25th

August to 30th September and J~ept in cold at ___g_if.tlrrent

t~ TOr cold pretreatment. The range 0~ temperature,

humidity and sunshine hourL during the period when anthers were

collected vis-a-vis ~requency o~ callusing is presented in Fig 1

and Table 1 •

Perusal o~ Fig 1 indicates that callusing ~requency

was maximum (78Z) in the anthers taken ~rom the panicles

collected Jn 16th ~ptembet- when the tempet~ature ranqed ~rom

37

Tabla 1 I Frequency of callusinQ in the anthers ccll•ct•d on

di-Fferent dates ( -from 25.8.92 to 30.9.92 )

Datr of No. of No. of I Tr•paraturl' Hu1idit y Total colll'ct ion anther cal luling callusing "ax. "in I 1 I Sunshint

inoculated antht!rs ( in 'c J hour

25.8.92 30 NIL NIL 29.8 21.4 83 8.52

26.8.92 400 63 15.7 27.2 20.5 88 2.23

28.8.92 1600 150 9.3 28.2 20.0 95 4.77

30.8.92 390 42 10.7 27.2 20.8 79 8.25

5.9.92 320 112 35.0 28.4 18.5 90 9.50

6.9.92 970 226 23.2 28.0 19.6 74 9.20

0.9.92 420 77 18.3 27.0 19.6 90 3.62

9.9.92 710 163 22.9 28.3 19.6 93 7.55

I 1. 9. 92 300 52 17.3 27.3 19.3 87 6.23

13.9.92 200 68 34.0 26.7 19.0 78 5.70

14.9.92 200 96 48.0 26.5 18.6 81 6.20

15.9.92 1600 290 18.1 24.8 18.0 93 4.75

16.9.92 205 160 78.0 26.3 17.2 83 3.10

17.9.92 1700 340 20.0 24.6 17.8 100 2.50

19.9.92 2500 270 10.8 26.6 17.2 77 8.70

23.9.92 so 4 5.0 20.8 19.6 89 5.00

30.9.92 600 20 3.3 24.4 18.0 DB 2.60

80

75

70

65

60

55 ""' ~ 50 bD c

..... 45 rn = .-< '; 40 C)

'E 35

t' 30 c Q)

& 25 Q)

"" "" 20

15

10

5

25 26 28 3 5 6 8 9 11 13 14 15 16 17 19 23 30

--------------~~Dates of inoculation

AUGUST SEPTEMBER Fig. 1 Frequency of callusing of different dates of inoculation

(from 25-8•92 to 30-9'92

l7.2-26.3°C. humidity 83%- 100% ~nd sunshine. 3.10 hour/day. In

addition, Fig.l also shows ~requency o~ callusinq ranginq ~rom

9.3 - 48% during Auqust 28th to September 14th.

3.1.1.3 Length o~ cold pretreatment

Cold pretreatment is known to exert pro~ound e~rect on

anther callusing. In the·present investigation it was observed

that ~requency

pretreatment (at

or callusing increased with the period

8-10° C). in both the media viz; G5

o-f cold

and

which were tested. Two to 4 % callusing was observed in the

anthers which were treated -for 1 day. Therea-fter, -frequency o-f

callusing increased with increase in ~ o-f cold pt-etreatment

and reached maximum (43.3% in G5 and 16% in E24 > a-fter 10 days o-f

pre- treatment. A-fter prolonged pre-treatment beyond 10 days, the

-frequency o-f callusing decreased abruptly -from 43.2% to 12.3% in

G5 medium a-fter 12 days. In case o-f E24 medium. it slowly

decreased with increase in days o-f cold pretreatment and reached

4% a-fter 26 days o-f cold pre treatment. None o-f the anther

callused in G5 medium a-fter 26 days o~ cold pre treatment

(Fig.2).

The

pr-ett-eatment

present study, thus. indicates that

10 days to the anthers

cold

be-fore

inoculation, is optimum -for obtaining maximum callusing in local

rice cultivf-s.

3R

3.1.1.4 Staging o~ micro5pores

Culture o~ anthers containing microspores at various

developmental stages ~ known to in~luence the ~requency o~

callusing. There~ore,

~evelopmental out a suitable stain

in order to inoculate microspores at

stage?, an attempt was -first made to -find J

to ascertain the exact developmental

staqe o~ the microspores. Anthers -from di-f-ferent parts o-f panicle

were stained with various stains and the results obtained are

pt-esented below:

3.1.1.4.1 Staging•with acetocarmine stain

Mict-ospore nucleus could not be seen distinctly in_/

sqash preparations o-f 2% acetocarmine. Even the addition o~

mordants did not help in distinguishing nucleus ~rom the

cytoplasm (Fig. 3). There-fore~ tilis stain was not ~ound suitable

~or staging o~ rice mic~ospores.

3.1.1.4.2 Staging with Feulgen stain

The nuclei were seen distinctly using this stain

(Fig. 4). They appeared magenta coloured while cytoplasm remained

colourless. But this technique was time- consuminq and required

minimum 2 to 5 hours. There~ore. it had limited application,

especially when large number o~ anthers were to be inoculated

everyday.

39

52

48

44

40

36

32

28 .,.... ~ v

24 lJI• c ...! tiJ

:3 20 ...... ...... Ill u

.._ 0

'>-u c Gl ':l tr Gl ... t.-

\ ,-

'I 2 4 6 8 'I 0 1 2 1 4

__. C.D 1 d I r a ~ r a a t m a n t d a y s '

1 6

0 in G 5

I in

18 20 2 2 24

medium

26

f i 9

• ~ : E f f e c t o f 'd a y s o f c o l d p r e t r e a t m e n t o n f r e q u e n c y

\ of callusing

3.1.1.4.3 Staginq with iron alum - haematoxylin stain

Haematoxylin with iron alum stained the microspore

nuclei very distinctly. Nuclei were stained deep brown against

colourless cytoplasm (Fig. 5). Usinq this staining procedure,

lan1e numb~r oF spikelets could b~ staged within a short period

because microspores could be stained within 2-5 minutes and thus

this stain was used routinely.

3.1.2 Effect of different culture media on callusing frequency

and callus proliferation

Altoqether 12 semisolid and 10 liquid media

were tried ~ wR&cK the induction o~ callus~ in the local

cultivars. Preliminary screenina reve~led callus induction in

all the media but, there was wide variatio~ in the ~reauency. In -------------------'--·-~-----· ----- ---

order to narrow down the number o~ media and to Find out the

responsive one (s), only those media were tested which induced 5%

or more callusinq. Three semisolid media viz •• G5 , E24 ~minimal

and two liQuid media viz., N6 and E 10G were ~ound promising and

thus these were used For Further study. Anthers o~ Khonorullo,

RCPL 1-lC, RCPL 1-2C~ RCPL 1-3C, Pusa 33 and DR 92 were

inoculated in semisolid G5 , E24 (both containing B5 basic salts)

& minimal, and liquid E10G (containina B5 basic salts) L modiFied

N6 media.

40

Fig. 3 I Microspore stained with 2'Y. acetocarmine stain ( X 750 )

,">

tiU

Fig. 4 I Microspore stained with t=eulg~ stain ( X 750 )

i

Fig. ~ I Microspore stained with iron-alum haematoxylin stain ( X 750 )

4

It was observed that in case o~ semisolid medium

the anther started browning during ~irst three to ~our weeks o~

inoculation. Subsequently~ callus mass bursted out o~ the anthers

and the anthers turned deep brown a~ter 40-45 days o~ inoculation

(Fig.t, 6 L 7). The calli were cream coloured with organised

sections which appeared as embryoids.

In most o~ the cases microspores started callusing

a~ter ~our weeks o~ inoculation and continued upto ~our months.

The percent callusing was 13. 46. 33. 8 and 0.4% in 1st~ 2nd,

~ 3rd, 4th and 5th months respectivlv a~ter inoculation • Maximum

I ~ I callusing was, however observed between 40 to 50 days o~

J

inoculation.

In case o~ liquid media (Fig. 8), anthers were ~ound

to split longitudinally thereby relefing microspores which were

seen as aggregates (Fig. 9). The microspores gave rise to calli

which remained attached to the anther wall in the initial stage

(Fig. 10) and were released a~terwards.

It was noticed that in most cases 200-300

microspores were released ~rom each anther a~ter splitting and

the maximum release was recorded between 4th to 6th day o~

culture. Three types o~ microspores viz •• highly elonqated- empty

; medium elongated- densely cytoplasmic and small unenlarged. in

the ratio o~ 5:4:1, respectively. were observed. Nuclear

41

~nJpaw

UOJ+e~aua6a~ o+ pa~~a~sue~+ aq o+ Apea~ Jtte3

tteM ~a4+ue a4+ ~o +no pa+s~nq Jtte~ 6UJM04S ~a4+u~

I

I

I

L

9

L-9

FiQB. B-10

B

.9

10

I

I

I

I

Callusing in liquid medium

Calli released in the medium ( single arrow) ( X 750 )

Longitudinal split anthers showing aggregates of microspores&( double arrow ) ( X 750 )

Anthers showing attached calli ( X 750 )

divisions occured mostly in the medium ~elongated and densely - . ~

cytoplasmic microspores a~ter 3rd day o~ culture and it continued

upto 16th day with wall ~ormation (Fig. 11-13). Further divisions

and wall ~ormation resulted in the ~ormation o~ enlarged ,--, /-----~---._""

multicellular r'~ generally composed o~ 50-60 cells -~-------

(Figs 14 & 15). The exine o~ the multicellular structur~

eventually bursted out and around 30th day. small calli were

released in the liquid mPdia. Globular and heart shaped calli

were observed a-fter 55 days o-f culture (Figs. (lb-~\~-< 17).

77"1. "J- In

qenerally

The overall -frequency o-f callusing ranqed -from 4.5 to

case o-f Khonorullo~ the ~requency

oood in almost all the media.

o-f callusing

However. liquid

I was\,

medium was the best where 57"1. callusinq was observed. In case o~

semisolid medium~ 50"1. callusinq was -found in G5 medium (Fiq. 18).

In some tubes callusing -frequency was as high as 98"1. in G5 medium

(Fig. 19).

In case o~ RCPL 1-lC, it was observed that liquid

medium~ E 10G was the best. where highest callusing (77''1.) was

observed. However, in semisolid medium, highest ~requency o-f

32.4% callusing was observed in E24 medium.

In RCPL 1-2C • the hiqhest callusino was recorded

in semisolid media where it was 45.5% in G5 • 3~).4% in E24 and

30.6% in minimal medium. In liquid N6 and E10G medium. the

42

Figs. 11-17 1 Various developmental stages o~ the microspores in liquid medium

" . '

11 1 Uninucleate microspore ( X 750 )

12 1 Microspore showing initial division ( X 750 )

13 1 Multicellular pollen ( X 750 )

14

17

1 Further divisions give rise to multicellular structure ( X 750 )

1 Individual multicellular pollen grains enclosed to show the densely staining cells ( X 750)

1 Microspore exine bursts out releasing microcalli ( X 750 )

1 Microcalli ready ~or trans~er to regeneration medium

~requency o~ callusing was 22.0 and 20.0% respectively.

In RCPL 1-3C. callusinq was equally good in both

semisolid and liquid medium. Fi~ty~our percent callusing was

observed in E24 semisolid (Fig. 20) and 58% in ElOG liquid

medium. However. only 5% callusinq was observed in G5 medium

which was generally good ~or other varieties.

In general, anthers o~ Pusa 33 did not respond

well in almost all the media and the ~requency o~ callusinq in

both semisolid and in liquid media ranqed ~rom 6.1% to 15% only.

DR92 anthers responded well in G5 semisolid (48%)

and N6 (31%) liquid medium. In minimal medium they did not

respond and the ~requency dropped to 4.5%. Data on the ~requency

o~ callusing in anthers o~ six varieties in semisolid and liquid

media at-e collated in~)ble 2.

Perusal o~ data on callusing o~ anthers in six

varieties (including parents and their progeny sister lines)

indicated a strong genotypic e~~ect. In qeneral, it was observed

that together with genotypic e~~ect , media containinq B5 salts

like G5, E24 and ElOG responded well to callusing o~ anthers in

local cult i vars except in Pusa 33 whet-e h ir--~hest ~t-equency o-f

callusing was observed in N6 medium. Khonorullo was in vitro

responsive variety whereas Pusa 33 was a recalcitrant one with

low ~requency o-f callus induction in almost all the media.

43

There~ore, apart ~rom presence /availability o~ appropriate

nutrients, genotype OT the donor plant exerts a

inTluence on callusing. Liquid media were relatively better

semisolid ones.

Proli~eration o~ calli

strong

than

Small pieces OT calli (weighing 10 to 15 mg) were

transTerred to N-19 and J-19 liquid medium TOr proliTeration. The

tiny callus grew into a big mass OT 2 to 3 q in 10 to 15 days

(Fiq.21}. Both the media were Tound to be equally good ~or

supporting proliTeration, but addition OT 2 mg/1 NAA in both the

media increased the ,rate OT callus growth in some cases.

Frequency OT regen~~ation was however,aTTected. ProliTeration 1n

dark or in low intensity light, was Tound better TOr

regeneration.

44

Fig•. 18-20 I Callus induction on semisolid media

18 I Callusing in Khonorullo on semisolid G5 medium

19 I Very high -frequency o-f callusing in Khonorullo on semisolid Gs medium

20 I Callusing in RCPL 1-3C on E24 medium

21 I Callus proli-feration in J-19 medium

(}oJ Table 2 : Frequency o~ callusing o~ anthers ~rom local cultivars

. L in dif~erent semisolid and liquid media *·

c-rl1LiSTrlijY.·-·

Cultivars

Khonorullo RCPL 1-1C RCPL 1-2C RCPL 1-3C Pusa 33 DR 92

65

50.0 20.4 45.5 5.0

12.0 48.0

Semisolid 7 .::::::.

Eb4

11.7 32.4 35.4 54.0 9.0

26.0

Media

7 t J6 Minimal

31.6 57.0 9.0 5.6

30.6 22.0 41.0 12.0 6.1 15.0 4.5 31.0

· Li-quid ·,\.

I

E10G

32.0 77.0 20.0 58.0 9.0 6.2

* Average o~ 3 experiments consisting o~ at/least 500 anthers in each J

** RCPL lines Pusa 33 and

are sister Khonorullo

lines selected ~rom a cross between

3.1.31 Regeneration and ccn~irmation c~ ploidy status

I

Anther - derived calli were trans~erred to regeneration

media ~or di~~erentiation. Reqenerability o~ calli was tested by

trans~erring them to regeneration media at various

a~ter their ~irst appearfnce. ~en to 15 days old

higher regeneration ~requency than older calli. Calli

intervali

calli gavel'

on trans~er

to reqeneration media started di~~erantiating within 10 days.

Green pigments were ~irst observed ~allowed by appear~nce o~

shoots and roots ,(Figs 22 26).

It w~s also observed that proli~erated calli tend~ to

l~se their regeneration capability as compared to directly

trans~erred ones. In Khonorullo and RCPL 1-2C , small calli were

proli~erated in both J-19 and N-19 media and a~ter 10-15 days

they were trans~erred to MSB regeneration medium together with

10-15 days old primary calli. Out o~ 10 proli~eratin~ calli, only

one regenerated in MSB media,whereas ~rom the primary calli

directly trans~en-ed to MSB medium, around 501. showed l regeneration and produced green plantlets. There~ore~ in ~urther

experiments• only primary calli were used ~or regeneration.

Regeneration o~ calli obtained ~rom di~~erent varieties in

various regeneration media is presented in Bable 3.

Table 31 Regeneration percentage o-f anthar-darived calli on

dif-ferent regeneration media

Khonorullo

Media

MSI MSA MSB MSBT MSC MSD MSBS RGA RG2 N6-H

Total no. 0~ 12 16 16 8 12 6 3 5 5 9 tubes with calli

Number 0~ tubes '4 7 8 e 3 1 (- {! ·-./

1 2 green pigment a-tion

Plant 33 43 50 (- 26 16 / 20 20 regenera- t -t ' .. __...,/

tion percentage \ . ~/

46

Table 3 continued RCPL 1-1C

Total no. o-F 17 10 16 9 8 12 6 15 9 10 tubes with calli

Number o-f 4 2 7 1 G 2 () 2 (j tubes with green pig-mentation

_) (_ \ I I

Plant rege- 23 20 43 11 16 13 -neration percentage

- Not observed

RCPL 1-2C

MSI MSA,MSB MSBT MSC MSD MSBS RGA RG2 N6-H

Total no. o-f 17 16 16 8 5 9 12 6- 12 9 tubes with calli

Number o-F 5 4 7 2 1 2 tubes with Qreen pig-mentation

Plant rege- 29 25 43 . 22 8 22 neration percentage

17

RCPL 1-3C

Total no. o-f 15 12 12 10 4 12 6 6 7 10 tubes with calli

Number oi= 2 2 4 2 1 1 1 1 tubes with qreen pig-mentation

Plant rege- 13 16 33 20 25 8 16 10 neration percentage

DR 92

Total no. o-f 6 5 9 4 5 6 6 6 tubes with calli

Number o-f 1 2 2 1 1 1 tubes with ~

qreen pig-mentation

Plant reg- 16 40 22 20 16 16 eneration pet·centage

- Not observed

48

Table 3 continued

Total no. tubes with calli

Number o-f tubes with qreen pig­mentation

Plant reg­neration percentage

o-f

- Not observed

MSI

10

1

10

Pus a 33

MSA MSB MSBT MSC MSD

12 9 5 6 Is j C....-/

1 '2 1 1

8 22 20 16

't>'O \1 ~' ~~...,. ~\)r !+t..f lot el ') J Y.,..t-1- M f'{;...:J\... ;

MSBS RGA RG2 N6-H

r--: l ~~- .....

6 /- -

1

16

l<))

It was observed that light intensity o-f 2500 to

3000 lux is suitable -for plant regeneration. A continuous light

-for 5 days a-fter transfe~ to regeneration media followed by 16

hr/8 hr liqht/dark was -found to be the best -for regeneration.

In Khonorullo, highest regeneration percentage

(50%) was observed in MSB medium (Figs 22 & 23) -followed by 43

and 33% in MSA and MSC. In regeneration media MSBT, MSBS and RGA

there was no regeneration response.

Almost all cultivars responded well in MSB

medium except DR 92 whet-e 40% regeneration was recorded in MSA

medium compared to 22% in MSB medium.

49

Forty three percent regeneration was recorded in RCPL

1-lC anthers in MSB regeneration medium ~allowed by 23, 20, 16,

13 and 11% in MSI, MSA, MSD, RGA and MSBT. There was no

reqeneration in MSC. RG2, MSBS and N6 media.

Forty three percent regeneration was observed in

RCPL 1-2C anthers on MSB medium followed by 29, 25, 22, 22 and B%

' in MSI, MSA, MSD, N6 and M~BS, respectively. MSBT, MSC, RGA and

RG2 media did not support regeneration. In cases o~ RCPL 1-3C

and Pusa 33 maximum regeneration o~ 33 and 22% respectivly was

observed in MSB.

It was observed that MSB , MSI and MSA supported

better regeneration than MSC, MSBS. MSBT, RGA, RG2 and N6, MSB

being the best ~or almost all local cultivars.

A~ter the plantlets attained 6 to 7 inches

height, they were trans~erred to Yoshida's liquid culture medium r----< and were covered with tubes to check evaporation (Figs 27-29).

A~ter about 15 to 20 days, they were trans-fer.red to pots for

~urther growth and maturity.

Ploidy status•

Root tip cytoloqy showed that most (70 to 75%)

o-f the regenerated plants showed diploid (Fiqs 30 and 31)

chromosome numbers, while 15 to 20Y. were haploids or mix~loids. f

50

Figs. 22-27 1 Various stages or plant regeneration rrom anther derived callus or local varieties

22 1 Initiation or greeen pigmentation in Khonorullo

23 1 Shoot rormation in Khonorullo

24 1 Leaf initiation in RCPL 1-1C

2~ 1 Plantlet or RCPL 1-1C

26 1 Plantlet in advanced stage in RCPL 1-1C and

27 1 Plantlet of Khonorullo on paperboat transrerred to Yosida's liquid medium

#

Figs. 28-29 1 Plantlets in Yoshida's liquid medium covered with tubes

28 1 Plantlets'o~ RCPL 1-1C

29 1 Plantlets o~ Khonorullo

Figs. 30-31 1 Squash preparation ~rom root tip o~ regenerated

plants showing diploid (2n=24) chromosomes

( X 3000)

.. ...... -~ ..

30

3.2 Production of double haploids

3.2.1 Selection of parents and hybridization

In order to trans~er cold tolerance to hiqh

yielding genetic background, IR 70, an elite breedinq line ~rom

IRRI1

and Khonorullo, a local cold tolerant cultiva~ ~ selected 1_'

for hybridization. IR 70 was used as ~emale whereas Khonorullo

was used as male. In all, 9 hybrid seeds were obtained.

3.2.2 Raising of F1 and their comparison with parents

The nine hybrid seeds were grown sep,rately in

the field. Data were recorded to compare the agrobotanic

characters o~ the Fl plants with their parents. Fl plants showed

hybrid vigour and performed better than both the parents in yield

and yield contributi~g characters (Table 4).

Days to 50% ~lowering (98 days) remained within

the range of both the parents which.was 109 and 91 days in IR 70

and Khonorullo, respectively. Lea~ length (29.0 em) was reduced

as compared to both the parents (34.0 and 36.5 in IR- 70 and

Khonorullo, respectively). Plant height remained within the range

o~ both the parents which was 87 em as compared to 60 and 131 em

in IR 70 and Khonorullo, respectively. Number of ear bearing

tiller increased remarkably to 7.1 as compared to 5.3 and 4.2 in

IR 70 and Khonorullo, respectively. Likewise, panicle length

51

Table - 4 : ~.f.._..JiClfD.&-- ~robotanic characters $IR 70 and Khonorullo 1~~heir Fl hybrid

Days to Plant Luf EBT Panicl! Panicl! &rain •t/ 100 se!d Yi!ld/ Total 1 Sound &rain Endrosp!rt 50 % H!ight l!ngth l!ngth •t. panicl!s .t. plant grain/ grain shap! Colour flo•ering ( Cl ) ( [I ) ( [I ) ( g ~ ( g ) ( g ) ( g ) panicle

Pl IR 70 109 60.0 34.0 5.3 21.0 0.90 0.31 1.70 O.b 125 9.1 s II 1 .... ~ ' 1' ~·.<. r---

F! IR 70 X t ~· r-"'· r ~,c•

Khonorullo 98 87.0 241.0 7.1 23.9 4.01 2.81 2.85 9.1 1b7 b3.2 " .~P~--~ -(i

\,_./ ··,·-.

P2 khonorullo 91 131.0 3b.5 4.2 23.5 3.50 2.9b 2.89 6.2 149 bO.B B /~-\/

,' ~~/ ~-f rl' ,I

(23.9 em) and panicle weight (4.01 g) also increased ~rom the

range o~ both the parents. Grain weight per panicle (2.81 g) and

100 seed weight (2.85 g) remained within the range o~ both the

parents. Yield per plant (9.1 g) increased remarkably and

exceeded both the parents (0.6 q in case o~ IR 70 and 6.2 g in

case o~ Khonorullo). Total qrains per plant (167) also increased

and was higher than the parents. Per cent sound grain (63.27.)

also increased as compared to both the parents which was 9.17. in

IR 70 and 60.87. in Khonorullo. Medium grains were observed in

case o~ F1 as compared to slender grains in IR 70 and bold in

Khonorullo. Light red coloured endosperm was observed which was a

mixed character o~ both the parents; IR 70 was white where as

Khonorullo, possessed deep red endosperm colour.

3.2~3 Culturing of anthers derived from Fl

3.2.3.1 CallusinQ

Anthers ~rom F1 plants o~ the cross IR 70 X

Khonorullo were cultured as described previously (time o~

collection o~ panicle and preparation o~ staining solutions and

procedure) in 65, E24, L8 and MP2 medium. Percentage o~ callusing

ranged ~rom 11.7 (in G~ semi solid ·medium) to 33.67. (in E24

semisolid medium). Highest percentage o~ callusing was observed

in one tube containinq L8 medium (81.6%). "In MP2 medium. the

~requeny o~ callusing was generally good (31.1X). Frequency o~

callus induction in di~~erent media is presented in Table 5.

Table 51 Frequency o~ CAllusing o~ F1 d•riv•d •nth•r• o~ IR 70 X

Khonorullo in di~~erent media

Media

65 E24 L8 MP2

Number o~ anthers 980 110 1585 170 inoculated

Total calli obta~ned 115 37 454 53

'l. callusing 11.7 33.6 28.6 31.1

3.2.3.2 Regeneratic;m

Calli obtained ~rom the above media were

trans~erred to MSB regeneration medium within 5 to !0 days o~

their appearence and kept in 2500 to 3000 lux light intensity at ./---"'\

t25 q j2oC temperature. Green pigmentation was observed a~ter 5 to ··H. '"----· .' 10 days o~ trans~er to the regeneration medium. The reqeneration

percentage obtained ~rom the calli induced in di~~erent media is

collated in !able 6.

53

Table 61 Regeneration percentage in the calli obtained ~rom F1

derived anthers o~ IR 70 X Khonorullo in MSB medium

Total no. or tubes with calli

Number or tubes with qreen pigmantation

Plant reqeneration Percentage

not observed.

G5

92

--.\ /_

Media

E24 L8 MP2

15 174 30

32

18 (

It was observed that green pigmentation and plant

regeneration was observed only in those calli which were obtained

rrom L8 medium (Table 6). Plantlets arter attaining 6 to 8 em

height were transrerred to ~lasks containing semisolid 1/2 MS

medium and kept until they attained 18 to 20 em height. They

were, then, transrerred to the Yosida · s 1 iquid ·medium ! and ~

ultimately to the pots arter attaining 30 to 35 em height. Double

haploids numbering 21 thus obtained, were grown to maturity in

the pots (Figs 32 and 33).

54

Figs. 32-33 1 Double haploid plants growing in pots

3.2.3.3 Evaluation o~ double haploid (DH) lines

Out o~ 21 plants regenerated, nine showed promising

characters and they were selected. Seeds ~rom these lines were

collected and ~ound to be di~~erent ~rom each other. Agrobotanic

characters o~ the parents and the double haploid lines are

presented in ~~iguers 34 to 67.

Panicle type oF IR 70 was compact (Fiq. 34)

whereas that o~ Khonorullo was open (Fig. 36). Among double

haploid lines, panicle type varied ~rom compact as in line nos.

1,6.15,19 and 21 (Figs. ,38.40,45,47 and 48) to open as in line

no. 7 13 and 16 (Figs. 42,44 and 48). Line no 10 (Fig. 43) t._

exhibited intermediate type o~ panicle. Secondary branching was

observed in line nos. 1,6,15,19 and 21 (Figs. 38,40,45,47 and

48) whereas it was absent in the parents. Straight panicle was

observed in line nos. 6,13,15,16 and ·21 (Figs. 40,44,45,46 and I . .

48) whereas line nos. 1,7,10 and 19 showed droopy pariicle (Figs.

38,41,42 and 47).

Shape and size o~ grains o~ each line varied

greatly. It ranged From bold ( Khonorullo) to slender, ( IR 70 ).

Line no. 1 and 21 showed slender grains with thin and short hair

on upper portion o~ lemma and palea. (Figs. 51 & 67). Line no. 6

showed bolder grains with qlabt·ous lemma and~~-~~-~--==:~d) (Fig. 53). Line no. 7 and 10 possessed slender grains with

55

glabrous lemma and P~·._.~ubescen~J(Figs. 55 & 57 ). In ------

contrast~ I ine with qlabrous

lemma and pale pubescencF (Figs. 59 & 65). Line no 15 and 21

showed slender grains with thin and short hair on upper portion

o~ lemma and palea (Fig. 61 and 67). In line no. 16, qrains were

round with short hair (Fiq. 63). A typical character~ where lemma

and palea remained ope~at the tip was observed in most o~ the

grains o~ line no. 13 (Fig. 59). This character was-not ~ound in

any o~ the parents.

The endosperm colour, ranged ~rom dark red

CKhonorullo colour) to white (IR 70 colour) in the parents,

whereas~ it was intermediate in the progenies (Fig 70). Line no.

10 and 21 showed white k••~•l coloured grains. In most o~ the

lines, (line nos. 1~6~7~13 and 15) light red coloured kernel was

observed, whereas in 1 ine no 19 the kernel colour was •fiUiiiFI!!-"

darker. In line no. 16, the kernel colour was dark red like

Khonorullo.

Individual anther derived double haploid lines o~

. ~ the cross IR 70 X Khonorullo showed uni~ormity~ many agronomic

characters. The range o~ variability among the plants o~ anther

culture derived lines were within the limits o~ variability o~

their respective parents.

56

l

Fig. 34

Fig. 35

Fig. 36

Fig. 37

I

I

I

I

Panicle o~ Parent 1~ IR 70

Husked and dehusked grains o~ IR 70

Panicle o~ Parent 2, Khonorullo

Husked and dehusked grains o~ Khonorullo

Fig. 38: Panicle o~ line no. 1 with husked and dehusked grains

Fig. 39: Closeup o~ husked and dehusked grains o~ line no. 1

Fig. 40: Panicle o~ line no. 6 with husked and dehusked grains

Fig. 41: Panicle o~ line no. 7 with husked and dehusked grains

Fig. 42: Panicle o~ line no. 10 with husked and dehusked grains

FiQ. 43: Closeup o~ husked and dehusked grains o~ line no. 10

Fig. 44: Panicle or line no. 13 with husked and dehusked grains

FlQ. 43: Panicle or line no. 15 with husked and dehusked grains

Fig. 46: Panicle or line no. 16 with husked and dehusked grains

Fig. 47: Panicle or line no. 19 with husked and dehusked grains

FiQ. 48: Panicle o~~ line no. 21 with husked and dehusked grains

FiQ. 49: Closeup o~ husked and dehusked grains o~ line no. 21

Fig. SO: Grains o~ double haploid (line no. 1) plant

Fig. Sl: A closeup o~ grains o~ line no. 1

Fig. S2: Grains o~ double haploid (line no. 6) plant

Fig. S3: A closeup o~ grains o~ line no. 6

FIQ. ~4: Grains o~ double haploid (line no. 7) plant

FIQ. 55: A closeup o~ grains o~ line no. 7

Fig. ~6: Grains o~ double haploid (line no. 10) plant

Fig. 57: A closeup o~ grains o~ line no. 10

Fig. SB: Grains o~ double haploid (line no. 13) plants

Fig. 59: A closeup o~ grains o~ line no. 13

Fig. 60: Grains o~ double haploid (line no.15) plants

Fig. 61: A closeup o~ grains o~ line no. 15

Fig. 62: Grain o~ double haploid (line no. 16) plants

Fig. 63: A closeup of grains of line no. 16

Fig. 64: Grains o~ double haploid (line no. 19) plants

Fig. 69: A closeup o~ grains o~ line no. 19

Line 19- IR 70 X KHONORULLO

Fig. 66: Grains o~ double haploid (line no. 21) plants

Fig. 67: A closeup o~ grains o~ line no. 21

Line 21- IR 70 X KHONORULLO

Figs. 68-69: Double haplod (DH) lines growing in pots

Fi;. 70: Comparision o~ grain shape and color o~ di~~erent double

haploid (DH) lines with their parents i.e., IR 70 and

Khonorullo

3.3 Field evaluation and perrormance or doubled haploid ( DH )

lines

Seeds obtained Trom the double haploid plants were (fi3 6'5- 69)

grown during next year TOr Tield evaluation and in potsAand data

were recorded to compare the agrobotanic characters with the

parents ( Table 8 ). Means, ranges~ genetic variability, analysis

oT variance, simple correlation coeTTicient and path coerTicient

analysis were worked out.

3.3.1 Means and ranges ror dirrerent characters

The mean values OT nine characters, their ranqes and SE

values Trom th~ Tield expriment conducted in lowland are

presented in TableL7' 8 and Fi~ 71 - 74.

The paren~ 2 Khonorullo attained maximum height

Tollowed by line no. 21 whereas least plant height was observed

in parent 1 ( IR 70 ). Maximum panicle length was observed

in parent 2 • Maximum and minimum grain weight per panicle

were observed in DH 1 and parent 2, respectively .. Similarly

maximum panicle weight was observed in parent 2 and minimum

weight was observed in parent 1. Maximum seed Tertility was

observed in line no.10 whereas parent 1 showed minimum seed I

Tertility. Maximum ear bearing tiller was observed in parent 2

and the least was in line no.6. 1000 grain weight was highest

in line no.7 and lowest in line no 6. Maximum spikelet per

57

Table 7. Mean• and ranges ~or di~~arant character•

Character Mean Range SE

Plant height 84.87 57.20 - 129.20 2.25

EBT 3.75 2.80 5.00 0.37

P/nicle length 20.24 17.04 23.00 0.60

Spikelet/Pyfnicle 140.64 77.00 -- 203.00 7.18

Pricle weight 2.43 0.78 3.52 0.66

Spikelet~~ertility 73.26 8.10 94.80 0.73

Grain weight/pfnicle 2.12 0.22 2.64 0.07

1000 grain weight 24.54 19.60 30.78 0.65

Grain yield 3.06 0.70 3.94 0.08

TAble 8. Comparison of ;rain yield And components in DH lines And parents under field condition of medium altitude ( 1000m msl )

line Plant EBT Panicle Spikelet/ Panicle Spikelet &rain weight/ 1000 grain orain yield Height ( ~o.) length panicle WEight fertility panicle weight [ t/ha ) ( Cl ) (, Cl ) ( n:. ) ( g• ) ( % ) ( X )

DH 1 87.00 3.80 22.00 ' 203. ~': 3.1 77.30 2.64 22.00 3.;~

DH 6 66.80 2.80 17.04 160.(1(; 2.74 87.50 2.60 19.60 3.27

DH 7 80.80 3.20 20.02 132.(;: 2.20 91.40 1. 90 30.78 2.97

DH 10 78.40 3.20 21.06 77 .c~· 2.20 94.80 2.06 30.20 ~ . ~ . ..Jo .~

DH 13 68.80• 3.60 19.18 119. (:: 2.38 68.20 2.30 22.80 ":" .... .,. .........

DH 15 80.00 4.00 20.16 178.0:: 2.32 72.42 2.20 21.64 3.15

DH 16 89.90 3.40 20.04 118. 0~: 2.42 81.82 2.28 26.80 3.~5

DH 19 95.90 4.00 20.16 133.0C 2.44 82.04 2.36 22.00 3.66

DH 21 100.60 4.20 20.02 159 .0(: 2.64 81.54 2.48 24.30 3.5~

IR 70 ( P1 ~ C) 57.20 5.00 20.00 123.0(· 0.78 8.10 0.22 25.60 0.70

Khonorullo i P2 - C ) 129.20 4.00 23.00 145.0(1 3.52 60.70 2.30 24.30 2.60

General tean 84.87 3.75 20.24 140.64 2.43 7.26 2.12 24.55 3.(16

SE 2.25 0.37 0.60 '7 .18 0.06 0.73 0.07 0.65 0.08

CD 5 % 6.43 1.05 1.72 20.53 0.17 2.10 0.19 0.86 0.23

CV ( l I 5.93 21.96 6.64 11.42 5.54 2.24 6.88 5.93 5.93

CJI c .... ,.. ID ::t D .... ...

'II'!. 0

"' D ... ., >-tU 0

l rig.71

E u

... c II .... a.

r1g.12

rig.71

rig. 12

120

100

90

80

70

60

50

40

30

20

1 0

140

130 120

11 0

100

90

80 70

60

50

40

30

20

10

6 7

6 7

1 0 1 3 1 5 1 6 1 9 21 p 1

OH lines

Ooubln haploid lines

Parents P1- IR 70 P2-

10 13 1 5 1 6 1 9 21 p 1 P2

Off linns

Variation in days to so% floysring of different lines yith respect to their parente Variation in Plant height of different lines yith respect to their parents

~ .0 bll

•r-4 Gl Ill

'0 Gl Gl Ill

0 0 0 ......

1 rig. 73

rig. 74

rig. 73

rig. 74

c: ... II

" Cl

, c: :I 0 II

~

35.0

30.0

25.0

20.0

15.0

10.0

5.0

6 7 10 1 '3 15 16 21 P2

DH lines ~Double haploid linea

100 P1: IR 70 ~ Parente:P2: Khonorullo

go

80

70

60

50

40

30

20

10

1 6 7 10 1 3 15 16 1 9 21 p 1 P2

DH lines

Variation in 1DOseed Yeight of different 'lines Yith respect to their parents Variation in % sound grain of different lines Yith respect to their parents

pi'lllir.:lP was ohsr:>r-verl in 1 inr 11n.l "''''~""' Pi'\S it mir1imum in

1 i ne no.lO. Maximum qrain yield was observed in line no.!

while parent 2 recorded the lowest grain yield.

3.3.2 Genetic variability

Phenotypic and genotypic coeFFicience oF variance ( PCV & GCV

heritability estimates ( h2 ) in broad sense )~ genetic

advance ( GA ) and genetic advance as percentage oF mean ( GA%

For diFFerent characters ~QEsrdgd have been presented in Table 10

in aenet·al~

vat· iat ion

Genotypic coeFFicient/oF variation ( GCV ) were

lot-vet- than thilt oF phenotypic cop-fficientL o-f

PCV ) fr'(' all cha.t·actet-s studied. Both GCV and PCV

were highest For the spikelet Fertility ( 32.54 and ·32~46 %

t·espect i ve 1 y Follbwed by grain weight /panicle

31.29 ~1. respectively ) and the 1 oN est ~es wet·e

%respectively ).

obset·ved in

panicle length ( 9.56 and 6.87 Heritability

in bt·oad sense ) w~s also hiqhPst i-ff J..... sp i kel Pt fertility

99.53 % Followed by panicle weight ( 96.24% ). grain yield

95.71 % grain weight /pan1cle ( 95.45 % ) and plant heiqht

93.65 'l. 25.01 % ) • Spikelet

/panicle exhibited maximum GA ( 72.15 'l. ) while it was lowest in

EBT ( 0.57 ) -followed by grain weight /panicle ( 1.57 ) and

panicle weight ( 1.61 . When thr:> qPnr:>tic advance was caculilted

as percentage oF mean. grain weiqht/panicle exhibited the highest

58

' . Table 9~ variability OT nine characters among DH lines and parents

Character Phenotypic Genotypic Heritability Genetic Genetic Advance Coefficient Coefficient I in broad sense } Advance as percentage of variation of variation ( 1 ) of aean.

( I } ( X l ( X I

Plant height 23.52 22.76 93.65 45.23 53.29

EBT 25.35 12.68 25.01 0.57 15.20

~itle length 9.56 6.87 51.67 2.42 11.96

Spik~le{ )Jnicle 2b.18 23.56 80.97 72.15 51.23

jlnicle 11eight 28.46 . 27.92 96.24 1.61 66.26

Spikelet/fertility 32.54 32.46 99.53 2.24 3.06

Grain lleightlpJ'nicle 32.03 31.2'1 95.45 1.57 74.06

1000 grain 11eight 15.41 14.22 85.20 7.80 31.77

Grain yield 28.b3 28.01 '15.71 2.03 66.34

GA '%. ( 74.06 '%. ~ollowed by grain yield ( 66.34 i. ) and panicle

weight ( 66.26 '%. ) • On the other hand, spikelet ~ertility showed

the least GA X 3.06 X ) ~ollowed by panicle length ( 11.96 i. ). ~·-

3.3.3 Analysis o~ variance

Highly signi~icant di~~erences ( P ~ 0.01 ) among

the genotypes were observed ~ot- yield and all other agronomic

components except in case o~ number o~ ear bearinq tillers which

was signi~icant at 5 /. level ( Table 10 ).

3.3.4 Simple correlation COTTicien~ among some major yield

contributing characters

Nine charactet-s) namel~ plant height. panicle

length, grain weight/panicle. panicle weight. spikelet

~ertility (%). ear bearing tillers/plant. 100 seed weight,

spikelet/panicle and yield/plant were subjected to analysis ~or

simple (phenotypic) correlation to ~ind out the extent o~

association among the characters. The results are presented in

Table 11.

Grain yield/plant showed positive signi~icant

relationship with panicle weight (r=0.70) only. However,

relationship o~ grain yield with plant height~ panicle length.

qrain weight/panicle and sptkelel/plant were positive but non-

signi~icant. The association o~ grain yield with spikelet

~ertility and 100 seed weiqht was negative. Signi~icant positive l

59

4 Tabl• 10 Analyata a~ varianc• a~ yt•ld and compan•nta

Source of vuiation

Replication

df Plut height

a.-4 3.209

EBT

0.336

Panicle length

1.034

L

Spikelet/ Puicle Spikelet 6rain 1000 panicle 11ight fertility ~eight/ seed

panicle ~tight

81.909 0.024 9.538$ 0.017 5.8871 -=:..-., __

6rain yield

0.0009

line 10 1890.531U / I

1.804t 11.479tt ~745.273p ~.324tt 2830.470tt 2.223tt 63.032tt 3.700tt ' !

--..... _ _.,..,../

Error 40 25.299 0.676 1.809 257.909 0.018 2.691 0.021 2.H8 0.033

t Significant at 51 II Significant at 11

df - Dtgree of frtedoa

ct . .-- "SS - "'an su1 of squares

Table 11. Simple correlation coe~~icients o~ nine characters among~inas under field condition

~ PL GW/P PWT SfTL EBT sw

PH 0.35 0.15 0.16 -0.(14 0.31 0.09

PL - 0.08 0.22 0.01 0.09 0.26 '

GW/P 0.79** - 0.22 0.17 - 0.64*

PWT - 0.22 0.20 - 0.·55

SFTL -0.32 0.64*

EBT - 0.37

sw

SPL/P

SPL/P e ____ ) 0.07 0.48

0.09 0.31

0.51 0.59

0.66* 0.70*

- 0.37 - 0.23

0.16 0.27

- 0.61 - 0.41

0.41

PH : Plant height~ PL : Panicle length ~ GW/P : Grain weiqht/panicle, PWT : Panicle weight, SFTL : Sp~kelet ~ertility~ EBT : Number o~ ear bearing tillers/plant~ SW : Seed weiqht~ SPL/P : Spikelet/panicle

c,y;p~ c.ya_~ .. '.,,I 7 t ... ). ··-r (' jl ~1 p cA.-(..( -~ ~

panicle weiqht (r=0.79)~ between spikelet/plant and panicle

weiqht (r=0.66) and also between 100 seed weight and spikelet

~ertility. A negative siqni~icant correlation was observed

between 100 seed weight and qrain weight/plant. All other

correlations were either o~ low magnitude or negative.

3.3.5 Direct and indirect effects of different characters on

grain yield

Path analysis was carried out to know the extent

o~ ~ect and indirect e~~ects o~ component characters

yield by partitioning the correlation coe~~icients. The

are presented in Table 12.

on seed

results

In low land condition~ panicle weight exerted the

highest positive in~luence on yield ~allowed by plant heiqht and ~

panicle length. The direct e~~ects 0~ seed weiqht.

spikelet/panicle and ear bearinq tillers on yield was in negative

direction.

60

Table 12 DIRECT AND INDIRECT EFFECTS OF EIGHT' CHARACTERS ON GRAIN YIELD UNDER LON LAHD CONDITION

--------------· PH Pl GW/P

PH 0.400 0.053 0.003

PL 0.140 .!.:..!..?.! - (1, 002

Gw/P 0.061 - 0.031 0.020

PWT 0.064 0.034 0.016

~WJ FTL EBT Sll SLP/P Correlation with yield

0.088 -0.001 - 0.020 - 0.026 - 0.013 0.48

0.122 o.ooo - o.oo6 - o.oao - o.ot7 o.31 '

0. 432 - o. 003 - 0.011 0.197 ·- 0.090 o. 59

0.548 - 0.003 - 0.013 0.170 - 0.118 0.70l

FTL - 0.016 0.001 - 0.004 - 0.120 0.015 0.021 - 0.196 0.065 - 0.23 ?

•' EBT 0.12b 0.013 0.003 0.109 - 0.005 - 0.065 0.112 - 0.028 0.27

SW 0.035 0.039 - 0.013 - 0.303 o.oo9 -:: - o.327 · o.1os(>::o~~ _,/!

SPL!P 0.030 0.014 0.010 0.363 - 0.005 - 0.010 0.186 - 0.178 0.41

Residual erfect=0.42

~1 Plant height ; Pl : Panicle length;~: Grain weight per panicl~-~ : Panicle weight ; ~: Seeo fertility ; EBT : Ear bearing t1ller; 511 : 100 seed weight; ~Spikelet per panicle

•

,-.

(,_ o~ ~~-,\ ~ ' ) ~' -----··--.. _____ _,_____ /

DISCUSSION

The agricultural productiyity needs to /-"'··········~. -·. . --· .. -. -~

securing -food as a~~,':r-~e~-~~~e)of population

be increased -for\ u~ ~ growth. Breeding ~

"' v should contribute to the expansion o-f yield potential and to the

improvement o-f the quality o-f products. The e-f-ficiency o-f

breeding depends on the e-f-ficient process o-f broadening the

genetic variability, selection, -fixation and rapid propagation.

Conventional breeding in which each process is

61

per-formed at population and individual plant level has yielded

excellent results. However. recent proqress in biological

sciences reveals that some stages o-f breedinq procedures can be

per-formed more e-f-ficiently by adopting tissue, cell culture and

molecular manipulation. ·-,.

The present i~vestiqation on androqenesis was ...

conducted with a view to bringing together the desirable

characters o-f both indica and japonica rice varieties. The

experimental results o-f the present investigation have been

discussed in the -following pages.

4.1. Standardization o-f conditions -for callusing o-f microspor•s

in local cultivars 1

sunshine

Environmental -factot·s such as temperature, humidity, . . ~e,.U- 't"'~u i1V"\ t:k ,.~«.)'~~~ h( ~O'f ~ ..rl<J..'"

hour etc. ~ signi-ficant1 ~~ androqenesis.'~~

f-- '---- 1 ....

Panicles collected between 15th to 17th September ( mean

temperature - minimum l8.2°c~ maximum 26.3°C; humidity 83 to

100 'Y.; average sunshine hours/day - 2.50 to 4.75 ) showed

maximum callusing. Panicles collected beyond these days did

not respond so well though the temperatu:·t:! -for cold

pretreatment and culture condition remained the same. This shows

that a temperature between 18.2 to 26.3° C , humidity between 83

to 100 'Y. and sunshine hours between 2.50 to 4.75 hours/day

rendet·j anthers in the best metabolic states -for the

62

callusing through androgenesis.

Cold shock to the anthers be~ore inoculation

is known to induce better to callusing and green

plant regeneration. This beneficial effect was first reported

by

et

Nitsch and Norrel 1973 ) in

al. ( 1975) in Oryza sat1va.

Datura innoxia and by Chale~-f

The -t~e~------v-or cold

pretreatment depends on the temperature used. Wanq

( 1974) demonstrated that shorter the period o~ tt·ea.tment

should be the temparature and vice versa. Cold shock ~or 10 days I at 8° C was ~ound to be the best for callusing as well as ~or the

green plant regeneration. The microspores subjected to this

temperature presumably reduce their metabolic activity which

results in the accumulation of larger perceritage of microspores

at the most suitable stage for e~~ecting the developmental shi~t

t-eguired ~or andt·oqenes_is. Cold pn?tt-eatment prolonqs the li~e

span o~ the anther and weight 0~ the anther also

during the cold pretreatment period ( Zhou and Cheng. 1983 ).

One of the impot·tant factors for haploid

production through androgenesis. is the culturinq of microspores

at the mid-uninucleate staqe o.f,development ( Chen.1977 ). When

cultured clt this stage. there i~ a shift from t~e normal pathway

o~ pollen development and a-ftet- t·epeated mitotic divisions o-f

the microspores. calli are formed. Thus. staginq of microspores

for correct identification of mid-uninucleate staqe helps in

( .. , ),)

obtaining high ~requency o~ callusing.

For staqinq o~ the microspores. acetocarmine stain

did not work satis~actorily. however. Feulqen reagent stained the

nuclus L

distinctly ( Gupta et ~1 .• 1987 ). but it takes 2 to 5

hours ar;.d hence this· techniquE·-had· limited application especially

when a large number o~ anthers are to be plated everyday. Acetic

acid iron-alum haematoxvl in stains the microspore nucleus

quickly ( within 2- 5 min) and the nucleus becomes very distin~t

i ( Gupta and Borthakur,1987 ).Th~ clarity o~ nuclear staging usinq

this stain may be due to the use o~ iron -alum which acts as a

mordant and haemato>~Yl in has lonq been known as a stain -for

nucleus I DNA.

4.2. Anther Microspore culture:

Induction o~ callusing in iaponica rice has been qenerally high.

Howevet-!1 it has not been so in indica. Consequently. work on

haploid product_ion on indica rice has been slow. In addition to

hormonal and other growth supplements Guha et al., 1970;

Niizeki and Dono. 1971 >. the concentration o~ nitrogen plays ~n

impot·tant t·ole in callusing Chu et a 1 • • 1975 Sonq et al ••

1978). In the present investigation. G5 • E24 and minimal

( semisolid ) media were ~ound more suitable than others.

The highest -frequency o~ callusing was recorded in E24 ( with 65

salts ) which contains 26.72 mM o~ total nitroqen as against 60

mM i~ MS (Murashige and Skoog~ 1962). Fi~ty ~our percent

callusing was observed in RCPL 1-3c in E24 medium. This

particular variety per~ormed equally well in almost all media. It

gave 41% , 12% and 58 X callusing in minimal, N6 and E10G media,

respectively. In the minimal medium where callusing was hiqh,

the concentration o~ nitrogen, phosphorous, potassium and calcium

was lowered to 1/3rd and micronutrients like zinc, boron, iodine, ----copper, molybdenum and cobalt were eliminated as suqqested

by Song et al.( 1978 ). It is thus clear that low concentration

o~ nitrogen is ~avourable ~or callus induction. TlilS is in

agreement wiht the results o~ Chu et al. ( 1975 ). They added

only 3.5 mM {NH4 >2 so4 to the basic medium that resulted in an

increased callus production. In addtion, they ~ound that NH4+ at

low concentration was advantageous to pollen callus ~ormation,

while high concentration inhibited the ~requency o~ callus

induction. It appears that total amount o~ N a~~ects the growth

o~ pollen callus, possibly by inhibition o~ enzymes the

Kreb·s cycle.

In 6 5 medium, t<honot-ullo and RCPL1-2C recorded 50 and 45.5%

callusing respectively. In 1 iquid medium, RCPL 1-1C showed

highest percentage ( 77% ) o~ callusing on E 10G medium. RCPL

l-3C showed 58/. callusing in medium ElOG which contains 35 mM

nitrogen and 47 mq/ 1 q lutamine. The addition o~ aminio acid,S

viz. glutamine increases the ~ormation o~ embryo and qreen

65

plantlets. This is in con~ormity with the results o~ Trottier et

al. (1993) in wheat where 50% embt-yo -formation and cn-een plant

regeneration was reported.

It can, therrore, be said that B5 and N6 basic media are

suitable ror callusinq o~ indica rice. However, small variations

have been observed in many cases. Genotypic di~~erences in

anther response to callusing have already been reported ( Guha

- Mukherjee, 1973 ; Mahesh war i et a 1 • ~ 1982; Thengane et a 1 • ,

1994). Khonorullo responded well to almost all media (57%,

sox, 32'1., and 11.7% in minimal and E 24'

respectively ) whereas in Pusa 33. anthers did not respond

well in almost all the media and the -frequency or callusinQ in

both semisolid and liquid media ranged ~rom 6.1 to 15 % only.

This also indicates that qenotypic dirrerence plays a vital role

in anther response to callusinq. Two types o~ calli, (embryogenic

and non-embryogenic ) were observed in cultivated rice (Raghava

Ram and Nabors, 1985; Thompson et al., 1986, Wang et al.,1974).

Embryogenic calli are capable o~ di-frerentiating into shoots

and roots whereas non - embryogenic calli mostly give rise to

roots. In this study, the embryogenic calli, on trans-fer to N6

medium without growth regul atot·s, proli-ferated and -formed

embryoids which developed coleoptile, scutellum and roots. These

structut-'es, in turn, gave rise to plantlels with shoots and

roots.

66

During regeneration, ·it was observed that proli~erating

calli tend to l~~e the capability o~ regeneration as compared to

the primary calli. This is evident ~rom the ~act that higher

response o~ regeneration ~rom the primary calli which ranged ~rom

22 X to SOX as compared to lOX in proli~erating calli. Plant

growth regulators like auxins and cytokinins play crucial role

in the process o~ androqenesis. This is in agreement with the

increase inproduction o~ regenerated plant, by a ~actor o~ 10 by

an early trans~er o~ callus in maize (Barloy and Beckert, 1993).

Earlier, 2,4-D was thought to be the only hormone essential ~or

androqenesis and it was invariably added to the culture medium

with or without cytokinin and/or other auxins (Niizeki and Dono,

1968; Harn, 1969; Nishi and Mitsuoka,1969; Guha et al.,1970; Iyer

and Raina, 1972; Woo and Tung, 1972; Guha-Mukharjee, 1973; Wang

et al.,1974). The inhibitary e~~ect o~ 2,4-D and that it can be

replaced by NAA was ~irst reported by Ni izeki and Dono ( 1971).

A~terwards it was also ~ound out that cytokinin is essential ~or

obtaining callus o~ high morphogenic potential ( Chen, 1977;

Chale~~ and Stolarz, 1981; Lee and Chen, 1982 ) • The

concentration o~ hormones required may not be uni~orm ~or all the

cultivars as genotypic di~~erences have been reported (Liang,

1978 ; Cornejo -Martin and Primo- Millo, 1981).

Other ~actors that inrluence anther response, are

67

composition o~ the gas mixture in the culture vessel ( Johansson

et al.,1982 and the density o~ anther inoculated in the culture

medium (Bajaj et al., 1977; Sundarland et al.,1981).

In the present study, it was observed that MSB was

the best regenerating medium as compared to other media. MSB

mTdium contained 0.5 mg/1 NAA and 3.0 mg/1 kinitin. The

regeneration percentage o~ di~~erent varieties in MSB medium

was 50 % in t<honorullo, 43 % in RCPL 1-lc and RCPL 1-2c, 33 %

in RCPL 1-3c and 22 % in DR 92 and Pusa 33.

Light intensity o~ 2500 to 3000 lux and a

continuous light ~or 5 days a~ter trans~er to regeneration media

~allowed by 16 hr/8hr liqht/dark period was the best ~or

regeneration.

The light conditions used ~or rice anther culture ranqe

~rom complete dadmess to continuous illumination. For callus

induction, inoculated anthers are typically placed in the dark

(Niizeki and Oono 1968; Harn 1969; Woo and Tung 1972; Y. Chen et

al., 1974). However, Nishi and Mi tsuoke ( 1969) and Ni i zek i and

Oono (1971) also reported callus induction under continuous

light. A typical current practice is to use dark incubation ~or

callus induction ~allowed by 16 hour light per day at 2000-3000

lux ~or plant regeneration (Chu et al., 1984; Crouqhan and Chu~

1991).

68

4.3 Ploidy status o~ plants derived through anther culture

The ploidy o-f plants derived -from anther or

microspore culture is highly variable and in most instances

diploidy predominates. In the present investigation also around

70 to 75% diploid plants were observed, 10 to 20% were haploids

and rest mixaploids. Haploid to pentaploid plants are reported in

D. sativa by Nishi and Mitsuoka (1969) and Niizeki and Dono

(1971). The range o~ diploids in reqenerated plants was 50 to 60X

against 30 to 40% haploids Dono, 1975; Mok and Woo 1976; Chen

and Lin, 1976 ). The regeneration o~ diploids

aF-4!-~known to be ~avoured over haploids through a selection

mechanism ( Dono ' 1978 and this is desirable ~or its

application to breeding. The higher the ~requency o~ diploids'/, '-"4

( the greater the e-f-fciency__-

The wide ranqe o-f ploidy levels seen in

androqenesis has been attributed to endomitosis and/or the

-fusion o-f various nuclei( Narayanaswamy and Chandy, 1971;

Engvild et al., 1972; Raquin and Pilet, 1978:; Engvild 1973 ;

Sunderland and Dunwell, 1974 ). In qeneral endomitosis would

cause diploidy and tetraploidy, whereas nuclear -fusion would

give rise to triploids and pentaploids. Chromosomal variation

and atypical mitosis leads to polyploidy and aneuploidy which

are common in plant tissue culture ( D'Amato, 1965; Torrey, 1967;

69

Murashige, 1974; Matthews and Vasi 1 ~ 1976 ) • Variation in the

ploidy o-f plants observed in anther culture appears to be a

-function o-f the developmental stage o-f the anthers at the time o-f

cultut-e, with higher ploidy levels mot-e prevalent in anther

culture -following microspore mitosis. This is expected in view

o-f the speci-fic timing o-f DNA synthesis with generative and

vegetative nuclei -following microspore mitosis and the -fusion o-f

nuclei at the 1c or 2c levels resulting in various ploidy levels.

4.4 Production o-f promising double haploid lines and their

evaluation

The use-fullness o-f in vitro selection during anthet-

culture was reviewed by Nabors and Dykes ( 1985). The selected

tolerance is liekly to be use-ful because it is inherited and

stable. Wenzel (1985) and Wenzel et al., (1987) also stressed

that cereals o-f-fer an ideal system -for isolated microspore

culture. In vitro selection against limiting -factors such as

water stress, cold tolerence etc. is a very e-f-fective way o-f

Qbtaining modi-fied variants o-f already highly adapted varieties.

Many researchers have con-firmed that pollen- derived 1 ines are

homozygous and about 90 /. o-f the plants produced are uni-form and

stable j,t< all major traits (

Rangaswamy, 1994).

Oono, 1975; Kumaravadivel and Sree

v{ ~~ The rapidity with which homozyqo~ can be . L

70

r~.: .. '""'t7 t... produced -i-n" anther cui ture is

~v:;s o-f this tech in i que. In

plant cells, a single cell can

hybrid plant, with rull genetic

one o-f the most important

view o-f the totipotency o-f

be considered as an intact --·- . ~t-J

diversity. In P.91-.I-e{culture /

o-f hybrid plants, ~ent geAOlyp~, 1 ml o-f medium,

may be su-f-ficient -for the cultivation o-f thousands o-f plantlets.

Haploid tissues are quite susceptible to change in ploidy

level during cell prol i -fer at ion and growth in vitro ( Helchers

and Bergman 1958 ) . In the present investigation, homozygosity

was observed in the double haploid lines as evidenced -from lack

o-f segregation within the lines.

insigni-ficant di-f-ference (CD)

signi-ficant di-f-ferences between the double haploid lines indicate •

to their origin -from di-f-ferent recombinant~ With a view to trans-ferring cold tolerance to high

yielding background, IR70 , an elite IRRI line and Khonorullo, a

local cold tolerant cultivar were crossed. The F1 hybrid showed

hybrid vigour and per-formed better than the parents. F 1 anthers

o-f the cross IR70 x Khonorullo were cultured in -four semisolid

( Gs , E24 LB and MP2) media. Percentage o-f callusing ranged

-from 11.7 ( in G5 medium ) to 33.6 ~ ( in E24 medium )

The results o-f this investigation show that higher

-frequency o-f callus induction and subsequent green plant

71

regen'eration -from anthers o-f the hybrid between two varieties

could be acheived by proper combination o-f growth regulators in

the induction medium. By and large LB and MP2 media were -found to

support callusing in IR lines. Green plant regeneration was

obtained -from calli inoculated mostly in LB medium ( with lmg/1

2,4-D and 3mg/l NAA ). Highest -frequency o-f callusing i.e., 33.6

'l. was observed in crossed anthers o-f IR70 x Khonorullo on E24

medium which contained 1 mg/1 2,4-D and 0.5 mg/1 IAA.

In anther culture, selection can start even in the

1st. ( DHl ) generation itsel-f, because it uncovers both the

genotypic and ~henotypic values even o-f additive genes in this

generation. Using / single seed descent ( SSD ) method o-f

breeding with 200 recombinant genotypes, one homozygous 1 ine

will be -found, with a probability o-f 68% -for characters coded by

48 independent genes (Fa-pou§nt-weh~ and Wellzel, 1990). The SSD

method is comparable with haploid method, but in SSD, selection

starts in F6 , whereas in DH progeny, one can success-fully select

at DHl. Repeated rounds o-f recombination in F2 and in F3 , result~

in too much recombination between desired linked genes. This is

the disadvantage o-f classical backcrossing where there is

possibility o-f important characters to be lost, because the

di-f-ference in the progeny are too small -for e-f-fective selection.

Haploids can also increase the e-f-ficiency -for most complex

breeding problems that is the trans-fer o-f quantitatively

72

q~g_~s to cultivars. In the pt·esent investigation nine out o-f ...._____--~~-----~--------

21 lines regenerated, showed promising characters. Seeds o-f these

lines showed di-f-ferences in agronomic characters -from each other

indicating -from di-f-ferent recombinants.

Panicles di-f-ferent lines showed mixed

characters1i.e., -from compact (in IR70) to open (in Khonorullo).

J.... Same was the case with shape and size o-f the grains. It ranged

-from bold (like Khonorullo) to slender (like IR70). The

endosperm colour ranged f-rom dark red (Khonorullo) to

white (IR70) in the parents. Among doubl~ haploid lines, DH no.

10 and 21 showed white kernel grains whereas all the other lines

showed light red coloured endosperm.

In the present study, IR70 and Khonorullo and

theit· Fl hybrids showed partial spikelet -fertility ( 8.1, 60.7

and 62.2%, respectively ) because o-f suboptimal temperature

prevailing throughout the li-fe cycle o-f the plants. In contrast,

the androgenic double haploid lines recorded a signi-ficant

increase in spikelet -fet·t i 1 i ty which ranged -from 77.3%

(line 1 ) to 94.8% ( line 10 ) . This was because o-f selection

-for desirable DH 1 ines. Maximum number o-f qrains per panicle

numbering 203 was recorded in DH line 1 as compared to 123 and

145 in IR70 and Khonorullo respectively. Increase in 1000 seed

weight was also observed in three DH lines (line no. 7, 10 and

16) as compared to the parents. Maximum 1000 seed weight was

73

recorded in line No.7 ( 30.8 gm.) and it ranged ~ram 19.6 to

30.8 gm in DH lines as compared to 25.6 and 24.3 gm in the case

o~ IR70 and Khonarullo, respectively. These results suggest that

sterility barrier

~ixation o~ ~ertile

(~or realising genetic

hamazyqous lines) in an

recombinants and

indica-japanica

hybridization programme could be overcome through F1 anther

culture technique.

4.5 Fiald evaluation and performance of double haploid (DH)

lines

Days to 50% ~lowering in double haploid lines

ranged ~rom 95 to 111 days in lines 10 and 19, respectively, and

it remained with in the limit o-f both the parents which was 95

days in case a~ Khanorullo and

(Fig. 71).

111 days in case o~ IR 70

Range o~ plant height also remained well within

the parents which was 57 em in case o~ IR 70 and 129 in case a~

Khonorullo. Plant height ranged ~rom 67 to 101 em in case o-f

double haploid lines(Fig. 72). Lea~ length was reduced in mast

cases but in line no. 19, 37 em lang lea~ was observed as

compared to 32 em in IR 70 and 27 em in Khanorullo.

Higher number o-f ear bearing tillers (EBT) was

observed in line nos. 15. 19 and 21 whereas others showed

74

redrction in number o~ EBT than the parents which

IR 70 and Khonorullo respectively.

is 5 and 4 in

Length o~ panicle remained within the ranqe o~

parents except in line no 6 and 13 where it was reduced to 17 and

19 em respectively • Panicle weiqht also remained well within the

range o~ the parents in all 1 ines. Yield on the other hand,

increased in all the DH lines (Table 8).

There was a signi~icant increase in 1000 seed

weight in lines 7 and 10 ( 30.8 and 30.0 g respectively) than the

parents which was 25.6 and 24.3 qm in IR 70 and Khonorullo,

repectively (Fig.73).

There was also a sharp increase in total qrain and

pe,fcent -fertility in many lines. In line no 1, there were 203

qrains as compared to 123 and 145 in IR 70 and Khonorullo

respectively. A record number o~ 94.8% ~ertile grains was

recorded in line no 10 ~allowed by 91.4% in line no 7 as compared

to 8.1 and 60.7% in IR 70 and Khonorullo respectively. All other

lines also showed increase in ~ertility over their parents

(Fig.74).

Slender grains were observed in line no 10 which

is a distinct character o~ IR 70. Medium type o~ grain was

observed in 1 ine nos 1, 7, 15 and 21 whereas bold qrains were

observed in rest o-f the lines.

75

White kernel grains (like IR 70) were observed in

1 ine no 10 and 21 Host o-f the other 1 ines showed 1 ight red

colour kernel except in 1 ine no 16 and 19 where deep red

colour (like khonorullo) kernel was observed. Panicle shape,

grain type and kernel colour o-f di-f-ferent double haploid lines,

together with their parents are presented in Figs. 34 - 49.

A wide genetic base with acceptable level o-f

productivity is essential in the c:r-op populations under

improvement. There-fore, the study o-f variability in crops is

essential TOr selecting the desirable genotypes. Knowledge OT the

nature and magnitude o-f genetic variation govern i nq the

inheritance o-f quantitative characters like yield and its

components is essential TOr e~~ecting genetic improvement. Since

only the genetic portion o-f the total variability contributes to

grain yield under selection, the importance to breeders o-f

in-formation about the parameters o-f the genotype - environment

complex is clear (Allard, 1960). Burton (1952) has suggested that

the genetic c:oeTTicient o-f variation together with the

heritability estimates would give the best picture o-f the amount

o-f advance to be expected -from selection.

polygenic: character is the ultimate

components.

Yield being a complex,

expression OT many

Highly signi-ficant di-f-ferences were observed among

the DH 1 ines and parents -for all the chat-acters studied. The

76

pheno~ypic coe-f-ficient o-f variation (PCV) was higher than the

genotypic coe-f-ficient o-f variation indicatinQ the larger

in-fluence o-f environment on the expression o-f characters.

Bhattacharjee and Mishra (1981) and Gomathinayagam et al. (1990)

have made the same observations in rice genotypes. All the

characters except number o-f ear bearing tillers/plant (EBT)

showed wide variation. In the present study the level o-f GCV and

PCV were not o-f high order.

The broad sense heritability estimates ranged -from

25.01~ in EBT/plant to 99.53/. in spikelet -fertility. High

heritability estimate was noticed with panicle weight, grain

yield, plant height, grain weight/panicle, spikelet -fertility,

1000 seed weight and spikelet/panicle while panicle length had

only moderate heritability (51.67'l.). According to Burton (1952) a

character having high GCV value with high heritability would be

more valuable -for selection. According to Johanson et al. (1955)

high heritability coupled with high genetic advance would be more

use-ful in predicting the results and e-f-fects o-f selection. High

heritability and genetic advance was shown only by

spikelet/panicle and plant heiqht indicating the preponderance o-f

additive gene action (Panse, 1957). The ranqe o-f genetic advance

as percentage o-f mean (GA'l.) was 3.06'Y. (spikelet -fertility) to

74.06/. (grain weight/panicle). The relatively lower estimates o-f

heritability and GA -for the EBT as observed in the present study

77

was reported by Bhattacharjee and Mishra (1981). The hiqher value

o~ heritability exhibited by plant height was in accordance with

the ~indings o-f Swamy~o and Gfud (1969). High heritability

coupled with high genetic gain observed in the present study was

also reported by Shukla et al. ( 1972) and Chaudhury et al. '> I

(1973). Since the heritability (Hk) is an estimate o-f the

heritable portion o+ the vat- i at ion , a

quantitative characters was usefu 1 as thay

select ion on the phenotypic perfonnance.

high ~~) value. in

pt-ov ide the basf o~

~~ b~i~icant positive association (amonq DH lines)

was sl::tewrr )ir<j yield/p 1 ant and panicle weight. There was also

positive, siqni~icant correlation between panicle weight and

qrain weight/panicle~ between spikelets/panicle and panicle

weight and between 100 seed weiqht and spikelet ~ertility. The

interrelationship between 100 seed weight and grain

weight/panicle was negatively signi~icant.

When the cor1elations were ~urther par~ioned, t.-. maximum po~itive direct e-ffect contribution was given by panicle

weight ~allowed by plant height and panicle length. Even thouqh

spikelet/panicle showed positive correlation, its direct

contribution was only on ihe negative side. Hundred seed weiqht

and EBT had direct neqative contribution while the remaininQ

traits were either in negative direction or o~ low magnitude.

~~ ~or panicle weiqht the

78

Except TOr panicle weight the indirect eTTect via grain

weight/plant and spikelet/panicle all other indirect eTTects were

also either very low or negative.

From the present study it is evident that the

perTormance OT DH lines possessing cold tolerance was quite -t~

satisTactory when compat-ed Woi.t.b. both the parents. The DH 1 ines

showed an increase in yield to the tune OT 79.237. over parent 1

(IR 70) and 22.857. over parent 2 (Khonorullo), respectively.

However, multilocational testing will be needed Tor

recommendation OT these 1 ines TOr cultivation in mid and low

altitude areas.

79

SUMMARY

Anther culture-mediated breeding has now become a

routine procedure used -for compressing breeding cycle in crop

plants in general and rice in particular. The major advantage o~

androgenesis is the reduction o~ time needed to obtain· pure

lines. Doubled haploid lines are completely homozygous and are

easier to study the genetic e-f~ects because dominance e~~ects are

eliminated. The technique is more use~ul in situations where

rapid generation advance is not possible because only one crop

can be grown every year.

80

Rice grown in high altitude areas o-f North Eastern__

Hills o-f India gets exposed to cold at -floz-ing/(1'"~-~=ni-.:;qJ phase

incomplete panicle exsertion, and -faces the problem o-f

asynchronous -flowering and poor seed setting. This results in

yield losses ranging -from 20 - 507. depending on severity o-f cold

stress. In addition, due to long crop season and severe winter,

only one crop o-f rice can be grown each year in these areas and

thus conventional breeding requires long time.

In order to use the technique o-f anther culture in

breeding rice -for these areas , we -first standardized conditions

-for anther callusing in local vari~ties o-f rice. All together,

six varieties and F 1-derived anthers (o-f IR70 x Khonorullo) were

tested -for anther callusing on 5 semisolid and 2 liquid media.

RCPL1-3c was -found the most responsive -followed by Khonorullo,

RCPL 1-2C and RCPL 1-1C. Apart -from -favourable climatic

conditions characterized by temperature (min 17.2•c, max 26.3°C),

humidity (83 1007.), sunshine hours (3.10/day); cold

pretreatment ~ (S•c -for 10 days), inoculation o-f anthers at

mid uninucleate stage and low concentration o-f nitrogen (26 to 35

mM) were the most important -factors required -for high -frequency

callus induction.

Early trans-fer o-f primary calli to regeneration

media was -found essential -for production o-f green plants. In

81

addition, exposure o~ calli to continuous light ~or ~irst ~ive

days a~ter trans~er to regeneration medium and then to a 16/8

hour 1 ight /dark period was ~ound to be the best ~or maximum

green plant regeneration. Majority o~ the regenerated plants were

~ound to possess diploid chromosome number indicating doubling o~

chromosomes during culture.

In order to trans~et- cold tolerance to high

yielding genetic background, anthers o~ F 1 plants obtained ~rom

cross, IR70 x Khonorullo; were cui tured and the double haploid

lines, thus obtained, were screened ~or desirable traits. Out o~

21 DH lines, 9 were ~ound promising and they were ~ield evaluated

~or yield and yield contributing characters. Among the 9 1 ines,

line no. 10 and 21 were white kernelled, a desirable character,

coupled with high spikelet ~ertility (94.8 and 81.54X,

respectively). Line no.1 was although red kernelled yet the

highest yielder. Field evaluation at Barapani (1000m msl) showed

line no.t, 19 and 21 as high yielders. Altogether_, DH lines

showed an increase o~ 79.2 and 22.85X in yield over parent

1(1R70) and parent 2 (Khonorullo), respectively.

Highly signi~icant di~~erences were observed among

the DH lines ~or the agronomic characters studied. The phenotypic

coe~~icient o~ variation was more than the genotypic coe~~icient

o~ variation indicating a high in~luence o~ environment. High

heritability estimates were recorded ~or several characters like

82

panicle weight, grain yield, plant height, grain weight/panicle,

spikelet ~ertility, no.o~ spikelet/panicle and 1000 grain weight.

High heritability and genetic advance, observed in plant height

and no. o~ spikelet/panicle, suqqested additive gene action.

Genetic advance as percentage o~ mean was highest _).R_.r' grain

weight/panicle.

Among the DH lines, yield/plant and panicle weight

showed signi~icant positive association. Characters, like panicle

weight and grain weight/panicle, no.o~ spikelet/panicle, panicle

weight, 100 seed weight and spikelet ~ertility showed signi~icant

positive correlation. When correlations were partitioned, maximum

positive direct e~~ect on yield was shown by panicle weight.

Plant height and panicle length were the next two higher

contributors. Hundred seed weight and number o~ ear bearing

tillers contributed negatively.

Field evaluation o~ the DH lines showed that these

lines possess cold tolerance and per~ormed better than both the

parents. Multilocational testing is being conducted at low and

mid altitude areas ~or identi~ying the high yielding DH lines

coupled with desirable attributes.

83

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